PPARα IS ESSENTIAL TO ORGAN SYSTEM FUNCTION AND SURVIVAL IN SEPSIS
S.W. Standage*1, 2, R. Waworuntu2, M.A. Delaney2, S. Maskal2, J.S. Duffield2, W.C. Parks2, and J.K. McGuire1, 2. 1Seattle Children’s Hospital, Seattle, WA, 2University of Washington, Seattle, WA
Objective: The peroxisome-proliferator activated receptor alpha (PPARα) is a nuclear hormone receptor that regulates metabolism and inflammation. PPARα is significantly downregulated in circulating leukocytes from children with sepsis. When subjected to sepsis by cecal ligation and puncture (CLP) PPARα null (KO) mice have much greater mortality than wildtype (WT) mice and demonstrate evidence of immune dysfunction. We sought to determine the cause of this difference.
Methods: We generated PPARα bone marrow chimeric mice in 4 groups: 1) WT mice with KO marrow, 2) KO mice with WT marrow, 3) WT mice with WT marrow, and 4) KO mice with KO marrow. We induced sepsis with CLP and measured survival for 7 days. Separately, non-transplanted WT and KO mice underwent CLP. Plasma biomarkers of organ injury were assayed and tissue collected at specific time points for histologic evaluation by a blinded veterinary pathologist as well as for gene expression analysis.
Results: KO mice had dramatically reduced survival compared to WT mice irrespective of the PPARα status of the bone marrow they received (3% vs 63%, p < 0.0001). No difference in survival was observed between KO mice that received WT vs. KO marrow or in WT mice receiving WT vs. KO marrow. In septic, non-transplanted mice at 24 hours, KO mice had significantly elevated cardiac troponin levels compared to WT mice (1.7 vs 0.4 ng/mL, p = 0.028). Markers of renal dysfunction, BUN and creatinine, were also raised in the KO mice (p = 0.017 and 0.034 respectively). A non-significant trend toward increased ALT levels was noted in KO mice. Cardiac histologic injury scores were much higher in the KO than in the WT specimens (rank-sum 120.5. vs 15.5, p = 0.001). Areas of frank myocardial necrosis were evident in the KO hearts. Renal injury scores were also elevated in the KO mice (p = 0.04), but were not as dramatic. Minimal changes were seen in the liver and lung. Expression of transcription factors and enzymes related to fatty acid oxidation was profoundly downregulated.
Conclusions: Mice lacking PPARα are more sensitive to sepsis primarily due to organ system failure rather than any immunologic perturbation. The heart and kidney seem to be most involved. These organs rely greatly on fatty acid oxidation for energy provision necessary to maintain adequate function. Suppression of those pathways in sepsis may account mechanistically for their dysfunction.
MPLA ENHANCES NEUTROPHIL RESPONSES TO INFECTION AFTER BURN
J. Bohannon, A. Hernandez, L. Luan, and E. Sherwood*. Vanderbilt University Medical Center, Nashville, TN
Infection is the leading cause of death in severely burned patients that survive initial injury. Prior sensitization with immune modulator monophosphoryl lipid A (MPLA) is known to induce resistance to subsequent bacterial challenge, associated with an enhanced ability to clear bacteria following systemic challenge with Pseudomonas aeruginosa. MPLA is relatively innocuous and is currently used in human vaccine preparations, making it well-suited for clinical usage to enhance immune responses to infection. Previous studies have shown that treatment of mice with MPLA enhances bacterial clearance, leading to improved survival in various models of sepsis including P. aeruginosa systemic and burn wound infections. The current study was aimed to better define the mechanisms responsible for improved bacterial clearance and survival in MPLA-treated burn-infected mice. Mice underwent severe burn injury to ~35% total body surface area, followed by systemic treatment with MPLA or lactated Ringers (LR; control) for two days. Mice were then inoculated with P. aeruginosa, topically or intraperitoneally, and responding phagocytic immune cells were measured in bone marrow, blood and sites of infection. MPLA increased the number of neutrophils in the blood, at the site of administration and at sites of infection. Conversely, neutrophil numbers in the bone marrow decreased following MPLA. Moreover, MPLA induces G-CSF, a cytokine necessary for the mobilization and expansion of neutrophils in mice. Neutralization of G-CSF prior to MPLA administration ablated MPLA-mediated survival and bacterial clearance in burn wound-infected mice (Fig 1). G-CSF was required for MPLA-induced mobilization of neutrophils out of the bone marrow and into the blood. G-CSF neutralization reversed MPLA-induced attenuation of plasma IL-6 levels in response to infection. This evidence suggests that G-CSF facilitates MPLA-induced mobilization of neutrophils from the bone marrow into systemic circulation, allowing for a more rapid response to infection.
ENDOTHELIAL PROGENITOR CELLS IMPROVE ENDOTHELIAL FUNCTION AND SURVIVAL IN SEVERE SEPSIS
H. Fan*1, A.J. Goodwin1, S. Guan2, 1, P. Halushka1, B. Zingarelli*3, and J. Cook*1. 1Medical University of South Carolina, Charleston, SC, 2Jilin University, Changchun, China, 3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Endothelial dysfunction is a critical factor in the transition from sepsis to multiple organ dysfunction syndrome (MODS). We examined the hypothesis that endothelial progenitor cells (EPCs) protect mice from cecal ligation and puncture (CLP)-induced sepsis and improve endothelial barrier function. EPCs were isolated and cultured from human cord blood. CD-1 mice underwent CLP and were then administrated EPCs (1×106 cells) or vehicle i.v. and SQ imipenem post-CLP. Survival, plasma AST, ALT, creatinine and IL-10 levels were determined. The level of the endothelial specific micro RNA, miR-126, was determined in circulating exosomes. Lung histopathology and vascular leakage were determined by H&E stain and Evans Blue dye. In subsequent in vitro studies, the effects of EPCs on the permeability of HUVECs was determined by transendothelial electrical resistance (TEER) while the effect of miR-126 on VCAM-1 expression in HUVECs was determined. EPCs significantly improved survival compared to vehicle controls (87% vs. 33%, P<0.05). EPCs reduced plasma AST and creatinine levels (45±5% and 55±6%, respectively) and increased IL-10 levels (3.3±0.5 fold). EPCs increased plasma exosome levels of miR-126 levels (3.1±0.8 fold). EPCs also decreased vascular leakage in the lung (55±2% decrease vs. vehicle treated CLP group) and reduced lung damage. EPCs markedly reduced HUVEC permeability as evidenced by increased TEER whereas addition of equivalent numbers of HUVECs as a control failed to alter permeability. Transfection of miR-126 mimic significantly decreased TNF-α-induced VCAM-1 expression (64±20% decrease vs. control). These data demonstrate that EPCs improve organ failure and survival in murine severe sepsis. The effect maybe a consequence of improved endothelial function mediated by altering endothelial specific miRNAs and/or anti-inflammatory cytokines. (Supported by NIH GM27673, GM67202)
PHARMACOLOGICAL TRANSLATION OF TRANSDERMAL NEURONAL STIMULATION IN SEPSIS
L. Ulloa*, J. Inclan-Rico, P. Morcillo, G. Pena, and R. Torres-Rosas. New Jersey Medical School Rutgers, Newark, NJ
Sepsis, a leading cause of death in the ICU, is characterized by inflammatory responses leading to multiple organ failure. The inhibition of specific cytokines has provided promising results in experimental sepsis, but this strategy has failed in multiple clinical trials. Our recent studies indicated that the vagus nerve induces a comprehensive mechanism to control inflammation at different levels and can improve survival in experimental sepsis (1,2). However, the clinical implications of this mechanism are limited by the surgical procedure required for direct nerve stimulation. We now report that transdermal neuronal stimulation with electroacupuncture prevents systemic inflammation and improves survival in experimental sepsis. Electroacupuncture inhibited the production of multiple inflammatory factors by inducing the production of dopamine from the adrenal medulla (3). Given that adrenal insufficiency is a common characteristic in septic patients, we analyzed whether adrenolectomized mice can mimic the clinical settings. Our results indicated that adrenolectomized animals were more susceptible to polymicrobial sepsis. From a pharmacological perspective, dopamine inhibited macrophage’s activation via D1-like dopaminergic receptor. D1 dopaminergic agonist inhibited systemic inflammation and rescued adrenolectomized animals from polymicrobial sepsis (3). Studies funded by the NIH-GM084125.
1. Ulloa L. Nat Rev Drug Discov (2006), 4: 673-684.
2. Peña G, et al. Journal of immunology (2011), 187: 718-725.
3. Torres-Rosas R, et al. Nat Med (In press)
FOREBRAIN CHOLINERGIC PATHWAYS CONTROL PERIPHERAL CYTOKINE RESPONSES
V.A. Pavlov*1, 2, H. Silverman1, 2, M. Dancho1, A. Regnier-Golanov3, M. Ochani*1, S.S. Chavan*1, E. Golanov4, V.F. Prado5, M.A. Prado5, and K.J. Tracey*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Hofstra North Shore-LIJ School of Medicine at Hofstra University, Hempstead, NY, 3Baylor College of Medicine, Houston, TX, 4The Houston Methodist Research Institute, Houston, TX, 5Robarts Research Institute, Department of Physiology and Pharmacology, Department of Anatomy & Cell Biology, The University of Western Ontario, London, ON, Canada
Brain cholinergic mechanisms play an important role in the neural control of peripheral cytokine release and inflammation (Nat Rev Endocrinol, 2012, 8:743). We have previously shown that activation of brain cholinergic signaling by the centrally-acting acetylcholinesterase inhibitor galantamine suppresses peripheral pro-inflammatory cytokine release through a neural, brain muscarinic receptor-mediated and vagus nerve-dependent mechanism (Brain Behav Immun. 2009, 23:41; Mucosal Immunol. 2013, Epub ahead of print). Here, we provide insight into the brain pathways involved in this regulation. We used mice with conditional knockout of the vesicular acetylcholine transporter - an important determinant of acetylcholine release, in basal forebrain cholinergic neurons. These VAChTNkx21-Cre-flox/flox mice and wild type VAChTflox/flox mice were treated with galantamine (3mg/kg, i.p.) or saline 1h prior to LPS (1mg/kg, i.p.) administration; mice were euthanized 1.5h post LPS and serum cytokines determined. Serum TNF and IL-10 levels in VAChTNkx21-Cre-flox/flox mice were significantly higher and lower respectively, as compared to VAChTflox/flox mice during endotoxemia. Moreover, in contrast to VAChTflox/flox mice, galantamine failed to reduce serum TNF in VAChTNkx21-Cre-flox/flox mice. Furthermore, we studied whether electrical stimulation of the medial septum, an important basal forebrain cholinergic region, altered cytokine levels during endotoxemia. Medial septum stimulation 1h prior to endotoxin injection significantly and stimulation parameter-dependently lowered serum TNF and other pro-inflammatory cytokine levels, as compared to sham-stimulated controls. These results demonstrate a previously unrecognized role of basal forebrain cholinergic pathways in the regulation of peripheral inflammation. Our findings are of interest for development of brain region specific modulation in novel anti-inflammatory approaches. This study was funded in part by NIH/NIGMS and CIHR.
MUSCLE CONTRACTION-INDUCED SIGNALING TO PROTEIN SYNTHESIS IS IMPAIRED DURING SEPSIS
J. Steiner and C.H. Lang*. College of Medicine at Penn State Hershey, Hershey, PA
Skeletal muscle atrophy and weakness induced by sepsis is due, at least in part, to a decrease in mTOR-mediated protein synthesis. Anabolic signaling to mTOR via nutrients and growth factors is impaired during sepsis preventing stimulation of protein synthesis. Electrically stimulated muscle contraction is a potential clinical therapy; however, whether sepsis alters contraction-induced anabolic signaling is unknown. The purpose of this study was to determine whether sepsis impairs mTOR signaling following muscle contraction. Cecal ligation and puncture was used to produce polymicrobial peritonitis in male C57BL/6 mice; time-matched, pair-fed controls (CON) were included for comparison. 24 hours after sepsis, the right hind limb was electrically stimulated via the sciatic nerve to evoke maximal muscle contractions (10 sets of 6 contractions), and the gastrocnemius muscle was collected 2 h later. Muscle contraction increased the phosphorylation of mTOR substrates central to initiation of protein translation including S6K1 Thr389 (8-fold), S6K1 Thr421/Ser424 (7-fold), ribosomal protein S6 (rpS6) Ser240/244 (8-fold) and 4E-BP1 Ser65 (11-fold) in CON mice. However, sepsis blunted the contraction-induced phosphorylation of S6K1 Thr389 (67%), S6K1 Thr421/Ser424 (46%), ribosomal protein S6 (rpS6) Ser240/244 (54%) and 4E-BP1 Ser65 (85%). Conversely, sepsis did not modulate protein elongation, as eEF2 Thr56 phosphorylation was decreased similarly by muscle contraction in CON and septic mice. mTOR-independent signaling remained intact as the phosphorylation of ERK Thr202/Tyr204 in both CON and septic muscle was increased similarly by muscle contraction. Sepsis also increased mRNA expression of IL-6 (72-fold) and TNF-α (6.5-fold), while only TNF-α was increased by contraction in both CON (3-fold) and septic (1.6-fold) mice. Injection of the mTOR inhibitor, Torin2, in separate mice indicated contraction-induced increases in S6K1, rpS6, and 4E-BP1 were mTOR-mediated. These findings demonstrate that resistance to muscle contraction-induced anabolic signaling during sepsis is predominantly mTOR-dependent and that therapies to attenuate muscle atrophy may need to focus on restoring mTOR activation in response to anabolic stimuli.
MYOSIN LIGHT CHAIN KINASE KNOCKOUT CONFERS A SURVIVAL ADVANTAGE IN POLYMICROBIAL SEPSIS ASSOCIATED WITH UPREGULATION OF ZO-1 AND CLAUDIN-15
A.C. Lorentz, Z. Liang, R. Mittal, and C.M. Coopersmith*. Emory University, Atlanta, GA
Background: Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the regulation of intestinal barrier function. We have shown that MLCK knockout (MLCK-/-) mice have decreased mortality and gut permeability after CLP (unpublished observations) although the mechanisms responsible for these observations are unknown.
Methods: MLCK-/- and C57Bl/6 (WT) mice underwent 2x25 CLP and were sacrificed 24 hours later. Jejunum, serum, and peritoneal fluid (PF) were collected and analyzed by immunohistochemistry, multiplex cytokine assay and Western Blot.
Results: MLCK-/- mice had increased levels of jejunal tight junction (TJ) proteins, ZO-1 and Claudin-15, expression 24 hours after CLP (1.0 ± 0.5 vs. 2.7 ± 1.4, and 1.0 ± 0.8 vs. 2.6 ± 1.0 Density/β-actin respectively, p<0.05 for both) by both Western Blot and immunohistochemical staining. Apoptosis and proliferation were similar between MLCK-/- and WT mice as measured by caspase-3 and BrdU respectively (6.9 ± 2.9 vs. 11.6 ± 7.9 apoptotic cells/100 crypts, and 731 ± 165 vs. 681 ± 103 proliferating cells/100 crypts, p=ns). Villus length was also similar between MLCK-/- and WT mice (304 ± 35 vs. 269 ± 46 μm, p=ns). IL-6 levels were lower in both serum and PF in MLCK-/- mice (63641 ± 71698 vs. 21567 ± 21582 pg/ml, and 55044 ± 30257 vs. 20118 ± 18315 pg/ml, p<0.05, 0.01, respectively). No differences were noted in IL-4, IL-10, IL-13, INFγ and TNF-α between MLCK-/- and WT mice.
Conclusions: The survival advantage conferred by knocking out MLCK is associated with alterations in TJ proteins ZO-1 and Claudin-15 but is independent of gut apoptosis and proliferation.
ELECTROPORATION-MEDIATED DELIVERY OF THE NA+/K+-ATPASE PUMP GENES CHANGES BRONCHIAL ALVEOLAR LAVAGE CELLULAR COMPOSITION AFTER LUNG CONTUSION INJURY
D.A. Machado-Aranda*, N. Talarico, V. Dolgachev, S. Madathilparambil, B. Thomas, M. Hemmila, and K. Raghavendran*. University of Michigan, Ann Arbor, MI
Objective: Lung Contusion (LC) is a leading cause of admission into the ICU after blunt thoracic trauma. Working with a murine model of LC, we have found a sequential reduction in the number of alveolar macrophages (aM∅∅) with time and these aM∅∅ are important in resolution of the acute inflammatory response. Additionally, electroporation-mediated induction of the Na+/K+-ATPase pump significantly improved lung injury and reduced inflammation. We set out to mechanistically examine how electroporation mediated gene transfer of the Na+/K+-ATPase modulates acute inflammation following LC.
Methodology: Following LC, plasmid DNA encoding for the α/β subunits of the Na+/K+-ATPase (LC + pump EP) were delivered into the trachea, and transthoracic electroporation was performed. A non-encoding DNA plasmid was used as the Control (LC + sham EP). At 24 hr after LC, qualitative and quantitative analyses of BAL cells were performed using Flow Cytometry. In additional experiments, gene transfer efficiency was assessed by co-delivery of luciferase reporter gene.
Results: LC + sham EP produced a significant increase in the number of cells not labeled with aM∅∅ or PMN differentiation markers. Importantly, the LC + pump group, showed an increase for PMN and aM∅∅ both for total numbers as well as live cell fraction. This change was particularly evident for aM∅∅. No significant differences were found in the number of necrotic or apoptotic PMN or aM∅∅. Following LC the integrity of the plasmids in the BAL diminished over time. Finally, LC resulted in a one order of magnitude decrease in reporter transgene expression compared to uninjured control.
Conclusions: Despite a reduction in transgene expression following LC, the enhanced recovery observed with overexpression of the Na+/K+-ATPase pump is in part related to increased recruitment and activation of the aM∅∅.
ELECTROPORATION GENE DELIVERY ATTENUATES ARDS
B. Emr1, M.C. Kollisch-Singule1, S. Roy1, L.A. Gatto3, M. Barravecchia2, J. Liu1, G. Wang*1, J. Satalin1, K. Snyder1, G. Nieman*1, and D.A. Dean2. 1SUNY Upstate, Syracuse, NY, 2University of Rochester, Rochester, NY, 3SUNY Cortland, Cortland, NY
Introduction: Acute Respiratory Distress Syndrome (ARDS) has mortality up to 40% and 3.6 million hospital days yearly. The initiating event is disruption of alveolar-capillary interface causing leakage of edema into alveoli.
Hypothesis: Electroporation mediated gene delivery of epithelial sodium channel (ENaC) and Na,K-ATPase into alveoli would improve alveolar clearance of edema and attenuate ARDS.
Methods: Pigs were anesthetized, instrumented, and secondary lung injury was induced. The superior mesenteric artery was clamped for 30 min and peritonitis induced by bowel perforation and fecal clot. All animals were ventilated with low tidal volume. Four hours after injury animals were randomized into two groups: 1. Treatment Na,K-ATPase/ENaC plasmid (n=5), 2. Empty plasmid (n=5). Plasmids were delivered to the lung using a bronchoscope. Then electric field was delivered with defibrillation across the chest with 8 square wave pulses. Following electroporation pigs were monitored for 48 hrs.
Results: PaO2/FiO2 ratio and lung compliance were significantly higher in the treatment group. Lung Wet/Dry Ratio, measure of pulmonary edema, was significantly lower in the treatment group. Relative expression of the Na,K-ATPase transgene compared to that of the endogenous b-actin protein was significantly higher throughout the lungs of animals receiving the treatment plasmid than those receiving the empty plasmid (6.15±0.89 vs 1.00±0.08, p<0.001). Quantitative histopathology revealed significant reduction in intra-alveolar fibrin demonstrating reduction in edema (fig). Analysis of bronchoalveolar lavage showed increase in surfactant protein B in the treatment group (p<0.02). Survival was improved in the treatment group (p<0.01).
Conclusion: Transfer of Na,K-ATPase/ENaC plasmids into alveolar cells improved lung function, reduced fibrin deposits, decreased lung edema, and improved survival. Gene therapy can attenuate ARDS pathophysiology suggesting a potentially exciting new therapy for ARDS patients.
PD-L1 REGULATES THE TREGS’ CAPACITY TO REPRESS SHOCK/SEPSIS INDUCED INDIRECT LUNG INJURY (IALI) BY RECRUITING PHOSPHATASE SHP-1
L. Tang1, J. Bai1, C. Chung*2, J. Lomas-Neira2, Y. Chen2, X. Huang2, and A. Ayala*2. 1Department of Emergency Medicine and Critical Care, Shanghai East Hospital/ Tong Ji University, Shanghai, China, 2Department of Surgery, Division of Surgical Research, the Alpert School of Medicine at Brown University/Rhode Island Hospital, Providence, RI
We recently reported that adoptively transferred (AT) exogenous CD4+CD25+ regulatory T cells (Tregs) to wild type (WT) mice can directly act to repress shock/sepsis induced experimental iALI and this is mediated in part by programmed cell death receptor 1 (PD-1). In this study, we further determine whether recipient mouse lacking PD-L1, one of the primary ligands for PD-1, contributes to the manipulation of the Tregs’ capacity to repress lung injury. To do this, Tregs isolated from the spleen of WT mice were AT into PD-L1-/- mice subjected to hemorrhagic shock [Hem] (during their resuscitation period) that were subsequently subjected to cecal ligation and puncture/septic challenge (24 h post-Hem) to induce iALI. We found that in PD-L1-/- recipient mice, AT WT-Tregs lost the ability to reverse the development of iALI seen in WT recipient mice (i.e., no reduction of lung injury indices assessed by histology and vascular leakage; failure to decrease the lung neutrophil influx [MPO activity] or the rise in lung apoptosis). Also a significant increase of interlukin-1β (IL-1β) and KC, but no changes of IL-6, IL-10 and IL-17A levels in lung tissues were seen in these mice compared with iALI mice without AT of Tregs. Furthermore, we noted that the lung tissue cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1), but not SHP-2, was recruited/activated with the AT of Tregs in PD-L1-/- iALI mice. Finally, through local depletion of CD4+ T cells or CD25+ (Tregs) in the lung, prior to inducing iALI, we found that SHP-1 activation was associated with the loss of Tregs’ protective effects in vivo. Collectively, our data reveal that PD-L1 is a critical modulator of Treg’s ability to suppress iALI and this appears to involve SHP-1 activation.
BLOCKING COLD-INDUCIBLE RNA-BINDING PROTEIN PROTECTS LIVER FROM ISCHEMIA-REPERFUSION INJURY
A.J. Godwin1, W. Yang*1, 2, A. Khader1, Z. Wang1, F. Zhang2, J. Nicastro1, G. Coppa1, and P. Wang*1, 2. 1North Shore-LIJ Hofstra School of Medicine, Manhasset, NY, 2Feinstein Institute of Medical Research, Manhasset, NY
Introduction: Hepatic ischemia-reperfusion (I/R) injury is an unavoidable complication, which may occur due to trauma, or during liver resections and transplantation. The pathogenesis underlying I/R is very complex; however, the generation of sterile inflammatory response is a key component of this condition. Cold inducible RNA-binding protein (CIRP) is a nuclear protein which has been recently identified as a novel inflammatory mediator. We hypothesized that CIRP was a potential mediator of inflammation after liver I/R, and that blocking CIRP would be protective on I/R-induced injury.
Methods: Male C57BL/6 mice were subjected to partial hepatic ischemia (70%) by microvascular clamping of the hilum of the left and median lobes of the liver for 60 min, followed by reperfusion. Anti-CIRP antibody (1 mg/kg body weight) or vehicle (normal saline) in 0.2 mL was injected via internal jugular at the beginning of the reperfusion. Blood and liver tissues were collected 24 h after I/R for histological evaluation and various measurements. Protein levels were measured by Western blotting or ELISA. The mRNA levels were analyzed by qPCR.
Results: There was a 25% and 69% increase of CIRP levels in the serum after hepatic I/R at 4 h and 24 h, respectively. Administration of anti-CIRP antibody significantly reduced the serum liver injury markers, aspartate aminotranferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), compared to the vehicle group (Table). The serum IL-6 levels in the anti-CIRP group were also significantly lower than the vehicle group (Table). Furthermore, the mRNA levels of IL-6, TNF-α, iNOS, and COX-2 were reduced in the anti-CIRP group in comparison to the vehicle group (Table).
Conclusions: CIRP is released into the circulation after hepatic I/R injury. Anti-CIRP antibody treatment attenuates liver injury induced by I/R through reducing inflammation. Thus, targeting CIRP offers potential therapeutic implications to the treatment of patients suffering from hepatic I/R injury.
ROLE OF SURFACTANT PROTEINS A AND D IN SEPSIS-INDUCED ACUTE KIDNEY INJURY
J. Liu, O. Abdel-Razek, Z. Liu, R.N. Cooney*, and G. Wang*. SUNY Upstate Medical University, Syracuse, NY
Sepsis is a major cause of acute kidney injury (AKI) with high rates of morbidity and mortality. Surfactant proteins A and D (SP-A, SP-D), members of C-type collectin family, play a critical role in host defense and regulating inflammation during infection. Recent studies indicated SP-A and SP-D expression in the kidney. The current study examines the role of SP-A and SP-D in the pathogenesis of sepsis-induced AKI.
Objectives: To investigate the role of SP-A and SP-D in a murine model of sepsis-induced AKI.
Methods: Wild-type (WT) and SP-A/SP-D double knockout (KO) mice were treated by cecal ligation and puncture (CLP) or sham surgery. Histological, cellular and molecular indices of kidney injury were analyzed and compared between WT and SP-A/SP-D KO mice at 6 h and 24 h after CLP treatment. N=8-10 mice/group. Differences were considered significant when p<0.05 by t-test or ANOVA.
Results: 24 h post-CLP, histological analysis shows kidney injury was more severe, kidney function was decreased, serum lactate and blood urea nitrogen(BUN) were higher in septic SP-A/SP-D KO mice than septic WT mice (p<0.01). Kidney wet weight and edema were also increased in septic SP-A/SP-D KO mice compared to septic WT mice 24 h after CLP (p<0.01). Furthermore, apoptotic cells increased significantly in the kidney tissue of septic SP-A/SP-D KO mice compared to septic WT mice at both the 6 h and 24 h time points after CLP. Molecular analysis revealed that the levels of caspase-3 (a biomarker of apoptosis) and NF-kB expression (a biomarker of inflammation) were higher and the level of BCL-2 (a biomarker as inhibitor of apoptosis) was lower in the kidney of septic SP-A/SP-D KO mice when compared to septic WT mice (p<0.01). Different levels of TNF-α and IL-6 in the peritoneal cavity (p<0.01) exist although no significant difference was found in the kidney between septic SP-A/SP-D KO and WT mice. In addition, in vitro studies indicate that bacteria opsonized with septic SP-A/SP-D KO mouse serum induced significantly less cytokine by peritoneal macrophages compared to septic WT mouse serum (p<0.01).
Conclusions: These results suggest SP-A and D attenuate kidney injury by modulating inflammation and apoptosis in sepsis-induced AKI.
HEMOLYSIS EXACERBATES HYPERFIBRINOLYSIS WHILE PLATELOLYSIS SHUTSDOWN FIBRINOLYSIS: EVOLVING CONCEPTS OF THE SPECTRUM OF FIBRINOLYSIS IN RESPONSE TO SEVERE INJURY
H.B. Moore1, E.E. Moore*1, 2, E. Gonzalez1, M.P. Chapman1, C.C. Silliman1, 3, A. Sauaia1, T.L. Chin1, K. Hansen1, and A. Banerjee*1. 1University of Colorado, Denver, CO, 2Denver Health, Denver, CO, 3Bonfils Blood Center, Denver, CO
Introduction: We have recently identified a spectrum of fibrinolysis in response to injury in which there is increased mortality in patients that have excessive fibrinolysis [hyperfibrinolysis (HF)] and impaired fibrinolysis (shutdown). The regulation of the fibrinolytic system after trauma remains poorly understood. Our group’s previous proteomic work identified elevated red blood cell degradation products in trauma patients manifesting HF. We therefore hypothesized that hemolysis (HL) was contributory to the pathogenesis of HF, and further investigated the potential role of the lysis of platelets (PL) considering their central role in the cell based model of hemostasis.
Methods: Citrated blood samples from healthy volunteers were evaluated with Thrombelastography (TEG). A 3cc tube of blood was spun to separate plasma and buffy coat from red blood cells (RBC). RBC were frozen in liquid nitrogen and vortexed to created mechanical non-dilution HL. Platelets from blood donors were prepared in a similar fashion. Assays were performed with citrated whole blood mixed with HL RBC, non HL RBC, PL, platelets, or plasma ex vivo. Tissue plasminogen activator (TPA) was added to promote fibrinolysis mimicking ischemic endothelium.
Results: The addition of TPA to whole blood increased fibrinolysis from median of 0.9%(Interquartile Range 0.4-1.3) to 5.7%(4.3-7). When HL RBC were added with TPA, fibrinolysis increased to 23.7%(22.3-27.1)p=0.008. PL decreased TPA mediated fibrinolysis to 0.1%(0.0-0.7)p=.028. Whole blood, non-lysed RBC, and plasma did not change lysis compared to TPA control. Transexamic acid also completely inhibited HL enhanced TPA mediated fibrinolysis and no differences were appreciated when mixed with PL.
Conclusion: Hemolysis is a potent enhancer of fibrinolysis. The association of HL and HF sets up a lethal double hit on coagulation capacity during hemorrhagic shock and resuscitation, which may explain why patients with HF have a high mortality. The shutdown of fibrinolysis with PL suggests that platelets contain potent inhibitors of fibrinolysis and are likely an important regulator of the fibrinolysis system. Elucidating the mechanistic role of targeted intracellular components of circulating blood components may result in a better understanding of the fibrinolytic response to trauma.
PLATELET DERIVED HMGB1 REGULATES PLATELET AGGREGATION AND MEDIATES ORGAN INJURY FOLLOWING TRAUMA AND HEMORRHAGIC SHOCK
M.D. Neal, R. Hoffman, P. Loughran, G. Chen, and T. Billiar*. University of Pittsburgh, Pittsburgh, PA
Objective: The regulation of platelet function is a key component of the host response to trauma and hemorrhage. Recently, high-mobility group protein 1 (HMGB1), a key danger signal and mediator of sterile inflammation has been shown to be released from platelets. However, the function of platelet HMGB1 and it role in the platelet response to sterile injury remain unexplored.
Methods: We identified HMGB1 in both mouse and human platelets using ELISA, flow cytometry, and immunostaining. Using cre-loxp technology, we generated transgenic mice specifically lacking HMGB1 on platelets (HMGB1Pf4-cre). Transgenic and wild-type (WT) mice were subjected to a model of trauma and hemorrhagic shock consisting of laparotomy, liver crush injury, and hemorrhage to a mean arterial pressure of 25mmHg for 60 min followed by resuscitation (THS-R). Lung and liver were harvested for histopathology and immunostaining for markers of injury. Platelet aggregation was measured by aggregometry. Bleeding times were measured using a tail vein bleed assay.
Results: THS-R resulted in significant platelet activation and sequestration of platelets into lung and liver in WT mice. HMGB1 was released by platelets following activation and localized to alpha granules. Strikingly, selective deletion of HMGB1 from platelets (HMGB1Pf4-cre) resulted in platelets that were resistant to sequestration with reduced adherence to microvascular endothelium. HMGB1Pf4-cre mice had markedly reduced liver injury compared to WT as measured by serum AST (850 ± 33.2 IU/L vs 505 ± 61.7 IU/L, p=0.02). Additionally, HMGB1Pf4-cre mice were protected from lung and liver injury as measured by histologic scoring, with markedly reduced microvascular thrombosis compared to WT. To address the mechanism behind these observations, we measured platelet function in response to known agonists (thrombin, ADP) and demonstrated that selective deletion of HMGB1 from platelets markedly reduced platelet aggregation.
Conclusions: We demonstrate for the first time that platelet HMGB1 is an essential mediator of platelet aggregation and that reduced aggregation via deletion of HMGB1 from platelets results in decreased organ injury following THS-R. Modulating HMGB1 release from platelets may represent a novel therapeutic target for preventing organ injury after trauma.
NEUTROPHIL EXPRESSION OF PROGRAMMED DEATH LIGAND (PD-L)-1 IS POTENTIATED FOLLOWING MIGRATION INTO LUNG TISSUE
J. Lomas-Neira*, X. Huang*, Y. Chen, S.F. Monaghan*, and A. Ayala*. Brown University/Rhode Island Hospital, Providence, RI
Programmed death (PD)-1 and its ligand, PD-L1/B7H1, are primarily known for their roles as negative regulatory molecules modulating T-cell activation and sustaining antigen tolerance. We recently show that expression of these molecules increases in plasma from septic and shocked/septic mice [our model for the development of indirect acute lung injury (iALI) in mice], and that PD-1 and PD-L1 gene deficiency improves their survival. While T-cell PD-1:PD-L1 biology is clearly involved, we and others have observed PD-1 and PD-L1 expression on innate immune cells, like neutrophils (PMN), as well as PD-L1 expression on non-immune cells, such as lung endothelial cells (EC) and epithelial cells. Thus, positioning PD-1:PD-L1 interactions as having a potential role(s) in not only leukocyte:leukocyte, but leukocyte:non-immune cell interaction in the pathogenesis of experimental iALI. To begin to understand the role PD-1 and/or PD-L1 expression may exert on PMN, we compared expression on peripheral blood PMN vs. PMN that had trans-migrated into lung tissue. PMN isolated from naïve and mice undergoing shock only, were used for controls. Flowcytometry was performed on whole blood and single cell suspensions of saline perfused lung tissue. PMN were characterized as Ly6G+ and expression of PD-1 or PD-L1 in peripheral blood vs. lung tissue was calculated as %PD-1+/Ly6G+ and %PD-L1(B7H1+)/Ly6G+. %PD-1+/Ly6G+ expression increased in both peripheral blood and lung tissue following shock/sepsis compared to controls. However, while %PD-L1(B7H1+)/Ly6G+ cells decreased in peripheral blood following shock/sepsis, this trend was reversed on the trans-migrated PMN. Interestingly, an increase in the total % of PD-1+/Ly6G+ and %PD-L1 (B7H1+)/Ly6G+ cells was seen in lung tissue vs. blood. While the functional significance of these changes in PD-1 and/or PD-L1 expression on these PMN populations has yet to be elucidated, data imply that the trans-migrated granulocyte, which appears in the injured lung in response to dual insults of shock/sepsis, may be the result of either a selective recruitment of a given PMN phenotype to actively diapedis or, alternatively, expression of an adapted phenotype that appears as a result of specific PD-1:PD-L1 interactions during diapedesis and/or interactions occurring within the lung tissue. Funding: GM107149 (A.A) & GM103652/8257 (J.L.N)
SPHINGOSINE 1-PHOSPHATE ATTENUATES BURN-INDUCED MICROVASCULAR HYPERPERMEABILITY
J.M. Guido, J.A. Elliott, J. Reynolds, M.H. Wu*, and S.Y. Yuan*. University of South Florida College of Medicine, Tampa, FL
Objective: Tissue damage following burn injury largely results from systemic inflammation. The end-organ effect of endothelial barrier breakdown is a critical obstacle to treating major burns. Sphingosine 1-phosphate (S1P) is an important regulator of vascular barrier function. The purpose of the study was to determine the protective effect of S1P on endothelial barrier function using a rodent model of scald burn.
Methods: Burn injury was induced in mice by applying boiling water to dorsal skin causing a third degree burn covering 25% of total body surface area. Plasma extravasation across mesenteric microvessels was measured in vivo using intravital microscopy. Endothelial cell-cell adhesive barrier function was evaluated in cultured mouse lung microvascular endothelial cell (MLMVEC) monolayers by measuring transendothelial electrical resistance (TER) with an electrical cell-substrate impedance sensor. The distribution of VE-cadherin, β-catenin, and F-actin was observed under a fluorescence microscope.
Results: Burn injury caused significant plasma extravasation across mesenteric microvessels. This extravasation was significantly attenuated by treatment with 1μM intravenous S1P. Treating MLMVECs with 1μM S1P decreased burn plasma-induced barrier dysfunction by reducing the drop in TER associated with application of burn plasma. In cultured MLMVECs burn plasma triggered a disruption of intercellular adherens junctions, characterized by dissociation of VE-cadherin and β-catenin and accompanied by F-actin stress fiber formation. These morphological changes were reversed by treatment with 1μM S1P.
Conclusion: Taken together, the data demonstrate that S1P has a protective effect on endothelial barrier function and significantly attenuates burn-induced microvascular leakage.
STIMULATION OF A7 NICOTINIC ACETYLCHOLINE RECEPTOR REDUCES TRAUMATIC HEMORRHAGE VIA THROMBIN-DEPENDENT PLATELET PRIMING IN SPLEEN
S.N. Sarangi1, J.R. Fritz2, H. Hoffman2, S.P. Valentino2, C.J. Czura2, K.J. Tracey*2, and J.M. Huston*2, 3. 1Cohen Children’s Medical Center, New Hyde Park, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY, 3North Shore-LIJ Health System, Manhasset, NY
Introduction: Uncontrolled hemorrhage is the leading preventable cause of death following traumatic injury. The cholinergic anti-inflammatory pathway protects against lethal systemic inflammation during hemorrhagic shock. Cholinergic stimulation also reduces blood loss and accelerates clot formation following traumatic injury. Cholinergic anti-inflammatory signaling requires a vagus nerve pathway to splenic macrophages expressing the α7 nicotinic acetylcholine receptor (α7nAChR). The molecular and cellular components required for cholinergic hemostatic effects are unknown. Here we evaluated whether cholinergic stimulation reduces traumatic hemorrhage in mice by priming circulating platelets in spleen via a signaling pathway involving α7nAChR.
Methods: Male 8-12 week-old mice (C57BL/6J, α7 knock-out or wild-type littermates) were pretreated with nicotine (2 mg/kg, ip) or saline vehicle 60 min before 2 mm distal tail transection and bleeding time measurement. To simulate tail injury ex vivo, systemic blood from nicotine or vehicle-treated mice was collected in separate experiments and stimulated with the platelet agonists thrombin and ADP. To quantify platelet activation, surface expression of P-selectin (CD62P) and glycoprotein IIb/IIIa (JON/A) was determined by FACS. To evaluate the spleen, animals underwent splenectomy or sham laparotomy five days before tail bleeding. Circulating soluble P-selectin concentrations were measured by ELISA.
Results: Nicotine significantly reduces bleeding time compared with vehicle treatment. Nicotine increases thrombin-dependent expression of P-selectin and glycoprotein IIb/IIIa on activated platelets. Nicotine cannot activate platelets without additional stimulation with thrombin. Combined with ADP stimulation, nicotine does not augment platelet activation. Nicotine fails to reduce bleeding or activate platelets following splenectomy, or in mice genetically deficient in α7nAChR. There is no difference in soluble P-selectin concentration following nicotine or vehicle treatment.
Conclusions: Cholinergic stimulation specifically enhances hemostasis at the site of tissue trauma and reduces hemorrhage via thrombin- and α7nAChR-dependent platelet priming in spleen. Further studies are needed to evaluate cholinergic stimulation as a hemostatic therapy to safely prevent lethal traumatic hemorrhage.
CLARITHROMYCIN EXPANDS CD11B+GR-1+ CELLS TO PROTECT AGAINST LPS-INDUCED LETHAL SHOCK
H. Namkoong1, 2, M. Ishii1, H. Fujii3, T. Asami1, K. Yagi1, H. Fujiwara1, F. Saitoh1, S. Tasaka1, N. Hasegawa1, S. Koyasu4, and T. Betsuyaku1. 1Keio University School of Medicine, Tokyo, Japan, 2Japan Society for the Promotion of Science, Tokyo, Japan, 3Graduate School of Medicine, University of the Ryukyus, Naha, Japan, 4RIKEN Research Center for Allergy and Immunology, Yokohama, Japan
Background: Macrolides are widely used for inflammatory diseases due to their anti-inflammatory properties. However, little is known about their precise immunological mechanisms and cell populations responsible for their effects.
Objectives: To identify and characterize cell populations which play a pivotal role in anti-inflammatory effects of macrolides.
Methods: Clarithromycin, a major macrolide antibiotic was administered intraperitoneally to C57BL/6 male mice once a day for three days. The mice were subsequently subjected to endotoxin shock.
Results: Clarithromycin markedly expanded splenic and lung CD11b+Gr-1+cells in normal mice. Pretreatment with clarithromycin improved mortality in mouse models of LPS-induced shock. Since we confirmed that more than 90% of Gr-1+ cells are CD11b+Gr-1+ cells, we substitute anti-Gr-1 antibody for depleting CD11b+Gr-1+ cells. Improved mortality by clarithromycin was cancelled by depleting Gr-1+ cells in endotoxin shock. Furthermore, adoptive transfer of sorted CD11b+Gr-1+cells obtained from clarithromycin-treated mice protected against LPS-induced lethality, which were associated with decrease of pro-inflammatory cytokines (TNF-α, IL-6, and IFN-γ) and increase of anti-inflammatory cytokine IL-10 in serum and bronchoalveolar lavage fluids. In vitro study, CD11b+Gr-1+cells obtained from clarithromycin-treated mice prohibited the proliferation of CD3+ T cells in the presence of anti-CD3 and anti-CD28, as compared to the control CD11b+Gr-1+ cells. Consistently, microarray analysis demonstrated that expressions of arginase-1, a key enzyme of inhibiting T cell proliferation, are much higher in CD11b+Gr-1+cells obtained from clarithromycin-treated mice compared to the control CD11b+Gr-1+cells. In addition, production of pro-inflammatory cytokines were significantly suppressed, while anti-inflammatory cytokine IL-10 was increased when bone marrow-derived macrophages or thiogllycolate-elicited macrophages were cocultured with CD11b+Gr-1+ cells obtained from clarithromycin-treated mice.
Conclusions: We have identified for the first time the unique population of immunosuppressive CD11b+Gr-1+ cells expanded by clarithromycin, which are essential for the anti-inflammatory properties of clarithromycin.
PERSISTENT INFLAMMATION AND T CELL EXHAUSTION IN SEVERE SEPSIS IN THE ELDERLY
S. Inoue*1, 2, Y. Komori2, 1, Y. Morishita2, 1, K. Suzuki3, T. Sato4, and S. Inokuchi1. 1Department of Emergency and Critical Care Medicine, Tokai University School of Medicine, Isehara, Japan, 2Institute of Innovative Science and Technology, Tokai University School of Medicine, Isehara, Japan, 3Department of Emergency and Disaster Medicine, Graduate school of Medicine, Gifu University, Gifu, Japan, 4Department of Immunology, Tokai University School of Medicine, Isehara, Japan
Introduction: Sepsis is known as a complex immunological response with hyperinflammation in the acute phase followed by immunosuppression. Although aging is crucial in sepsis, the impact of aging on inflammation and immunosuppression is still unclear. The purpose of this study was to investigate the relationship between inflammation and immunosuppression in aged patients and mice after sepsis.
Methods: Fifty-five patients with severe sepsis and 30 healthy donors were prospectively enrolled, and 90-day survival was compared between elderly (≥65 years) and adult (18-64 years) septic patients with serial measurement of serum IL-6. Within 24 h after diagnosis of severe sepsis, peripheral blood mononuclear cells were stimulated ex vivo to measure expression of the activation maker CD25 by T cells, IL-2 in supernatant, and proliferation by CFSE. In the mouse study, young (6-8 weeks) and aged (20-22 months) C57/B6 mice were subjected to cecal ligation and puncture (CLP), and survival was compared after 7 days with serial measurement of serum IL-6. Expression of negative co-stimulatory molecules, CD25, IL-2 of CD4+ T cells in the spleen, lymph nodes, and peripheral blood, was measured.
Results: The survival rate in elderly sepsis patients and aged septic mice was significantly lower than that in adult patients and young septic mice (60% vs. 93% in septic patients, 0% vs. 63% in septic mice, P < 0.05). Serum IL-6 levels in elderly sepsis patients and aged septic mice were persistently higher than those in adult patients and young septic mice. Expression of negative co-stimulatory molecules on CD4+ T cells in the spleen, lymph nodes, and peripheral blood, was significantly higher in aged mice than in young mice (P < 0.01). Ex vivo stimulation assay, decreased CD25 expression on CD4+ T cells with impaired proliferation and IL-2 production was observed in elderly patients and aged septic mice than in adult patients and young septic mice.
Conclusions: Persistent inflammation and T cell exhaustion may be associated with decreased survival in elderly patients and mice after sepsis.
AMYLOID PLAQUES ARE INCREASED IN THE BRAIN OF TBI SURVIVORS AT 12 MONTHS AFTER INJURY
J. Gatson*, T. Harris, C. Stebbins, J. Minei*, and S.E. Wolf*. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Traumatic brain injury (TBI) is a major risk factor for the development of Alzheimer’s disease (AD). The link between TBI and Alzheimer’s-like dementia has been studied extensively. For example, studies have shown that amyloid plaques are present in the brain as early as 2 hours after injury and persist in subjects that survived up to 2.5 years after injury as detected in post mortem brain tissue.
Methods: We have initiated a clinical study to assess whether amyloid plaques accumulate in the brain at a faster rate in TBI survivors. In this study we utilized the F18 positron emission tomography (PET) amyloid tracer florbetapir that has been shown to bind to aggregated Aβ peptides in amyloid plaques with high affinity and displays good agreement with amyloid plaque labeling at autopsy. Florbetapir F18 is currently used in many centers for research in AD and has been approved to aid in the clinical diagnosis of AD. In this study, mild, moderate, or severe TBI patients (N=12) (emergency department Glasgow Coma Scale [GCS] 3-15) were enrolled and following hospital discharge (~1 month after injury) underwent the florbetapir F18 PET imaging procedure and neuropsychological testing (Mini-Mental State Examination [MMSE] test).
Results: Compared to the baseline scans, the severe TBI subjects exhibited increases in amyloid plaques in various regions of the brain at the 12 month time-point. For example an increase was observed in the frontal cortex (14%), parietal cortex (10%), posterior cingulate (26.5%), hippocampus (22%), and thalamic (21%) brain regions.
Conclusion: This pilot study is novel and will elucidate whether amyloid plaques increase at a faster rate over time. Findings from this study could lead to the design of interventional therapies to decrease plaque formation after TBI in an attempt to lower long-term cognitive deficits. As a future direction, we will scan these participants at 2 and 5 years after injury.
DELAYED SKIN HEALING IN AGED MICE IS DUE TO DEFICIENT RECRUITMENT OF PROGENITOR CELLS INTO THE WOUND BED: A STORY OF MYELOID-MESENCHYMAL CELLS COMMUNICATION
S. Amini-Nik1, 2, A. Abdullahi1, N. Yu1, C. Belo1, and M.G. Jeschke*1, 2. 1University of Toronto, Sunnybrook Research Institute, Toronto, ON, Canada, 2University of Toronto, Department of Surgery, Toronto, ON, Canada
Introduction: Duo to numerous physical and cognitive changes which elderly experience, they are more exposed to fire and burn injury. This is more important nowadays as advances in health care and economic prosperity are contributing to a longer life span. Once burned, deficient healing, in particular skin healing, contributes to high morbidity and mortality in elderly patients. We hypothesized that altered mesenchymal progenitor cells play a critical role in deficient healing of elderly patients due to a) decreased number of these cells and b) deficient requirement into the wound bed.
Methods: We compared skin healing characteristics of old patients (as well as old mice) with the young ones. Young (8 weeks old) and old mice (>52 weeks) were subjected to 30% full thickness burn and their skin healing were monitored up to two weeks post burning.
Results: We observed a delayed healing in aged mice (and human) together with higher mortality. Lack of granulation tissue, deficient collagen deposition, lack of myeloid lineage cells and altered keratinization were observed in aged mice in compare with young mice. Beside lack of F4/80+ myeloid cells in the wound bed of aged mice, our in vitro study revealed an altered differentiation of their bone marrow into myeloid lineage. We then explored the underlying mechanism of this deficient healing. Flow cytometry analysis revealed a lack of mesenchymal stem cells (MSCs) in the wound bed of elderly patient (as well as old mice) when compared with their young counterpart. While in old mice, the border of healing bed were enriched for Sca1+ cells in compare with the young mice, we observed a significant reduction in the number of this progenitor cells toward the center of wound.
Conclusion: Here, we report that lack of MSCs in the wound bed of old mice led to a delayed healing. This attribution was also observed in elderly patients in compare with young ones. Decreased MSCs was accompanying with the lack of myeloid lineage cells in wound bed of aged mice, leading to an undesirable niche for migration of mesenchymal progenitors into the wound bed. Our data suggest that Wnt/β-catenin signalling plays role in this myeloid-mesenchymal interaction and thus local modulation of Wnt/β-catenin signalling might be a critical step in the management of elderly burned patients.
PLASMA HUMAN MITOCHONDRIAL DNA (MTDNA) CONCENTRATIONS ARE ELEVATED IN PEDIATRIC SEPSIS
R. Aneja*, T. Walko, A. Au, V. Hsue, J.D. Hong, R.S. Clark, and J.A. Carcillo. University of Pittsburgh and Children’s Hospital of Pittsburgh, Pittsburgh, PA
Introduction: The pro-inflammatory cytokine response after sepsis has been well characterized and recently the role of damage-associated molecular patterns (DAMPs, also known as alarmins) has been recognized. MtDNA is a novel DAMP that is released into the extracellular milieu subsequent to cell death and injury and acts via TLR-9, a pattern recognition receptor of the immune system that detects bacterial and viral DNA. We hypothesized that cell death in septic children would lead to elevated plasma human mtDNA concentrations.
Methods: This IRB-approved study included children who were admitted to the Children’s Hospital of Pittsburgh PICU with severe sepsis between December 2009 to December 2010. In addition to the collection of demographic and clinical data, we quantified free mtDNA in blood samples from 38 children with severe sepsis, and 4 critically ill children (who were not septic) subjects that were designated as controls. We designed PCR primers that amplify bases between 421 and 781 encoding the mitochondrial gene cytochrome c oxidase I (Ref Seq: NC_012920). A standard curve was created by using a cloned plasmid DNA, which was serially diluted to prepare a series of calibrators with known concentrations (Chiu et al., 2003). All statistical analyses were performed using Mann-Whitney test.
Results: The mean age of the cohort was 5.2 y and 22 (57%) children were male. As compared to critically ill non-septic children (n=4), we observed a significant elevation of plasma mtDNA in the severe sepsis cohort (1.24E+06 vs 1.19E+08 copies/ml respectively). Furthermore, significantly elevated concentrations of plasma mtDNA levels were noted in females as compared to males (2.3E+08 vs 6.5E+07 copies/ml). Although there was no significant difference in mtDNA concentrations as it relates to organ failure index (OFI), patients with organ failure (OFI ≥2) demonstrated increased plasma concentrations of mtDNA as compared to patients with OFI <2 (1.1E+08 vs 4.8E+07 copies/ml).
Conclusions: The elevated serum concentrations of mtDNA, a novel DAMP, support the notion of mtDNA as an endogenous danger signal in septic children. Further validation of this data is required in a larger cohort. Our ongoing work is focused on identifying the pathways in the secondary injury response that are activated by this novel danger signal. (Supported by R01GM108618-01, R01GM098474)
ENHANCED PROTECTIVE IMMUNITY AND EXTRAMEDULLARY MYELOPOIESIS IN NEONATAL SEPSIS BY ABLATION OF CASPASE-1 SIGNALING
L.F. Gentile, A.L. Cuenca, A. Cuenca, E. Vanzant, D.C. Nacionales, R. Ungaro, L. Moldawer*, P.A. Efron*, and S. Larson. University of Florida, Gainesville, FL
Greater than one million newborns die each year from sepsis that occurs in the first 4 weeks of life. Mortality rates are highest in premature and low birth weight infants. We have shown that in neonatal mice, sepsis survival relies primarily on the innate immune response for protection. The inflammasome plays a central role in the regulation of innate immunity, inflammation and pyroptosis. Adult sepsis studies suggest that the absence of caspase-1 improves survival primarily by attenuating inflammatory responses. However, we suggest that the inflammasome is also an integral component of host protective immunity and myelopoiesis.
Neonatal (5-7 days) B6 and B6caspase-1-/- knockout mice underwent a low-lethality cecal slurry (CS) model of intra-abdominal sepsis (LD 25-40). Blood and peritoneal washes were collected at various time points after sepsis for analysis.
Caspase-1 deficient neonatal mice exhibited increased survival after sepsis compared to B6 mice (p<0.001). Additionally, caspase-1 null mice had decreased levels of inflammatory cytokines (IL-1β,IL-1α, IL-6, IFN-γ, TNF-α) in the serum and peritoneum 18 hours after sepsis. In contrast, caspase-1 null mice exhibited increased splenic LSKs (Lin-ska+c-kit+) 36 hours after sepsis (p<0.001), with increased concentrations of G-CSF and M-CSF in the peritoneum (p<0.001). This was associated with increased macrophages (CD11b+Ly6G-F4/80+) (p<0.05), increased phagocytosis by neutrophils (CD11b+Ly6G+) (p=0.003), and improved bacterial killing (p=0.01) in the peritoneum post-sepsis.
In this study, we show that caspase-1 deficient neonatal mice have improved survival in a low-lethality model of polymicrobial sepsis. Additionally, we show that the absence of caspase-1 signaling leads to increased extramedullary hematopoieis, with increased myelopoiesis via increased production of growth factors. These findings were associated with increased recruitment and activation of innate immune effector cells, and an improved ability to clear infection compared to wild-type mice. In contrast to adults, the neonatal spleen resembles a hematopoietic organ rather than a clearance organ; therefore, we propose that the increased survival, protective immunity and extramedullary hematopeisis may be secondary to the absence of caspase-1 induced pyroptosis in the neonatal spleen.
A NOVEL MECHANISM REGULATING THE TRANSITION FROM ENDOTHELIAL BARRIER INJURY TO REPAIR PHASES FOLLOWING ENDOTOXEMIC LUNG INJURY
G. Liu*, X. Ye, and S. Liu. The Feinstein Institute for Medical Research, Manhasset, NY
Little is known about the mechanisms regulating the transition from endothelial barrier injury to repair phases. This study unveiled an “NF-κB to AP (activator protein)-1 switch” mechanism that regulates this transition. Wild type (WT) and transgenic (TG) mice with EC-restricted and inducible expression of a mutant I-κBα (I-κBαmt) were injected with saline or E. coli LPS (5 mg/kg, i.p.). Lung endothelial permeability and nuclear levels of AP-1 family of proteins, C-fos and C-jun, were measured. Transition from endothelial barrier injury to repair phases occurred at 24 hours post-LPS. Targeted and stage-specific inhibition of endothelial intrinsic NF-κB activity by doxycycline-induced I-κBαmt expression in ECs at 24 hours increased nuclear levels of C-fos and C-jun proteins in TG, but not WT lungs. EC-targeted NF-κB inhibition also increased AP-1 binding activity. Thus, inhibition of endothelial NF-κB pathway at transition phase activates AP-1 pathway (NF-κB-to-AP-1 switch). Molecular analysis revealed that EC-targeted NF-κB blockade at 24 hours activated c-Jun NH2-terminal kinase (JNK), which was associated with increased C-jun phosphorylation and increased nuclear level of C-jun protein. Pretreatment of mice with JNK inhibitor, SP600125, reduced tissue level of phospho-c-Jun in 24 hour TG lungs, suggesting that JNK activation mediates the NF-κB-to-AP-1 switch. Blockade of the NF-κB-to-AP-1 switch mechanism by overexpressing a dominant negative C-jun, TAM67, at 24 hours enhanced EC apoptosis and augmented endothelial permeability in TG, but not in WT lungs. The numbers of apoptotic ECs (% of total cells), for Vector-con, TAM-con, WT-vector, WT-TAM, TG-vector and TG-TAM groups were 0.25±0.01, 0.26±0.01, 1.78±0.08, 1.60±0.15, 1.64±0.13 and 3.37±0.21%. Lung Evens blue dye leakage index (ng/mg tissue) for Vector-con, TAM-con, WT-vector, WT-TAM, TG-vector and TG-TAM groups were 21.4±1.8, 21.3±2.2, 87.7±7.3, 86.6±8.6, 50.5±3.7 and 118.3±7.5. Our data demonstrates an NF-κB-to-AP-1 switch mechanism that accelerates the transition from endothelial barrier injury to repair by inhibiting EC apoptosis and by promoting endothelial barrier repair. (Supported by AHA grant 12GRNT1214002).
A NOVEL ROLE FOR PROGRAMMED CELL DEATH RECEPTOR LIGAND-1 (PD-L1) IN SEPSIS-INDUCED GASTROINTESTINAL DYSFUNCTION
Y. Wu, X. Huang, C. Chung*, and A. Ayala*. Rhode Island Hospital/Brown University, Providence, RI
Sepsis remains the leading cause of death in critically ill patients. Evidence from several groups implies that intestinal barrier dysfunction is a key contributor to morbid events associated with sepsis. Recent studies have indicated that co-inhibitory molecule PD-L1 is involved in the regulation of intestinal immune tolerance and/or inflammation. In a recent study we showed that PD-L1 gene deficiency reduced morphological evidence of sepsis-induced intestinal injury. We hypothesized that PD-L1 expressed on intestinal epithelial cells (IECs) has a role in sepsis-induced intestinal barrier dysfunction. To test this, wild-type (WT) C57BL/6 and PD-L1-/- mice underwent cecal ligation and puncture (CLP) or sham surgery. Initially, we found that IECs from naive WT mice constitutively expressed PD-L1 mRNA and protein; however, CLP significantly up-regulated IEC PD-L1. PD-L1 gene deficiency significantly decreased intestinal epithelial permeability (in vivo ligated loop model with FD4), reduced IL-6, TNF-α and MCP-1 levels as well as prevented the decline in the expression of tight junction proteins (ZO-1) in the ileum after sepsis. To the extent that these in vivo changes are reflection of the response to local inflammatory mediators released during sepsis, we comparatively examined the in vitro response of Caco-2 cells, a human intestinal epithelial cell line, to treatment with/without recombinant human TNF-α and/or IFN-γ in the presence/absence of anti-PD-L1 antibody (Ab). Results showed that cytokine stimulation significantly increased PD-L1 expression and decreased Caco-2 cell monolayer permeability. Furthermore, anti-PD-L1 Ab treatment significantly reduced cytokine-induced Caco-2 monolayer hyperpermeability. Taken together, our study indicates that PD-L1 appears to play a novel role in the pathophysiology of sepsis-induced intestinal barrier dysfunction.
CXC CHEMOKINE CONCENTRATION DICTATES THE HEPATOCYTE PROLIFERATIVE RESPONSE AFTER INJURY
G.C. Wilson, H. Nojima, C. Freeman, R.M. Schuster, J. Blanchard, M.J. Edwards, and A.B. Lentsch*. University of Cincinnati Medical Center, Cincinnati, OH
Introduction: Liver dysfunction in hemorrhagic or septic shock results from hepatic ischemia/reperfusion injury (I/R) and the ensuing inflammatory response. CXCR2 ligands propagate this inflammation and are detrimental to the recovering liver after I/R. However in models of hepatectomy, the opposite is seen where CXCR2 ligands facilitate hepatocyte proliferation and liver regeneration. We sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver repair after I/R injury and partial hepatectomy.
Methods: Murine models of partial I/R and partial hepatectomy were used to study liver repair. Tissue and serum levels of CXCR2 ligands were assessed by enzyme-linked immunosorbent assay (ELISA). Hepatocyte proliferation and liver regeneration were assessed by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and normalized liver mass.
Results: Tissue expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), were significantly increased after both I/R and partial hepatectomy. However, after I/R injury expression of these ligands was more than 30-fold greater than after hepatectomy. Interestingly, when we compared the ischemic and non-ischemic liver lobes after I/R, the same pattern of ligand expression was found; ischemic lobes expressed more than 30-fold higher levels compared to non-ischemic liver lobes. In both models, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, exogenous MIP-2 and KC were administered in amounts that replicated serum levels of MIP-2 and KC found in I/R to mice undergoing partial hepatectomy. Mice treated with this “high” dose had reduced amounts of hepatocyte proliferation and liver regeneration compared to a “low” dose, that actually increased hepatocyte proliferation and liver mass.
Conclusion: The data suggest that the concentration of CXC chemokines regulates the hepatic proliferative response such that relatively low levels of ligands induce significant liver repair, whereas high expression levels diminish this response. The data also suggest CXC chemokines, signaling through CXCR2, function as a rheostat for hepatocyte proliferation and liver regeneration.
STAT3 INHIBITION BLOCKS MUSCLE WASTING IN A MOUSE MODEL OF BURN-INJURY INDUCED CACHEXIA
A. Bonetto1, F. Pedroso3, P.B. Spaulding2, L. Koniaris1, and T. Zimmers*1. 1Indiana University School of Medicine, Indianapolis, IN, 2University of Miami School of Medicine, Miami, FL, 3Columbia University Medical Center, New York, NY
Burn injury causes chronic inflammation and hypermetabolism with muscle wasting. Patients with burn injury exhibit sustained high serum IL-6. IL-6 is highly correlated with muscle loss and mortality in burn, trauma and sepsis. IL-6 over-expression causes cachexia in mice, while neutralization of IL-6 reduces muscle wasting in various settings. We have shown that IL-6 induces STAT3 activation in skeletal muscle, and further that STAT3 is necessary and sufficient for muscle wasting downstream of IL-6 and in cancer cachexia. Here we use a mouse model of 20% TBSA burn to probe the causal roles of STAT3 and IL-6 in burn cachexia.
Twelve week-old C67BL/6 male mice were anesthetized, shaved, Naired and subjected to contact burn injury on the dorsum with heated brass plates. Plasma IL-6 measured at 7, 14, 21 and 30 days after injury were ~30pg/ml, roughly 4-9 times sham. Consistent with high IL-6 signaling, burn injury induced STAT3 activation in quadriceps muscles as early as 6 hours, which remained high at 14 days. Muscle mass at 14 days was reduced 15-20% and tibialis myofiber diameters were reduced 14%. Analysis of the muscle transcriptome at 14 days revealed that the most highly regulated canonical pathways were genes involved in the immune system and genes involved in adaptive immunity. Overall, the burn muscle transcriptome correlated positively with other datasets from mouse models of cachexia and negatively with mouse models of disuse atrophy. We also observed a high concordance of gene expression with a model of muscle wasting from excess IL-6 (p=7.6e-59), and elevated STAT3 target gene expression. Given this evidence, we sought to inhibit STAT3 to reduce muscle wasting. Mice were subjected to tibialis injection and electroporation with plasmids encoding shSTAT3 or SOCS3, a STAT3 target gene and inhibitor of gp130/JAK/STAT3 activation, and then burned. Fourteen days later, shSTAT3 and SOCS3 expressing myofibers revealed normal cross sectional area, in contrast to control transfected fibers which were 15-25% smaller than normal.
These results indicate that STAT3 inhibition prevents muscle wasting after burn injury. Given prior data from others documenting both STAT3 and STAT3 target gene activation in human muscle after burn injury, inhibition of this transcription factor and/or stabilization of SOCS3 might reduce muscle wasting in patients suffering from burns.
RESTORATION OF ENDOTHELIAL BARRIER FUNCTION BY RESIDENT ENDOTHELIAL CELLS IN ENDOTOXEMIC LUNG INJURY
X. Ye, S. Mao, G. Liu, and S. Liu. The Feinstein Institute for Medical Research, Manhasset, NY
Endothelial barrier disruption is a hallmark of acute lung injury. However, the origin and function of cells restoring endothelial barrier function remain unknown. This study defined the role of resident endothelial cells (RECs) in endothelial barrier repair. EC-rtTA-WT-BM or EC-I-κBα-WT-BM chimera with REC-restricted expression of reverse tetracycline transactivator (rtTA) or REC-restricted expression of a mutant I-κBα was created. Wild type (WT) and chimeras were injected with saline or E. coli LPS (5 mg/kg, i.p.). The relationship between REC proliferation and endothelial barrier recovery was analyzed. Effect of targeted inhibition of REC proliferation on endothelial barrier recovery was examined. Endothelial barrier recovery was associated with a markedly increased REC proliferation. REC proliferation peaked at 48 hours, which is consistent with 48 hours being active barrier repair phase. Histological examination revealed an increased number of proliferating RECs that were localized to microvascular endothelial layer in lungs at active barrier repair phase. Doxycycline-induced I-κBαmt expression at 48 hours inhibited endothelial NF-κB activity in RECs, but not in bone marrow endothelial progenitor cells, indicating a REC-targeted and stage-specific inhibition of NF-κB activity. Blockade of REC intrinsic NF-κB activity at 48 hours inhibited REC proliferation. Numbers of proliferating RECs (% total proliferating cells) in lungs of EC-rtTA-WT-BM-Con, EC-I-κB-WT-BM-Con, EC-rtTA-WT-BM-LPS and EC-I-κB-WT-BM-LPS groups were 0.0±0.0, 0.0±0.0, 48.6±3.4 and 28.0±2.3%. Targeted inhibition of REC proliferation at 48 hours augmented endothelial permeability and impeded endothelial barrier recovery. Lung Evens blue dye leakage index (ng/mg protein) for WT-Con, EC-I-κB-WT-BM-Con, WT-LPS and EC-I-κB-WT-BM-LPS groups were 20.5±1.3, 20.4±1.1, 59.5±1.1 and 69.6±4.7. Targeted inhibition of REC intrinsic NF-κB activity at 48 hours had no effects on markers of inter-endothelial junction disruption and tissue levels of inflammatory cytokines. Our data demonstrates that REC proliferation plays a critical role in the restoration of endothelial barrier function following endotoxemic lung injury. (Supported by AHA grant 12GRNT1214002).
MELANOCORTIN-1-RECEPTOR POLYMORPHISMS ARE ASSOCIATED WITH POST-BURN HYPERTROPHIC SCARRING
R.F. Sood, A.M. Hocking, L.A. Muffley, M. Ga, S. Honari, M. Caceres, A.P. Reiner, A. Rowhani-Rahbar, and N.S. Gibran. University of Washington Regional Burn Center, Harborview Medical Center, Seattle, WA
Objective: Hypertrophic scarring (HTS) is a common and debilitating late sequela of burn injury. Known risk factors including skin color and race suggest a genetic mechanism. Increasing evidence links melanocortin signaling to anti-inflammatory and wound-repair functions, with functional mutations in the melanocortin 1 receptor (MC1R) gene leading to increased inflammatory responses. Single-nucleotide polymorphisms (SNPs) in MC1R are common in racial groups predisposed to HTS. We hypothesized that MC1R SNPs would be associated with HTS.
Methods: At-risk adults (age ≥18) admitted to our burn center were enrolled between 2007-2013. Blood was drawn for DNA isolation and patients were evaluated ≥2.5 months post-discharge for scar assessment using the Vancouver Scar Scale (VSS). Genotyping was performed for 5 MC1R SNPs (see Table) by polymerase chain reaction. A multivariate Poisson regression model with robust variance estimates was constructed including age; sex; burn size, depth, and site; number of operations; ethnicity; race; and MC1R genotype as covariates.
Results: Of 425 subjects (median age 40; median burn size 6.8% body surface area), 77% were white, 70% were male, and 80% developed HTS (total VSS score ≥6). After controlling for known clinical risk factors, heterozygosity for the I155T polymorphism and homozygosity for the R151C or R163Q polymorphisms were found to be independent risk factors for HTS (see Table).
Conclusion: To our knowledge, this is the first study to demonstrate a significant association between genetic polymorphisms and post-burn hypertrophic scarring. Our findings suggest that MC1R polymorphisms may cause dysfunctional inflammatory responses to injury that lead to post-burn hypertrophic scarring.
S-NITROSYLATION OF THE CXXC ZINC FINGER MOTIF IN SIRT1 MEDIATES AND ENHANCES NITROSATIVE STRESS IN ENDOTOXIN-INDUCED LIVER APOPTOSIS AND INFLAMMATION: PROTECTIVE ROLE OF S-NITROSOGLUTATHIONE REDUCTASE
S. Shinozaki1, 2, K. Chang1, 3, M. Sakai1, N. Shimizu1, K. Shimokado2, J. Stamler4, and M. Kaneki*1. 1Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Charlestown, MA, 2Department of Geriatrics and Vascular Medicine, Tokyo Medical and Dental University Graduate School, Tokyo, Japan, 3Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 4Institute for Transformative Molecular Medicine, Case Western Reserve University, Cleveland, OH
Inducible nitric oxide (NO) synthase (iNOS) plays a pivotal role in cellular and organ damage in inflammatory disorders, including endotoxemia. S-nitrosylation is the reversible covalent attachment of NO moiety to reactive cysteine thiols and has emerged as a major mediator of nitrosative stress. Nonetheless, little is known about the molecular targets of nitrosative stress. Moreover, iNOS can both up- and down-regulate inflammation presumably in a cellular context-dependent manner. Yet, the molecular mechanisms by which iNOS enhances inflammation remain largely unknown. Sirt1 downregulates apoptosis and inflammation by deacetylating p53 and p65 nuclear factor-κB (NF-κB). Here, we show that S-nitrosylation of the evolutionally conserved Cys-X-X-Cys (CXXC) motif in Sirt1 disrupts Zn2+ binding, thereby inactivating Sirt1, and, in turn, increasing acetylation and activity of p53 and p65 NF-κB in cultured cells and liver of lipopolysaccharide (LPS)-challenged mice. S-nitrosylation, but not oxidation, of the CXXC motif represents a major reversible thiol modification of Sirt1. The CXXC motif in Sirt1 is required for NO-mediated acetylation of p53 and p65 NF-κB in cultured cells. LPS caused Sirt1 S-nitrosylation, acetylation and activation of p53 and p65 NF-κB, and apoptosis and inflammatory response (e.g., TNF-α and TLR-4 induction) in mouse liver, which paralleled iNOS expression. LPS-induced these changes were prevented by iNOS deficiency, while they were aggravated by gene disruption of S-nitrosoglutathione reductase (GSNOR), a negative regulator of S-nitrosylation. Our data show that GSNOR is a physiological Sirt1 denitrosylase that attenuates nitrosative stress and subsequent apoptosis and inflammatory response. Our data indentify a novel positive feedback loop comprised of iNOS→Sirt1 S-nitrosylation→acetylation/activation of p65 NF-κB/p53, which leads to enhanced and sustained inflammation and dictates cell fate. Overall, these findings indicate that S-nitrosylation/denitrosylation of the CXXC zinc finger motif of Sirt1 serves as a nitrosative stress-sensitive switch capable of mediating and enhancing inflammatory pathophysiology. The study further defines the focus of potential therapies for nitrosative stress-mediated organ damage.
MD-2 PLAYS A CRITICAL ROLE IN RECOGNIZING ISOFORMS OF HMGB1 IN TLR4-DEPENDENT HMGB1 SIGNALING
H. Yang*1, Z. Ju1, A.A. Ragab1, S.S. Chavan*1, D. Gero3, C. Szabo3, T. Billiar*2, K.J. Tracey*1, and Y. Al-abed1. 1Feinstein Institute, Manhasset, NY, 2Department of Surgery, University of Pittsburgh, Pittsburgh, PA, 3Department of Medicine, University of Texas, Galveston, TX
High mobility group box 1 (HMGB1) is a 25 KDa nuclear protein involved in regulation of gene transcription. Once released outside of the cells, HMGB1 acts as a pro-inflammatory cytokine and mediates activation of innate immune responses. Although HMGB1can interact with multiple receptors, many pro-inflammatory cytokine effects of HMGB1 are mediated via TLR4/MD-2 receptor complex. HMGB1 has three cysteine residues located at 23, 45 and 106 position (C23, C45 and C106) and it is extremely redox-sensitive. Its redox status dictates its chemokine or cytokine-inducing properties. In order to interact with TLR4/MD-2 complex, HMGB1 needs both an intra-molecular disulfide bond (C23-C45) and a C106 thiol (disulfide form). HMGB1 with cysteines in all-thiol form is important for chemotaxis via CXCL12 and CXCR4 receptor. HMGB1 with cysteines in all-oxidized form does neither (Yang et al, Mol Med 2012, Venereau et al, JEM 2012). It is known that TLR4 activity and interaction with its ligands depend on the extracellular adaptor myeloid differentiation factor 2 (MD-2). MD-2 is important for distinguishing different species of LPS in TLR4-mediated LPS signaling (Meng et al, JBC 2009). But it is previously unknown the role of MD-2 in HMGB1-TLR4 interaction and subsequent inflammatory responses. Given the critical role of MD-2 in specific innate immune recognition of LPS for TLR4 signaling, we reasoned that MD-2 may similarly be able to bind HMGB1 to facilitate TLR4-dependent cytokine production. Here we have identified that to signal via TLR4/MD2 system, only the cytokine-inducing disulfide form of HMGB1 binds to MD-2 directly and with high affinity (apparent Kd = 12 nM) and does not bind to TLR4 alone, as revealed by biosensor-based surface plasmon resonance analysis (BIAcore). Non-cytokine-inducing HMGB1, generated by redox modification of HMGB1 including all thiol, all oxidized or mutation of C106 to other amino acids, renders HMGB1 binding to MD-2 (BIAcore analysis). Thus, our findings demonstrate that MD-2 plays a critical role in recognizing isoforms of HMGB1 in TLR4-dependent signaling (Supported by grants from NIH, NIGMS RO1GM62508 to KJT and RO1GM098446 to HY).
RESVERATROL RESTORES KIDNEY MITOCHONDRIAL FUNCTION FOLLOWING HEMORRHAGIC SHOCK
H. Wang1, 2, Y. Guan2, 3, M. Karamercan*2, 4, L.B. Becker4, 3, J.A. Baur5, and C.A. Sims2, 3. 1Qilu Hospital of Shandong University, Jinan, China, 2The Trauma Center at Penn, University of Pennsylvania, Philadelphia, PA, 3The Center for Resuscitation Science, University of Pennsylvania, Philadelphia, PA, 4Department of Emergency Medicine, Gazi University School of Medicine, Ankara, Turkey, 5Institute for Diabetes, Obesity, and Metabolism and Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
Objective: Altered mitochondrial function may be central to the development of acute kidney injury following hemorrhagic shock (HS). Resveratrol (RSV), a naturally occurring Sirtuin-1 (SIRT1) activator, has been shown to promote mitochondrial function and reduce oxidative damage in a variety of aging-related diseases. We hypothesized that RSV treatment during resuscitation would ameliorate HS-induced kidney mitochondrial dysfunction and decrease oxidative damage.
Methods: Using a decompensated HS model, male LE rats (n=6 per group) were sacrificed prior to hemorrhage (Sham), at severe shock, and following either lactated Ringer’s (LR) Resuscitation or LR+RSV Resuscitation (RSV: 30mg/kg). At each time point, blood samples were assayed for arterial blood gases, lactate, BUN and serum creatinine. Mitochondria were also isolated from kidney samples in order to assess individual electron transport complexes (CI, CII, and CIV) using high-resolution respirometry. Total mitochondria ROS were measured using fluorometry and lipid peroxidation was assessed by measuring 4-hydroxynonenal by Western blot. qPCR was used quantify mRNA from PGC1-α, SIRT1, and proteins known to mitigate oxidative damage and promote mitochondrial biogenesis.
Results: RSV supplementation during resuscitation restored mitochondrial respiratory Complex II and IV dependent respiratory capacities (Figure 1A). Compared to LR, RSV enhanced SIRT1 and PGC1-α expression and significantly increased both SOD2 and catalase expression following resuscitation (Figure 1B). RSV treatment resulted in decreased oxidative stress with reduced mitochondrial ROS production and lipid peroxidation. RSV did not elevate the mRNA expressions of mitochondrial biogenesis factors. Although RSV was associated with decreased lactate production, pH, BUN and serum creatinine values did not differ between resuscitation strategies.
Conclusion: Resuscitation with RSV restored renal mitochondrial function and decreased oxidative injury following HS.
C1 INHIBITOR AND ITS EFFECT ON TRAUMA-HEMORRHAGIC SHOCK
J.A. Bonitz, Y. Qin, L.M. Prescott, T. Murphy, E. Feketeova, and E.A. Deitch*. Rutgers University-New Jersey Medical School, Newark, NJ
Background: Trauma/hemorrhagic shock (T/HS) is one of the major consequences of battlefield injury as well as civilian trauma. Complement activation has been linked with ischemia-reperfusion mediated organ dysfunction since its level has been found to be increased early in trauma patients. In addition, since its level of activation has been correlated with level of injury, complement inhibition was chosen as the therapy to test our hypothesis that Complement 1 Inhibitor (C1 Inh), as part of resuscitation therapy, would limit T/HS induced multiple organ dysfunction syndrome (MODS) in a rodent hemorrhagic shock model.
Methods: Rats over a dose-response curve with trauma/sham-shock (T/SS) or T/HS (30mmHg x 90min), were administered C1 Inh (25IU/kg, 50IU/kg, 100IU/kg, or 200IU/kg) or vehicle during volume resuscitation. Lung injury (permeability to Evans Blue dye; EBD), gut injury (permeability of Fluorescein isothiocyanate-dextran; FD4), polymorphonuclear leukocyte (PMN) activation (respiratory burst (RB) activity), and red blood cell (RBC) deformability (elongation index; EI) were all tested.
Results: T/HS-induced increased gut permeability was abrogated by all the doses of C1 Inh tested, while only the 200IU/kg dose reduced the increase in lung permeability (Table). The increase in T/HS induced RBC deformability was abrogated in all the doses with the 100IU/kg dose being the most effective. Similarly, the PMN activity was abrogated in the 50IU/kg and 100IU/kg groups with the 100IU/kg dose group being the most effective. The values for the T/SS+vehicle group were similar to the T/SS+C1 Inh groups. Thus for clarity, only the T/SS+vehicle group will be shown.
Conclusion: Although all doses of C1 Inh prevent gut injury, the higher doses of 100IU/kg and 200IU/kg were found to have an improved effect on RBC deformability, PMN activation, and lung injury respectively. Further studies need to be done to observe the effect of C1 Inh on biologically active lymph.
TARGETING HEPATIC ENDOPLASMIC RETICULUM (ER) STRESS AND ITS INTERACTION WITH MITOCHONDRIA FOR THE TREATMENT OF METABOLIC DERANGEMENT AFTER MAJOR BURN INJURY
L. Diao1, 2, E. Bogdanovic2, X. Dai2, A. Abdullahi2, S. Amini-Nik2, and M.G. Jeschke*1, 2. 1Institute of Medical Science, University of Toronto, Toronto, ON, Canada, 2Sunnybrook Research Institute, Toronto, ON, Canada
Background: Stress induced hypermetabolism and insulin resistance are commonly seen and are associated with increased morbidity and mortality in severely burned patients. Our previous studies indicated that burn induced hepatic ER stress leads to mitochondrial dysfunction and contributes to post-burn hypermetabolism and insulin resistance. We therefore asked the question whether molecular ER chaperones that reduce ER stress could alleviate post-burn mitochondrial dysfunction and associated hypermetabolism and insulin resistance.
Material and Methods: A liver cancer cell line (HepG2) was used for in vitro and a 30% scald burn mice model for in vivo studies. Chemical chaperones (PBA and TUDCA) that are known to attenuate ER stress were used to study whether reduced ER stress may contribute to restoration of mitochondrial biogenesis and thus ameliorate hypermetabolism and insulin resistance. qRT-PCR, western blotting, and immunohistochemical analysis were conducted to determine hepatic ER stress (BiP, IRE-1α, PERK, ATF6, CHOP, XBP-1s), Akt and mTOR complex activity, PGC-1α and its upstream signaling modulators (AMPKα, SIRT1, PKA). Structural changes in liver upon ER stress and chaperone treatment were observed by electron microscopy.
Result: Thapsigargin induces ER stress and impairs mitochondrial biogenesis and cell metabolism via mitochondria-associated ER membrane (MAM) in multiple signaling pathways. In vitro, thapsigargin induced ER stress in HepG2 cells, concomitantly suppressed Akt-mTOR complex and inhibited PGC-1α and its upstream regulatory signaling (AMPKα, PKA, SIRT1). In vivo model of 30% total body surface area scald burn in mice confirmed such hepatic ER stress and accompanying suppressed Akt-mTOR signaling and inhibited PGC-1α and its upstream regulatory signaling. In vitro and in vivo, pre-treatment with PBA or TUDCA attenuated ER stress and improved such metabolic signaling and mitochondrial biogenesis. TUDCA showed more potent protective effect than PBA in vivo.
Conclusion: Our data indicate that molecular chaperones attenuate ER stress and improve hepatic metabolic signaling and mitochondrial biogenesis. These agents may thus have therapeutic potential for metabolic dysfunction.
ROLE OF LEUKOTRIENE B4 1 (BLT1) IN LIVER INJURY AND REPAIR FOLLOWING HEPATIC ISCHEMIA/REPERFUSION INJURY IN MICE
H. Ohkubo, Y. Ito, K. Kojo, M. Watanabe, and M. Majima. Kitasato University Graduate School of Medicine, Sagamihara, Japan
Aims: Leukotriene B4 (LTB4) is a potent chemoattractant for leukocytes including macrophages. Recruited macrophages play a critical role in liver repair after acute liver injury. The objective of the present study was to examine the role of LTB4 receptor 1 (BLT1) signaling in liver repair after hepatic ischemia/reperfusion (I/R) injury.
Methods: BLT1-knockout mice (BLT1-KO) or their wild counterparts (WT) were subjected to 60 min of partial hepatic ischemia followed by reperfusion. Serum samples and liver tissue for histology were analyzed at 6, 24, 48, and 96 h after reperfusion. The mRNA expressions of growth factors were determined by RT-PCR.
Results: ALT levels in WT with a peak at 6 h were declined thereafter and returned to controls at 96 h. In BLT1-KO, ALT levels also peaked at 6 h, but those at 48 and 96 h were higher than WT. Necrotic area in WT peaked at 24 h, and reduced gradually, while that in BLT1-KO was remained high until 96 h. The proliferation of hepatocytes as indicated by proliferating cell nuclear antigen (PCNA) expression in BLT1-KO was delayed. The hepatic mRNA levels of VEGF, VEGFR-1, and epidermal growth factor (EGF) were suppressed in BLT1-KO. Recruitment of CD11b/VEGFR1-positive macrophages expressing EGF in injured liver was attenuated in BLT1-KO at 48 h.
Conclusions: These results indicate that BLT1 signaling plays a role in liver repair during hepatic I/R through enhancement of EGF on recruited macrophages.
STIMULATION OF AMPK PATHWAY IN THE CNS ATTENUATES INFLAMMATION AND ACUTE LUNG INJURY IN SEVERE SEPSIS
N. Mulchandani1, W. Yang*2,1, F. Zhang2, J. Nicastro1, G. Coppa1, and P. Wang*2,1. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2Feinstein Institute of Medical Research, Manhasset, NY
Introduction: Sepsis and septic shock are serious problems that have required millions of healthcare dollars. Many studies have linked the involvement of the central nervous system (CNS) in the septic process, primarily via the vagus nerve. Despite this, no clear molecular mechanism has been defined. AMP-activated protein kinase (AMPK) is a protein with an anti-inflammatory activity and a central role as an energy sensing enzyme. AMPK is expressed in multiple tissues, including the hypothalamus. We hypothesized that pharmacologic activation of AMPK in the CNS with 5-aminoimidazole-4-carboxamide riboneucleotide (AICAR) would attenuate inflammatory response during severe sepsis, particularly in lung tissue.
Methods: C67BL/6 adult male mice underwent intracerebroventricular (ICV) injection of 20 ng AICAR (n=8) or vehicle (2 μL normal saline, n=8), followed by cecal ligation and puncture (CLP) 30 min post-ICV. Blood and tissues were collected 20 h after CLP. Protein, antigen, and mRNA levels were evaluated using Western blot, ELISA assay, and qPCR respectively.
Results: Mice treated with AICAR were shown to have a 71%, 95%, and 94% reduction in serum TNF-α, IL-1β, and IL-6, respectively, when compared to vehicle (Table). Lung tissues, a primary site of damage in severe sepsis, revealed reductions in protein levels of cytokines TNF-α, IL-1β, and IL-6 by 49%, 55%, and 87% respectively (Table) as well as mRNA levels of TNF-α and IL-1β by 72% and 73% (p<0.05) respectively. The mRNA levels of chemokines KC and MIP2 decreased 68% and 81% respectively, in lung tissues (p<0.05). AICAR treatment group had improved morphologic structure on H&E staining and reduced apoptosis by TUNEL assay in lung tissues when compared to vehicle.
Conclusion: Activation of AMPK in the CNS reduces pro-inflammatory cytokine levels systemically and in the lungs. In addition, it decreases chemokine levels and tissue damage in the lungs. Thus, centrally located AMPK has a crucial role in regulating inflammation during severe sepsis.
MESENCHYMAL STEM CELLS REVERSE TRAUMA AND HEMORRHAGIC SHOCK INDUCED BONE MARROW DYSFUNCTION
A.V. Gore, L.E. Bible, W.D. Alzate, D.H. Livingston*, A.M. Mohr*, and Z.C. Sifri*. Rutgers- New Jersey Medical School, Newark, NJ
Introduction: Lung contusion (LC) followed by hemorrhagic shock (HS) results in persistent bone marrow (BM) dysfunction lasting for up to seven days after injury. Mesenchymal stem cells (MSC) are multipotential cells that can hasten healing as well as exert protective immunomodulatory effects. We hypothesize that MSC can attenuate BM dysfunction following combined LCHS.
Methods: Male Sprague-Dawley rats (n=5-6/group) underwent LCHS ± injection of MSC. Following LC, rats were subjected to 45 minutes of HS (MAP of 30-35). Allogeneic MSCs (5 x 10^6 cells) were injected iv in 1mL IMDM media following the return of shed blood. Animals were sacrificed after seven days and peripheral blood (PB) and BM were analyzed. BM cellularity and growth of hematopoetic progenitor cell (HPC) colonies (CFU-E, BFU-E, CFU-GEMM) were evaluated. Flow cytometry to determine mobilization of HPCs to PB and plasma G-CSF levels were measured via commercial ELISA. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.
Results: As previously shown, LCHS resulted in a 13% decrease in BM cellularity with 22, 30, and 24% decreases in CFU-GEMM, BFU-E and CFU-E colony growth respectively as compared to naïve seven days following injury. The addition of MSCs returned all BM colony growth to naïve levels. While there was no difference in the percentage of HPC mobilized to PB (naive 0.05±0.5 vs LCHS1.0±2.5, p>0.05), G-CSF remained elevated seven days following LCHS whereas addition of MSC returned G-CSF to naïve levels. (Table)
Conclusion: One week following LCHS there is persistent BM dysfunction manifested by decreased BM cellularity, HPC colony growth and elevated plasma G-CSF levels. The addition of MSC following acute traumatic injury and shock reverses this BM suppression and returns G-CSF levels to normal. Further study into the immunomodulatory effects of MSC following trauma and hemorrhagic shock is warranted.
SIRT1 AGONIST MINIMIZES INJURY AND IMPROVES THE IMMUNE RESPONSE FOLLOWING TRAUMATIC SHOCK
J.A. Lucianox, S. Darwiche, B. Kautza, S. Martinez, S. Stratimirovic, and B.S. Zuckerbraun*. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA
Background: Survival from traumatic injury requires a coordinated and controlled inflammatory and immune response. Mitochondrial and metabolic responses to stress have been shown to play a role in these inflammatory and immune responses. We hypothesized that activation of mitochondrial biogenesis pathways via a Sirt1 agonist would lead to decreased tissue injury and partially ameliorate the immunosuppression seen following trauma.
Methods: C57Bl/6 mice were subjected to a multiple trauma model involving pseudofracture, crush injury, laparotomy, and 30% blood volume hemorrhage. Mice were pretreated with either 100mg/kg/day Srt1720 via oral gavage or vehicle for 3 days prior to trauma and extended until the day of sacrifice. Mice were euthanized at 4 and 48hrs after trauma at which time blood and organs were collected. Splenocyte responses were measured following stimulation with the T-cell mitogen anti-CD3ε (1μg/μL) and mitochondrial complex IV activity was evaluated.
Results: As expected, mice treated with Srt1720 had significantly higher mitochondrial complex IV activity following traumatic injury (p<.01). Pre-treatment with Srt1720 led to protection from hepatic injury and to a blunted degree of immunosuppression following trauma. Four hours post trauma, animals pre-treated with Srt1720 were noted to have reduced liver injury as measured by serum ALT levels (82.67 vs 193.07 U/I, p=.01). Immune function was measured via splenocyte responses 48 hours following trauma. Treatment with the sirt1 agonist minimized trauma-induced decreases in splenocyte proliferation (p<.05). The depression of splenocyte release of IFN-gamma following traumatic injury was furthermore attenuated in splenocytes harvested from Srt1720 pre-treated mice (p<.01).
Conclusions: Pretreatment with a Sirt1 agonist prior to traumatic injury protects against liver injury and partially minimizes the post-injury immunosuppression.
DELAYED TREATMENT WITH THE C5A-NEUTRALIZING SPIEGELMER® NOX-D20 IMPROVES OUTCOME IN EXPERIMENTAL SEPSIS
A. Vater1, N. Shushakova2, K. Hoehlig1, C. Maasch1, K. Buchner1, W.G. Purschke1, M. Huber-Lang3, and S. Klussmann1. 1NOXXON Pharma, Berlin, Germany, 2Phenos GmbH, Hanover, Germany, 3Department of Traumatology, Hand, Plastic, and Reconstructive Surgery, Center of Surgery, University of Ulm, Ulm, Germany
Background: Bacterial sepsis is among the most common hospital acquired conditions resulting in the need for intensive care and frequently leading to death. The formation of the anaphylatoxin C5a in sepsis has been shown to hyperactivate the immune system which subsequently may cause cell and organ damage and even fatal multi-organ failure. We hypothesized that selectively blocking C5a with an L-oligonucleotide-based aptamer would attenuate the inflammatory response and improve outcome in experimental sepsis.
Methods: Using an in vitro selection process we have generated NOX-D20, a plasma-stable mirror-image (L-)oligonucleotide (so-called Spiegelmer), that directly binds murine and human C5a and thereby inhibits signaling on both its receptors (C5aR and C5L2). In the cecal ligation and puncture (CLP)-induced murine sepsis model NOX-D20 therapy was initiated 0, 3, 6 or 12 h after CLP in order to show proof of efficacy for an interventional treatment of sepsis for this drug candidate.
Results: Mice with CLP-induced polymicrobial sepsis survived significantly longer when treated with NOX-D20 compared to vehicle treatment. Furthermore, pro-inflammatory cytokine and chemokine release, infiltration of neutrophils into the abdominal cavity and capillary leakage were significantly reduced. Noteworthy, treatment was efficacious even when initiated up to 6 h after CLP surgery.
Conclusion: Because of its efficacy in the mouse CLP model and the excellent safety profile of other members of the Spiegelmer class in healthy volunteers and patients, the C5a-neutralizing Spiegelmer NOX-D20 represent a promising candidate for clinical development. Targeting complement by NOX-D20 may add a new tool to the current armamentarium that currently consists of antibiotics, surgery and supportive care.
ENDOTHELIAL GLYCOCALYX (EG) AFTER SEVERE HEMORRHAGIC SHOCK (HS) FOLLOWED BY PLASMA VOLUME EXPANSION WITH NORMAL SALINE
L.N. Torres, J. Sondeen, C. Salgado, C. Valdez, M.A. Dubick*, and I. Torres Filho. US Army Institute of Surgical Research, San Antonio, TX
Background: Although an intact EG excludes larger molecules, its structure is semi-permeable to anionic macromolecules, such as albumin. Protection or restoration of EG might be an important therapeutic goal. Since generally blood products are not available at far forward battlefield locations, non-compressible injuries are currently managed by giving crystalloids such as 0.9% NaCl solution (normal saline) as resuscitation fluid. For the military, questions continue to arise regarding efficacy of normal saline since it is the most common resuscitation fluid in the battlefield. We investigated the effects of resuscitation with normal saline (n = 3) on EG thickness, permeability and coagulation after HS, and compared to SHAM (n = 5) and HS non-treated rats (n = 5).
Methods: SHAM rats were equally instrumented but not bled/resuscitated. Sixty-five venules of cremaster muscle were studied in anesthetized rats subjected to 1h of HS (40% estimated total blood volume) followed by 1h resuscitation with 45ml/Kg normal saline or no treatment. EG thickness, blood flow and permeability were quantified using intravital microscopy. Dextrans bound to fluorescein isothiocyanate or Texas Red were injected to estimate EG thickness in vivo. Blood hemodynamics and analysis as well as coagulation tests (ROTEM) were measured.
Results: SHAM rats showed stable systemic parameters throughout the experimental period of 2 hours. Preliminary analysis showed that resuscitation with normal saline decreased the hematocrit and total plasma protein compared to baseline levels. Treatment with normal saline improved mean arterial pressure, but induced significant EG shedding (compared to SHAM rats) and increase in microvascular permeability (compared to HS non-treated rats). Clotting times were longer and clot strength decreased in the normal saline group compared to SHAM and HS non-treated rats, possibly by dilution of coagulation factors.
Conclusion: Infusion of isotonic salt solution may entail net loss of albumin from the vascular space and wash out of EG components, which may have a deleterious impact in the microcirculation functions in our rat model of HS. The increase in permeability at microvascular level may lead to secondary systemic repercussions associated with HS.
Supported by US Army Medical Research & Materiel Command.
UNCOUPLED SKELETAL MUSCLE MITOCHONDRIA CONTRIBUTE TO INCREASED METABOLIC RATE IN SEVERELY BURNED ADULTS
C. Porter*1, 2, D. Herndon*1, 2, T. Chao1, 2, N. Bhattarai1, 2, M. Chondronikola1, 2, P. Reidy1, M. Borack*1, B.B. Rasmussen1, and L. Sidossis1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals for Children, Galveston, TX
Elevated metabolic rate is a hallmark of the stress response to burn injury. Increases in ATP consuming reactions only explain around 50% of this response. Uncoupled mitochondrial respiration, proton transfer not linked to ATP synthesis, is a means of generating heat in vivo. Here, we determined skeletal muscle mitochondrial function in healthy and severely burned adults. We hypothesize that uncoupling of skeletal muscle mitochondria contributes to increased metabolic rate in burn survivors.
Indirect calorimetry was used to estimate metabolic rate in burn patients. Quadriceps muscle biopsies were collected from 7 severely burned adults (72±20% of total body surface area burned) and 8 healthy adults. Leak, coupled and uncoupled mitochondrial respiration were determined in fresh, permeabilized myofiber bundles.
Metabolic rate was significantly greater than predicted values for burn patients (+65±11%, P<0.001). Coupled mitochondrial respiration was lower in burn patients vs. controls (22.6±5.2 vs. 66.2±8.4 pmol/sec/mg; P<0.001). The ratio of leak respiration to coupled respiration was lower in burn patients vs. controls (1.25±0.26 vs. 2.15±0.24; P<0.05), whereas the ratio of coupled respiration to uncoupled respiration (where ATP synthase is inhibited by oligomycin), was higher in burn patients vs. controls (0.54±0.11 vs. 0.32±0.03; P=0.05).
Increased metabolic rate in severely burned adults is accompanied by derangements in skeletal muscle mitochondrial function. Mitochondria from burn victims are more uncoupled, indicating greater heat production within skeletal muscle. We conclude that skeletal muscle mitochondrial dysfunction contributes to increased metabolic rate in burn victims.
FUNCTIONAL DIFFERENCE OF HUMAN SP-B GENETIC VARIANTS IN BACTERIAL PNEUMONIA MODEL USING HUMANIZED SP-B TRANSGENIC MICE
G. Wang*, J. Liu, S. Javidiparsijani, O. Abdel-Razek, Z. Liu, and R.N. Cooney*. SUNY Upstate Medical University, Syracuse, NY
Objective: Pneumonia is the leading cause of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). One of the most frequent pathogens is bacterial Staphylococcus aureus (S. aureus). Surfactant protein B (SP-B), a member of Saposin-like family of proteins (SAPLIP), plays a critical role in both lowering surface tension and antimicrobial activity in the lung. Genotyping studies demonstrate patients with the C allele of SP-B gene are susceptible to bacterial pneumonia. In this study we utilize an in vivo mouse pneumonia model to elucidate functional difference of human SP-B genetic variants.
Methods: Humanized transgenic (hTG) mice, which express either C or T allele of human SP-B genetic variants without mouse SP-B gene expression, were generated and used in this study. Pneumonia was induced using S. aureus (CFU: 5x107/mouse) intratracheal injection in hTG SP-B mice. Lung tissues and bronchoalveolar lavage fluids (BALF) were harvested 24h after exposure to S. aureus or saline. Histological, cellular and molecular changes in the lung of infected hTG mice were studied.
Results: Infected hTG SP-B-C mice (human SP-B C allele) showed more severe lung injury (p<0.01) and inflammation (p<0.01) in the lung compared to infected hTG SP-B-T mice (human SP-B T allele) 24 hrs after infection. The bacterial CFU was higher (p<0.01) in BALF from infected hTG SP-B-C mice (1.4+0.35x103 CFU/lung), compared to infected hTG SP-B-T mice (4.8+0.28x102 CFU/lung). Infected hTG SP-B-C mice demonstrate increased apoptosis and NF-kB expression in the lung (p<0.05) and compared to infected hTG SP-B-T mice. IL-6 and TNF-α levels were higher in BALF from infected hTG SP-B-C mice compared to infected hTG SP-B-T mice. Furthermore, the levels of surfactant phospholipid and surfactant proteins A and B in BALF, which function lowering surface tension in the alveoli of lung, decreased significantly (p<0.05) in infected hTG SP-B-C mice, when compared with infected hTG SP-B-T mice.
Conclusion: The C allele of human SP-B genetic variants confers increased susceptibility to S. aureus pneumonia and lung injury compared to the T allele SP-B variant. These demonstrate that hTG SP-B mice are an excellent tool for studying functional difference of human SP-B genetic variants.
THE ROLE OF CXCL10 IN THE PATHOGENESIS OF EXPERIMENTAL SEPTIC SHOCK
L. Luan1, D.S. Herzig2, 3, J. Bohannon1, T. Toliver-Kinsky2, Y. Guo1, and E. Sherwood*1. 1Vanderbilt University, Nashville, TN, 2The University of Texas Medical Branch and Shriners Hospital for Children, Galveston, TX, 3Ludwig Maximilians University, Munich, Germany
Introduction: The chemokine CXCL10 is produced during infection and inflammation to activate the chemokine receptor CXCR3, an important regulator of lymphocyte trafficking and activation. The goal of this study was to assess the contributions of CXCL10 to the pathogenesis of experimental septic shock in mice.
Methods: Severe sepsis was induced by cecal ligation and puncture (CLP) in mice resuscitated with lactated Ringer’s solution and, in some cases, the broad spectrum antibiotic Primaxin. Studies were performed in CXCL10 knockout mice and mice treated with anti-CXCL10 IgG. Endpoints included leukocyte trafficking and activation, core body temperature, plasma cytokine concentrations, bacterial clearance and survival.
Results: CXCL10 was present at high concentrations in plasma during CLP-induced septic shock (Figure A). Survival was significantly improved in mice treated with anti-CXCL10 IgG (Figure B) and CXCL10 knockout (CXCL10KO) mice (Figure C) compared to controls. CXCL10KO mice and mice treated with anti-CXCL10 IgG showed attenuated hypothermia, lower concentrations of IL-6 and MIP-2 in plasma and lessened NK cell activation compared to control mice. Compared to control mice, bacterial burden in blood and lungs was lower in CXCL10-deficient mice but not in mice treated with anti-CXCL10 IgG. Treatment of mice with anti-CXCL10 IgG plus fluids and Primaxin at 2 or 6 hours after CLP significantly improved survival compared to mice treated with non-specific IgG under the same conditions.
Conclusions: CXCL10 plays a role in the pathogenesis of CLP-induced septic shock and could serve as a therapeutic target during the acute phase of septic shock.
GENOMIC ANALYSIS OF INDIVIDUAL LEUKOCYTE POPULATIONS AFTER SEVERE TRAUMA
E. Vanzant, M. Lopez, H. Baker, S. Brakenridge, A.M. Mohr, D.C. Nacionales, R. Ungaro, A. Bihorac, C. Leeuwenburgh, L.F. Gentile, B.E. Szpila, A.L. Cuenca, F. Moore, L. Moldawer*, and P.A. Efron*. University of Florida, Gainesville, FL
We recently reported the genomic response of total circulating leukocytes after severe trauma(ST) and their potential predictive value. Unfortunately, analysis of this data is complicated by the dramatic changes in the differential leukocyte count after ST. In this study, we examined the genome-wide responses to injury in individual leukocyte populations(neutrophils(PMN), monocytes(MO) and T-lymphocytes(T)).
Microarray data from the Inflammation and Host Response to Injury Glue Grant database from isolated blood leukocyte populations was obtained from 244 ST patients(ISS>15, no TBI, in shock requiring blood transfusion, age>16) and 18 healthy controls. Trauma responsive genes(TRG) were determined in each subset to be TRG if significant(p<0.001, t-test) compared to controls at any time point(.5, 1, 4, 7 and 14 days after ST) and concurrent fold change of >1.5. Analysis consisted of using TRG to compare significant individual gene fold changes(p<0.05), and canonical and biofunctional pathway differences(IPA) between ST patients and controls in each cell subset.
3,211 TRG were identified of which, only 185 were common among all cell types. PMNs, MOs and Ts contained 2,038, 550 and 1672 TRG, respectively. Common TRGs among subsets were involved in pathways for cytokine receptor interactions, hematopoietic cell lineage, T-cell receptor signaling, TLR signaling and antigen processing and presentation. At early time points(<7d) there was downregulation of chemotaxis pathways but upregulation of phagocytosis pathways in PMNs. MOs demonstrated downregulation of maturation and activation pathways. PMNs/MOs both revealed upregulation of individual MDSCs genes and downregulation of innate immunity(ie. antigen presentation/PAMP detection). At later(>7d) time points, Ts revealed genomic patterns consistent with humoral immune dysfunction.
PMNs, MOs and Ts display mostly unique TRG and pathways after ST. PMNs contained the most TRG, proving that their contribution to total leukocyte TRG analysis was not due to their being the most common leukocyte. Although there were some common overall genomics changes, individual leukocyte populations respond differently at earlier and later time points after ST. The pattern displayed after 7-14 days is consistent with our description of the Persistent Inflammation Immunosuppression Syndrome(PICS) and may explain some of the increased long-term consequences of ST.
SUPPRESSED GENE EXPRESSION OF NLRP1 AND NLRP3 IN MONOCYTES IS RESPONSIBLE FOR THEIR IMPAIRED FUNCTIONALITY AFTER TRAUMA
B. Relja, J. Horstmann, K. Wilhelm, K. Jurida, and I. Marzi*. University Hospital of the Goethe-University, Frankfurt am Main, Germany
Objectives: The IL-1beta-producing activity of monocytes is suppressed after major trauma. The generation of IL-1beta requires its synthesis, the transcription of inflammasome components and their activation. Deregulated NLRP1 and/or NLRP3 inflammasome assembly is associated with several inflammatory diseases, resulting from altered processing of the pro-inflammatory IL-1beta. The present study was designed to determine which NLRP1 and/or NLRP3 inflammasome components are responsible for depressed monocyte function after trauma.
Methods: From ten healthy volunteers and twenty severely injured trauma patients [ISS≥16] CD14+-monocytes were isolated by microbeads daily up to day 10. Functional analysis of monocytes was performed daily by their ex vivo in vitro LPS-stimulation (10μg/ml, 24h) and subsequent evaluation of the IL-1beta-release by ELISA. Inflammasome activation was verified by protein analysis of cleaved IL-1beta and caspase-1. Gene expression of inflammasome components was analyzed daily before and after LPS-stimulation. Subsequently, isolated monocytes after admission were transfected with expression plasmids encoding for the lacking components identified by gene analyses.
Results: LPS-induced IL-1beta-release in monocytes from healthy volunteers was significantly suppressed after trauma, with lowest levels at the day of admission (p<0.05). The protein expression of processed IL-1beta and caspase-1 correlated with the IL-1beta-release. IL-1beta, caspase-1 and asc gene expressions were comparable in both, trauma patients and controls after LPS-stimulation during the 10-days course. In contrast, gene expression of nlrp1 and nlrp3 was reduced in trauma patients compared to controls after LPS-stimulation. Transfecting monocytes that were isolated from trauma patients after admission with nlrp1 or nlrp3 recovered the expression of these components as well as the LPS-induced IL-1beta-release to comparable levels of monocytes from healthy volunteers.
Conclusions: These results indicate that nlrp1 and/or nlrp3 may represent the lacking components in monocytes that are responsible for their suppressed activity after trauma. Regaining all components of NLRP1 and/or NLRP3 inflammasomes in monocytes recovers the monocyte activity after trauma, suggesting new mechanistic targets for balanced immune reaction after trauma.
IMMUNE PHENOTYPING THE RESPONSE TO LUNG S. PNEUMONIAE AND P. AERUGINOSA INFECTIONS FOLLOWING INJURY
K. Shimizu*1, L. Wanke-Jellinek1, 2, P. Gao1, J. Keegan1, and J.A. Lederer*1. 1Brigham and Women’s Hospital, Boston, MA, 2Technical University Munich, Munich, Germany
Background: Traumatic injuries induce systemic effects on the immune system and alter the response to infections. In this study, we used an unbiased systems biology approach to determine how injury affects immune cell responses to bacterial infections caused by clinically-relevant sepsis-causing pathogens - S. pneumoniae and P. aeruginosa. We hypothesized that these bacteria may cause different types of immune responses in uninjured vs. injured mice. Since there is limited information about the cellular immune response to lung infections, we centered this study on using bronchial alveolar lavage (BAL) as an approach to measure the kinetics of the cellular response to infection.
Methods: Mice underwent burn injury and were infected with S. pneumoniae and P. aeruginosa at 1 day after injury. BAL samples were collected before infection and at 4hrs, 1day, 2days, and 4days after infection. Flow cytometry was used to measure a variety of cell types including neutrophils, monocytes/macrophages, dendritic cells, B cells, T cells, NK cells.
Results: First, we found that CD11c+F4/80 cells represented the majority of cells in BAL and were present in significantly higher numbers in the lungs of burn mice. However, after infection, neutrophils became the predominant population followed by Gr-1+ macrophages. Important differences in cell responses between burn and sham mice were observed in several cell subtypes, including γδ T cell receptor (TCR)+ cells and macrophages. Sham mice showed increases in γδ T cells on day 1 after infection, while in burn mice γδ T cells did not increase until day 4. In addition, sham and burn mice showed markedly different cellular responses to S. pneumoniae and P. aeruginosa. For example, sham mice showed high 4 hr neutrophil reactivity to P. aeruginosa infection, but burn mice did not, whereas burn mice showed higher neutrophils at 4 hrs in response to S. pneumoniae than sham mice.
Summary: We characterized the differences in 17 immune cell subset responses to lung infection in sham and burn mice over a 4 day period. Among these cell subsets, we found significant injury-associated differences in neutrophils, Gr-1+ macrophages, γδ T cells and CD11c+ macrophages. As a whole, these findings suggest that these immune cell subsets may be central to the differences in susceptibility to infection in injured versus uninjured mice.
METABOLOMIC PROFILING OF POST-SHOCK PLASMA DEMONSTRATES A DRAMATIC INCREASE IN UNMEASURED ANIONS WHICH CONTRIBUTE TO ACIDOSIS
E.D. Peltz1, 2, E.E. Moore*1, 2, T.L. Chin1, K. Hanse1, and A. Banerjee*1. 1University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 2Denver Health Medical Center, Denver, CO
Severe acid-base disturbances occur during profound shock and are correlated with poor patient outcomes and risk of mortality. The severity of acidosis may not be fully explained by anaerobic metabolism and lactate production alone. Unmeasured plasma anions are the most common component of metabolic acidosis in trauma patients admitted to the ICU. While candidates for these anions have been proposed the identity of these substances remains largely unknown.
Hypothesis: Liquid chromatography (LC) / gas chromatography (GC) with mass spectrometry analysis of the post-shock plasma metabolome will reveal previously unrecognized anions and systems biology contributing to post-shock acidosis.
Methods: Plasma was collected from 13 severely injured trauma patients (age 43±13 years, 62% male, 85% blunt injury, median ISS 26) and from 5 healthy control donors (age 50±15.8 years, 40% male). Samples were processed by Metabolon (Durham, NC). After a standard solvent extraction samples were analyzed by LC or GC and mass spectrometry for metabolite identification.
Results: Trauma patients demonstrated early acidosis with mean pH 7.19 ± 0.13, base deficit 12.7 ± 5.48 mmol/L and lactate 8.29 ± 5.76 mmol/L. We identified 24 anions which increased in relative concentration between 1.42 and 635 fold following trauma; p< 0.02 vs control plasma. These anions which behave as acids at physiologic pH include 11 mono-carboxylates, 6 di-carboxylates, 2 tri-carboxylates, 2 amino acids, 2 phosphatidic acids and 1 sulfate.
Conclusion: Metabolomic profiling of post-shock plasma reveals previously undemonstrated systems biology and metabolites contributing to acidosis associated with severe traumatic injury. These anions which represent downstream products of protein, lipid, carbohydrate and nucleotide metabolism as well as metabolic substrates for the Krebs cycle and energy production may significantly contribute to post-injury acidosis. The precise contribution to the post-shock acid-base milieu may be elucidated by future quantitative investigations of individual metabolites.
2B4: A POTENTIAL TARGET FOR RESTORING IMMUNE COMPETENCE DURING SEPSIS
R. Mittal, M. Wagener, Z. Liang, J.D. Lyons, L. Margoles, C.M. Coopersmith*, and M.L. Ford. Emory University, Atlanta, GA
Objective: Immune suppression and increased expression of coinhibitory receptors on immune cells during sepsis are increasingly being implicated as mechanisms underlying sepsis mortality. The role of the coinhibitory receptor 2B4 (CD244) in sepsis is currently unknown.
Methods: Sepsis was induced by cecal ligation and puncture (CLP). WT sham and septic mice were sacrificed and spleens were collected 24 hours (n = 9-10/group) post-surgery so that 2B4 expression on immune subsets could be assayed using flow cytometric analysis. WT and 2B4-/- mice were then followed for 7 days post-CLP for survival (n = 22/group). Additionally, splenocytes from septic WT and 2B4-/- animals were analyzed 24 hours after CLP (n = 9-10/group).
Results: First, to determine which cell subsets express 2B4 during sepsis, immune cell subsets (CD11c, NK1.1, CD4, and CD8) in WT sham and WT CLP mice were assayed. Our data revealed an increased frequency of 2B4+ cells within the CD8+ T cell compartment in septic animals as compared to sham controls (3.6%±0.9 vs. 1.0%±0.1;p = 0.002) but not within the CD4+ T cell, dendritic (CD11c) or NK cell compartments. No differences were noted in CD48 expression (the ligand for 2B4) on these cell subsets. Given phenotypic upregulation of 2B4 on CD8+ T cells following sepsis, the next step was to determine whether 2B4 plays a functional role in sepsis. We observed that 2B4-/- mice exhibit markedly improved 7 day survival following CLP compared to WT (81.8% vs. 13.6%; p<0.0001). Therefore, in order to identify possible mechanism(s) by which loss of 2B4 protected animals from death following sepsis, we assessed T cell, dendritic cell and NK cell subsets in septic WT and 2B4-/- animals. 2B4-/- mice exhibited increased dendritic cell frequencies (0.9%±0.1 vs. 0.4%±0.04, respectively; p = 0.003) and increased CD3+ T cell frequencies (20.2%±1.2 vs. 15.2%±2.0; p = 0.05).
Conclusion: Loss of 2B4 coinhibitory signals potently protects animals from death following sepsis. This effect may be mediated through increased functionality of CD8+ T cells resulting in increased immune competence and reduced immune dysregulation following sepsis.
USE OF ALUM AS AN INNATE IMMUNE ADJUVANT IN NEONATAL SEPSIS: WHAT IS THE ROLE OF CASPASE 1?
A.L. Cuenca, A. Cuenca, L.F. Gentile, D.C. Nacionales, R. Ungaro, L. Moldawer*, P.A. Efron*, and S. Larson. University of Florida, Gainesville, FL
Background: Sepsis is a leading cause of infant mortality, especially in premature and low birth weight neonates. We have previously shown that sepsis survival in neonatal mice relies primarily on the innate immune response for protection. Adjuvant activation via the TLR4 receptor has been shown to improve outcome (Cuenca et al. I&I 2011 Jul;79(7):2746-54). Unfortunately, TLR agonists such as LPS and resiquimod are contraindicated due to sepsis-like symptoms they induce. Other clinically-useful adjuvants are commonly used in infants, including alum, which is presumed to activate innate immunity via caspase-1 activation and the inflammasome. Here we tested whether pretreatment of neonatal mice with alum could improve survival to polymicrobial sepsis, and whether the improved host response was caspase-1 dependent.
Methods: Wild-type C57BL/6 (WT) and C57BL/6caspase-1-/- knockout (KO) mice received a low (LD25-40) or high (LD50-85) dose cecal slurry (CS) model of intra-abdominal sepsis. Neonates received either no pretreatment (control) or 20 ug alum, via i.p. injection, 24 hrs prior to CS administration. In addition to survival, bone marrow and splenic hematopoietic stem cells were harvested 36 hours post CS injection and were stained for lin-sca1+c-kit+ (LSK) cells by flow cytometry.
Results: Caspase-1 KO mice exhibited increased survival after sepsis when compared to WT mice in response to both low and high dose CS (p<0.001). In addition, both WT and KO mice pretreated with alum had 100% survival compared to groups receiving CS alone. WT mice pretreated with alum had significant expansion of LSKs 36 hours after sepsis in both spleen and bone marrow (p<0.0001). However, LSK expansion was significantly attenuated in caspase-1-/- KO mice (p<0.01).
Conclusions: We demonstrate here that alum pretreatment improves survival to low and high dose polymicrobial sepsis in neonates, even in the absence of caspase-1. In contrast, the expected expansion of bone marrow and splenic hematopoiesis in response to sepsis is dependent upon endogenous caspase-1. We conclude that the adjuvant effects of alum are both dependent and independent of caspase-1 in neonates; however, improved survival is independent of the inflammasome.
CD39 IMPROVES SURVIVAL IN MICROBIAL SEPSIS BY ATTENUATING SYSTEMIC INFLAMMATION
Z. Németh1, 2, B. Csóka2, G. Törö2, B. Koscsó2, E. Kókai2, L. DiFazio1, and G. Haskó*2. 1Morristown Medical Center, Morristown, NJ, 2Rutgers-New Jersey Medical School, Newark, NJ
Introduction: With its high mortality rates, sepsis remains a major challenge in surgical intensive care units (SICU-s). Changes in levels of ATP, ADP, AMP and adenosine have profound effects in various immune diseases. We hypothesized that the ATP phosphohydrolyzing CD39 ectonucleotidase or apyrase would affect septic immune responses.
Methods: The most clinically relevant model of sepsis was induced by subjecting CD39 knockout (KO) and wild-type (WT) mice to cecal ligation and puncture (CLP). In another set of experiments, WT mice were treated with the CD39 mimicking enzyme, apyrase or the selective CD39 inhibitor, POM 1. Survival rates, cytokine levels from blood, peritoneal lavage fluid and vital organs were measured. Intracellular mechanisms of tissue injury and apoptosis were examined in lung, heart, kidney, thymus and spleen.
Results: Both genetic and pharmacologic inactivation of CD39 resulted in increased pro-inflammatory cytokine and chemokine concentrations in the blood and peritoneum of septic mice. Animals treated with the specific CD39 inhibitor POM 1 showed decreased survival rates as opposed to the improved survival of the CD39-mimicking apyrase treated mice after 7 days of CLP. Septic CD39KO mice had evidence of more severe injury in the heart and lung. Using CD39 bone marrow-chimeric mice we found that CD39KO→CD39WT chimeras that have KO bone marrow and WT parenchyma exhibited higher pro-inflammatory cytokine responses than CD39WT→CD39WT mice 16h after CLP.
Conclusions: Nucleotide phosphohydrolysis by CD39 or apyrase results in pulmonary and cardiac protection as well as improved survival of septic mice suggesting apyrase treatment may have a therapeutic role in sepsis.
AMITRIPTYLINE TREATMENT RESULTS IN DECREASED IMMUNE CELL NUMBERS AND FUNCTION FOLLOWING BURN INJURY
E. Gulbins, B.L. Johnson, and C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Introduction: Currently over 10% of the U.S. population are taking anti-depressants. Numerous anti-depressants currently on the market inhibit acid sphingomyelinase (ASM), an enzyme that is known to mediate leukocyte function and homeostasis. Amitriptyline is one such drug. Severe burn injury can lead to an immunosuppressive state that is characterized by decreased leukocyte function and numbers as well as increased susceptibility to infection. Based upon the intersection of these facts, we hypothesized that amitriptyline-treated, burn-injured mice would have an altered immune response to injury as compared to control injured mice.
Methods: CF-1 mice were pre-treated with multiple doses of 10mg/kg amitriptyline. This dose is shown to reduce ASM levels by 30-50%. Drug- or vehicle (normal saline)- treated mice were subjected a 22% TBSA scald injury or sham injury. Immune cells from spleen, thymus, and bone marrow were harvested on post burn day one. Cells were enumerated and characterized by flow cytometry. Alternatively, mice were treated with peptidylglycan (PepG) 1 day after burn to characterize immune response.
Results: We first determined that amitriptyline treatment resulted in a 30-40% decrease of Asm-activity and splenocyte ceramide levels. Following sham- or burn injury ± amitriptyline, we observed the typical reduction of lymphocyte precursors in the bone marrow and thymus as well as mature leukocytes in the spleen. Interestingly, numbers of these cells were further reduced in injured mice treated with amitriptyline. Additionally, of the CD4+ and CD8+ T cells remaining 1 day after injury, cellular activation was significantly decreased in the amitryptiline treated burn group compared to the vehicle-burn group. We also observed that amitriptyline treatment reduced neutrophil recruitment following PepG stimulus in both sham- and burn-injured mice.
Conclusions: These data suggest ceramide alterations can play a significant role in immune suppression and potential susceptibility to infections after injury.
FMS-LIKE TYROSINE KINASE-3 LIGAND ACTIVATES PLASMACYTOID DENDRITIC CELLS TO PROMOTE NEUTROPHIL RESPONSE TO INFECTION AFTER BURN INJURY
L. Bae, W. Cui, M. Vinish, and T. Toliver-Kinsky*. University of Texas Medical Branch, Galveston, TX
Patients with severe burn injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for detection of infection and activation of innate and acquired responses by other immune cells. DCs are significantly decreased in burn patients early after injury, and development of sepsis is associated with a persistent depletion of DCs. In an experimental model of burn wound infection, enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) significantly enhances neutrophil migration, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC), and cDCs have previously shown to be required for some of these effects of FL after injury. The objective of this study was to determine the contribution of the pDC subset to the protective effects of FL after burns. We report that FL treatment after burn injury increases survival, bacterial clearance, and neutrophil migration to infection, and depletion of pDCs at the initiation of treatment prevents these effects of FL. Additionally, FL directly stimulates pDCs to produce a neutrophil chemotactic factor(s). Such enhancement of neutrophil migratory capacity by pDCs is a novel function that is induced by FL. These findings suggest a unique mechanism that may be modulated to enhance immune responses to infection and improve outcome after burn injury.
ACTIVATED PROTEIN C RELIEVES ER STRESS BUT NOT MORTALITY IN MOUSE KIDNEY INJURY
C.S. Leibowitz, C.L. Mayer, A. Motomochi, B.C. Lee, K. Sivalogan, S. Kurosawa*, and D.J. Stearns-Kurosawa*. Boston University School of Medicine, Boston, MA
Introduction: Endoplasmic reticulum (ER) stress-induced cell and organ injury accompanies inflammatory challenge and is relieved by activated protein C (APC), which is anti-coagulant and cytoprotective. Shiga toxin-2 (Stx2) from food-borne enterohemorrhagic E.coli causes potentially lethal hemolytic uremic syndrome with acute kidney injury (AKI) in severe infections. Stx2 is a ribotoxin that causes renal tubular injury with AKI in mice. This is complicated by glomerular thrombi in patients and nonhuman primate (Papio) models. Therapy options are limited by lack of early injury biomarkers.
Objective: We hypothesized that APC would be protective and ER stress molecules would report Stx2-induced renal injury. Experiments were designed to take advantage of differential responses to Stx2 in mice (renal tubular AKI) and baboons (coagulopathy and AKI).
Methods: Mice were challenged with lethal Stx2 (1ng i.p; days 0,3) +/- APC (20 ug i.p; days 0-3) or saline with periodic phlebotomy. Kidney mRNA for qPCR was harvested at early euthanasia (day 2-3) or at endpoint (n≥5/group). Baboons received lethal Stx2 (50 ng/kg) +/- APC (2.4 mg/kg) at 24 or 48 hours after challenge and pathophysiology was monitored by established methods.
Results: Stx2 challenged mice developed terminal AKI with elevated BUN (Stx2, 81.0±15.7 vs saline, 31.3±10.4 mg/dL, p<0.001) and kidney AKI markers NGAL (37.3-fold; p<0.01) and KIM1 (69.5-fold; p<0.001). On day 2, ER stress markers HSP40 and spliced XBP1 mRNA were increased (2.8±0.6-fold,p<0.01; 1.79±0.72-fold, p<0.05) and on day 3, ER stress marker CHOP increased 8.74-fold (p<0.01). At endpoint, kidney anti-apoptotic Bcl2 and pro-apoptotic DR5 mRNA were decreased (0.44±0.18-fold, p<0.001) and increased (6.38±3.71-fold, p<0.01), respectively. APC co-treatment of Stx2 mice delayed rising BUN (p<0.05) and abrogated kidney CHOP mRNA (0.32±0.17-fold, p<0.001), but did not alter mortality (5.1±1.1 vs 5.6±0.5 days). In contrast, baboons were rescued from lethal Stx2 by delayed APC treatment, accompanied by significantly delayed BUN and creatinine increases.
Conclusions: This study identifies ER stress responses to be early indicators of Stx2-induced cell injury in vivo and possibly of value as early biomarkers. Reduced ER stress by APC did not associate with improved mortality in the mouse model, but such treatment was successful in the more complex baboon model.
L-ARGININE AND NITRIC OXIDE ATTENUATE IL-1B-INDUCED NF-KB ACTIVATION IN CACO-2 CELLS
Q. Meng*, A. Sharif, M. Cooney, T. Ahmed, and R.N. Cooney*. SUNY Upstate Medical University, Syracuse, NY
Introduction: L-arginine (L-Arg) is the main precursor of nitric oxide (NO) and substrate for inducible nitric oxide synthase (iNOS). Administration of dietary L-Arg reduces sepsis-induced systemic inflammation in clinical trials. In intestinal epithelia, NF-κB mediates IL-1β activated pro-inflammatory gene expression and mucosal inflammation. Our lab has shown L-Arg attenuates the induction of NF-κB activity in intestinal epithelia (Caco-2) cells by IL-1 β. This study examines the mechanisms by which L-Arg mediates IL-1β -induced NF-κB activation in Caco-2 cells.
Methods: Caco-2 cells were transfected with a NF-κB promoter luciferase vector and exposed to increasing concentrations of L-Arg (0 to 20mmol); the nitric oxide donor sodium nitroprusside (SNP, 0-10 mM) in serum-free media for 4 h, and then stimulated IL-1β (0 to 5 ng/ml) ± a inhibitor of NOS, Nω-Nitro-L-arginine (L-NNA 0-5 mM) for 4 h. Luciferase activity was expressed as fold-induction, normalized to protein. mRNAs of NF-κB (p65 subunit) and iNOS and protein of NF-κB levels were measured by RT-PCR and Western blot, then normalized to GAPDH. Data are expressed as means ± SE, n=8/group, and analyzed with t-test (P<0.05).
Results: IL-1β increased NF-κB promoter luciferase vector activity (6-fold). Expression of NF-κB mRNA (1.4 fold) and protein (1.5 fold) was also increased in a time- and dose-dependent fashion by IL-1 incubation. L-Arg and SNP attenuated IL-1β-induced increase in NF-κB luciferase activity and expression (mRNA and protein). L-NNA administration attenuated the inhibitory effects of L-Arg on IL-1β- inducible NF- κB luciferase activity and expression of NF-κB mRNA and protein. iNOS mRNA level was increased by IL-1β and this induction was reduced by administration of L-Arg.
Conclusions: IL-1β stimulates inflammation in intestinal epithelial cells by multiple mechanisms: upregulating NF-κB expression (mRNA and protein, increased iNOS expression (mRNA) and stimulating NF-κB activation. The inhibitory effects of L-Arg on NF-κB activation by IL-1β appear to be mediated by iNOS since suppression of NO production by the iNOS inhibitor, L-NNA, diminishes L-Arg’s ability to attenuate IL-1β inducible NF-κB activation.
AMPK ACTIVATORS PRODUCE DIFFERENT RESPONSES IN SMALL AND LARGE MUSCULAR ARTERIES
Y. Huang1, A.S. Miner2, 1, B. Sharma2, G. Chen2, T. Makinde1, R.W. Barbee*3, 5, and P.H. Ratz*4, 5. 1Dept. of Emergency Medicine, Virginia Commonwealth University, Richmond, VA, 2Dept. of Biochemistry, Virginia Commonwealth University, Richmond, VA, 3Depts of Emergency Medicine and Physiology, Virginia Commonwealth University, Richmond, VA, 4Depts. of Biochemistry and Pediatrics, Virginia Commonwealth University, Richmond, VA, 5VCURES, Richmond, VA
Regulation of arterial contraction plays a critical role in determining cardiac output distribution during hemorrhage and resuscitation. Many contractile agents are mobilized during hemorrhage, but their relative activities in arteries from different peripheral organs remains to be fully established. Moreover, the relative ability of different arteries to respond to metabolic stress and to activators of the metabolic sensor/effector, AMP kinase (AMPK), is not known. Thus, we examined the ability of different agents (angiotensin II (Ang II), phenylephrine (PE), U-46619 and vasopressin (VP)) to contract different peripheral arteries (renal (RA), mesenteric (MA), femoral (FA) and epigastric (EA)) in the presence and absence of specific (AICAR and A769662) and non-specific (berberine (BBR), resveratrol (RSV), simvastatin (SIMV) and metformin (MET)) AMPK activators. Also, to mimic a metabolic stress similar to ischemia, some tissues were exposed for 30 and 60 min to STARVE (a hypoxic (pO2 < 20 mmHg) and glucose-free solution). Artery rings were prepared and secured to a wire myograph for tension analyses. Tissues were quick-frozen and processed to measure AMPK phosphorylation (AMPK-pT172) as an index of activation. The order of contractile potency to PE was RA>FA>EA>MA. Compared to MA and RA, FA and EA responded more strongly and potently to Ang II, U-46619 and VP. In FA but not EA, AICAR caused a significant rightward shift in the PE-induced concentration-response curve (CRC) and a 3-fold increase in AMPK-pT172. In EA but not FA, MET and SIMV produced an ~2-fold increase in AMPK-pT172. BBR produced an ~2-fold increase in both arteries. For comparison, 30μM MET, BBR and SIMV produced an ~2-3-fold increase in AMPK-pT172 in liver slices. While MET had no effect on PE-induced tension in EA and FA, SIM strongly inhibited PE- and VP-induced contractions. In FA and EA, BBR caused a significant rightward shift in a PE-, but not VP-, induced CRC. RSV (30μM) induced a rightward shift in a VP-, but not PE-, induced CRC. A769662 had no effect on a PE-induced CRC in FA or EA, and produced a >½ log rightward shift in the VP-induced CRC in EA. In FA and EA, STARVE produced a strong 5-6-fold increase in AMPK-pT172. Together, these data reveal significant differences in responses to AMPK activators in large (FA) and small (EA) musculocutaneous arteries. Support: DOD Grant #W81XWH-12-1-0525
EX VIVO VASCULAR REACTIVITY AS A SURROGATE FOR THE TRAUMA-HEMORRHAGE (T-H) SURVIVAL MODEL: COMPARISON OF SOLUBLE ESTROGENS EE-3-SO4 VS. E2-SO4 POTENCY
M. Jinno1, W.J. Hubbard1, J. Robertson1, M. Miller2, and I.H. Chaudry*1. 1University of Alabama at Birmingham, Birmingham, AL, 2Texas A&M University, College Station, TX
Severe hemorrhage remains the most prevalent cause of death in combat. It is difficult to treat injuries, especially under battlefield conditions. In 2005 DARPA launched the Surviving Blood Loss program to explore treatments which could enable warfighters to survive otherwise fatal hemorrhage. We have observed survival for 6 hr after 60% blood loss in rats and pigs, with no intervention other than iv administration of soluble estrogen (E2) delivered in a non-resuscitative volume. However, our T-H survival model is technically complex, costly, time-consuming and requires many animal subjects. In lieu of intact animals, we have exploited isolated vascular rings as an in vitro surrogate of the cardiovascular physiology of E2 treatment. For this study, the isolated rat’s aortic rings were placed in an aerated Krebs-HCO3 organ bath at 37°C. Force transducers connected to a data acquisition system measured ring tension. In additional studies, aortic ring viability was confirmed before/after the experiment with KCl (70mM). Results (n=6/group): 1) shows that in vitro vascular reactivity correlates with higher 6-hr survival rate with 17β-ethinyl-estradiol-3-sulfate (EE-3-SO4) vs. 17β-estradiol-3-SO4 (E2-SO4); and 2) ascertain that EE-3-SO4 lowers vascular resistance via nitric oxide (NO) production. Comparing EE-3-SO4 and E2-SO4 in a logarithmic, dose-escalating fashion, EE-3-SO4 was more potent at all doses vs. E2-SO4 (Fig). Furthermore, that mode of action for EE-3-SO4’s vascular relaxation is through the nitric oxide synthase (NOS) pathway, as determined using the NOS inhibitor L-nitrosyl arginine methyl ester (L-NAME). Thus, 1) the vascular reactivity under appropriate conditions is a powerful in vitro tool to explore the pharmacology and physiology of soluble estrogens; 2) EE-3-SO4 is a superior drug for use in treating severe blood loss; 3) EE-3-SO4 acts through the NOS pathway to lower vascular tension and reduce resistance; and 4) the likely target is endothelial NOS (eNOS), since eNOS and E2 receptors are coupled in a cell-surface complex and removal of the endothelium from rings ablates this action (DARPA W911NF-10-1-0130).
SILENCING GENE EXPRESSION IN MACROPHAGES AND DENDRITIC CELLS FOR ANTI-INFLAMMATORY TREATMENT
C. Ye*, P. Shankar, and M.N. Swamy. Texas Tech University Health Science Center, El Paso, TX
Macrophages and dendritic cells are key immune cells that regulate immune response as well as play important roles in inflammatory disease treatment. Small interfering RNAs (siRNAs), which induce potent gene silencing by degradation of cognate mRNA, can be used for suppressing inflammatory cytokine production by these immune cells, provided a practical delivery strategy can be developed for in vivo use. Here, we show that macrophage and dendritic cells express the nicotinic acetylcholine receptor (AchR) on their surface, which can be specifically targeted by a short peptide derived from the rabies virus glycoprotein (RVG). The peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce HMGB1 gene silencing specifically in human macrophages and dendritic cells in humanized mouse sepsis model. The results showed that RVG-9R/siHMGB1 effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis and rescued humanized mice from CLP-induced mortality. We also tested the RVG-9dR delivery of TNF-α siRNA to macrophages and dendritic cells for arthritis treatment in CAIA mouse model. The results showed that RVG-9R/siTNF-α can reduce paw thickness scores as well as synovia TNF-α level, histological analysis also showed the treated mice were protected from severe arthritis. These results suggest that RVG-9dR can be harnessed as a tool for siRNA delivery to macrophages and dendritic cells that could potentially be a clinically viable strategy for anti-inflammatory treatment.
MARKERS OF IMMUNE SUPPRESSION FOLLOWING BURN INJURY
C.P. Henson*, L. Luan*, and E. Sherwood*. Vanderbilt University, Nashville, TN
Infection is responsible for many complications following severe burn injury. Alterations in lymphocyte populations and function have been described in patients with severe sepsis unrelated to burn injury. The net effect of these changes appears to be immunosuppressive, predisposing patients to late infection, and this is subsequently associated with worse outcomes. T lymphocytes are the primary effectors of the cellular adaptive immune system, and altered expression of co-inhibitory receptors by T lymphocytes (PD-1, PD-L1, etc) has been implicated in immune dysfunction. Following the initial resuscitation of the patient with burn injury, repair and regenerative processes predominate, and this may lead to inhibition of the immune response similar to what is seen in late sepsis. We hypothesized that changes in immune cell activity are also present early in burn injury, and if such changes do occur, they are likely to promote immunosuppression and lead to the development and progression of late infection. We enrolled patients with large total body surface area burns, requiring admission to our dedicated burn ICU, and evaluated leukocyte numbers, co-inhibitory receptor expression and plasma cytokine profiles. Compared to age- and sex-matched controls, CD4+ and CD8+ T lymphocyte counts were decreased significantly in burn patients at the time of admission and remained decreased out to 14 days post-burn (Figure). PD-1 expression was significantly increased on CD8+ T cells on day 14 post-burn but not at earlier time points. A trend towards increased numbers of pro-inflammatory CD14+CD16+ monocytes was also observed in burn patients and pro-inflammatory monocytes showed increased expression of the co-inhibitory ligand PD-L1. In addition to these changes in lymphocyte subset composition, there was a trend towards altered expression of pro- (IL-6, IL-8) and anti-inflammatory (IL-10) cytokines in patients following burn injury, suggesting early immune dysfunction in this population.
ACTIVATED PROTEIN C MEDIATES TRAUMA PATIENT PLASMA INDUCED ENDOTHELIAL BARRIER INJURY THROUGH S1P RECEPTOR SIGNALING
B. Miyazawa and M.J. Cohen*. University of California San Francisco, San Francisco, CA
Background: In addition to its well-described anti-coagulant effect, activated Protein C (aPC) also has several cytoprotective functions that are still not well understood. We have previously shown an endothelial barrier breakdown after treatment with trauma patient plasma, which can be attenuated by aPC. Activated Protein C has been shown to mediate barrier dysfunction through the sphingosine-1-phosphate (S1P) family of receptors. Therefore, we hypothesized the patient plasma signals through S1P receptors, activating RhoA, which binds to its kinase ROCK, which is involved in barrier disruption.
Methods: An electrical cell-substrate impedence sensing system (ECIS) was used to assess the permeability of monolayers of HUVECs. A decrease in transendothelial electrical resistance indicates a loss in cell junction adherence, reflecting an increase in permeability. Plasma samples were prospectively collected from trauma patients upon arrival to a Level I trauma intensive care unit.
Results: Treatment of HUVEC monolayers with trauma patient plasma resulted in a maximal decrease in resistance of 43.3%. One hour pretreatment with aPC attenuated this decrease in resistance, resulting in a maximal decrease of only 7.5%.One hour pretreatment with the sphingosine kinase inhibitor DMS similarly attenuated the decrease in resistance, resulting in a maximal decrease of 8.7%. One hour pretreatment with a ROCK inhibitor (Y-27632) also attenuated the decrease in resistance, resulting in a maximal decrease of 28.7%.
Conclusion: Trauma patient plasma does indeed appear to signal through S1P receptors to induce endothelial barrier breakdown via RhoA activation. Furthermore, aPC attenuates barrier disruption similarly to DMS, suggesting a mechanism by which, after injury, aPC can signal through S1P receptors to preserve the endothelial integrity.
MECHANISMS OF C5A INDUCED CARDIOMYOCYTE DYSFUNCTION
M. Kalbitz2, 1, M. Huber-Lang*1, F. Gebhard*1, and P. Ward*2. 1University of Ulm, Ulm, Germany, 2University of Michigan, Ann Arbor, MI
Myocardial dysfunction in sepsis is correlated with high lethality. There is evidence that the innate immune system, and in particular, the powerful complement activation product, C5a, and its interaction with both C5a-receptors (C5aR and C5L2), are involved in the development of septic cardiomyopathy. We have previously shown that addition of C5a to cardiomyocytes (CM) results in defective contractility and relaxation by incompletely understood mechanisms. We hypothesized that interaction of C5a with its receptors, C5aR and C5L2 contribute to cardiac dysfunction due to defective regulation of intracellular calcium ([Ca2+]i) and reactive oxygen species (ROS).
[Ca2+]i and ROS measurements in single CMs isolated from young adult male rats and from wild type, C5aR-/-, and C5L2-/- mice were performed by flow cytometry. mRNA for sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2) and sodium-calcium exchanger (NCX) were measured by real-time PCR employing left ventricular homogenates. Protein levels of SERCA2 and NCX were evaluated by western blots. Cardiac hemodynamic function in mice was determined with echocardiograms (ECHO).
Presence of C5a results in time- and dose-dependent increases in [Ca2+]i and ROS in CMs. Rises in CM [Ca2+]i after CLP were correlated with reduced levels of two calcium regulating proteins, SERCA2 and NCX in mouse CMs. Cardiac dysfunction in sepsis was significantly attenuated in C5aR-/- and C5L2-/- mice.
Previously we showed that CMs contain C5aR and that addition of C5a produces defects in CM contractility and relaxation. The current studies indicate that C5a addition to CMs produces a buildup of [Ca2+]i and ROS, both being correlated with ventricular dysfunction and arrhythmias, together with reductions in calcium handling proteins. Collectively, these alterations may explain, at least in part, the cardiomyopathy of sepsis.
TLR3-MEDIATED INFLAMMATORY RESPONSE AFTER GLOBAL MYOCARDIAL I/R VIA ACTIVATION OF TLR4-MEDIATED NF-κB AND TRIF/IRF SIGNALING PATHWAYS
N.G. Yousif*, Y. Zhai, H. Slimani, L. Ao, D. Fullerton, and X. Meng*. University of Colorado, Aurora, CO
The myocardial inflammatory response augments ischemia reperfusion injury (I/R), and TLR3 plays role in the regulation of myocardial inflammatory response, the importance of this response in vivo during acute inflammatory processes has not been fully understood. Our previous studies found that TLR4-mediated MCP-1 production contributes to the mechanism of myocardial injury following global myocardial I/R. We test the hypothesis that myocardial tissue TLR4-mediated NF-κB and TRIF/IRF signaling pathways activated by TLR3 after global myocardial I/R. Synergeneic heterotopic abdominal heart transplant was performed in mice (WT, TLR4-/-, TLR3-/-) for global myocardial I/R by using microsurgical techniques for vascular anastomoses. The donor’s thoracic aorta was anastomosed end-to-side to the recipient’s infrarenal abdominal aorta, and the donor’s pulmonary artery was anastomosed to the recipient’s inferior vena cava. Donor hearts were subjected to 4 h global ischemia followed by 4 h reperfusion. Our result shows that TLR3−/− donor heart significantly attenuated myocardial NF-κB binding activity both the levels of phosphorylated IκBα/IκBα after global myocardial I/R. In addition to reduced myocardial injury and decrease the inflammatory response. Further, global myocardial I/R increased levels of TLR4, TRIF and IFN-β in the myocardium. Interestingly the TLR4-activation signaling was significantly prevented by TLR3 deficiency. We concluded that the use of antibody directed against TLR3 might serve as a therapeutic clinical option in the treatment of cardiac injury induced by global myocardial I/R.
NEPHRILIN PEPTIDE MODULATES A NEUROIMMUNE STRESS RESPONSE IN RODENT MODELS OF BURN TRAUMA AND SEPSIS
D. Mascarenhas1, A. ElAyadi2, B.K. Singh1, A. Prasai2, D. Herndon*2, S. Hegde2, and C.C. Finnerty*2. 1Mayflower Orgn Research & Education, Sunnyvale, CA, 2University of Texas Medical Branch & Shriners Hospitals, Galveston, TX
Introduction: Sepsis occurs three times more often in burns than in other types of trauma, suggesting an overlap or synergy between underlying immune mechanisms in burn trauma and sepsis. Nephrilin peptide, a designed inhibitor of mTORC2, has previously been shown to modulate a neuroimmune stress response in rodent models of xenobiotic and metabolic stress. Here we investigate the effect of nephrilin peptide administration in different rodent models of burn trauma and sepsis.
Methods: Using a well-validated rat scald burn model we injected rats daily either with saline or 4 mg/kg body weight nephrilin. Animal tissues were analysed at 24 hours post-burn (Day 1) and on Day 14, in order to capture acute and delayed phases of the NSR. Nephrilin was similarly tested in the cecal ligation and puncture (CLP) mouse model of polymicrobial sepsis after surgical stress, measuring mortality in the Day 1-7 range.
Results: In the rat scald burn model, daily subcutaneous bolus injection of 4 mg/kg nephrilin significantly reduced the elevation of kidney tissue substance P, S100A9 gene expression, PMN infiltration and plasma inflammatory markers in the acute phase, while suppressing plasma CCL2 and insulin C-peptide, kidney p66shc-S36 phosphorylation and PKC-beta and CGRP in dorsal root ganglia at 14 days (chronic phase). In the mouse cecal ligation and puncture model of sepsis, nephrilin fully protected mice from mortality between surgery and day 7, compared to 67% mortality in saline-treated animals, while significantly reducing elevated CCL2 in plasma.
Conclusions: mTORC2 may modulate important neuroimmune responses in both burn trauma and sepsis. Applicability of Research to Practice: Traumatic stress is associated with secondary complications such as sepsis and organ failure, which lead to morbidity and death. The mechanism has never been elucidated. This work provides a novel mechanistic explanation and intervention.
R-107 ATTENUATES SEVERITY OF ACUTE LUNG INJURY IN CONSCIOUS OVINE MODEL OF CHLORINE GAS INHALATION
O. Fujiwara1, E. Lopez1, A. Salzman2, G. Southan2, and P. Enkhbaatar*1. 1The University of Texas Medical Branch, Galveston, TX, 2Radikal Therapeutics, West Tisbury, MA
Background: In the U.S. more than 14 million tons of chlorine gas is produced and transported to urban areas at risk. Exposures can be seen in both civilian and warfare environment, causing acute lung injury and death. To date there is no specific therapy for it. We hypothesized that novel agent R-107 containing nitric oxide donor, peroxynitrite modulator and oxygen superoxide scavenger effectively ameliorates chlorine-induced acute lung injury.
Methods: Previously instrumented (Swan Ganz, femoral artery and left atrium catheters) eight female sheep, were anesthetized and ventilated with 140ppm chlorine gas for 30 minutes in the approved facility with the strict monitoring for possible leak. Urine output was measured via Foley catheter. We studied randomly (double blinded) allocated two groups of sheep: 1) Control: Injured, treated with vehicle (PEG400) (n=5); and R-107, injured treated with R-107 (n=3). After the injury sheep were awakened, placed on mechanical ventilator and monitored for 48 hours. R-107 was started 1h after injury (2ml IM and 0.625ml/h via venous line for 47 hours). Analgesia was provided with long acting buprenorphine.
Results: All sheep survived 48 hrs in the treated group, while 3 of 5 sheep survived in control group. Pulmonary gas exchange evaluated by measuring PaO2/FiO2 ratio was markedly improved by the treatment and it was 298 ±69 in the treated group and 155± 60 in the control group at 48 h. This was associated with a significant (p<0.05) attenuation of pulmonary shunt fraction in the treated group (0.23±0.04) vs. control (0.44±0.07) at 30 h. Airway peak pressure was also significantly reduced by R-107, which was 23.0 ±1.2mmHg and 36.3±4.5mmHg (p<0.01), in treated and control groups, respectively. Accumulated fluid net balance was significantly higher in control (4343 ±1663ml) vs. treated sheep (567±501ml), p<0.05.
Conclusion: R-107 effectively attenuates acute lung injury possibly by inhibiting pulmonary shunt fraction and microvascular hyper-permeability. Future studies are warranted increasing the sample sizes and exploring underlying mechanisms.
Support: SHC84050, NIH5U01ES021154-03
GENDER DIMORPHISM OF THE CARDIAC DYSFUNCTION IN SEPSIS
J. Chen1, F. Chiazza2, M. Collino2, S.M. Coldewey3, N.S. Patel1, and C. Thiemermann*1. 1William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 2University of Turin, Department of Drug Science and Technology, Turin, Italy, 3Hannover Medical School, Department of Anesthesia and Intensive Care Medicine, Hannover, Germany
Development of cardiac dysfunction is associated with increased mortality in patients with sepsis. Increasing evidence shows that gender determines the degree of the inflammatory response of the host and that females tolerate sepsis better than males. However, little is known about whether there is gender dimorphism in the cardiac dysfunction and the underlying mechanisms that occurs in animals or patients with sepsis.
To investigate this, ten weeks-old male or female C57BL/6 mice were subjected to co-administration of lipopolysaccharide (LPS)/peptidoglycan (PepG). At 18 hours, cardiac function was evaluated by echocardiography. LPS (3 mg/kg)/ PepG (0.1 mg/kg) caused a significant reduction in ejection fraction (EF) in both genders. However, EF was significantly higher in female mice in comparison with males after the septic insult (Figure 1). Compared with male mice injected with LPS (3 mg/kg)/ PepG (0.1 mg/kg), western blotting analysis of myocardium from females injected with LPS/PepG revealed i) significant decreases in nuclear translocation of the NF-κB subunit p65 and inducible nitric oxide synthase (iNOS) expression; ii) profound increases in phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). However, gender differences of the cardiac dysfunction after septic insult were abrogated by the severe injury induced by high dose of LPS/PepG (LPS, 9 mg/kg, PepG, 1 mg/kg) co-administration.
In conclusion, cardiac dysfunction caused by LPS/PepG was less pronounced in female than in male animals. This cardioprotection against sepsis in females is attributable to decreased activation of NF-κB and iNOS expression, and activation of PI3K/Akt/eNOS survival pathway. However, the gender dimorphism of the cardiac dysfunction after septic insult could be override by severe injury.
ADVANCED AGE IS AN INDEPENDENT PREDICTOR OF COMPLICATED OUTCOMES AND MORTALITY AMONG SEVERELY INJURED PATEINTS IN HEMORRHAGIC SHOCK
S. Brakenridge*, E. Vanzant, A. Bihorac, C. Leeuwenburgh, F. Moore, L. Moldawer*, and P.A. Efron*. University of Florida, Gainesville, FL
Objective: Advancing age is a known risk factor for increased morbidity and mortality with many disease processes. We performed an analysis of the Inflammation and Host Response to Injury Collaborative Research Program cohort to determine if age is a significant clinical determinant of complicated recovery and chronic critical illness (CCI), which are the precursors to persistent inflammatory, immunosuppressive and catabolic syndrome (PICS), and increased mortality.
Methods: Young (<55 yrs) and aged (≥55 yrs) patients from a prospective, multi-institutional cohort of severely injured blunt trauma patients in hemorrhagic shock were analyzed to assess for the relationship of age on patient outcomes. Multivariate logistic regression models were developed to determine if advanced age is an independent risk factor of complicated recovery, and 28-day mortality.
Results: The cohort consisted of 1,783 trauma patients in hemorrhagic shock. There was no difference in injury severity between elderly (n=481) and young (n=1302) patients (p=0.19). However, aged patients had significantly more baseline comorbid conditions (p<0.001) and showed evidence of more severe shock with lower systolic blood pressure (p<0.001), and increased lactate (p=0.03) compared to young patients. Aged patients had more severe organ dysfunction (Marshall MOF score 5.4 vs. 4.8, p<0.001), longer ICU LOS (13 vs. 10 days, p<0.001), and higher rates of infection (51.4% vs. 44.8%, p=0.016). Advanced age was also associated with increased ventilator days, VAP, non-infectious complications, tracheostomy, complicated recovery, CCI and 28 day mortality (p<0.01). Among survivors, the aged cohort was significantly more likely to be discharged to skilled nursing or long-term care facilities rather than home (p<0.001). Age ≥55 years is an independent predictor of complicated outcome (OR 2.24, 95% CI 1.64-3.06, p<0.001) and 28-day mortality (OR 2.90, 95% CI 1.95-4.32, p<0.001) after controlling for injury severity, shock severity, transfusion requirements and comorbidities.
Conclusion: This analysis of injured patients in hemorrhagic shock reveals that age is a primary criterion of risk for poor outcomes after severe trauma, and may actually be the greatest risk factor affecting the development of CCI, PICS and dismal long-term outcomes.
RESUSCITATION WITH HEXTEND OR HESPAN DECREASES RESUSCITATION VOLUME AND PULMONARY INFLAMMATION IN A MURINE MODEL OF TRAUMA AND HEMORRHAGE
J.M. Sutton, R.S. Hoehn, P.L. Jernigan, A.T. Makley, S.G. Miller, J. Richter, L.W. Friend, R.M. Schuster, W. Dorlac, T.W. Gerlach, A.B. Lentsch, and T.A. Pritts*. Division of Trauma and Critical Care, Department of Surgery and Institute for Military Medicine, University of Cincinnati Medical Center, Cincinnati, OH
Background: Initial studies investigating the resuscitative efficacy of hetastarch-containing fluids suggested increased morbidity and mortality, but more recent studies in trauma patients have, to some extent, alleviated these initial concerns. We hypothesized that resuscitation with hetastarch-based fluids would result in lower fluid requirements and comparable endpoints of resuscitation.
Methods: Mice underwent splenectomy followed by pressure-targeted hemorrhage for 60 minutes and resuscitation with either crystalloid (0.9% normal saline [NS] or lactated Ringer’s solution [LR]) or hetastarch-based (6% hetastarch in NS [Hespan] or LR [Hextend]) fluids. Mice were sacrificed at intervals to evaluate resuscitation parameters via arterial blood gas, systemic inflammation via serum cytokine analysis, localized lung inflammation via histology, and survival.
Results: Resuscitation with hetastarch-containing fluids required significantly less volume to reach target blood pressure than resuscitation with their respective crystalloids (all p < 0.001, Figure 1A). Base deficit and lactate did not differ between groups. Resuscitation with Hespan or Hextend significantly attenuated pulmonary inflammation as evidenced by reduced numbers of inflammatory cells on histology compared to lungs from mice resuscitated with their respective crystalloid (Figure 1B). No differences in survival were noted between resuscitation strategies.
Conclusion: The use of hetastarch-containing fluid for resuscitation after trauma and hemorrhage results in use of lower resuscitation fluid volumes and decreased pulmonary inflammation compared to crystalloid alone. Hetastarch-containing fluids should be considered a therapeutic alternative to initial crystalloid resuscitation.
COLD-INDUCIBLE RNA-BINDING PROTEIN (CIRP) MEDIATES NEUROINFLAMMATION IN CEREBRAL ISCHEMIA
M. Zhou*, W. Yang*, X. Qiang, and P. Wang*. Hofstra North Shore-LIJ School of Medicine and The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: After cerebral ischemia, excessive release of proinflammatory mediators exacerbates brain injury. Cold-inducible RNA- binding protein (CIRP) is a newly discovered proinflammatory mediator released into the circulation during hemorrhagic and septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.
Methods: Stroke was induced by middle cerebral artery occlusion (MCAO) in adult male C57BL/6 mice. In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. Levels of CIRP and tumor necrosis factor-α (TNF-α) were assessed by RT-PCR and Western blot analysis.
Results: Mouse brain levels of CIRP and TNF-α were significantly increased 30-48h after MCAO. CIRP-deficient mice were protected from MCAO, and had decreased brain infarct volume, lower TNF-α levels and reduced microglial activation. In vitro, hypoxia induced the expression, translocation, and release of CIRP from microglial BV2 cell, together with increased levels of TNF-α. Treatment of BV2 cells with recombinant murine (rm) CIRP induced the production of TNF-α which was inhibited by neutralizing antisera to CIRP. rmCIRP also activated the NF-κB signaling pathway in BV2 cells. Conditioned medium from BV2 cells exposed to hypoxia triggered the apoptotic cascade in neuroblastoma SH-SY5Y cells by increasing caspase activity and decreasing Bcl-2 expression. Pretreatment of BV2 cells with antisera to CIRP abrogated the pro-apoptotic effect of the conditioned medium.
Conclusion: Extracellular CIRP is a detrimental proinflammatory factor that induces neuronal apoptosis after cerebral ischemia. Development of an anti-CIRP therapy may benefit patients with brain ischemia.
ADMINISTRATION OF MITOQ FOLLOWING HEMORRHAGE ALTERS ENDOGENOUS ANTIOXIDANT ACTIVITY IN A RAT MODEL OF HEMORRHAGIC SHOCK AND RESUSCITATION
R.D. Powell*, I.H. Mckillop, T.T. Huynh*, and S.L. Evans*. Carolinas Medical Center, Charlotte, NC
Introduction: Oxidative stress associated with hemorrhagic shock and resuscitation (HSR) results in production of superoxide radicals and other reactive oxygen species (ROS) leading to cell damage and multiple organ dysfunction. MitoQ (MQ) is a mitochondrial-targeted antioxidant. We sought to determine if MQ affects outcomes in a rat model of HSR via changes in hepatic antioxidant activity and/or ROS production.
Methods: HSR was achieved in male rats by arterial blood withdrawal to a MAP of 25±2.0mmHg for 1-hr prior to resuscitation. MQ (5mg/kg) or saline (0.9%) was administered (iv) 30-mins prior to resuscitation, followed by IP administration (MQ; 20mg/kg) immediately after resuscitation (n=5/group). Morbidity was assessed based on cumulative markers of animal distress (0-10 scale). Rats were sacrificed 2-hrs after procedure completion and liver tissue collected and processed for histology (H&E), or assayed for lipid peroxidation (TBARS) or endogenous antioxidant activity (catalase, glutathione peroxidase and superoxide dismutase).
Results: HSR significantly increased morbidity, lipid peroxidation and catalase activity vs Sham animals. Conversely, no difference between Sham and HSR was measured in glutathione peroxidase (GPx) or superoxide dismutase (SOD) activity. MQ administration reduced morbidity vs HSR (5.8±0.3 vs 7.6±0.3; p<0.05) with no significant difference in hepatic necrosis in MQ vs HSR. Analysis of oxidative stress demonstrated increased hepatic lipid peroxidation in MQ vs HSR (12.0±01.1 vs 9.4±0.73 (μM); p<0.05), and GPx activity (3794±3.1 vs 2286±2.7(μmol/min/ml); p<0.05). Conversely, catalase activity in MQ was reduced vs HSR (3919±2.3 vs 5364±3.9(μmol/min/ml); p<0.05), and no significant difference in SOD activity was measured.
Conclusions: Our data demonstrate MitoQ treatment following hemorrhage significantly limits morbidity and differentially regulates hepatic antioxidant activity.
SEPSIS SURVIVAL IS INFLUENCED BY TBI
M.D. Goodman, S. Yang, E.F. Midura, and C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Traumatic brain injury (TBI) contributes to almost one third of injury related deaths each year and creates significant morbidity in the surviving trauma population. This neurologic insult causes an inflammatory response in the brain that leads to the breakdown of the blood-brain barrier (BBB), passage of these inflammatory mediators into the systemic circulation and alteration of the immune response to infection. As increased inflammation and increased pro-inflammatory responses are often associated with higher mortality in sepsis, we hypothesized that TBI would negatively impact survival during sepsis.
Methods: A weight drop model of TBI was used to induce injury in naïve mice. One day, one week and two weeks after TBI, sepsis was induced using a cecal ligation and puncture (CLP) model. Control mice were only subject to CLP.
Results: Survival at each time point are displayed in Figure. Survival was improved in the one-day and two-week post TBI CLP groups and unchanged in the one-week group. The one-day post TBI group demonstrated significantly decreased levels of splenic CD4, B-cells and macrophages with similar populations compared with controls in the serum. The one-week group and two-week groups had similar splenic and serum immune cell populations compared with control mice.
Conclusion: Altogether, we observed that 1) one day post-TBI there is a hypo-inflammatory response to sepsis which results in improved survival, 2) one week post-TBI the immune system has normalized and there is no effect on sepsis survival and 3) two weeks post-TBI the immune system remains normalized however there is improved survival in septic mice.
A UNIQUE RODENT MODEL OF CONCURRENT COMBINED INJURIES (POLYTRAUMA)
R.M. Akscyn1, T.A. Gavrikova1, J.L. Franklin1, M.G. Schwacha*2, and J.L. Messina*1. 1University of Alabama at Birmingham, Birmingham, AL, 2University of Texas Health Science Center at San Antonio, San Antonio, TX
Polytrauma is a term indicating a combination of injuries to more than one body part or organ system. Polytrauma is common in modern warfare and in automobile and industrial accidents. The combination of injuries can include burn injury, fracture, hemorrhage, trauma to the extremities, and trauma to specific organ systems. To investigate the effects of combined injuries, we have developed a new and highly reproducible model of polytrauma. This model combines burn injury with gastrointestinal (GI) tract trauma. Male Sprague Dawley rats were subjected to a 20% total body surface area scald burn, or a single puncture of the cecum with a G30 needle, or the combination of both injuries (polytrauma). Unlike many ‘double hit’ models the injuries in our model are concurrent.
Previous studies demonstrated that either the burn injury or GI trauma alone were highly survivable. However, when these injuries were combined mortality was greatly increased (~70% within 48h, vs. 5-10% for each injury alone). Our current results demonstrate this model of polytrauma causes an inability to properly maintain blood glucose levels with early hyperglycemia followed by hypoglycemia by 24h. Thermal dysregulation and weight changes were observed with animals becoming severely hypothermic and body weight changes that differed following polytrauma versus single injuries alone. Only polytrauma resulted in a significant increase in hepatic endoplasmic reticulum stress. Increases in proinflammatory gene expression were dependent upon the injury, but increased to a greater extent following polytrauma (IL-6, IL-1β, MCP-1, PAI-1). As animal models have been developed to study injury it has been suggested that models utilizing combinatorial injuries may be needed to more accurately model the human condition. Our previous data demonstrated that polytrauma induced hepatic insulin resistance and acute liver damage. Together with the present data, we believe our model is ideal for studying the complex sequelae of polytrauma which differ from single injuries. Insights gained from this model may predict better treatment options and improved prognosis for patients.
MINOCYCLINE REDUCES THE LEVELS OF PHOSPHORYLATED SAF-1 AND SERUM AMYLOID-A IN THE HIPPOCAMPUS AFTER MILD TBI
M. Liu*, D.L. Carlson*, J. Minei*, S.E. Wolf*, and J. Gatson*. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Following a mild traumatic brain injury (TBI) event, heightened neuro-inflammation persists for hours, days, and weeks. Subsequent activation of the transcription factor, serum amyloid A activation factor 1 (SAF-1), has been shown to regulate the transcription of pro-inflammatory factors such as serum amyloid A (SAA). In neuro-degenerative disorders such as Alzheimer’s disease, SAA has been implicated in exacerbating pro-inflammatory pathways such as cytokine signaling and the activation of microglia. In this study we proposed to characterize the activity of this pathway after mild TBI (mTBI) and determine the efficacy of minocycline.
Methods: In this study, using the controlled cortical impact device we produced a mild-to-moderate closed head injury in young adult C57 BL/6 mice. At 5 minutes after injury, the mice were injected intraperitoneally with either placebo or minocycline (30 mg/kg). Mice were also treated at 12, 24, 48, and 72 hours after injury. At day 1, 7, and 30 after injury, the brain was harvested and processed. Western analysis and immunohistochemistry (IHC) was used to detect the levels of total and phospho-SAF-1, SAA, and activated microglia in the hippocampus.
Results: In this study, on day 1, 7, and 30 after injury there was a significant increase in the phosphorylation of SAF-1 (Day 1, p=0.05; Day 7, p=0.04; Day 30, p=0.01) and the levels of SAA (Day 1, p=0.03; Day 7, p=0.02; Day 30, p=0.05) within the dentate gyrus of the hippocampus compared to the control animals. Treatment with minocycline significantly decreased the phosphorylation of SAF-1 (p=0.001), which led to a decrease in the levels of SAA (p=0.008) on day 30 after injury. Treatment with minocycline also lowered the levels of activated microglia (p=0.05) and astrocytes (p=0.04).
Conclusions: Activation of the SAF-1 signaling pathway after mTBI may be a key component in the chronic activation of microglia and heightened inflammation in the hippocampus after TBI, resulting in long-term memory deficits. As a therapeutic strategy, by targeting this pathway we hypothesize that chronic neuro-inflammation will be reduced, resulting in better neurological outcomes.
THE ADRENOMEDULLIN ANTIBODY HAM1101 PROTECTS AGAINST CLP- INDUCED KIDNEY BARRIER DYSFUNCTION
O. McCook1, F. Wagner1, A. Scheuerle3, M. Gröger1, A. Bergmann2, F. Hein2, J. Struck2, P. Radermacher1, and K. Wagner1. 1Uniklinikum Ulm, Ulm, Germany, 2Adrenomed AG, Hamburg, Germany, 3Uniklinikum Ulm Dept Pathology, Ulm, Germany
Background: Septic shock causes diffuse microvascular leakage leading to hypovolemia and tissue edema. We showed that the adrenomedullin antibody HAM1101 reduced norepinephrine requirements, increased urine flow, ameliorated kidney dysfunction and organ injury in resuscitated murine CLP-induced septic shock . Both adrenomedullin and the “3rd gaseous messenger“  H2S, have been implicated in endothelial barrier dysfunction, the former promoting and the latter preventing fluid extravasation [3,4]. In the kidney, constitutive expression of the H2S producing enzymes CBS and CSE is associated with organ protection . VEGF increases endothelial vascular permeability, whereas Angiopoietin1 (Ang1) protects against this VEGF-induced plasma leakage . Therefore, we investigated if the beneficial effects of the HAM1101 therapy also translate into improved kidney barrier function.
Methods: 15 hours after CLP and random assignment to vehicle or HAM1101, anesthetized, mechanically ventilated, and instrumented mice underwent 5 hours of fluid resuscitation and norepinephrine support to maintain target hemodynamics . Thereafter, the left kidney was removed, formalin-fixed and embedded in paraffin. Immunohistochemistry for CSE, CBS, VEGF, Ang1, and albumin was performed on deparaffinised sections using appropriate 1° antibodies and 2° anti-rabbit or anti-goat IgG AP, Dako REAL chromogen, and HE counterstaining. Slides were analyzed using the Axio Vision software, data are mean densitometric sums red.
Results: HAM1101 significantly attenuated the loss of both CBS and CSE. While it significantly reduced VEGF expression, Ang1 expression was increased. This effect coincided with a marked reduction of extravascular albumin accumulation.
Conclusion: Administration of HAM1101 ameliorated the septic shock-induced vascular leakage, most likely due to preserved CBS and CSE expression. These results confirm the important role of microvascular leakage in the pathogenesis of sepsis, and suggest an interplay between endogenous adrenomedullin and H2S formation under these conditions.
1. Wagner et al, Intensive Care Med Ex 2013;1:2
2. Lowicka & Beltowski, Pharmacol Rep 2007;59:4-24
3. Kaufmann et al, J Physiol 2001;534:527-533
4. Wang et al, Am J Respir Cell Mol Biol 2012;47:491-496
5. Wang, Physiol Rev 2012;92:791-896
6. Gavard et al, Developmental Cell 2008;14:25-36
INCREASED PREVALENCE OF DSDNA IN PATIENTS FOLLOWING ECMO TREATMENT
D. Moore1, A. Khoury2, B. Linz1, R. Maile1, B. Cairns1, and A. Charles1. 1University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Howard University, Washington D.C.
Acute respiratory distress syndrome (ARDS) occurs due to an over active immune response and leads to an increased mortality rate of 25-45% of patients. ARDS occurs as sequelae of pneumonia, sepsis, pancreatitis and multiple traumas. Extracorporeal membrane oxygenator (ECMO) provides cardiopulmonary support for patients suffering with ARDS, enhancing survival rates. Although ECMO treatment results in increased survival, its effect on immune cells in the patients is currently unknown. The study objective was to measured double-stranded DNA (dsDNA), released after tissue damage and a known mediator of immune function. In this preliminary study, we examined the changes that occur in human peripheral blood due to ECMO treatment. Longitudinal blood samples were taken from patients with severe respiratory failure (n=2) treated with ECMO. Samples were obtained from patient arterial blood and ECMO circuit, before and after the membrane oxygenator. dsDNA levels were quantified using an ELISA. Results showed an increase in the concentration of patient dsDNA from baseline (<100ng/ml) to 2hrs (900ng/ml) after initiation of ECMO. There was a decrease of patient and circuit dsDNA levels to baseline at 50hrs and 120hrs. Blood collected from patients had higher levels of dsDNA than blood collected from circuit. These results suggest that ECMO may concentrate dsDNA within the blood. This increase of dsDNA (a known damage-associated molecule pattern) molecules in patient blood may potentiate patient survival by stimulating the innate system by activation of toll-like receptors.
TSLP CONTRIBUTES TO EARLY MORTALITY IN SEPSIS
E.F. Midura, J. Kuethe, B.L. Johnson, and C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Introduction: Almost half of ICU deaths are related to sepsis induced multi-system organ failure. The balance between the pro- and counter-inflammatory response in sepsis is an important area of current research. It has been shown that IL-7 signaling through γδ T-cells and IL-17 improves outcome in sepsis. Thymic Stromal Lymphopoietin (TSLP) is produced in response to pro-inflammatory mediators, has been well studied in asthma and allergy models and appears to enhance the Th2 response in these models. Although TSLP activates many different immune cell subtypes and forms a heterodimer with the IL-7 receptor, its role in sepsis remains unclear. We believe TSLP is a potent pro-inflammatory cytokine, and therefore we hypothesized TSLP provides a potential therapeutic target in sepsis and blockade of TSLP will result in improved survival.
Methods: A Cecal Ligation and Puncture (CLP) model was used to produce sepsis in naïve mice. At the time of CLP, mice were treated with either a neutralizing TSLP antibody or an isotype control and their survival, bacterial load and neutrophil response were analyzed.
Results: Mice treated with TSLP neutralizing antibody had significantly improved survival compared to isotype controls. Treated mice also had significantly lower bacterial loads in their serum and peritoneal cavity. Furthermore, increased peritoneal neutrophil populations were seen in treated mice as well as an increase in the oxidative burst of these neutrophils.
Conclusion: From these experiments, we conclude that 1) inhibition of TSLP is associated with improved survival in sepsis, 2) TSLP reduces bacterial clearance and 3) TSLP hinders neutrophil recruitment and activity during sepsis. Future study is needed to better understand the interaction between TSLP and other inflammatory mediators that leads to the diminished immune response and increased mortality.
ELEVATED BASE DEFICIT ON ADMISSION IS ASSOCIATED WITH ALTERED INFLAMMATORY RESPONSE AND WORSE OUTCOMES IN BLUNT TRAUMA PATIENTS
O.M. Abdul-Malak*1, K.W. Almahmoud*1, A.M. Zaaqoq*1, 2, D. Barclay1, Y. Vodovotz*1, 3, T. Billiar*1, 3, and R.A. Namas*1, 3. 1University of Pittsburgh, Pittsburgh, PA, 2Texas Tech Health Sciences Center, Amarillo, TX, 3McGowan Institute for Regenerative Medicine, Pittsburgh, PA
Background: Base deficit (BD) post-trauma is a classical marker of tissue hypoperfusion, volume resuscitation and injury severity. Hypothesizing that elevated admission BD can be associated with an altered inflammatory response, we carried out an extensive time course analysis of circulating inflammation biomarkers as a function of admission BD (above and below 4 mEq/L) in a subset of blunt trauma patients.
Methods: From a cohort of 472 blunt trauma survivors, we identified 154 patients after excluding patients with alcohol intoxication and who had received pre-hospital IV fluids. From this sub-cohort, 84 patients had a BD≥4 mEq/L (age: 45.9±2, M/F 55/29, ISS: 23±1.2, Glasgow coma scale [GCS]: 12.6±1.3) and 70 patients with BD<4 mEq/L (age: 49.3±2.4, M/F 55/15, ISS: 21±1.3, GCS: 13±1.2). Serial blood samples were obtained from all patients (3 samples within the first 24 h and then from days 1 to 7 post-injury). Twenty-three inflammatory mediators were assayed using Luminex™. Plasma lactate and creatine phosphokinase (CPK) data were collected from inpatient electronic database. Inflammation biomarker data were analyzed using Two-Way ANOVA (p<0.05) and area under the curve (AUC) analysis.
Results: There was no significant difference in age, ISS, and GCS between the two sub-groups. However, ICU length of stay (LOS), total LOS, and days on ventilator were significantly different in the BD≥4 group vs. the BD<4 group. Moreover, the BD≥4 group had elevated admission levels of lactate (3.4±0.3 mMol/L) and CPK (1735±189 U/L) when compared to BD<4 group (lactate: 2.1±0.1 mMol/L; CPK: 660±80 U/L). Circulating levels of CC chemokines were significantly elevated within the first 24 h post-injury in the BD≥4 group vs. the BD<4 group. Interestingly, multiple mediators of the innate and adaptive immune response in the BD≥4 group exhibited a biphasic response, being elevated within 24 h and after day 2 post-injury, with a >1.5 fold increase in AUC when compared to BD<4 group.
Conclusion: These results suggest that admission BD can segregate patients into distinct groups with differential clinical outcomes associated with an upregulation of multiple components of the immune system. Importantly, our results suggest the utility of admission BD as an early potential biomarker to identify patients who are prone to develop worse outcomes post-trauma.
MITOCHONDRIAL COMPLEX I RESPIRATION IN CHILDREN WITH BURN INJURY TREATED WITH AND WITHOUT METFORMIN
A. Ali1, 2, C.C. Finnerty*1, 2, O.E. Suman*1, 2, D. Herndon*1, 2, M. Wetzel1, 2, L. Sidossis1, 2, and C. Porter1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX
Introduction: Metformin has been postulated to attenuate hyperglycemia and insulin resistance following severe burn injury. However, the impact of metformin treatment on skeletal muscle mitochondrial function in this population is unknown. Since metformin has been implicated as an inhibitor of mitochondrial complex I, we sought to determine the impact of metformin treatment on complex I respiration and mitochondrial respiratory control in burned children
Methods: Lower limb skeletal muscle biopsies from 15 children (ages 7-18 years) with burns covering 30-60% of the total body surface area were collected shortly after burn injury. Patients were divided into those receiving standard of care (n=8), and those receiving standard of care plus metformin at a dose of 1 gram per day (n=7). Mitochondrial respiration was determined following the addition of substrates and to saponin permeabilized myofibers. Statistical analysis was performed using Mann-Whitney rank sum tests. Significance was accepted when p<0.05.
Results: No significant differences were noted between groups in age, percent total body surface area burned, percent full thickness burned, or duration post burn muscle samples were collected. Complex I respiration per mg of muscle was significantly lower in metformin treated patients (14.1±5.0 vs. 26.0±13.2 pmol/mg/sec; p<0.05) as was maximal mitochondrial respiration (18.1±8.8 vs. 37.±18.8 pmol/mg/sec; p<0.05). When normalized to maximal mitochondrial respiration, Complex I respiration was not different between groups (0.73±0.14 vs. 0.83±0.16 pmol/mg/sec; p>0.05). The respiratory control ratio (ADP sensitivity) and substrate control ratio (succinate sensitivity) were similar in both groups.
Conclusions: Metformin treatment does not appear to qualitatively alter mitochondrial complex I function or respiratory control in burned children. However, total mitochondrial oxidative capacity was significantly lower in metformin treated patients. In light of these preliminary findings, further research is needed to elucidate the efficacy and safety of metformin treatment on whole body glucose homeostasis and skeletal muscle mitochondrial function patients with burns.
DISRUPTION OF GENES ENCODING 4E-BP1 AND 4E-BP2 DOES NOT ALTER BASAL OR SEPSIS-INDUCED PROTEIN SYNTHESIS IN SKELETAL MUSCLE
C.H. Lang*, J.L. Steiner, and S.R. Kimball. Penn State College Medicine, Hershey, PA
Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male C57Bl/6 mice under basal conditions and after 24 h of sepsis induced by CLP. At 12 months of age, body weight, body composition and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo basal rates of global protein synthesis for gastrocnemius, heart and liver did not differ between DKO and WT mice. Also, there was no genotype-specific difference in the mRNA content in gastrocnemius for MuRF1, atrogin-1, IGF-I, TNF and IL-6 under basal conditions. Sepsis decreased protein synthesis as well as AKT, S6K1 and 4E-BP1 phosphorylation in skeletal muscle of WT mice. These sepsis-induced decreases were comparable in WT and DKO mice, except there was no 4E-BP1 phosphorylation in DKO mice because of the lack of this protein. Importantly, eIF4G-eIF4E binding was selectively decreased in WT but not DKO mice in response to sepsis. Muscle mRNA for MuRF1 and atrogin-1 (markers of muscle proteolysis) as well as TNF and IL-6 (inflammatory cytokines) were higher in DKO than WT septic mice. The sepsis-induced decrease in muscle IGF-I was comparable between WT and DKO mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis also did not differ between WT and DKO mice. The sepsis-induced reduction in body weight was the same in WT and DKO mice. These data suggest that sepsis-induced changes in mTOR signaling in muscle may be mediated by multiple down-stream mechanisms independent of 4E-BP1/BP2. (GM38032)
ROLE OF HYPOGLYCEMIA AND HYPOXIA IN RENAL ARTERY AMPK AND CONTRACTILE ACTIVITY
A.S. Miner2, 1, R.W. Barbee*1, 3, and P.H. Ratz*2, 3. 1Depts of Emergency Medicine and Physiology, Virginia Commonwealth University, Richmond, VA, 2Depts of Biochemistry and Pediatrics, Virginia Commonwealth University, Richmond, VA, 3VCURES, Richmond, VA
Peripheral organ blood flow is reduced to preserve heart and brain flow during hemorrhage. During resuscitation, appropriate blood flow must be re-established to peripheral tissues. Thus, peripheral arterial contraction regulation plays a critical role in homeostatic mechanisms engaged during hemorrhagic insult. The renal vascular bed demands over 25% of normal cardiac output distribution for normal function, but is non-essential and under vasoconstrictor tone during hemorrhage. Thus, we examined the effect of the metabolic sensor, AMP kinase (AMPK), on regulation of renal artery (RA) contraction induced by the cumulative addition of the vasoconstrictors phenylephrine (PE), vasopressin (VP), and K+-depolarization (KCl) in the presence and absence of the AMPK activators, 2mM AICAR and 30μM A769662. We also examined the effect of metabolic starvation (STARVE; 0mM added glucose, pO2 < 30mmHg) as a surrogate for hemorrhage on contractile tension and activation of AMPK. To measure isometric tension, RA from NZW rabbits were cleaned, denuded of endothelium, and placed between clips using a wire myograph system. As an index of AMPK activation, AMPK-pT172 was measured in RA quick-frozen and homogenized for processing using Western Blot analysis. AICAR produced a rightward shift in the concentration-response curves (CRCs) produced by each contractile agonist, and depressed the maximum contractions produced by VP and KCl. A769662 produced a weaker rightward shift in RA contracted by KCl and PE, and produced a weak but non-significant depression of the maximum VP-induced tension. Both STARVE and AICAR produced an ~3-fold increase in AMPK-pT172. Like AICAR, STARVE inhibited contractile tension induced by PE. Together, these data support the hypothesis that AMPK regulates RA contraction and is modulated by low glucose and hypoxic states.
Supported by DOD Grant #W81XWH-12-1-0525.
TRICHOSTATIN A, A HISTONE DEACETYLASE INHIBITOR, PROTECTS AGAINST SEPSIS VIA ACETYLATION OF PATTERN RECOGNITION RECEPTORS
S. Kim*, J. Choi, J. Park, and S. Lee*. Sungkyunkwan University, Suwon, Republic of Korea
Sepsis is a multifactorial disease characterized by an overwhelming activation of the immune system. Pattern recognition receptors (PRRs) signaling plays a critical role in host defense and uncontrolled activation of PRRs signaling may result in inflammatory disorders such as sepsis. Histone deacetylase (HDAC) regulates gene transcription by acetylation of histone protein and emerging evidence showed that HDAC inhibitors also possess an anti-inflammatory property through acetylation of non-histone protein. However, the functional role and molecular mechanism of HDAC inhibition on the PRRs signaling remains unknown. Therefore, the aim of this study was to investigate the effect of trichostatin A (TSA), a HDAC inhibitor, on expression and activation of PRRs. Male C57BL/6 mice were intraperitoneally administered with TSA (1, 2, and 5 mg/kg) or vehicle (5% dimethyl sulfoxide in saline) 1 h before cecal ligation and puncture (CLP) and animals were sacrificed at 3, 6, 12, and 24 h after the surgery. TSA improved survival rate and attenuated multiple organ dysfunction. Toll-like receptor (TLR)2, TLR4, and receptor for advanced glycation end-products (RAGE) protein expressions significantly increased after CLP and these increases were attenuated by TSA. Acetylated (Ac)-TLR2, Ac-TLR4, and Ac-RAGE protein expressions significantly increased after CLP. TSA augmented Ac-TLR2 and Ac-RAGE protein expressions while it did not affect Ac-TLR4 protein expression. Binding of high-mobility group box (HMGB)1 with TLR2 and RAGE significantly increased after CLP and these increases were attenuated by TSA. However, neither CLP nor TSA did affect the binding of HMGB1 with TLR4. TSA decreased CLP-induced nuclear translocation of nuclear factor kappa-B and mRNA expressions of tumor necrosis factor-α, interleukin-6, and inducible nitric oxide synthase. Our findings suggest that TSA ameliorates CLP-induced septic injury through upregulation of PRRs acetylation and inhibition of HMGB1 and PRRs interaction.
PRE-EXISTING CHRONIC KIDNEY DISEASE WORSENS MYOCARDIAL DYSFUNCTION CAUSED BY SEPSIS IN MICE
J. Chen, J. Kieswich, M.M. Yaqoob, and C. Thiemermann*. William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
Patients with chronic kidney disease (CKD) requiring dialysis have a higher risk of infection and sepsis. Dialysis patients with sepsis have a 100-fold higher mortality than the general population. Cardiovascular disease is the leading cause of death in patients with CKD. The cardiac injury caused by ischemia-reperfusion is greater in uremic rats when compared to non-uremic controls. The severity of cardiac dysfunction is an important predictor of mortality among patients with sepsis. It is unknown whether pre-existing CKD worsens cardiac outcome in animals or man with sepsis. This study was designed to investigate the effects of lipopolysaccharide (LPS) and peptidoglycan (PepG) on cardiac performance in mice with CKD.
Male C57BL/6 (4-6 weeks) mice were subjected to a two-stage, subtotal (5/6th) nephrectomy (SNX). After 8 weeks, cardiac function was evaluated by echocardiography. When compared to age-matched control mice, SNX for 8 weeks resulted in a significant rise in urea (from 8.37 ± 0.58 to 43.35 ± 3.41 mmol/L) and creatinine (from 31.66 ± 0.54 to 79.17 ± 8.02 μmol/L) and, hence, severe uremia (CKD). In mice without CKD, intraperitoneal co-administration of high dose LPS (6 mg/kg) and PepG (0.1 mg/kg) for 18 hours caused a profound decline in ejection fraction (EF) from 74.34 ± 0.86% (control) to 33.29 ± 0.92% (n=7), while low dose LPS (2 mg/kg) had no effect (72.15 ± 0.91%, n=7). In contrast, administration of low dose LPS (2 mg/kg) to mice with CKD caused a profound reduction in EF (to 40.38 ± 2.77%, n=7)(Figure 1). Thus, the impairment in cardiac function caused by low dose LPS in CKD mice was similar to the one caused by high dose LPS/PepG in mice without CKD.
In conclusion, the presence of CKD aggravates the cardiac dysfunction caused by LPS in the mouse.
DOXYCYCLINE ATTENUATES LIPOPOLYSACCHARIDE-INDUCED MICROVASCULAR HYPERPERMEABILITY
K. Wiggins-Dohlvik1, H. Stagg1, M. Han2, H. Alluri2, C. Anasooya Shaji1, R.P. Oakley1, M.L. Davis1, 2, and B. Tharakan1, 2. 1Scott and White Memorial Hospital, Temple, TX, 2Texas A&M Health Science Center, Temple, TX
Lipopolysaccharide (LPS) is known to incite intense inflammatory reactions. The pathophysiology involved therein has yet to be fully elucidated, but breakdown of endothelial cell adherens junctions is integral. We found matrix metalloproteinase-9 (MMP9) plays a large role in this process and doxycycline, a known MMP inhibitor, can attenuate damage in vitro. We hypothesized that in vivo, LPS administration causes increased microvascular permeability and doxycycline mitigates such fluid leak. To test this, Sprague Dawley rats were divided into the following groups: sham (IV NS), IV LPS, and IV LPS with IV doxycycline pretreatment (n=5). Fluorescein isothiocyanate-albumin was administered IV and a laparotomy was performed. Mesenteric postcapillary venules were examined with intravital microscopy and images were obtained. Fluoroscein intensities were measured intra- and extravascularly. Statistical analysis was conducted using Student’s t-test and ANOVA. Intravital microscopy revealed LPS induced permeability when compared to sham (p < 0.05): this increase was attenuated with doxycycline (p < 0.05; Figure 1). We conclude endotoxemia induces hyperpermeability in vivo and this can be negated with doxycycline. IV LPS injection is a sterile process and offers no anti-bacterial target for doxycycline. Through this utilization of doxycycline’s anti-MMP properties, these data illuminate the possible role of MMPs in the pathophysiology of LPS-induced microvascular hyperpermeability.
NEUTRALIZING THE COMPLEMENT COMPONENT C5A PROTECTS AGAINST LUNG INJURY AND EXTRAPULMONARY ORGAN INJURY IN PNEUMOCOCCAL PNEUMONIA-INDUCED SEPSIS
H. Müller-Redetzky2, U. Kellermann2, T. Tschernig1, S. Wienhold2, M. Polikarpova2, K. Hellwig2, A. Vater4, C. Maasch4, S. Klussmann4, M. Menger3, N. Suttorp2, and M. Witzenrath2. 1Institute of Anatomy and Cell Biology, Saarland University, Faculty of Medicine, Homburg, Germany, 2Charité - Universitätsmedizin Berlin, Dept. of Infectious Diseases and Pulmonary Medicine, Berlin, Germany, 3Institute for Clinical and Experimental Surgery, Saarland University, Faculty of Medicine, Homburg, Germany, 4NOXXON Pharma, Berlin, Germany
Rationale: Severe community acquired pneumonia (sCAP) and resultant sepsis cause high lethality despite adequate antibiotic treatment. S. pneumoniae is the most prevalent causative pathogen of sCAP. Uncontrolled inflammatory host responses likely contribute to unfavorable outcome by aggravating lung and extrapulmonary organ injury. The complement system is involved in the immune response against infection but also contributes to hyperinflammation and vascular barrier failure mainly mediated by the complement fragment C5a, an anaphylatoxin. We thus aimed to investigate the regulation of C5a in pneumonia and hypothesized that in pneumococcal pneumonia neutralizing C5a by NOX-D19, a PEGylated L-RNA aptamer (Spiegelmer), would ameliorate pulmonary vascular barrier failure, modulate the inflammatory host response and protect against extrapulmonary organ failure in sepsis.
Methods: Mice were infected with S. pneumoniae. NOX-D19 was applied i.p. 24 and 48 h post infection. Measurements of pulmonary permeability, pulmonary and blood leukocytes, and levels of IL-1β, G-CSF, KC and IL-6, bacterial load in lung, spleen and blood, markers of liver and kidney function (AST, BUN) and histological analyses of fibrin deposition and apoptosis in the liver were performed.
Results: C5a levels increased during pneumonia in lung and blood reaching a maximum 24 h post infection. Pulmonary leukocyte counts and cytokine levels were increased, and moderate pulmonary vascular permeability and bacteraemia had developed 24 h post infection, reflecting severe pneumonia. Lung failure and septic extra pulmonary organ injury developed within 48 h post infection, displayed by a further increase of pulmonary permeability and blood cytokine levels, as well as increased AST and BUN. Hepatic fibrin deposition reflected microcirculatory failure that was accompanied by apoptosis of hepatocytes. NOX-D19 attenuated pulmonary permeability, reduced blood cytokine levels, protected against liver injury as judged by reduced AST levels, and abrogated hepatic fibrin deposition as well as apoptosis of hepatocytes 48 h post infection.
Conclusion: Neutralizing C5a by NOX-D19 protected against lung and extrapulmonary organ failure in pneumococcal pneumonia-induced sepsis. C5a neutralization thus may offer an adjuvant treatment strategy in pneumococcal sepsis.
ANTIMICROBIAL RESISTANCE PROFILE IN COMPLICATED INTRA-ABDOMINAL INFECTIONS IN DEVELOPING COUNTRY
N. Shahzad, A.R. Alvi, R. Quratulain, and J. Farooqi. Aga Khan University Hospital, Karachi, Pakistan
Introduction: Morbidity and mortality rates associated with complicated intra-abdominal infections remain exceedingly high. They account for increased cost of care and prolonged hospital stay. Hemodynamic support, source control and early administration of appropriate empiric antibiotics are cornerstones of early management. Selection of empiric antibiotics is based upon microbiologic profile and resistance patterns of commonly encountered microbes in hospital and community. In developing countries, limited local microbiological susceptibility information is available. This can lead to selection of empiric antibiotics that result in either under treatment or unnecessarily broad coverage.
Objectives: Objective of our study was to find out frequency of various micro-organisms and their resistance profile to help decide empiric antibiotics to be used in complicated intra-abdominal infections.
Methodology: We conducted review of medical records of adult patients admitted from Jan 2011 to Dec 2013 with complicated intra-abdominal infections. Data was collected regarding demographics, source of infection, healthcare association of infection and culture reports.
Results: Mean age of 317 patients included in the study was 51+/-18 years. Healthcare associated infections were present in 44% of patients. Common sources of infection were biliary (30%) and large bowel (22.7%). Post laparotomy intra-abdominal infection accounted for 25% of cases. Common organisms reported were E. Coli (56.2%), Enterococci (28.4%) and Klebsiella Pneumoniae (15.1%). Fungal infection was encountered in 13.2% of patients. Fungal infection was significantly more common in healthcare associated infections. 75% of E Coli and 55% of Klebsiella Pneumoniae were Extended Spectrum Beta Lactamase (ESBL) producers. Cabapenem resistance was present in 8% of Klebsiella Pneumoniae isolates. Frequency of Vancomycin Resistant Enterococci (VRE) was 10%.
Conclusion: High prevalence of ESBL producing gram negative rods and resistance to various broad spectrum antibiotics needs to be considered while planning empiric antibiotics therapy in complicated intra-abdominal infections. Need of empiric anti-fungal coverage in cases of healthcare care associated infections needs to be evaluated.
APOE MEDIATES SEPTIC MORTALITY IN MICE BY REGULATING MACROPHAGE PHENOTYPE AND ACTIVITY
M.H. Lee and H. Harris*. University of California San Francisco, San Francisco, CA
Introduction: Decreased plasma apoE is associated with decreased Th1 cytokine levels, tissue bacteria counts, and septic mortality in rodents through an uncertain cellular mechanism. Macrophages are pleiomorphic immune cells that play a critical role in host defense against severe infections, adopting a pro-inflammatory (M1) versus an anti-inflammatory (M2) phenotype depending on the circumstances. While macrophages are critical for cytokine secretion and bacterial clearance, macrophages also produce apoE and express LDL (apoE) receptors. Given the central role of macrophages in sepsis, we examined the effects of varying apoE concentrations on macrophage differentiation (phenotype) and function during sepsis in mice.
Objective: To determine whether apoE mediates septic mortality by regulating macrophage phenotype and activity.
Methods: Peritoneal macrophage phenotypes were examined following cecal ligation and puncture (CLP) in mice expressing various combinations of apoE and NKT cell concentrations using gene expression analyses, flow cytometry, cell sorting, quantitative bacteriology assay, and phagocytosis assays.
Results: Mice with reduced apoE and NKT cell levels showed lower M1: M2 macrophage ratios (p<0.0001), higher phagocytic activities (p<0.0001), and enhanced bacterial clearance (p<0.01). A novel, highly activated group of peritoneal macrophages were discovered in septic mice with reduced apoE and NKT cells while septic wildtype mice lacked this group of macrophages.
Conclusion: Reduced levels of apoE and/or NKT cells decrease sepsis-induced mortality in mice through a macrophage-dependent mechanism. Specifically, decreased apoE levels in sepsis are associated with increased M2 macrophages, phagocytic activity and bacterial clearance. Further investigation regarding how apoE and NKT cells regulate macrophage differentiation and activity may yield novel therapeutic insights for combating severe bacterial infections.
CHARACTERIZATION OF THE CD8 T CELL RESPONSE IN A MURINE MODEL OF SEPSIS
M. Serbanescu, A. Hadley, L. Margoles, R. Mittal, J. Lyons, Y. Ali, M.L. Ford, C.M. Coopersmith*, and K. McConnell*. Emory University School of Medicine, Atlanta, GA
Apoptosis of CD8 T cells in sepsis results in increased susceptibility to infection and bystander-activated CD8 Te/M (effector/memory) can act in an innate-like manner decreasing pathogen load. Nonetheless, how CD8 T cells respond to sepsis and whether they are driven by bystander-effects by cytokines or TCR-mediated Ag-specific signals remains unclear. We sought to characterize activation and trafficking of CD8 T cells in sepsis through detailed immunophenotyping and by comparison of host and TCR-restricted CD8 T cells in an adoptive transfer model.
Male C57BL/6 mice were made septic via CLP. Mice were sacrificed at 6, 24, 48, 72, and 120h for collection of blood and spleen. Expression of CD69, CD25, CD11a, and CD44 on CD8 T cells was measured by flow cytometry. In a separate experiment 1x106 Thy1.1 CD8 T cells specific for the Ova-peptide (OT1s) were transferred to C57BL/6 mice followed by CLP 24h later. Spleens were harvested at 48h and CD44 and CD69 was measured on endogenous CD8 T cells and OT1s.
Spleens of septic mice had higher frequencies of CD8 CD69+ cells at 6h-48h (peak at 24h, 7.78% vs 1.89%), CD11aHI CD8aLO cells at 72h and CD8 CD25+ cells at 120h (p<.05). The percent and number of CD44HI and CD44HICD11aHI cells was lower in sepsis at 24h-72h (p<.05). In blood, septic mice had higher frequencies of CD8 CD69+ cells at 6 and 24h, and CD11aHICD8aLO cells and CD8 CD25+ cells at 48 and 72h. Cell counts of CD44HI and CD44HICD11aHI CD8 T cells were lower at 24-72h (p<.05) but frequencies were unchanged. In adoptive transfer studies, the percent of endogenous CD8 CD69+ cells increased and CD44HI cells decreased, whereas percent of CD69+ OT1s increased but the percent of CD44HI OT1s did not change.
The CD8 T cell response in sepsis mirrors aspects of both Ag-specific and bystander activation. The upregulation of CD69 on endogenous and OT1 CD8 T cells suggests a cytokine-driven process while the increases in CD25+ and CD11aHICD8aLO CD8 T cells suggests that a subset of cells may respond in an Ag-specific manner. Yet in both activation processes the percent of Te/M cells (CD44HICD11aHI) is expected to increase; the paradoxical decline in frequency and number of these cells indicates a dysregulated response, while the differential response of the OT1 cells and CD11aHICD8aLO subset suggests that in sepsis a subset of Ag-specific cells may be spared from immune dysregulation.
DYNAMICS OF MOLECULAR CIRCADIAN CLOCK RELATED MARKERS IN MURINE GUT MUCOSA IN RELATION TO MELATONIN FOLLOWING MAJOR THERMAL INJURY
S. Amuda, M. Kim, A. Ali, and W.M. Al-Ghoul*. Chicago State University, Chicago, IL
Recent evidence points to circadian dysregulation as a key player in pathophysiological susceptibility or protection in relation to gut inflammation. We have observed substantial variability in the degree of gut tissue inflammation following major injury which was possible to medicate by exogenous melatonin administration (Al-Ghoul, et al., Int J Biol Sci 17;6(3):282-93, 2010). Here, we test the hypothesis that major thermal injury is associated with changes in molecular clock and melatonin expression in intestinal tissues and underlying gut associated lymph tissues (GALT). Towards this end, we examined the expression of CLOCK and PER-2 molecular clock partners alongside the melatonin synthesizing enzyme AA-NAT in cellular homogenates of ileum and colon mucosa as well as spleen and MLN of control mice as compared to thermally injured (TI) and TI mice that received melatonin treatment (TI+Mel). All animal treatments were in accordance with IACUC and NIH guidelines. Accordingly, adult male mice were fed ad libitum, kept under 12:12 light:dark cycle, and treated and sacrificed at Zeitgeber time 5 to 6. Thermal injury was administered as a hot water scald (90deg. C, 9sec.) on the shaved back while under deep pentobarbital anesthesia and covered 20-25% body surface area. Levels of CLOCK, PER-2, and AA-NAT were assessed by fluorescent immunohistochemistry and flow cytometry. Thermal injury was associated with a consistent decrease in CLOCK immunoreactivity in large, but not small cells, in all tissues examined. Contrariwise, both AA-NAT and PER-2 increased in MLN and spleen, but not in ileum and colon. These effects were consistently largely reversed by melatonin treatment. This differential effect may be due to the trafficking of T cells in ileum and colon mucosae milieu, thus implicating T cells in dynamic circadian clock related changes. Our work was supported by NIGMS-MBRS-SCORE grant #1SC3GM099632-02.
MILD HYPOTHERMIA ON ADMISSION IS AN INDEPENDENT PREDICTOR OF POOR OUTCOMES AFTER TRAUMA
M. Lin-Duffy1, X. Tang2, F.K. Toy1, and N.S. Becker*1. 1Geisinger Wyoming Valley Department of Trauma Surgery, Wilkes-Barre, PA, 2Geisinger Center for Health Research Department of Biostatistics, Danville, PA
Introduction: Hypothermia (traditionally, temperature < 35°C) is associated with increased mortality after trauma. Few studies assess the effect of mild hypothermia (35-36°C) on outcomes. This retrospective study examines whether mild hypothermia predicts poor outcomes and longer length of hospital stay (LOS), ICU stay (ICU LOS), and ventilator dependence (VD).
Methods: The NTDB was obtained for the years 2010-2011. All patients >age 13 with traumatic injury and recorded admission temperature were included and stratified by temperature. Outcomes including mortality, LOS, ICU LOS, and VD were compared. Multivariate analyses were used to adjust for variables previously shown to affect outcomes.
Results: The study analyzed 1,162,743 records from 2010-2011. The majority of patients (73.0%) were normothermic (36-38°C), 23.0% were mildly hypothermic (35-36°C), 3.0% were moderately hypothermic (33-35°C), and 0.25% were severely hypothermic (less than 33°C). Patients who were older, more severely injured, and in shock were more likely to be hypothermic (all p values < 0.001). Hypothermic patients had higher mortality, longer ICU LOS, and VD (all p values < 0.001). After adjustment, in all subsets, hypothermia remained an independent predictor of mortality (Table 1). Hypothermia was also an independent predictor of longer LOS, ICU LOS, and VD.
Discussion: Hypothermia on admission, even if mild, is an independent predictor for mortality in trauma patients. In hypothermic patients who survive, LOS, ICU LOS, and VD are significantly longer. More studies need to be done to determine if re-warming patients expeditiously will improve outcomes.
IMPORTANT ROLE OF HSP70 IN COMPLEMENT COMPONENT C5A-INDUCED DELAYED APOPTOSIS OF NEUTROPHIL GRANULOCYTES
R. Wiegner1, M. Perl2, S. Denk1, S. Braumueller1, and M. Huber-Lang1. 1Department of Orthopedic Trauma, Hand, Plastic, and Reconstructive Surgery, University Hospital Ulm, Ulm, Germany, 2Clinic for Surgery, Trauma Surgery and Orthopedics at the BGU Murnau, Murnau, Germany
Despite high serum levels of pro-apoptotic factors, lifespan of neutrophil granulocytes (PMN) in patients during sepsis and after severe trauma is significantly prolonged, which may be associated with remote organ damage. As the complement system is activated early after trauma, we recently investigated the influence of activated complement component C5a on PMN apoptosis. We were able to show that C5a partially increases PMN lifespan in a phosphatidylinositide-3 kinase (PI3K)-dependent way. Heat shock protein 70 (Hsp70) is known to have various anti-apoptotic effects and its expression can be regulated by PI 3-kinase. Hence, we investigated the role of Hsp70 in C5a-induced prolonged survival of PMN.
PMN from healthy male volunteers were isolated and apoptotic events were induced via the intrinsic (hydrogen peroxide, H2O2) or the extrinsic (Fas agonist, CH11) pathway in the presence or absence of C5a. Intracellular levels of Hsp70 were measured by ELISA. A specific, low molecular C5a receptor (C5aR) antagonist served as control, and Hsp70 was inhibited by an adenosine derivative inhibitor. The percentage of apoptotic PMN after 2 h was determined by Annexin-V/7-Aminoactinomycin staining as well as quantification of caspase-3 activity.
Intracellular levels of Hsp70 increased considerably after incubation with C5a even after induction of apoptosis via the extrinsic pathway. As expected, stimulation of the death receptor Fas with CH11 as well as incubation with H2O2 significantly increased PMN apoptosis. The amount of apoptotic cells was reduced by addition of C5a in a time- and dose-dependent manner; this effect was fully reversible by specific blockade of the C5aR. Preincubation with the Hsp70 inhibitor resulted in a significant increase in PMN apoptosis.
The anaphylatoxin C5a seems to prolong PMN lifespan both via the intrinsic and extrinsic pathway. Our results indicate that this protection from apoptosis might not only be dependent on PI3K activation, but also by upregulation of Hsp70 expression which in turn may reveal numerous anti-apoptotic effects. If direct modulation of Hsp70 function could lead to a better outcome after severe tissue trauma needs to be addressed in future studies.
HEME OXYGENASE-1 CONTRIBUTES TO IMMUNE SUPPRESSION IN THE LATE PHASE OF SEPSIS
J. Kang, S. Yoon, J. Kim, J. Choi, and S. Lee*. Sungkyunkwan University, Suwon, Republic of Korea
Introduction: Sepsis progresses from an early/acute hyperinflammatory to a late/chronic hypoinflammatory phase with immunosuppression. Suppression of immune responsiveness impairs effective removal of infectious organisms, thus intensifying all the clinical complication of sepsis and increasing susceptibility to secondary infection. Septic patients also possess many signs of deficient immune response including ineffective antigen presentation and T cell hypo-responsiveness. Heme oxygenase-1 (HO-1) has been known as an antioxidant and anti-inflammatory molecule. Recently, the immunoregulatory capacity of HO-1 in adaptive response has received attention. Despite robust studies in the role of HO-1, its exact functional mechanism and interaction with cell populations providing immune regulation remains unclear. Therefore, this study investigates the role of HO-1 as a possible cellular regulator in immunosuppression during sepsis.
Methods: C57BL/6 mice were subjected to experimental sepsis by cecal-ligation and puncture (CLP). Hemin (HO-1 inducer; 20 mg/kg) or zinc protoporphyrin (ZnPP; HO-1 inhibitor; 25 mg/kg) were administered intraperitoneally 24 h and 36 h after CLP, respectively.
Results: HO-1 protein expressions in lung, liver, spleen, and thymus significantly increased 12 h after CLP and these levels remained until 72 h after CLP. ZnPP improved survival rate and enhanced bacterial clearance in CLP mice, whereas hemin worsened these phenomena. ZnPP inhibited apoptotic loss of splenic CD4 and CD8 T cells, increase of Bim and FADD protein expressions, and elevation of caspase-8 and caspase-3 activities in CLP mice. Regulatory T cell, programmed death-1, and cytotoxic T-lymphocyte antigen-4 expressions significantly increased after CLP and ZnPP attenuated these increases. CLP suppressed splenic interleukin (IL)-2 and interferon-γ production while it increased IL-4, IL-10 and IL-17 generation and Th17 cell population. These alterations were attenuated by ZnPP. Finally, ZnPP improved survival in CLP followed by the induction of Pseudomonas aeruginosa pneumonia, a clinically relevant secondary infection model of sepsis.
Conclusion: Our findings suggest that HO-1 overproduction contributes to immune paralysis in the late phase of sepsis through T cell apoptosis and regulatory T cell dysfunction.
THE EFFECT OF TRAUMATIC INJURY ON CIRCULATING TESTOSTERONE AND INFLAMMATORY MEDIATORS
R. Harris-Morris, M. Rani*, T.L. Holloway, J.G. Myers, and M.G. Schwacha*. University of Texas Health Science Center, San Antonio, TX
Introduction: Previous studies have implicated sex steroids, and specifically testosterone, in the deleterious immunological and inflammatory aspects of traumatic injury. This study aimed to examine the effect of trauma on plasma testosterone and inflammatory cytokine levels during the first week after injury.
Methods: To study this, a prospective observational study was designed. Following IRB approval, 33 severely injured trauma patients were enrolled along with 5 healthy normal volunteers that served as controls. The plasma levels of testosterone, cortisol and corticosterone in trauma subjects were evaluated at admission, day 2 and day 7 by EIA and plasma cytokines (IL-6, MCP-1, MIP-1β) were evaluated by Bioplex assay.
Results: In the subjects, the average age was 41±3 years and the ISS was 25±1 (mean ± SEM). Eight-five percent of the injuries were blunt and two-thirds of the subjects were male. Mortality was 10%. A profound drop in circulating testosterone levels (~35%) was observed at admission that further declined to ~15% of normal by day 7. This was paralleled by 2.5-fold and 7.5-fold increases in cortisol and corticosterone levels, respectively that returned towards normals by 7 days. These changes in circulating steroid levels were associated with up to a 10-fold increase in circulating levels of IL-6, MCP-1 and MIP-1β at admission. Plasma cytokine levels returned towards normal by 7 days.
Conclusions: These findings show an apparent inverse relationship between testosterone and inflammatory mediators and glucocorticoids, suggesting that androgens may not be causative in driving post-trauma inflammation in humans.
PHARMACOLOGIC SIRTUIN 1 ACTIVATION PRESERVES MITOCHONDRIAL MASS AND FUNCTION IN RENAL EPITHELIAL CELLS UNDER HYPOXIC INJURY
A. Khader1, 2, W. Yang*2, 3, J.M. Prince2, 3, J. Nicastro2, 3, G.F. Coppa2, 3, and P. Wang*2, 3. 1Elmezzi Graduate School of Molecular Medicine, The Feinstein Institute for Medical Research, Manhasset, NY, 2Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 3Center for Translational Research, The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Renal injury as a result of ischemia-reperfusion (I/R) is a clinical problem with no specific treatment. Sirtuin 1 (Sirt1) is an energy-sensing enzyme with multiple physiologic functions. We have recently demonstrated that pharmacologic Sirt1 activation, with SRT1720, was renoprotective in a murine model of renal I/R. The mechanism for Sirt1’s protective effect in renal I/R remains elusive. We hypothesize that by enhancing mitochondrial biogenesis, Sirt1 activation, replaces the loss of mitochondrial mass resulting from I/R induced oxidative injury.
Methods: Human kidney cells (HK-2) were subjected to 24 h of 1% hypoxia followed by 24 h of reoxygenation. Cells were treated with 0.25 μM or 2 μM of SRT1720 at the start of reoxygenation. Mitochondria were stained with MitoTracker green and mitochondrial mass was determined by Flow Cytometry. Another set of experiments were conducted with the hypoxia mimetic, cobalt chloride, at 4 μM for 4 h followed by a 24 h recovery.
Results: Under normoxic conditions, administration of 0.25 μM and 2 μM of SRT1720 increased mitochondrial mass in a dose dependent manner by 46.4% and 66.7%, respectively, as measured by mean fluorescent intensity (MFI) (Figure). Consistently, there was a 51.7% and a 65.8% increase in mitochondrial mass with administration of 0.25 μM and 2 μM of SRT1720, respectively, after hypoxia-reoxygenation. Similarly, there was a 21.2% and 39.1% increase in mitochondrial mass with 0.25 μM and 2 μM of SRT1720 after exposure to cobalt chloride.
Conclusion: Pharmacologic stimulation of Sirt1 preserved mitochondrial mass after hypoxic injury likely through the stimulation of mitochondrial biogenesis. Thus, an improvement in overall mitochondrial function and cellular energy metabolism is a likely mechanism for the protective effect of SRT1720 in renal I/R. Further experiments to delineate the role of mitochondrial biogenesis after hypoxic injury are currently underway.
THE ANTIFUNGAL DRUG CASPOFUNGIN SPECIFICALLY INTERACTS WITH GLUCAN IN CANDIDA ALBICANS BIOFILMS
M. Kruppa1, D. Lowman1, T.R. Ozment*1, P. Rice2, and D.L. Williams*1. 1East Tennessee State University, Johnson City, TN, 2University of Colorado, Denver, CO
Invasive fungal infections caused by Candida species have increased over the past decade. The mortality rate for fungal sepsis can be as high as that for septic shock. Candida albicans is one the most common fungal pathogens in immunocompromised patients, aged patients (>65 y.o.) and ICU patients. The ability of C. albicans to form biofilms has a profound effect on the ability of the organism to cause disease. This is due, in part, to the fact that biofilms are very resistant to antifungal therapy. Nett et al. have reported that C. albicans biofilm glucan plays a prominent role in resistance of biofilms to anti-fungal drugs (Antimicrob. Agents Chemother. 51:510, 2007). However, little is known about C. albicans biofilm glucan or how this glucan interacts with antifungal drugs. To address these deficits in our knowledge, we isolated glucan from C. albicans biofilms. The biofilm isolate was identified as a (1→3,1→6)-β-glucan by 1D and 2D NMR. The ability of biofilm glucan to interact with antifungal drugs was examined by labeling caspofungin with a fluorescent tag, i.e. AlexaFluor 488. We examined the binding interactions of caspofungin-488 with C. albicans biofilm glucan using a flow cytometric analysis. A reference β-glucan, UV killed C. albicans and heat killed C. albicans served as controls. Casofungin-488 interacted with C. albicans biofilm glucan with characteristics of a single specific binding site and half maximal concentration of ~20 μg/ml. Caspofungin-488 also interacted with the reference glucan, but with lower binding affinity. Interestingly, caspofungin bound non-specifically to UV killed C. albicans yeast, but it bound specifically to heat killed C. albicans yeast. Heat killed C. albicans show increased exposure of cell wall β-glucan when compared to UV killed C. albicans. This suggests that increased exposure of glucan in the C. albicans cell wall facilitates caspofungin binding. In conclusion, our data confirm the presence of (1→3,1→6)-β-glucan in C. albicans biofilms. Of greater importance, our data indicate that glucan isolated from C. albicans biofilms, as well as glucan exposed in the C. albicans cell wall, specifically interacts with caspofungin. We speculate that the ability of C. albicans biofilm glucan to interact with caspofungin may alter the efficacy and bioavailability of this antifungal drug.
DIFFERENTIAL PRE-PROGRAMMING OF INNATE IMMUNITY MODULATES SUBSEQUENT SEPSIS MORTALITY
R. Yuan1, 2, K. Chen1, and L. Li1, 2. 1Department of Biological Science, Virginia Tech, Blacksburg, VA, 2Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA
Background: Sepsis is the leading cause of morbidity and mortality in most intensive care units. No effective therapy is currently available due to the lack of understanding of the complex dynamics of immune modulation. We hypothesize that human innate immune environment prior to the unset of sepsis may significantly influence the subsequent outcome of morbidity and mortality. Super low-grade endotoxemia is a common phenomenon in humans and may skew host immune environment either conducive or preventative for subsequence sepsis mortality.
Method: To test this hypothesis, we pre-injected the male C57BL/6 mice with PBS, super-low dose LPS, or low dose LPS, prior to the procedure of cecal ligation and puncture (CLP). Mortality was closed monitored over a four-day period. In a separate trial, blood, peritoneal cells and spleens were harvested for analyses 24 hours and 4 days after surgery. Bacterial counts, quantities as well as functions of macrophages and neutrophils were analyzed.
Result: We observed that septic mortality were significantly increased in mice pre-challenged with super-low dose LPS, while significantly decreased in mice pre-challenged with low-dose LPS as compared to mice challenged with PBS control. Mice pre-challenged with low dose LPS showed significantly reduced bacteria load in blood and massive PMN infiltration into the peritoneum 4 days after the surgery. We also observed elevated iNOS expression in macrophages and enhanced neutrophil NET formation from mice pre-challenged with low dose LPS. In contrast, super-low dose LPS caused opposite effects in macrophages and neutrophils.
Conclusion: Taken together, our data indicate that host innate environment can be preferentially skewed by varying dosages of LPS, and as a consequence, can affect the mortality risks of subsequence sepsis.
CHARACTERIZATION OF WOUND MYELOID CELLS IN A MOUSE MODEL OF BURN INJURY
M. Rani*, Q. Zhang, and M.G. Schwacha*. University of Texas Health Sciences Center San Antonio, San Antonio, TX
Introduction: Burn-related immunopathology is a complex phenomenon that is poorly understood. Myeloid cells, (i.e., neutrophils, monocytes and macrophages) play an important role in the post-burn immune response; however, their role in regulating the burn wound inflammatory response remains unclear.
Methods: C57BL/6 male mice were subjected to a major burn or sham treatment. One, three and seven days thereafter, skin samples were collected and single cells were isolated by digestion. The isolated cells were characterized for myeloid cell subsets by flow cytometry and skin cytokine levels were determined.
Results: A profound infiltration of the burn wound with CD11b+ monocytes and Gr1+ neutrophils (2-4-fold) was observed at day 1 after injury and was elevated (17-34-fold) by day 7. Macrophages (F4/80+) numbers at the wound site were decreased at day 1; however, they were elevated by day 7 post-injury. Further analysis of the macrophages in the wound revealed that the F4/80+CD11blow subset was decreased, while the F4/80+CD11bhigh subset was increased. An influx of myeloid-derived suppressor cells (i.e., CD11b+Gr1+) into the wound was also observed at all time points. The infiltration of the burn wound with myeloid cells was paralleled by a strong inflammatory response with elevated levels of cytokines (IL-1β, IL-6 and TNF-α) and chemokines (MCP-1, MIP-1α, MIP-1β).
Conclusions: Various myeloid cell subsets infiltrate the burn wound site as early as one day after injury and remain elevated 7 days post-injury. This cellular infiltration is associated with a profound inflammatory response. These findings suggest a causative relationship between myeloid cells and burn wound inflammatory complications.
Grant information: NIH grant GM079122
EXOSOMES ISOLATED FROM HSPA12B DEFICIENT MICE PROMOTE ENDOTHELIAL CELL DYSFUNCTION AND INFLAMMATORY RESPONSES IN MACROPHAGES DURING SEPSIS
X. Zhang, X. Wang, T. Ha, C. Lu, J. Kalbfleisch, R. Kao, D.L. Williams*, and C. Li*. East Tennessee State University, Johnson City, TN
Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 family. HSPA12B is predominately expressed in endothelial cells and it plays an important role in the induction of angiogenesis. Exosomes have been demonstrated to play an important role in cell-cell communication. We examined the role of exosomes isolated from septic HSPA12B deficient mice on inflammatory responses in macrophages and tight junction integrity in endothelial cells. Exosomes were isolated from septic wild type (WT) and HSPA12B deficient mice. Sham surgery served as control. Exosomes from septic WT mice stimulated TNF-α and IL-6 (p<0.05) production in J774 macrophages compared with sham exosomes. However, TNF-α and IL-6 production in J774 cells treated with exosomes from septic HSPA12B-/- mice were significantly greater than in WT septic exosomes treated cells. The levels of TRAF6 ubiquitination and IκBα phosphorylation in macrophages treated with HSPA12B-/- septic exosomes were greater (p<0.05) than in macrophages treated with WT septic exosomes. Exosomes from septic wild type mice induced the loss of tight junction proteins (ZO-1 and Occludin) in endothelial cells (HUVEC). However, exosomes from septic HSPA12B-/- mice induced greater loss of tight junction proteins. HSPA12B-/- septic exosomes also stimulated eNOS expression in HUVEC. In addition, the levels of microRNA-146a, which negatively regulate NF-κB signaling, microRNA-126, which inhibits the expression of adhesion molecules, and miR-26 which regulates angiogenesis, were attenuated in exosomes from septic HSPA12B-/- mice. The data indicate that exosomes released into the serum of HSPA12B-/- mice potentiate septic sequelae. We conclude that HPSA12B exerts a protective role during sepsis.
PROTEASOME INHIBITION AFTER BURN INJURY
P.G. Vana, H. LaPorte, Y.M. Wong, L. Ke-He, R.H. Kennedy, R.L. Gamelli*, and M. Majetschak*. Loyola University Chicago, Maywood, IL
Background: Proteasomes are major non-lysosomal proteases and thought to contribute to the development of hypermetabolism after burns, which is associated with adverse outcomes and prolonged recovery periods. The consequences of proteasome inhibition on the development of hypermetabolism after burns, however, are unknown. Thus, we tested the effects of the FDA approved proteasome inhibitor bortezomib (Bor) in a scald burn injury model in rats.
Methods: Rats were assigned to the following groups: 1) Sham injury (sham)-vehicle (veh), n=10; 2) Sham/Bor, n=13; 3) 30-40% TBSA dorsal scald burn (burn)-veh, n=10; 4) Burn-Bor-early, 1st dose 2h after injury, n=20; 5) Burn-Bor-late, 1st dose 48h after injury, n=13. Bor (0.1mg/kg) was administered ip 3 x weekly. Body weights and resting energy expenditures (REE) were measured on days 0 (pre-burn), 7, 14, 42 post-burn. Blood and pectoralis muscle were harvested at day 42. Muscle extracts were prepared and used to measure proteasome activity using fluorogenic peptides in combination with epoxomicin (proteasome inhibitor). Proteasome content was measured by ELISA. Blood was analyzed for blood chemistry. Statistics: ANOVA/Dunnett post-hoc, Log rank test.
Results: Specific proteasome activity (activity/ng of proteasome) in muscle extracts was reduced with Bor treatment (31.60±7.73% of untreated; p<0.05). Survival was 100% and 92% in groups 1 and 2 (p>0.05). After burns, survival was 80% in group 3, 35% in group 4 (p<0.01 vs. group 3) and 69% in group 5 (p>0.05 vs. group 3). Median survival for group 4 was 45h and >42 days for all other groups. In group 3, REE was significantly increased on days 7, 14 and 42. In groups 4 and 5, REE remained unchanged on days 7 and 14, followed by a significant increase on day 42. There were no differences in blood chemistry parameters among the groups.
Conclusions: Proteasome inhibition with bortezomib delays development of hypermetabolism after burns. Proteasome inhibition during the very early post-burn period, however, results in significant early mortality. These findings suggest that normal proteasome function during the early inflammatory response to burn injury is essential for survival and further support the assumption that proteasomes contribute to the development of burn induced hypermetabolism. Our data highlight the risk of modulating an essential proteolytic pathway after severe burn injury.
CHARACTERIZATION OF HUMAN SAA, BUT NOT SAA1, AS A POSITIVE REGULATOR OF HMGB1 RELEASE
W. Li*2, 1, S. Zhu*2, 1, J. Li1, H. Yang*1, K.J. Tracey*1, P. Wang*2, 1, A. Sama2, 1, and H. Wang*2, 1. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2North Shore University Hospital, Manhasset, NY
Background: The pathogenesis of sepsis remains poorly understood, but is partly attributable to dys-regulated inflammatory responses that are propagated by early cytokines (e.g., TNF and IFN-γ) but sustained by late-acting proinflammatory mediators (e.g., HMGB1). Early cytokines not only directly stimulate macrophages/monocytes to release HMGB1, but also alter the expression of liver-derived acute phase proteins (APPs), which may also participate in the regulation of HMGB1 release.
Objectives: To elucidate the mechanisms underlying the regulation of HMGB1 release, we sought to search for other proinflammatory mediators that could contribute to HMGB1 release.
Methods: Blood samples were collected at various time points (0, 12, 24, 48, and 72 h) from septic patients, and serum levels of HMGB1 and multiple other cytokines/chemokines were determined by Western blots and Antibody Arrays. Cytokines that positively correlated with HMGB1 levels were evaluated for activities in stimulating HMGB1 release in murine macrophage and human monocyte cultures.
Results: In 8 out of 23 septic patients, serum HMGB1 levels appeared to positively correlate with the serum levels of several sepsis surrogate markers (e.g., IL-6, GRO/KC, IL-8, MCP-1, and RANTES), as well as the clinical scores - circulating HMGB1 levels returned to baselines when these patients recovered from the illness. In a subset of septic patients, our anti-HMGB1 IgGs paradoxically cross-reacted with a 12 kDa protein, which was identified as a member of the human serum amyloid A (SAA) family by mass spectrometry analysis. Despite the overwhelming sequence identity between human SAA and SAA1, their capacities in stimulating HMGB1 release were surprisingly distinct. Specifically, human SAA (Cat. No. 300-13, PeproTech), but not SAA1 (Cat. No. 300-53, PeproTech), paradoxically cross-reacted with some anti-HMGB1 antibodies, and effectively induced HMGB1 release in both murine macrophage and human monocyte cultures.
Conclusions: These experimental data revealed a novel mechanism underlying the regulation of HMGB1 release, and shed light on the development of novel SAA-targeting therapy for human sepsis.
[Supported by the National Center of Complementary and Alternative Medicine (R01AT005076) and the National Institute of General Medical Sciences (R01GM063075)].
MONOCYTE GENOMIC SIGNATURES CORRELATE WITH DISCHARGE DISPOSITION AND MORTALITY IN SEVERELY INJURED TRAUMA PATIENTS
M.J. Delano2, R.E. Plevin2, L. Moldawer*1, H. Baker1, C. Lopez1, S. Arbabi*2, G.E. O’Keefe*2, R.V. Maier*2, and J. Cuschieri*2. 1University of Florida, Gainesville, FL, 2University of Washington, Seattle, WA
Objectives: Trauma patient discharge disposition is associated with increased mortality. Patients discharged to home have an improved survival compared to those discharged to inpatient rehabilitation (Rehab) or skilled nursing facility (SNF). Patients that require a SNF have a 13% mortality risk following hospital discharge. We sought to determine whether peripheral blood leukocyte genomic alterations correlate with discharge disposition to home, Rehab or SNF and outcome.
Methods: We reviewed the clinical and genomic data in the Inflammation and Host Response to Injury multicenter trauma database. Trauma patients with age ≥ 18 yrs were classified by discharge disposition into home, Rehab, and SNF groups. Univariate analysis was performed to evaluate the effects of disposition on patient outcome measures. Blood leukocytes were separated into neutrophils, monocytes and lymphocytes. Leukocyte genomic analysis was completed using Affymetrix Glue Grant Human Transcriptome (GG-H) Arrays™ obtained at seven standardized time points in 244 patients.
Results: Patient clinical course and complication rate were associated with discharge disposition. Discharge to SNF was associated with increased MSOF, ARDS, VAP, prolonged hospital and intensive care unit LOS and nosocomial infection rates (p<0.01). Twelve hours after injury discharge to SNF was associated with significant monocyte and not neutrophil and lymphocyte genomic alteration. Monocyte genomic expression over time revealed 1058 probe sets representing immune competence: cytokine signaling (Th1/Th2 cytokines and interferon signaling), pathogen recognition (TLR signaling, HLA recognition), and metabolic health: cellular replication (gene transcription, protein synthesis, histone compatibility), that were altered between discharge groups (FDR 0.05). Lymphocytes and neutrophils revealed modest genomic alteration over time.
Conclusion: We have demonstrated that trauma patient blood monocyte genomic expression differs significantly after injury and correlates with discharge disposition. The genomic signature patterns correlate with increased complications and long term morbidity/mortality. Monocyte genomic alterations identify innate immunity as the potential driving force for late morbidity/mortality and provide potential targets for therapeutic intervention to reduce long term morbidity and mortality.
SURFACE FLUORESCENCE STUDIES OF TISSUE MITOCHONDRIAL REDOX STATE IN EX-VIVO LUNG PERFUSION
T.R. Schaid1, 2, A.H. Abdelhafeez1, 3, M. Ranji4, 3, R. Love1, S. Audi5, 3, S. Kaul1, 2, F. Bashiri4, E. MasoudiMotlagh4, F. Salehpoor4, E. Jacobs*1, 3, and J.C. Densmore*1, 3. 1Medical College of Wisconsin, Milwaukee, WI, 2Children’s Research Institute, Milwaukee, WI, 3Clinical and Translational Science Institute, Milwaukee, WI, 4University of Wisconsin Milwaukee, Milwaukee, WI, 5Marquette University, Milwaukee, WI
Background:Ex vivo lung perfusion (EVLP) provides an opportunity to assess and rehabilitate lung allografts. Currently 80% of donated lungs are unfit for direct transplantation. As long-term allograft failure rates approach 50% and are secondary to early ischemia, measuring early mitochondrial dysfunction via NADH and FAD autofluorescence provides a unique opportunity to identify and alter lung bioenergetics. We hypothesize that changes in lung mitochondrial metabolism can be detected in ex vivo lung perfusion via surface NADH and FAD autofluorescence.
Methods: Adult sheep (n=2) lungs and heart were harvested en bloc and cannulated for EVLP with ventilation. Pulmonary artery and left atrial pressure were continuously monitored and maintained between 3 and 5 mmHg. Every 30 min, perfusate samples were obtained for blood gas analysis and lactate measurement. Lung surface autofluorescence of NADH and FAD was measured using a fiberoptically-coupled UV laser/gated spectrophotomer with appropriate filters. Once steady state was achieved, pentachlorophenol (PCP, 4 mM), an electron transport chain (ETC) uncoupler, was added to the perfusate. Subsequently, potassium cyanide (KCN, 4 mM), an ETC complex IV inhibitor, was added to the perfusate. Lung biopsies were obtained at baseline, prior, and following addition of each toxin for cryo-imaging.
Results: Administration of PCP resulted in an average NADH signal drop of 8.5% (± 2.5) with an FAD signal reduction by 2.5% (± 2.5). This corresponded to a 6.5% (± 0.5) drop in NADH/FAD redox ratio. After administering KCN, NADH signal increased by 8% (± 4) with a 6.5% (± 0.5) drop in FAD signal, corresponding to a redox ratio increase of 14% (± 2). Cryo-imaging showed an average reduction in NADH/FAD ratio by 19.5% (± 16.5), validating our real time detection of PCP’s oxidative effect. Perfusate analysis revealed significantly increased production of lactate after the administration of PCP (123% ± 23).
Conclusions: This study provides evidence that flourometric measurement of NADH and FAD signals can be used to detect alterations in mitochondrial redox state in EVLP. The opposite effects of PCP (oxidized mitochondria) and KCN (reduced mitochondria) on the ETC and hence NADH/FAD ratio delineate the dynamic signal range. Surface flourometry does reflect the state of the lung mitochondria, and improvements in dynamic range are feasible.
EXAGGERATED INFLAMMATORY AND THROMBOTIC GENE EXPRESSION IN VISCERAL ADIPOSE TISSUE OF PATIENTS WITH INTRA-ABDOMINAL SEPSIS
M.E. Starr*1, B.A. Zwischenberger1, J.W. Harris1, 2, B.M. Evers1, 2, P.K. Chang1, and H. Saito*1, 3. 1University of Kentucky, Department of Surgery, Lexington, KY, 2University of Kentucky, Markey Cancer Center, Lexington, KY, 3University of Kentucky, Department of Physiology, Lexington, KY
Background: Adipose tissue is now recognized as an important source of inflammation. We previously reported, using mouse models, that visceral adipose tissue is a major source of inflammatory cytokines and pro-coagulant factors during endotoxemia and that adipose tissue expression of certain cytokines correlates well with plasma levels.
Objective: The purpose of this study was to verify whether patients with intra-abdominal sepsis exhibit exaggerated inflammatory and thrombotic gene expression in visceral adipose tissue and to evaluate the differences in inflammatory profiles among the various human abdominal fat sources.
Methods: Patients undergoing an abdominal procedure with suspected intra-abdominal sepsis were enrolled in this study. Visceral adipose tissues (mesenteric fat, epiploic appendages, and omentum) were collected. Patients undergoing elective abdominal procedures for non-inflammatory conditions served as controls. Histological analyses were performed on formalin-fixed tissues. Quantitative real-time RT-PCR analysis was utilized to determine the expression level of inflammatory and thrombotic factors.
Results: Histologically, adipose tissues from patients with sepsis exhibited signs of polymorphonuclear cell margination and migration and an abundance of reactive mesothelial cells. All sources of visceral adipose tissue expressed significantly higher levels of proinflammatory cytokines interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), and pro-coagulant factors thrombospondin-1 (Thbs-1), and plasminogen activator inhibitors (PAI)-1 and -2 in patients with sepsis compared to control. Among these genes, IL-1α and PAI-1 showed particularly strong expression in the mesenteric adipose tissue (8-fold and 14-fold increase, p<0.01, respectively.) Interestingly, while similar gene expression was observed for each gene across the adipose depots, the omentum from patients with severe sepsis expressed extraordinarily high levels of IL-1α (4-6 fold) and PAI-2 (8-17 fold) compared to the other depots.
Conclusion: Inflammatory and thrombotic gene expression is strongly induced in visceral adipose tissues of patients with intra-abdominal sepsis, validating our mouse models. Further studies will elucidate whether these responses are an inciting event or sequela of intra-abdominal sepsis.
THE NEUROPROTECTIVE EFFECTS OF CO-ULTRAPEALUT IN A MOUSE MODEL OF SPINAL CORD INJURY
D. Impellizzeri, R. Crupi, I. Paterniti, G. Bruschetta, E. Esposito*, and S. Cuzzocrea*. Department of Biological and Environmental Sciences, University of Messina, Messina, Italy
Traumatic injuries to the spinal cord frequently cause permanent neurological disabilities and yet there is no effective therapeutic option to improve functional recovery. Spinal cord injury (SCI) is well-known to induce the formation of reactive astrocytes and the infiltration of immune cells in the vicinity of the lesion site, but whether SCI also induces the production of new neurons in vivo remains controversial. Neurogenesis has been described in various regions of the central nervous system. Significant spontaneous neuroplasticity occurs over the weeks and months following brain or spinal cord trauma leading to some functional recovery. Moreover, studies have shown that spinal neurogenesis occurs to a limited extent after SCI, but that it could be stimulated by experimental intervention. In that regard, in a recent study, we have demonstrated that treatment with a new composite, a formulation including palmitoylethanolamide (PEA) and the antioxidant compound luteolin (Lut), subjected to an ultramicronization process, co-ultraPEALut, significantly reduced inflammatory secondary damage associated with SCI. Thus, the aim of this study was to investigate the neuroprotective effect of co-ultraPEALut in the injury-induced neurogenesis in a mouse model of SCI. SCI was induced in mice through spinal cord compression by the application of vascular clips (force of 24 g) to the dura via a four-level T5 to T8 laminectomy. The animals were sacrificed and the spinal cord were collected. Chronic exogenous administration of co-ultraPEALut increased bromodeoxyuridine (BrdU) and doublecortin immunoreactive cells in the spinal cord of SCI subjected mice. This neuronal development was correlated with synaptic plasticity, identified using the Golgi impregnation method to quantify dendritic spines in spinal cord. In addition, co-ultraPEALut treatment also increased the expression of different neurotrophic factors such as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3). The results indicate that co-ultraPEALut could have a role on birth, survival, and differentiation of new neurons and maturation of spines in the spinal cord and could be a therapeutic target in traumatic diseases.
ALPHA-ENOLASE, A GLYCOLYTIC ENZYME ELEVATED IN HEMORRHAGIC SHOCK, POTENTIATES FIBRINOLYSIS, AND IS REVERSIBLE BY TRANEXAMIC ACID
E. Gonzalez1, 2, E.E. Moore*1, 2, H.B. Moore1, 2, M.P. Chapman1, C.C. Silliman1, 3, and A. Banerjee*1. 1University of Colorado, Aurora, CO, 2Denver Health Medical Center, Denver, CO, 3Bonfils Blood Center, Denver, CO
Introduction: Alpha-enolase(ENO) is a glycolytic enzyme expressed in cells of most tissues. We recently published that in a proteome analysis of a rodent model of hemorrhagic shock, ENO has +2.4-fold increase during hemorrhagic shock. ENO can serve as a cell surface receptor for plasminogen. The effects of this interaction on fibrinolysis have not yet been quantified. We hypothesized that ENO would potentiate the fibrinolytic effect of tissue plasminogen activator(tPA) in whole blood as measured by thrombelastography.
Methods: Whole blood from ten healthy volunteers was collected in citrated tubes. In order to compare the effect of ENO on fibrinolysis, a paired TEG analysis was performed in whole blood samples with tPA(50ng/ml) alone in parallel with samples with tPA and ENO(50mcg/m). Samples were re-calcified with calcium chloride. Tranexamic acid(TXA), a lysine analogue which is a fibrinolysis inhibitor, was added to TEG samples. TEG variables were compared by Student’s t-test for statistical significance.
Results: ENO significantly increased tPA induced fibrinolysis by 22.1% (41.4% with tPA alone vs. 63.5% with tPA + ENO, p=0.02), as measured by LY30(percent of clot lysed 30 minutes after reaching maximum clot strength). The TEG-variables R-time, angle and MA were not statistically different between samples with tPA alone and tPA and ENO. TXA reversed the effect of both tPA and alpha-enolase on fibrinolysis, decreasing the LY30 to baseline.
Conclusion: This novel description of quantifiable fibrinolysis achieved by ENO occurred in the presence of tPA at levels previously measured in trauma patients. These data provide a novel insight into the yet unclear pathophysiology of hyper-fibrinolysis seen in trauma-induced coagulopathy, and serves as a platform for further study.
NEUTROPHIL SCAVENGER RECEPTOR CLASS A (SRA) EXPRESSION IS INCREASED BY TLR2 STIMULATION
T.R. Ozment*, B. Graves, J. Kalbfleisch, and D.L. Williams*. East Tennessee State University, Johnson City, TN
Sepsis is a serious consequence of critical illness. Due to the complex pathophysiology of the disease, effective therapies for sepsis remain elusive, and the mortality rate remains high. Scavenger receptor A (SRA) is best known for its role in atherosclerosis. Though SRA binds modified LDL, it also binds pathogen associated molecular patterns including LPS, lipoteichoic acid and fungal glucans. Thus, SRA is considered an innate immune pattern recognition receptor. We have shown that SRA plays a role in mediating morbidity and mortality in polymicrobial sepsis. SRA deficient mice have improved survival, decreased inflammation, and lower bacterial burdens than wild type mice. Neutrophils are integral to the host response to infection. The reactive oxygen species, hydrolytic enzymes and neutrophil extracellular traps produced by neutrophils lead to pathogen clearance. However, these neutrophil mediators are non-specific, and they can cause tissue damage. Organ dysfunction is one consequence of sepsis, and neutrophil infiltration has been linked to organ damage. We have discovered that SRA, a receptor normally associated with macrophages, is expressed by circulating and bone marrow neutrophils. Of greater significance, neutrophil SRA expression is increased in polymicrobial sepsis. To determine the mechanism responsible for increased neutrophil SRA expression in sepsis, we treated MPRO cells, a murine neutrophil cell line, or murine bone marrow cells with ultra-pure LPS (Toll-like receptor (TLR) 4 ligand), Sigma LPS (mixed TLR ligand), Pam3CSK4 (Pam3, TLR2 ligand), Pam3 and LPS, fungal glucan (Dectin-1 ligand) or media alone for 24 h. SRA expression was measured by flow cytometry. In both cell types, Pam3 was the only mediator to significantly increase SRA expression. In MPRO cells Pam3 increased SRA expression by 535% compared to media alone (p<0.05). In bone marrow neutrophils, Pam3 increased SRA expression by 273%, and Pam3+LPS increased SRA expression by 97.0% (p<0.05) compared to media alone. Neither type of LPS or glucan increased SRA expression. We conclude that the increase in neutrophil SRA expression in sepsis is mediated by TLR2 stimulation, but not by TLR4 or Dectin-1. This suggests that neutrophil SRA up regulation is not a generic inflammatory response to infection but occurs in response to specific stimuli.
MECHANISMS UNDERLYING NEUTROPHIL DERIVED MICROVESICLE GENERATION
B.L. Johnson, J. Kuethe, E.F. Midura, and C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Introduction: During infections, a key element of the early immune response is mediated by neutrophils participating in the anti-microbial response. Microvesicles (MV) from activated and apoptotic neutrophils are of varying composition, size and extracellular expression of phosphatidylserine serine (PS). We previously demonstrated that neutrophil derived MVs (NDMVs) are generated at infectious foci in critically ill patients and that PS NDMVs can have a divergent effect on leukocytes. However, signaling pathways activated during NDMV production remain relatively unexplored. In this current study, we investigated the role of LPS on NDMV generation.
Methods: Bone marrow derived neutrophils were isolated and cultured ex vivo with LPS as well as a panel of pharmacological mediators to include: pan-Caspase inhibitor, Caspase-9 inhibitor, and ML-7 (myosin light chain kinase inhibitor). MVs were either labeled with Ly6G (a neutrophil specific antibody) and analyzed by Nanosight tracking technology, or labeled with Ly6G and Annexin V (a phosphatidylserine specific antibody) and analyzed by flow cytometry.
Results: We observed a 2-fold increase in NDMVs following LPS treatment compared to spontaneous NDMV generation. LPS treatment increased both Annexin(-) and Annexin(+) populations of NDMVs. LPS co-incubation with pan-caspase inhibitor decreased LPS elicited production of both Annexin(-) and Annexin(+) NDMVs by 56%. Treatment with Capase-9 inhibitor similarly suppressed LPS induced Annexin(-) and Annexin(+) NDMV generation by approximately 45%. Additionally, co-treatment with ML-7 completely ameliorated any LPS induced Annexin(+) NDMV generation, while enhancing Annexin(-) NDMV generation 3-fold higher than LPS treatment alone.
Conclusion: We demonstrate that neutrophil exposure to LPS results in NDMV formation through activation of caspase 9 and Myosin light chain kinase to generate NDMVs. Our data continue to build a framework depicting the molecular mechanisms regulating NDMV generation.
ANGIOTENSIN II AND VASOPRESSIN SERVE AS THE DEFENSE SYSTEM AGAINST RAT ANAPHYLACTIC SHOCK
T. Shibamoto*1, M. Wang1, 2, Y. Kuda1, M. Tanida1, and Y. Kurata1. 1Kanazawa Medical University, Uchinada, Japan, 2The Fourth Affiliated Hospital of China Medical University, Shenyang, China
Anaphylactic shock is sometimes life-threatening, but the defense system against this circulatory failure was not fully understood. Ameliorating roles of angiotensin (ANG) II and vasopressin in anaphylactic hypotension were investigated in anesthetized ovalbumin-sensitized Sprague-Dawley rats. The sensitized rats were randomly allocated to the following pretreatment groups (n=7/group): (1) control (non-pretreatment), (2) ANG II synthesis inhibitor captopril, (3) ANG II receptor antagonist losartan, and (4) V1a vasopressin receptor antagonist. Anaphylactic shock was induced by an intravenous injection of the antigen. The systemic arterial pressure (SAP), central venous pressure (CVP), portal venous pressure (PVP) and portal venous blood flow (PBF) were measured, and splanchnic vascular resistance (Rspl: (SAP-PVP)/PBF) was determined. In the control group, SAP markedly decreased, followed by a gradual recovery toward baseline. Rspl transiently decreased immediately after antigen, and then increased 1.5-fold at 15 min and thereafter. The pretreatment with either losartan, captopril or V1a receptor antagonist augmented the initial fall of SAP and attenuated the SAP recovery along with augmentation of the late increase in Rspl. The 2-hr survival rate was significantly smaller in either pretreatment group than in the control group (100%). Plasma levels of ANG II and vasopressin increased to 3.8- and 9.8-fold, respectively, at 30 min after antigen in the control group, whereas captopril pretreatment inhibited the increase in ANG II. In conclusion, inhibition of ANG II or vasopressin exacerbates anaphylaxis-induced hypotension in anesthetized rats.
THE ALTERATION OF LIPID METABOLISM IN DIFFERENT DEGREE OF LPS-INDUCED SEPTIC MICE
T. Irahara1, N. Sato*2, K. Inoue3, M. Fujitani3, M. Manio3, K. Otake1, K. Koike*2, and T. Fushiki3. 1Graduate School of Emergency and Critical Care Medicine, Nippon Medical School, Tokyo, Japan, 2Department of Primary Care and Emergency Medicine, Kyoto University, Kyoto, Japan, 3Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Objectives: The change of energy metabolism during sepsis has been known, however the kinetics of lipid predominance in different degree of sepsis have not been studied well. The objective of this study was to investigate the lipid metabolism in septic mice.
Methods: Septic mice were made by giving various dose of lipopolysaccharide(LPS) intraperitoneally to C57BL/6 mice. Indirect calorimetry by respiratory gas analysis was performed for 72 hours, then carbonhydrate oxidation(CHO) and fatty acid oxidation(FAO) were calculated. Other parameters such as rectal temperature, food intake, body temperature, spontaneous motor activity(SMA) were also measured. Mice were sacrificed at several points during the experiment, then blood and tissue samples were collected. Lipid levels of those samples were determined using colorimetric kits.
Results: Septic mice showed the decrease of rectal temperature, food intake, body weight, and SMA. Indirect calorimetry showed the increase of FAO and the decrease of CHO depending on the degree of sepsis. These changes were significant in acute phase. It suggests the energy metabolism during sepsis change from glucose into lipid predominance. Lipid levels of blood and tissue changed greatly up and down during sepsis. It suggests the lipid is carried to liver as energy substance in acute phase, but is exhausted in late phase due to the decrease of food intake.
Conclusions: The lipid metabolism dramatically changes depending on the degree of sepsis. It may lead to multiple organ failure from malnutrition of vital organ, and influence the prognosis. Intervention for this mechanism can become one of the treatments for severe sepsis.
APOE AND NKT CELLS MEDIATE SERUM CREATININE LEVELS IN MICE DURING SEPSIS
S. Kasravi and H. Harris*. University of California San Francisco, San Francisco, CA
Introduction: It has been proposed that renal failure is a large contributor to mortality in septic mice. Previous work in our lab has showed that Apolipoprotein (apoE) E and Natural Killer T (NKT) cells modulate the effects of pro-inflammatory signals following infection likely through altered antigen presentation. Decreased apoE and/or NKT cells result in dramatically decreased septic mortality and decreased levels of Th1 cytokines in mice. The favorability of survival in apoE and/or NKT cell deficient mice could potentially be due to the interruption of an inflammatory pathway which leads to kidney failure. Serum creatinine concentrations as a measure of GFR can be used as indicators of renal damage in septic mice.
Objective: To determine whether ApoE and NKT cells contribute to kidney damage in septic mice.
Methods: We measured concentrations of serum creatinine 24 hours after septic insult via cecal ligation and puncture (CLP) in mice expressing various combinations of low and normal apoE and NKT cell concentrations. The resulting data were compared by a t-test to determine two-tailed p values.
Results: Sepsis causes significant upregulation of serum creatinine compared to sham in wildtype mice undergoing cecal ligation and puncture (0.7 mg/dl v. 1.8 mg/dl, p = 0.006), while mice deficient in ApoE and/or NKT cells demonstrate an almost 3-fold decrease in serum creatinine concentrations 24 hours following septic insult compared to septic wildtype mice (See figure).
Conclusions: Reduced levels of apoE and/or NKT cells significantly decreased concentrations of serum creatinine in septic mice. These data support the hypothesis that renal failure is a significant contributor to septic mortality and suggest that inhibition of renal failure in apoE/NKT cell deficient mice may explain their improved survival.
PRE-CONDITIONING WITH SUPER-LOW DOSE ENDOTOXIN LEADS TO LOW-GRADE INFLAMMATION AND REDUCED WOUND REPAIR
K. Chen1, R. Yuan1, 2, S. Geng1, and L. Li1, 2. 1Department of Biological Science, Virginia Tech, Blacksburg, VA, 2Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA
Subclinical super-low dose endotoxemia is prevalent in humans with adverse health conditions, and it correlates with the nagging persistence of chronic inflammatory diseases such as atherosclerosis, diabetes, and neurologic inflammation. In this report, we aim to test the hypothesis that super-low dose LPS may program host innate immunity into a low-grade inflammatory state that is detrimental for effect wound repair. To test this hypothesis, we pre-injected the male C57BL/6 mice with PBS or super-low dose LPS for 10 days, prior to the generation of excisional skin wound. The wound closure was closely monitored and documented for 15 days. In a separate trial, we harvested spleen, bone marrow, peripheral blood, and skin tissues, for both histological and immunological analyses through tissue staining, sectioning and flow cytometry. We observed that mice pre-conditioned with super-low dose LPS had significantly reduced wound closure as compared to mice injected with PBS. Histological analyses indicated that pre-conditioning with super-low dose LPS significantly reduced TGFβ levels and collagen content in regenerated skin tissues. Flow studies showed that pre-conditioning with super-low dose LPS significantly increased the population of neutrophils as well as the pro-inflammatory CD11bLy6C+ monocytes. Taken together, our study demonstrates that super-low dose LPS can skew innate immune environment into a low-grade inflammatory state and impede effective wound healing.
REDUCTION OF NATURAL ACTIVATED PROTEIN C PREVENTS COAGULOPATHY IN A PRECLINICAL ANIMAL MODEL OF ACUTE TRAUMATIC COAGULOPATHY
M.L. Guerreiro1, 2, J.L. Tremoleda1, C. Thiemermann1, 2, and K. Brohi1. 1Bart’s and The London School of Medicine and Dentistry, Queen Mary University, London, United Kingdom, 2William Harvey Research Institute, London, United Kingdom
Background: Acute traumatic coagulopathy (ATC) identified in the first hour of trauma patients is associated with worse outcomes and increased risk of death. Identification of critical coagulation pathways involved in ATC is important for future targeted drug discovery. Previous studies have demonstrated an activation of the thrombomodulin-protein C pathway during ATC. We examined the effect of reduced thrombomodulin generation in an experimental model of ATC.
Methods: Both wild type (WT) and modified Knock-in TM pro/pro mice (TMKI) were subjected to trauma (laparotomy with muscle injury and bilateral tibia-fibula fracture) and haemorrhage (40± 5% of estimated blood volume) to a target mean arterial blood pressure of 30 ± 5 mmHg. This model was developed to elicit coagulopathy as a response to trauma and bleeding thus mimicking the coagulopathy present in the injured patient. The animals were kept euthermic and were not resuscitated. After 60 minutes blood samples were taken and analysed via thrombelastometry tests.
Results: All animals were shocked when compared with controls (lactate ≥ 7 mg/dL and ≤ 3 mg/dL; p<0.001). After trauma and haemorrhagic shock (THS), WT mice developed a significant coagulopathy, as measured by a reduction in clotting amplitude at 5 minutes (CA5’) (THS time 0 vs time 60 = 46.7± 1.37 vs 27.6±2.57 mm, p<0.0002) and in maximal clot firmness (MCF) (THS t0 vs t60 =62.7±1.1 mm vs 46± 3.3 mm, p<0.001). In the TMKI group, which entails a 1000 fold reduction in aPC production there was a correction of the coagulopathy (CA 5’: TMKI time 0 vs time 60 = 42.6± 2.3 vs 41.6±1.5 mm, p<0.0002 and MCF (TMKI t0 vs t60 =56.8±2 mm vs 55.1± 1 mm, p<0.001.
Conclusion: Severe trauma shock induced a significant coagulopathy characterized by the generation of fragile clots. The reduction in activated protein C generation encompassed an improvement in the experimental coagulopathy therefore the protein C pathway seems to play an important role in acute traumatic coagulopathy.
FULL THICKNESS BURNS RESULT IN ESTROGEN INDUCED INCREASES IN BURNED SKIN LEVELS OF SONIC HEDGEHOG (SHH), A SIGNALING PROTEIN CONTROLLING DIVISION OF ADULT STEM CELLS
J.G. Wigginton*1, P.E. Pepe1, D. Maass1, J.W. Simpkins2, V. Warren1, J. Minei*1, J. Gatson*1, S.E. Wolf*1, and A. Idris1. 1UT Southwestern Medical Center, Dallas, TX, 2University of West Virginia, Morgantown, WV
Introduction: Prior findings from our lab note that burned skin, including the “dead tissue” in 3° burns, reveals a substantial, rapid and sustained pattern of inflammation that is significantly blunted by a single, post-burn dose of estrogen. Others have noted that eschars contain stem cells that possess the potential for future skin regeneration. Based on an understanding that 3° burned skin could be manipulated, and that estrogens exert influence on a variety of stem cells, we hypothesized Sonic Hedgehog (SHH) (a signaling protein that controls and directs differentiation of both embryonic and adult stem cells) might be present in the eschar, and that estrogens might increase levels of SHH.
Methods: 48 male rats were randomized into 3 groups: 1) sham/burn (n=4); 2) burn/placebo (n=20); 3) burn/17β-estradiol (E2) (n=20). Burned rats received a 40% 3° TBSA dorsal burn, fluid resuscitation and one dose of E2 or placebo (0.5 mg/kg IP) 15 minutes post-burn. 8 animals from each of the two burn groups (burn/placebo; burn/E2) were sacrificed at 24 hours and 7 days (sham group at 7 days only), with 4 each of the two burn groups sacrificed at 45 days. Burn skin tissue samples were analyzed by ELISA for SHH.
Results: At all measured time points, estrogen significantly increased levels of SHH in 3° burned skin.
Conclusions: Early, single-dose estrogen administration following severe burn injury significantly elevates levels of SHH in third degree burn tissue. This may represent an extremely novel and important pathway for skin regeneration in burn patients in the future.
LEUKOTRIENE B4 RECEPTOR 1 (BLT1) SIGNALING ENHANCES ENDOTOXIN-INDUCED LIVER INJURY AND LIVER MICROCIRCULATORY DYSFUNCTION
K. Kojo, Y. Ito, H. Ohkubo, M. Watanabe, and M. Majima. Kitasato University Graduate School of Medicine, Sagamihara, Japan
Aims: Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils, which are essential for liver injury during endotoxemia. In particular, neutrophil activation occurring in the extra-sinusoidal space is critical for the induction of liver injury. The objective of the present study was to examine the role of LTB4 receptor 1 (BLT1) signaling in neutrophil-mediated liver injury in response to endotoxin.
Methods: BLT1 knockout mice (BLT1-/-) and wild-type mice (WT) were treated with endotoxin (LPS, 0.1 mg/kg, ip) and galactosamine (Gal, 700 mg/kg, ip). Blood and liver tissues were collected at 0, 3, 5, and 6 hours after Gal/ET administration for various measurements. Liver microcirculation was observed using in vivo microscopy.
Results: The hepatic levels of mRNA for BLT1 and 5-lipoxygenase (5-LOX) were increased after Gal/ET. In WT mice, ALT levels were elevated with time after Gal/ET, and reached to maximal levels at 6 h. In BLT-/- mice, ALT levels were lower than WT mice. Tunnel-positive cells in BLT1-/- -livers treated with Gal/ET were decreased as compared with those in WT-livers. Accumulated and extravasated neutrophils were suppressed in BLT-/- livers. In vivo microscopic analysis revealed that impaired sinusoidal perfusion with adhesion and extravasation of leukocytes was suppressed in BLT-/- mice, which was associated with reduced levels of hepatic mRNA expression of TNF and ICAM-1. There were no differences in mRNA levels of CXCL1, CXCL2, and CXCR2 between WT mice and BLT1-/- mice.
Conclusions: BLT1 signaling plays an important role in liver injury during endotoxemia by affecting leukocyte-sinusoidal endothelial interaction.
THE EFFECT OF OXYGEN TENSION ON REACTIVE OXYGEN SPECIES PRODUCTION IN BLOOD EX VIVO
F.T. Billings*, A.T. Bikineyeva, J.L. Gamboa, and S.I. Dikalov. Vanderbilt University, Nashville, TN
Excess reactive oxygen species (ROS) damage tissues due to extensive lipid and protein oxidation. Molecular oxygen is the primary substrate for ROS production, and oxygen tension is tightly regulated in aerobic organisms. Anesthesiologists hyper-oxygenate patients during surgery. We exposed whole blood from healthy volunteers to hypoxia (target pO2 = 30mmHg, Hb O2 sat = 50%), normoxia (80mmHg, 95% sat), hyperoxia (200mmHg, 100% sat), or severe hyperoxia (550mmHg, 100% sat) for 30 minutes and measured intra- and extracellular ROS production using site specific electron spin probes and electron paramagnetic resonance to test the hypothesis that increased oxygen tension increases ROS production in blood.
The target hypoxic, normoxic, and hyperoxic oxygen tensions were achieved (Figure 1A). ROS production, measured by TMH electron spin probe, increased in proportion to oxygen tension from 4.8 + 2.1 pmol/ml blood during hypoxia to 24.8 + 4.3 during severe hyperoxia (MWU P=0.03; pO2 vs. ROS Spearman correlation r=0.77, P=0.001; Figure 1B). Oxygen tension did not affect extracellular ROS production, measured by cell impermeable CAT1H probe (r=-0.06, P=0.86, Figure 1C). To further examine the affect of oxygen tension on intracellular ROS production we added cell impermeable superoxide dismutase (SOD) to TMH probe in additional experiments. Extracellular elimination of superoxide with SOD did not affect ROS production or the effect of oxygen tension on ROS production (r=0.89, P=0.003), further suggesting that increased oxygen tension increases intracellular but not extracellular ROS.
Hyperoxia increase ROS production in blood ex vivo. The source of excess ROS production appears to be intracellular, possibly from mitochondria, a common source of ROS implicated in oxidative stress. Hyperoxia does not appear to affect phagocytic NADPH oxidase in healthy volunteer blood, an important extracellular source of ROS. Subsequent studies are required to determine the cell type and molecular source of hyperoxia-induced ROS production in blood and to determine if hyperoxia increases ROS production or oxidative damage in solid organs.
FIBRINOLYTIC ACTIVITY IN PLASMA AFTER POLYTRAUMA AND HEMORRHGE IN RATS
X. Wu, D.N. Darlington, and A.P. Cap. US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: We have recently developed an Acute Coagulopathy of Trauma (aCOT) in rat that mimics the clinical hypocoagulation in both civilian and military patients with severe trauma and hemorrhage. This polytrauma and hemorrhage model allows for the study of changes in plasma fibrinolysis that may, in turn, contribute to the development of coagulopathy.
Objective: To determine if fibrinolytic activity in plasma changes after polytrauma and hemorrhage.
Method: Sprague-Dawley rats (300-400g, n=8) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and by fracturing the right femur. The rats were bled to a mean arterial pressure of 40mmHg and held there until 40% of the blood volume was removed. Hemorrhage was completed between 30-60min. No fluid resuscitation was given. All rats survived the 240 min experimental period. Blood samples were taken before (time 0) and after trauma at 30, 60, 120 and 240min. Rat tissue plasminogen activator, plasminogen activator inhibitor-1, d-dimer, α-Macroglobulin and thrombin activatable fibrinolysis inhibitor were measured by immunoassay.
Results: Prothrombin time significantly increased over the 4hr period thereby validating the model of coagulopathy. Rat tissue plasminogen activator was significantly elevated by 30min, but fell below control levels by 2-4hrs. However, plasminogen activator inhibitor-1 (PAI-1) rose significantly over time to be significantly elevated at 2-4hrs. D-dimer slowly rose over time and was significantly elevated by 4hrs. α-Macroglobulin fell significantly by 2hrs. Thrombin activatable fibrinolysis inhibitor levels in plasma did not change over time.
Conclusion: Polytrauma and hemorrhage in rat showed an initial rise in fibrinolysis as seen by the rise in tissue plasminogen activator by 30min and the elevation in d-dimers. However, the steady rise in PAI-1 over time suggests that fibrinolysis is inhibited by 4hrs suggesting that fibrinolysis may contribute to an early aCOT and generate a compensatory anti-fibrinolytic response (PAI-1). This suggests that anti-fibrinolytic drugs like tranexamic acid may be most effective if administered early after trauma. This project was funded by MRMC.
A CORRELATION BETWEEN INFECTION AND PULMONARY ENDOTHELIAL DAMAGE DETECTED IN BRONCHOALVEOLAR LAVAGE (BAL) SAMPLES OF TRAUMA PATIENTS WITH PNEUMONIA
L. Ma, X. Yao, R.M. Huebinger*, S.E. Wolf*, J. Minei, and Q.S. Zang*. University of Texas Southwestern Medical Center, Dallas, TX
Objective: The aim of this study was to examine whether levels of endothelial markers correlate with the degree of airway bacterial infection in BAL samples of trauma patients that developed ventilator or community acquired pneumonia.
Methods: BAL samples of trauma patients diagnosed with pneumonia were collected. Airway infection was determined by standard bacterial culture. Levels of von Willebrand factor (vWF), an endothelial marker, were measured by ELISA assay. Total protein levels were also compared. To further determine whether pulmonary endothelial damage is induced in pneumonia-related sepsis, alveolar expression of vWF was examined in an animal model. Sepsis was produced in SD rats infected by S. pneumoniae (4x106 CFU/rat). Lung tissue was harvested and vWF was examined by immunohistology.
Results: In BAL fluid collected from pneumonia patients, increases in vWF and total protein levels were found to associate with the extent of bacterial infection. In animal model, a dramatic decrease in vWF expression was found in alveolar endothelium in septic rats.
Conclusion: Our data suggest that airway bacterial infection induces damage of endothelial vasculature in the lung. Previous studies suggest that plasma vWF is associated with sepsis mortality. Further investigation is warranted to determine whether BAL vWF can be used as a prognosis marker for lung injury severity and occurrence of pneumonia sepsis in trauma patients.
A COMPARISON OF THE LEUKOCYTE GENOMIC RESPONSES TO STERILE OR INFECTIOUS INFLAMMATION
B.E. Szpila, L.F. Gentile, D.C. Nacionales, M. Lopez, E. Vanzant, R. Ungaro, A.L. Cuenca, A. Rountree, A.M. Mohr, S. Brakenridge*, F. Moore*, H. Baker, L. Moldawer*, and P.A. Efron*. University of Florida, Gainesville, FL
Objectives: Trauma and sepsis are leading causes of morbidity and mortality in the ICU setting. Both are characterized by dramatic host responses presumably to specific molecular patterns referred to as damage-associated (DAMPs) and pathogen-associated molecular patterns (PAMPs) with injury and infection, respectively. Using three well accepted murine models, including severe trauma and hemorrhagic shock (polytrauma (PT)), sepsis (cecal ligation and puncture (CLP)) and PT followed by infection (PT+ Pseudomonas pneumonia (Pp), we determined the unique genomic leukocyte responses to sterile vs non-sterile inflammation.
Methods: 6-9 wo B6 mice underwent PT (hemorrhagic shock + lower extremity fracture + laparotomy & cectomy), CLP, or PT + intranasal Pp induction 24 hours after surgery. Mice were euthanized at 2h, 1d, and 3d post PT/CLP and their blood and serum were collected. The Pp group was euthanized at 1d post Pp. Total blood leukocytes were isolated and genome-wide expression was performed on their isolated RNA. Expression patterns were compared between PT, CLP, PT+Pp and naïve control mice at p<0.001 (F test).
Results: Peak genomic dysregulation for the PT and CLP models occurs at 2 hr and 1 d, respectively. During these time points, 1938 genes in the PT model and 3816 genes in the CLP model significantly differ vs control. By day 1, the genomic expression in PT mice begins to return to baseline with only 131 genes that are significantly different from control; however, upon pneumonia induction, expression of 1622 genes again differ from control. On further analysis, we found that 297 genes show similar changes between only the 1d CLP and PT+Pp populations and only 48 genes have similar changes in expression between 1d CLP and 1d PT. The function of these genes varies but includes caspase cascades, cell cycle regulators, metabolite synthesis, DNA repair, calcineurin effects, natural killer mediated toxicity, and endothelial migration.
Discussion: Polytrauma and sepsis each produce dramatic changes in the host genome, although the patterns of gene expression vary dramatically. Subsequent pneumonia in the trauma model leads to further dysregulation of the transcriptomic responses. The findings suggest that although there are shared core genomic responses to trauma and to sepsis in the mouse, much of the response is stimulus-specific.
EFFECTS OF THE SLOW-RELEASING SULFIDE DONOR GYY4137 DURING RESUSCITATED SEPTIC SHOCK IN ATHEROSCLEROTIC SWINE
S. Hafner1, J. Matallo1, M. Gröger1, U. Wachter1, O. McCook1, M. Wood2, M. Whiteman3, M. Georgieff1, E. Calzia1, P. Radermacher1, and M. Wepler1, 4. 1Sektion APV, Klinik für Anästhesiologie, Universitätsklinikum Ulm, Ulm, Germany, 2Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom, 3University of Exeter Medical School, St Luke, Exeter, United Kingdom, 4Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA
Background: The slow-releasing sulfide donor GYY4137  was shown to blunt LPS-induced hypotension and inflammatory response in a rat model of endotoxic shock . These data originate from un-resuscitated rodent models without any pre-existing co-morbidities. Since impaired endogenous H2S formation is referred to as a key factor of atherosclerosis , we investigated the effects of GYY4137 during long-term, resuscitated, fecal peritonitis-induced septic shock in swine characterized by hypercholesteremia and consecutive ubiquitous atherosclerosis .
Methods: Up to now, 11 anesthetized and instrumented animals received either GYY4137 (10 mg/kg, n=7) or vehicle (n=4) at 12 and 18 hours after induction of peritonitis. Systemic hemodynamics, left heart function (pressure-conductance catheters), kidney blood flow (ultrasound flow probes) and function, and metabolic status were measured before as well as at 12 and 24 hours of sepsis. Free sulfide concentrations were determined hourly with combined gas chromatography/mass spectrometry (GC/MS). Data are median (quartiles).
Results: GYY4137 did not affect the norepinephrine infusion rate required to achieve target hemodynamics (vehicle 1.2 (0.6;1.3) vs. GYY4137 1.1 (0.9;1.2) μg/kg/min). Sepsis decreased creatinine clearance (vehicle 38 (11;53) vs. GYY4137 46 (39;69) ml/min) and increased plasma creatinine levels (vehicle 155 (135;206) vs. GYY4137 130 (116;159) μmol/l at 24 hours of sepsis), again without intergroup difference. Neither kidney blood flow nor arterial base excess/lactate levels showed any statistical inter-group difference. GC/MS revealed comparable levels of free sulfide in animals treated with GYY4137 or vehicle.
Conclusion: In swine with pre-existing atherosclerosis, GYY4137 failed to attenuate hypotension or kidney dysfunction during septic shock.
Supported by the Perspektivförderung Innovationsfond Medizin Land Baden-Württemberg
1. Li et al, Circulation 2008;117:2351-60
2. Li et al, Free Radic Biol Med 2009;47:103-13
3. Mani et al, Circulation 2013;127:2523-34
4. Wepler et al, Intensive Care Med Exp 2013;1:9
A NOVEL MODEL OF MULTISYSTEM TRAUMA DEMONSTRATES INCREASED MORTALITY AFTER INFECTION IN YOUNG MICE
R.E. Southard*, S. Ghosh, R.D. Winfield*, I. Turnbull, J. Hilliard, C. Davis*, A. Fuchs, and N. Karlow. Washington University in St Louis, St Louis, MO
Background: Animal models of trauma are necessary to study the mechanisms by which injury affects the immune system and other systems. Models using mild and single system injuries have not consistently been shown to induce immune suppression in animals comparable to what is seen in patients after severe trauma. More severe injury models are problematic because of the need for close monitoring and resuscitation of small animals. The hemorrhagic shock model is time- and labor-intensive, which limits its ability to assay an adequate number of animals simultaneously for a reasonable statistical analysis. We sought to create a multisystem model of severe injury which would demonstrate a phenotype of immune suppression when mice were subsequently subjected to infection with intratracheal Pseudomonas aeruginosa.
Methods: Mice (male C57BL/6J, age 6-8 weeks) were subjected to all of the following injuries: laparotomy with crush injury of the left lateral liver, pseudofracture of both hind limbs, and removal of 15% of the total blood volume. Pneumonia was then induced 48 hours after injury by intratracheal injection of Pseudomonas aeruginosa (6x10^6 CFU.)
Results: Mice routinely survived the primary injury (>80 %.) Mice subjected to multisystem injury were more likely to die (PT+Pa, 69% mortality) from Pseudomonas aeruginosa pneumonia compared to their uninjured counterparts (sham+Pa, 33% mortality, p=0.02.)
Conclusion: We have demonstrated a novel model of trauma which induces immune suppression after injury as assessed by a clinically relevant route of infection. This represents an ideal model of blunt traumatic injury because it can be performed rapidly and reproducibly, allowing multiple animals to be injured and subsequently assayed in a single experiment. This model of multisystem trauma induces immune suppression in young C57/BL6J mice which makes it suitable for use in future studies of the effects of trauma on the immune system.
AGE-DEPENDENT CHANGES OF METABOLIC SIGNALING PATHWAYS IN THE MYOCARDIUM IN EXPERIMENTAL SEPSIS IN MICE
Y. Inata, P. Hake, G. Piraino, J.R. Ledford, M. O’Connor, and B. Zingarelli*. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
The development of multiple organ failure and the outcome of sepsis are significantly impacted by the age of the patient. Myocardial dysfunction is a recognized manifestation of sepsis. However, the molecular mechanisms have not yet been defined. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls autophagy, i.e. the disposal of defective organelles, and metabolic recovery through mitochondrial biogenesis. Here, we tested the hypothesis that metabolic signaling pathways are altered in the myocardium during sepsis and are age-dependent. Polymicrobial sepsis was induced in male young (2-3 months old) and old mice (11-13 months old) by cecal ligation and puncture (CLP). Mice were euthanized at 6 and 18 hrs after CLP and the heart was harvested. At Western blot analysis, there was a time-dependent increase of nuclear translocation of the active catalytic subunit pAMPKα in young mice (1.93±0.08 U of relative intensity at 18 hrs after CLP), but not in old mice (0.63±0.12 U of relative intensity). Since AMPK regulates mitochondrial function through peroxisome proliferator-activated receptor γ co-activator α (PGC-1α), we also evaluated the nuclear expression of this co-factor. Young mice exhibited a significant increase of PGC-1α nuclear expression at 18 hrs after CLP (1.88±0.53 U of relative intensity) when compared to the expression of old mice (0.73±0.62 U, P<0.05). We next determined the capacity to form autophagic vesicles by evaluating the conversion of the light chain (LC3B) I of the autophagosome to the LC3B-II isoform. Under basal conditions the LC3B-I/LC3B-II ratio was similar in sham mice of both ages. After sepsis, young mice exhibited an increase of LC3B-II conversion in the heart at 18 hrs (1.18±0.13 U of relative intensity), suggesting formation of autophagic vesicles. However, LC3B-II conversion was significantly reduced in old mice (0.38±0.03 U of relative intensity) when compared to the young group (P<0.05). Thus, our data suggest that during sepsis metabolic repair mechanisms are activated in the heart and involve AMPK-dependent pathways. However, this restorative process diminishes in old age. (Supported by NIH R01GM067202).
PERIOPERATIVE ENTERAL IMMUNONUTRITION AS A GREEN ALTERNATIVE WAY TO REDUCE THE ACUTE PHASE RESPONSE IN ABDOMINAL SURGERY: RESULTS OF A META-ANALYSIS
C. Matuschek1, E. Boelke*1, P. Gerber1, M. van Griensven*2, K. Kammers3, W. Budach1, and K. Orth1. 1Heinrich Heine University, Dusseldorf, Germany, 2LMU, Munich, Germany, 3Johns Hopkins University, Baltimore, MD
Background: Perioperative enteral immunonutrition is thought to reduce the acute phase response and postoperative morbidity in patients undergoing major abdominal surgery. This meta analysis assessed the clinical effects of preoperative enteral immunonutrition in cancer patients undergoing major abdominal surgery.
Methods: In this meta-analysis a total of 284 patients were randomly assigned to receive either immunonutrition or a regular diet. The primary endpoint was the incidence of surgical-site infection. Secondary endpoints were rates of infectious complications, overall postoperative morbidity and C-reactive protein (CRP) levels on 3-4 days after surgery. Published relative risks were basis of the meta-analysis. Meta-analysis of the effect sizes on clinical outcome and mediator response was performed using a random effects model based on parameter estimates of relative risks and their standard errors.
Results: Of 284 randomized patients, 137 were allocated to the control group and 147 received immunonutrition. Clinical data were comparable in both groups. The acute phase response did not vary between groups. Furthermore there was no significant difference in the clinical outcome regarding infections complications and overall postoperative morbidity.
Conclusion: Preoperative enteral immunonutrition failed to show any significant advantage in terms of early clinical outcomes or change of the systemic acute-phase reaction.
PHARMACOLOGICAL ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE PATHWAY BY METFORMIN AMELIORATES KIDNEY INJURY IN HEMORRHAGIC SHOCK IN OLD RATS
B. Zingarelli*, P. Hake, J.R. Ledford, G. Piraino, and M. O’Connor. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Hemorrhagic shock is a predictor of poor outcome in elderly trauma patients as it is associated with high rates of organ failure. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis by modulating mitochondrial biogenesis and autophagy. Phosphorylation of the α-subunit by the upstream liver kinase B1 (LKB1) is essential for its activation. We hypothesized that pharmacological activation of AMPK activity by the LKB1 activator metformin ameliorates hemorrhage-induced kidney injury in old rats. Hemorrhagic shock was induced in anesthetized male rats (18-24 months old) by withdrawing blood to a mean arterial pressure of 50 mmHg. After 3h, rats were rapidly resuscitated by infusing the shed blood and were treated with vehicle or with metformin (100 mg/kg intraperitoneally) at the time of transfusion. Kidneys were harvested 3h after resuscitation. After hemorrhagic shock, kidney injury was characterized by severe tubulo-interstitial injury and was associated with elevated levels of plasma creatinine (1.33±0.18 mg/dL) when compared to sham age-matched rats (0.26±0.09 mg/dL, P<0.05). Myeloperoxidase activity (MPO), which was measured to evaluate neutrophil infiltration, was significantly high (7.6±1.2 U/100 mg tissue) after hemorrhagic shock in vehicle-treated rats when compared to sham animals (3.9±0.9 U/100 mg tissue, P<0.05). Treatment of old rats with metformin significantly (P<0.05) reduced MPO activity in the kidney (3.5±1.2 U/100 mg tissue). At molecular analysis, the nephron-protective effect of metformin was associated with increased autophagy as demonstrated by increase in the conversion of the light chain (LC3B) I to LC3B-II. Since AMPK regulates mitochondrial function through peroxisome proliferator-activated receptor γ co-activator α (PGC-1α), we also evaluated the expression of this co-factor. There was an increase in nuclear translocation of PGC-1α in the metformin-treated group, thus suggesting the possibility of mitochondrial regeneration in the kidney. Treatment with metformin at the time of resuscitation also improved mean arterial blood pressure when compared to vehicle-treated rats. Thus, these results suggest that AMPK activation may improve metabolic recovery and may be a potential therapeutic approach in hemorrhagic shock in elderly patients. (Supported by NIH R01 AG-0227990).
ALCOHOL INTOXICATION ALTERS SYSTEMIC IMMUNE RESPONSES IN ADULT BURN INJURY VICTIMS
E.B. OHalloran, S.R. Carter, C.S. Davis, L. Ramirez, J.M. Albright, R.L. Gamelli*, and E.J. Kovacs*. Loyola University Medical Center, Maywood, IL
Background: About 50% of the adult burn population hospitalized annually has a positive blood alcohol content (BAC) at presentation. Intoxication at the time of burn injury has been associated with greater mortality and higher complication rates. We aimed to investigate if being intoxicated at the time of burn elicits a systemic immune response and to characterize the effects of alcohol on systemic inflammatory mediators following thermal injury.
Methods: In a prospective study conducted from 2007 to 2013, serum samples were collected from patients admitted to the burn intensive care unit within 15 hours of injury. Thirteen patients were studied, and demographic and injury-specific information were recorded. Serum was analyzed for immunomodulating proteins via multiplex bead array.
Results: On admission, nine patients had BAC levels of zero while four had detectable BAC levels. The two groups were similar in terms of demographics, total body surface area of burn, and Baux score. Those intoxicated at the time of injury required more resuscitation in the first 24 and 48 hours after injury than those not intoxicated (122.0 vs 66.1 and 253.7 vs 127.4 mL/kg, p < 0.05) and required systemic antibiotic therapy for more days (24.0 vs 6.1 days, p < 0.05). In the serum, the concentrations of several immune mediators were increased in the intoxicated group, including interleukin (IL)-1β, IL-1RA, IL-10, IL-12, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interferon γ, monocyte chemotactic protein 1, macrophage inflammatory protein 1a, and tumor necrosis factor α (p < 0.05 for all). A greater than ten-fold increase in serum IL-6 and greater than four-fold increase in IL-8 were seen in intoxicated patients when compared to the non-intoxicated group.
Conclusions: Patients intoxicated at the time of burn injury require more aggressive resuscitation in the immediate post-injury period, are subjected to more systemic antibiotic days, and demonstrate elevation of many pro- and anti-inflammatory immunomodulators. While the source of these immune mediators remains to be determined, the observed elevation in intoxicated patients may correlate with previously reported morbidity and mortality in these patients.
PATTERNS OF GENE EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND OUTCOMES FROM PATIENTS WITH SEPSIS SECONDARY TO COMMUNITY ACQUIRED PNEUMONIA
P. Severino2, E. Silva2, G. Baggio-Zappia1, M. Brunialti1, L.A. Nucci1, O. Rigato3, 1, I.D. Silva1, F.R. Machado1, and R. Salomao*1. 1Universidade Federal de Sao Paulo, Sao Paulo, Brazil, 2Hospital Israelita Albert Einstein, Sao Paulo, Brazil, 3Hospital Sirio Libanes, Sao Paulo, Brazil
Mechanisms governing the inflammatory response during sepsis have been shown to be complex, involving cross-talk between diverse signaling pathways. Current knowledge regarding the mechanisms underlying sepsis provides an incomplete picture of the syndrome, justifying additional efforts to understand this condition. Microarray-based expression profiling is a powerful approach for the investigation of complex clinical conditions such as sepsis. In this study, we investigate whole-genome expression profiles in mononuclear cells from survivors (n=5) and non-survivors (n=5) of sepsis. To circumvent the heterogeneity of septic patients, only patients admitted with sepsis caused by community-acquired pneumonia were included. Blood samples were collected at the time of sepsis diagnosis and seven days later to evaluate the role of biological processes or genes possibly involved in patient recovery. Principal Components Analysis (PCA) profiling discriminated between patients with early sepsis and healthy individuals. Genes with differential expression were grouped according to Gene Ontology, and most genes related to immune defense were up-regulated in septic patients. Additionally, PCA in the early stage was able to distinguish survivors from non-survivors. Differences in oxidative phosphorylation seem to be associated with clinical outcome because significant differences in the expression profile of genes related to mitochondrial electron transport chain (ETC) I-V were observed between survivors and non-survivors at the time of patient enrollment. Global gene expression profiles after seven days of sepsis progression seem to reproduce, to a certain extent, patterns collected at the time of diagnosis. Gene expression profiles comparing admission and follow-up samples differed between survivors and non-survivors, with decreased expression of genes related to immune functions in non-survivors. In conclusion, genes related to host defense and inflammatory response ontology were up-regulated during sepsis, consistent with the need for a host response to infection, and the sustainability of their expression in follow-up samples was associated with outcomes.
VARIATION IN THE GUT MICROBIOME IS ASSOCIATED WITH DIFFERENTIAL IMMUNE RESPONSE FOLLOWING SEVERE BURN
R.M. Huebinger*1, D. Maass*1, L. Ma1, D.L. Carlson*1, J. Song*1, M. Rout2, Y. Zhang2, M.S. Allen2, S.E. Wolf*1, and R.C. Barber2. 1UT Southwestern Medical Center, Dallas, TX, 2University of North Texas Health Science Center, Fort Worth, TX
The intestinal tract is an important organ in the recovery from burn injury having roles in digestion, metabolism, and inflammation. Recent evidence suggests that resident bacteria in the gut play a crucial role in regulating key inflammatory factors. Additionally, we previously identified considerable microbiome variation in patients following severe burn. In this study, we systematically changed the gut microbiome in an animal model and examined subsequent cytokine expression following severe burn. Adult male Sprague-Dawley rats were divided into treatment groups (depleted, supplemented, untreated, sham). Rats in the depleted group were gavaged with antibiotics daily for one week prior to injury. The supplemented group was gavaged with 3x10^9 cfu of Lactobacillus reuteri (ATCC 23272) daily for one week. After pre-treatment, rats were anesthetized with isoflurane and given a 40% TBSA injury and resuscitated with lactated Ringer’s solution, then sacrificed at 24 hours. Cytokines (TNF-α, IL-6, and IL-10) were analyzed in serum and lung tissue lysates. The microbiome of each treatment group were sequenced for the 16S rRNA region using the ION Torrent PGM. Cytokine expression was significantly reduced in the bacteria supplemented+Burn relative to Burn only (Serum: TNF-α p<0.001; IL-6 p<0.001; IL-10 p=0.005; Lung: TNF-α p=0.007; IL-6 p<0.001; IL-10 p<0.001). Expression was also reduced in the bacteria supplemented+Burn relative to the bacteria depleted+Burn group (Serum: TNF-α p<0.001; IL-6 p<0.001; IL-10 p=0.015; Lung: TNF-α p=0.001; IL-6 p=0.001; IL-10 p=0.001). Histological analysis of the lung with TUNEL staining showed reduced apoptosis in the bacteria depleted group versus burn only group. Through alteration of the gut microbiome prior to injury, we changed cytokine expression. In addition, different levels of apoptosis in lung tissue were exhibited across groups relative to their microbiome. These data show that the microbiome in the gut plays a role in regulating inflammation after severe injury. Additionally, these data show that the gut microbiome may be an important clinical target for treatment.
THE INTEGRATION OF INJURY-SITE INFLAMMASOME ACTIVATION WITH SYSTEMIC CYTOKINE PRODUCTION SERVES AS A CLINICAL INDEX TO PREDICT SEPSIS IN THERMALLY INJURED PATIENTS
P. Chen1, M. Stanojcic1, and M.G. Jeschke*2, 3. 1Sunnybrook Research Institute, Toronto, ON, Canada, 2Division of Plastic Surgery Department of Surgery, University of Toronto, Toronto, ON, Canada, 3Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada
Introduction: Since the detection of clinical sepsis usually occurs at a stage where irreversible damage to patients has already occurred, the goal is to discover indices that can predict sepsis in advance before the infection worsens to the point of no return. Most of the published predictors are based solely on the hosts’ pro-inflammatory (PI) response against the invading microbes, and their efficacy are controversial. The aim of this study is to develop novel tissue and serum derived biomarkers to predict sepsis before sepsis or septic shock occurs.
Methods: Burned site-derived leukocytes isolated from patient adipose tissues were assayed for IL-1β production through flow cytometry to assess inflammasome activation. In addition, blood of burned patients was collected to measure various plasma cytokines (39 total) via a multiplex immunoassay kit. Finally, the inflammasome and cytokine profiles of burned patients that eventually develop sepsis are compared to the thermally injured, non-septic counterparts (either infected or non-infected). All patient specimens used for analysis were collected within 96-hours post burn injury.
Results: Greater inflammasome activation was observed in burned patients that develop sepsis than the non-septic group, indicated by a 2-fold higher production of IL-1β by the burn site-derived leukocytes. In contrast to the elevated inflammation detected at the injury site of septic patients, there is a significantly higher systemic AI profile in their blood relative to the non-septic group, characterized by a 5- and 7-fold increase of plasma IL-10 and IL-1RA, respectively (p<0.05 for both).
Conclusion: In the septic burn-patients, although a greater degree of inflammation is detected at the site of injury in contrast to the non-septic group, this response is counteracted by the aberrant elevation of IL-10 and IL-1RA levels in the blood. The clash of the immune functions between the wound site and the peripheral organs appears to foreshadow sepsis and can be used to predict burn patients with high risk of developing sepsis.
A POSSIBLE ROLE OF CD14 IN ESTRADIOL-PROVIDED PROTECION OF CARDIAC MITOCHONDRIA DURING BURN TRAUMA
X. Yao, L. Ma, D. Maass, J.G. Wigginton, S.E. Wolf*, J. Minei, and Q.S. Zang*. University of Texas Southwestern Medical Center, Dallas, TX
Objective: Our recent investigation suggests that 17beta-estradiol provides cardiac protection via suppression of mitochondria-derived danger-associated molecular patterns (DAMPs) in burn trauma. Previously, we obtained results showing CD14-dependent mitochondrial damage in burn serum-challenged adult cardiomyocytes. Thus, the aim of this study was to examine the response of CD14 to 17 beta-estradiol in the heart of a burn injury rat model.
Methods: SD rats were given a third-degree scald burn comprising 40% TBSA. 17 beta-estradiol (0.5 mg/kg) or control vehicle was administered subcutaneously 15 minutes post-injury. Heart tissues were harvested 24 hours later. Using the heart tissue lysates, expression of CD14 was determined by Western blot, and the association between CD14 and lipopolysaccharide binding protein (LBP) was determined by immunoprecipitation.
Results: Burn injury significantly up-related the expression of CD14 in the heart. The interaction between CD14 and LBP appeared constant. Treatment with 17 beta-estradiol suppresses burn-induced increase in CD14 expression.
Conclusion: Our data suggest that post-burn administration of estradiol protects the heart via remedy of mitochondrial damage through a CD-14-mediated pathway.
DIFFERENTIATION OF BURNED SKIN FROM NORMAL SKIN AS EARLY AS 1 HOUR AFTER INJURY USING OPTICAL ABSORPTION
H. de Pedro1, C. Chang1, F. Zarnani1, D. Maass2, J.G. Wigginton2, A. Idris2, and R. Glosser1. 1University of Texas at Dallas, Richardson, TX, 2University of Texas Southwestern Medical Center, Dallas, TX
The mortality and morbidity of burn victims can be reduced if the treatment includes both excision of the burn area and providing therapy that limits the secondary evolution and extension of burn injury. Early excision here means removal of burned tissue in less than 24 hours. Excision of wounds in less than 24 hours has been limited by the great difficulty in defining wound margins and depth that early after burn. Due to the dynamic properties of burn wounds after injury, rapid removal of burned tissue becomes critical for survivability. Thus, the process of removing burned tissue requires some means of discriminating between healthy and burned cells. This is conducted by excising the affected tissue until reaching tissue that bleeds, which signifies the presence of viable tissue. In this study, we used the optical absorption to differentiate between unburned and burned skin. Burns causes rapid protein denaturation and cell damage which will eventually cause a change in its optical properties. In this study, the Mason-Walker scald method was used to produce full thickness burns on Sprague-Dawley rats. The skin samples were harvested, posthumously, 1 hour, 12 hrs and 24 hours after injury. The experimental results show that the optical absorbance of normal skin differs from burned skin. This difference can be seen for all the burned skin samples that are harvested in the intervals from 1 hour to 24 hours after burn injury. Results shows that the absorbance ratio at 550 nm / 400 nm for normal skin is 0.84 ± 0.02 while the burned samples gives us 1.06 ± 0.03, 1.11 ± 0.01 and 1.09 ± 0.02.
DIFFERENTIAL WBC GENE EXPRESSION IS ASSOCIATED WITH PNEUMONIA DEVELOPMENT AFTER BURN INJURY
P. Nainar1, 2, A.S. Kudlicki1, B. Fongang1, X. Li1, B.D. Arnoldo3, R.L. Gamelli4, N.S. Gibran5, M.G. Jeschke*6, M. Klein5, H. Baker*7, C. Lopez7, L. Moldawer7, R.G. Tomkins*8, D. Herndon*1, 2, and C.C. Finnerty*1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriner’s Hospital, Galveston, TX, 3UT Southwestern, Dallas, TX, 4Loyola University, Chicago, IL, 5University of Washington, Seattle, WA, 6University of Toronto, Toronto, ON, Canada, 7University of Florida, Gainesville, FL, 8Massachusetts General Hospital for Children, Boston, MA
Background: Sepsis is the second leading cause of mortality following burn injury, with pulmonary sepsis being the most common cause of systemic sepsis. In survivors of burn injury, pneumonia in the acute period following burn injury is associated with more ventilator days, complications from mechanical ventilation, length of ICU and hospital stays, and prolonged morbidity. Here we established the differential gene expression in white blood cells in patients who developed pneumonia within 20 days of burn injury.
Hypothesis: Development of pneumonia in the acute period following burn injury is associated with differential leukocytic gene expression.
Methodology: Blood samples [n=249] were taken from patients admitted to participating burn units within 96hrs of injury; patients were divided into two cohorts - no pneumonia (or other infections) or & diagnosed pneumonia during first 20 days post burn. Leukocytic gene expression was measured with Affymetrix HGU133Plus2.0 arrays. Differential gene expression was determined by KS tests (p<0.05). Fold change of 1.5 was used as cut off. Functional analysis of the significantly different genes was done using Ingenuity pathway analysis.
Results: 1000 probe-sets showed significant difference between both groups. Of the 1000, 234 were up-regulated and the remaining 766 were down-regulated in patients who developed pneumonia. Functional analysis showed that genes associated with the following functions were up-regulated: phagocytosis, inflammatory response, chemotaxis / recruitment, apoptosis, migration and response to bacterial infections. At the same time significant decreases in genes associated with binding of blood cell subsets and cell death are seen in the patients with pneumonia.
Conclusions: Significant differential expression is seen in gene expression in white blood cells of patients developing pneumonia in the acute phase following burn injury. The identified genes may be used to develop a biomarker panel that will enable early identification of pneumonia following burn injury.
CD68 GENE EXPRESSION IS ELEVATED IN FAT AFTER BURN
J. Song*, M. Saeman, K. Despain, J. Minei*, and S.E. Wolf*. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Hypermetabolic response disrupts organ function and body homeostasis in burn patients. We previously showed that severe burn associated insulin resistance is associated with endoplasmic reticulum (ER) stress. Macrophage infiltration in fat contributes to obesity and/or diabetes induced insulin resistance. We thus hypothesized that macrophage infiltration in fat tissue plays a role in mediating insulin resistance after burn. The aim of the study is to determine the macrophage changes in fat following severe burn.
Methods: Six C57BL6 adult mice received 25% TBSA scald burn under general anaesthesia. Epididymal white adipocyte tissue and interscapular brown adipocyte tissue were collected 24 hours after burn. Total RNA were extracted for cDNA formation. Gene expression including adipocyte marker adiponectin, ER stress marker Hspa5, brown adipocyte tissue marker UCP-1, macrophage markers CD68, CD14 and F4/80, and housekeeping control gene GAPDH were examined by real time qPCR. Six animals without burn served as controls.
Results: qPCR results showed that adiponectin expression was 5 times higher, and macrophage markers CD14, CD68 and F4/80 were increased in white adipose compared to brown adipose before injury. Gene expression for UCP-1 was significantly higher in brown than white adipose (p<0.05). Following severe burn, expression of adiponectin significantly decreased, while Hspa5 increased in both white and brown adipose (p<0.05). Expression of CD68 was significantly elevated in brown adipose (p<0.05), but not in white adipose. No changes in CD14 and F4/80 were seen in either white or brown fat after burn.
Summary: Severe burn decreased adiponectin expression and increased Hspa5 in fat, indicating lipolysis and ER stress following severe burn. Expression of macrophage/monocyte marker CD68 increased after injury, suggesting macrophage infiltration in fat tissue after burn injury. However, this change was seen only in brown and not white fat; other mechanisms of regulating metabolism in white and brown fat in response to severe burn should be further investigated.
DIFFERENTIATION OF BURNED SKIN FROM NORMAL SKIN USING FLUORESCENCE LIFETIME SPECTROSCOPY
H. de Pedro1, C. Chang1, F. Zarnani1, D. Maass2, J.G. Wigginton2, A. Idris2, and R. Glosser1. 1University of Texas at Dallas, Richardson, TX, 2University of Texas Southwestern Medical Center, Dallas, TX
Treatment of burned victims requires the removal of affected tissues since complications arises due to the response of the body to the presence of burned tissue. The process of removing burned tissue requires discriminating between healthy and burned cells. Currently this procedure is done by surgically scraping the affected area until reaching tissue that bleed, which will signify the presence of viable tissues. This study aims to determine the differences between healthy and burned tissues spec
ABERRATIONS IN IMMUNE AND HORMONAL EXPRESSION IN BURN AND ER STRESSED MICE
M. Stanojcic1, 2, E. Bogdanovic1, 2, and M.G. Jeschke*3, 4. 1Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada, 2Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada, 3Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 4Department of Surgery, Division of Plastic Surgery and Department of Immunology, University of Toronto, Toronto, ON, Canada
Introduction: Thermal injury is an extreme trauma that causes a hyperinflammatory and hypermetabolic response with a predisposition to sepsis and organ dysfunction. The liver plays a central role in post-burn complications and has shown to result in endoplasmic reticulum (ER) stress, apoptosis and mitochondrial dysfunction. By assessing serological measures, it serves to shed light on underlying tissue processes and pathophysiology. The following study investigates the immune and hormone profile during ER stress-induced hepatic dysfunction. Furthermore, it will elucidate how the immunometabolic profile changes over time.
Methods: Balb/c mice (4-6 wks old) were intraperioneally injected with Tunicamycin (1μg/g body weight) and 24-hr later received a dorsum scald burn (30% TBSA, 98°C for 10s). Mice were divided into the following groups: sham, burn, tunicamycin and burn+tunicamycin (burn+T); and sacrificed during the acute (1-day) and late (4-6 days) phases after burn. Cardiac blood was collected and a multi-analyte Milliplex (Millipore, MA) platform was used to measure inflammatory and hormonal cytokines in plasma. Data was analyzed using Mann-Whitney and students t-test for differences between experimental groups and time points.
Results: As an indicator of inflammation during the acute phase, IL-6 was elevated in both the burn and burn+T groups, relative to controls. Interestingly, burn+T showed aberrations in IL-1α and C-peptide suggesting robust pro-inflammatory response and metabolic dysfunction. When comparing later periods, G-CSF and IL-6 were significantly elevated in burn and burn+T relative to controls. Furthermore, burn+T group had the following immune and metabolic factors elevated relative to controls: IL-10, IL15, IP-10, KC, RANTES and PYY. However, when comparing these factors to the burn group, the only differences were in IP-10 and GIP, suggesting ongoing chemokine signaling and metabolic discourse. Lastly, as expected, early vs. late phase comparison revealed a significant decrease in numerous immune and metabolic factors.
Conclusion: Hepatic ER stress results in a time dependent metabolic up-regulation and hyperinflammation. Collectively, these results support the two-hit model of burn and ER stress in the liver as a representative method of investigating hepatic dysfunction.
DEXMEDETOMIDINE SUPPRESSES INFLAMMATORY MEDIATORS LEVEL IN PATIENTS OF CARDIAC SURGERY USING CARDIOPULMONARY BYPASS
T. Kawasaki*1, C. Kawasaki2, and T. Sata1. 1University of Occupational and Environmental Health, Kitakyushu, Japan, 2Sugioka Memorial Hospital, Fukuoka, Japan
Objective: Cardiac surgery with cardiopulmonary bypass is associated with the development of a systemic inflammatory response that can often lead to dysfunction of major organs. We hypothesised that the highly selective α2 adrenergic agonist, dexmedetomidine attenuates the systemic inflammatory response during cardiopulmonary bypass.
Methods: This prospective randomized study included patients who underwent elective cardiac surgery with cardiopulmonary bypass. The dexmedetomidine group received dexmedetomidine administration after aortic cross-clamping (1 μg/kg for 10 minutes after aortic cross-clamping, and 0.5 μg/kg/.hr i.v. intraoperatively), while the control group received the same volume of saline. Blood samples were taken before the induction of anesthesia, 1h after aortic occlusion, 1 h after declamping the aorta, 4 h after declamping the aorta, post operative day 1, and postoperative day 3. Plasma HMGB1 levels and IL-6 levels were measured by using enzyme-linked immunosorbent assay.
Results: Plasma HMGB1 level increased significantly during (5.1±2.2 vs 16.6±7.3 ng/ml) and after (vs 14.3±8.2 ng/ml) cardiopulmonary bypass in control groups. In dexmedetomidine group, plasma HMGB1 level increased significantly during cardiopulmonary bypass (4.0±1.9 vs 10.8±2.7 ng/ml). Dexmedetomidine infusion suppressed increased plasma HMGB1 and IL-6 levels during and after cardiopulmonary bypass (p<0.05). These suppressive effects of dexmedetomidine might be due to the inhibition of NF-κB activation.
Conclusion: Our findings suggest that intraoperative dexmedetomidine inhibits inflammatory responses associated ischemia-reperfusion injury during cardiopulmonary bypass. To regulate the inflammatory response by dexmedetomidine infusion may be a beneficial treatment for prevention of postoperative complications in patients of cardiac surgery with cardiopulmonary bypass.
BIODISTRIBUTION AND FUNCTIONAL EFFECTS OF A PKC-DELTA INHIBITORY PEPTIDE IN SEPSIS-INDUCED LUNG INJURY
M. Mondrinos1, 3, P.A. Kennedy1, 2, J. Wu2, M.B. Kauffman1, S.T. Baker2, L.C. Knight4, M.R. Wolfson1, 2, and L.E. Kilpatrick*1, 3. 1Center for Inflammation, Translational and Clinical Lung Research, Temple University School of Medicine, Philadelphia, PA, 2Department of Physiology, Temple University School of Medicine, Philadelphia, PA, 3Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, 4Department of Radiology, Temple University School of Medicine, Philadelphia, PA
Introduction: There are no specific pharmacologic therapies available for ARDS management. We are testing a PKC-δ inhibitory peptide (PKCδ-TAT) as a potential therapeutic. Previously we reported lung protection upon intratracheal (IT) delivery of PKCδ-TAT in sepsis, however biodistribution facilitated by membrane-permeant TAT-conjugation and functional mechanisms of this peptide are not known. Here we present the effects of IT delivery on organ distribution, cellular uptake in the lung, therapeutic efficacy and functional modulations of endothelial cells (EC) and alveolar macrophages (AMΦ) in sepsis-induced lung injury.
Methods: Cecal ligation and puncture (CLP) was used to model sepsis-induced indirect lung injury in rats. Organ biodistribution was tracked by radiolabeling PKCδ-TAT with technetium (Tc99m), while spatial lung distribution and cellular uptake was assessed with a rhodamine tag (TMR), allowing co-visualization with cell lineage markers. Peptide biodistribution was measured across a range of 50-500 μg/kg, then at 4 and 24 hrs post-CLP. Therapeutic efficacy was assessed at 24 hours post-CLP by histopathology/IHC, MPO activity, PKCδ phosphorylation, and AMΦ phenotype and phagocytic capacity.
Results: Lung uptake of Tc99m-PKCδ-TAT upon systemic (tail vein) delivery was negligible (<1% initial dose/g (ID/g)), while lung retention of >50% ID/g was seen at 30 minutes post-IT delivery, with minimal uptake in liver or kidney (<1% ID/gram). TMR-PKCδ-TAT studies demonstrated spatially uniform lung distribution with localization in type I cells, MΦ and EC. Distribution trends were similar at 50 and 500 μg/kg Tc99m-PKCδ-TAT, but 200 μg/kg PKCδ-TAT was optimal in therapeutic studies, with a significant reduction in CLP-induced lung MPO activity (>2-fold) and decreased ICAM-1 and phospho-PKCδ (Tyr311) staining. Spatial lung distribution and reduction of MPO activity were not altered when PKCδ-TAT was delivered 4 hrs post-CLP; however lung distribution was impaired at 24 hrs post-CLP. PKCδ-TAT restored CLP-induced phagocytic dysfunction in AMΦ to sham levels and reduced elevated CD206-positive AMΦ numbers by 2-fold.
Conclusion: IT delivery of PKCδ-TAT facilitates preferential distribution to the lung and selective modulation of target cell types, thereby attenuating sepsis-induced acute lung inflammation and dysfunction in EC, neutrophils and AMΦ.
HEAT SHOCK PROTEIN A12B DEFICIENCY POTENTIATES CARDIAC DYSFUNCTION IN POLYMICROBIAL SEPSIS
X. Zhang, X. Wang, T. Ha, C. Lu, J. Kalbfleisch, R. Kao, D.L. Williams*, and C. Li*. East Tennessee State University, Johnson City, TN
Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 family. HSPA12B is predominately expressed in endothelial cells and it plays an important role in the induction of angiogenesis. This study examined the role of HSPA12B in cardiac function during polymicrobial sepsis. Endothelial HSPA12B knockout (n=6) and wild type (WT, n=6) mice were subjected to cecal ligation and puncture (CLP)-induced sepsis. Sham surgical operation served as sham control (n=6). Cardiac function was examined by echocardiography before and 6 hour after CLP. In WT mice, CLP sepsis resulted in cardiac dysfunction. Specifically, ejection fraction (EF%) and fractional shortening (FS%) were reduced by 34.8% and 43.1% (p<0.05), compared with control. In HSPA12B deficient mice with sepsis, EF% and FS% were improved by 19.9% and 22.5%, compared to control septic mice. Immunohistochemistry and Western blot showed that sepsis increased expression of adhesion molecules in the heart. The levels of ICAM1 and VCAM1 in HSPA12B deficient septic mice were higher than in septic controls. We also observed greater macrophage infiltration in the heart of HSPA12B deficient mice with sepsis. In addition, sepsis significantly increased the levels of the pro-inflammatory cytokines TNF-α and IL-6 in the serum of HSPA12B deficient mice. Our data indicate that HSPA12B deficiency potentiates septic cardiomyopathy. The mechanisms involve increased expression of adhesion molecules, increased pro-inflammatory cytokine production and increased infiltration of macrophages into the myocardium during sepsis. We conclude that endothelial HSPA12B plays a beneficial role in sepsis by maintaining cardiac function and attenuating pro-inflammatory responses.
CHARACTERIZATION OF LEFT VENTRICULAR FUNCTION DURING SEPTIC SHOCK IN ATHEROSCLEROTIC SWINE
J. Matallo1, P. Radermacher1, O. McCook1, E. Calzia1, M. Wepler2, and S. Hafner1. 1University of Ulm, Ulm, Germany, 2Massachusetts General Hospital, Boston, MA
Introduction: Septic cardiomyopathy is characterized by decreased left ventricular (LV) ejection fraction and compensatory dilation to maintain stroke volume . This mechanism is referred to be dependent on iNOS activation-induced excess NO production, resulting in increased end-diastolic volume (EDV) due to improved diastolic relaxation [2,3]. However, these data originate from studies in patients and/or animal models without pre-existing cardiac co-morbidity and/or vasopressor support. Therefore we tested the hypothesis that pre-existing coronary artery disease and/or ongoing norepinephrine (NE) infusion may blunt the typical sepsis-induced modification of LV function.
Materials and Methods: In anesthetized and mechanically ventilated swine with hypercholesteremia, ubiquitous atherosclerosis and coronary heart disease [4,5], fecal peritonitis was induced by inoculation of autologeous feces into the abdominal cavity (n=8) or sham procedure (n=5). Resuscitation comprised colloid fluid resuscitation and continuous i.v. norepinephrine (NE) titrated to maintain mean blood pressure at pre-shock values. Before, and at 12 and 24 h of sepsis, LV function was analyzed using pressure-conductance catheters. Data are median (quartiles).
Results: In sham-operated animals all cardiac function parameters remained unchanged during the 24-h observation period. Despite fluid resuscitation and i.v. NE sepsis caused progressive hypotension, but together with the unchanged stroke volume NE-induced tachycardia resulted in a sustained increase of cardiac output. Neither LV end-diastolic volume nor ejection fraction significantly changed, while LV end-diastolic pressure progressively increased and the diastolic relaxation constant (Tau) decreased.
Conclusion: During resuscitated septic shock, reduced LV ejection fraction and compensatory ventricular dilation were not present in swine with pre-existing coronary artery disease. This effect was most likely due to the ongoing NE infusion as well as the atherosclerosis-related oxidative stress and reduced endogenous NO production, ultimately resulting in compromised LV diastolic relaxation.
1. Parker et al, Ann Intern med 1984;100:483-90
2. Zanotti et al, Chest 2010;138:848-55
3. Barth et al, Crit Care Med 2006;34:307-13
4. Thim, Dan Med Bull 2010;57:B4161
5. Wepler Intensive Care Med Exp 2013;1:9
CIGARETTE-SMOKE INDUCED CHRONIC PULMONARY INFLAMMATION AGGRAVATES LUNG INJURY AND PULMONARY DYSFUNCTION AFTER BLUNT CHEST TRAUMA
F. Wagner1, M. Gröger1, O. McCook1, J. Matallo1, A. Scheuerle1, B. Jung2, E. Calzia1, M. Georgieff1, P. Radermacher1, and K. Wagner1. 1Ulm University Hospital, Ulm, Germany, 2Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
Objectives: Blunt chest trauma is a frequent observation in multiple injured patients, independently contributing to mortality (1). Chronic cigarette smoke (CS) exposure was shown to be associated with an increased risk to develop ARDS after blunt chest trauma (2), but the mechanism of this effect remains unclear. Therefore we investigated the effect of pre-existing CS-induced chronic pulmonary inflammation on lung injury, systemic inflammation and pulmonary after experimental blunt chest trauma.
Methods: Blunt chest trauma was induced by blast wave (3) in anesthetized and chronically CS-exposed (4) C57BL/6 mice (n=8) as well as healthy controls (n=8). Immediately after trauma, mice were instrumented and mechanically ventilated. Lung mechanics (quasistatic pressure-volume relationships) and oxygenation were assessed at the beginning and after 4 hours of pressure-controlled, PaCO2 titrated, lung-protective mechanical ventilation. Blood and lung tissue were harvested for cytokine concentrations (multiplex ELISA), cleaved caspase-3 (immunoblotting), nuclear factor-kB (NF-kB) activation (electrophoretic mobility shift assay), nitrotyrosine (immunohistochemistry) and lung histology (hematoxylin-eosin staining).
Results: CS-exposed mice presented with an increased activation of caspase-3, NF-kB (p<0.05) and nitrotyrosine formation (not significant) as well as enhanced IL-18 levels in lung tissue. This coincided with increased alveolar membrane thickening and pulmonary infiltration of immune cells. In CS-exposed mice post-traumatic pulmonary compliance was increased, most likely due to emphysematous, and oxygenation was impaired.
Conclusion: Chronic CS exposure results in increased susceptibility for lung tissue apoptosis and inflammation after blunt chest trauma, thereby leading to alveolar membrane thickening and consecutively impaired oxygenation.
Supported by the KFO 200 (DFG Ra 396/9-2)
1. Shah CV: Crit Care Med. 2008;36:2309-15
2. Calfee CS: Am J Respir Crit Care Med 2011;183:1660-5
3. Wagner F: J Trauma. 2011;71:1659-67
4. Wollin L: Pulm Pharmacol Ther. 2010;23:345-54
INFLAMMATION BIOMARKER PATTERNS DIFFERENTIATE SUSCEPTIBILITY TO NOSOCOMIAL INFECTION IN BLUNT TRAUMA PATIENTS
R.A. Namas*1, 2, Y. Vodovotz*1, 2, K.W. Almahmoud*1, O.M. Abdul-Malak*1, A.M. Zaaqoq*1, 3, R. Namas*1, 4, A.B. Peitzman1, J. Sperry*1, and T. Billiar*1, 2. 1University of Pittsburgh, Pittsburgh, PA, 2McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 3Texas Tech Health Sciences Center, Amarillo, TX, 4Wayne State University, Detroit, MI
Background: Traumatic injury results in an altered inflammatory response that can lead to nosocomial infection (NI) through mechanisms not yet fully elucidated. We carried out an extensive time course analysis of circulating inflammation biomarkers in a large cohort of blunt trauma patients to gain insights into the trauma-induced inflammatory response associated with NI.
Methods: In a cohort of 472 blunt trauma survivors, 127 patients (27%) were diagnosed with NI with an average day of diagnosis between 7 to 11 days post-injury. To perform a pairwise, case-control study with 1:1 matching, 44 NI trauma patients (27 males and 17 females; age: 48 ± 3; ISS: 26.3 ± 1.7) were identified and compared to 44 no-NI (27 males and 17 females; age: 47.3 ± 2; ISS: 26.1 ± 0.9) selected by matching the demographics and predominant injury characteristics. Plasma was sampled 3 times within the first 24 h and then from days 1 to 7 post-injury, and assayed for 25 inflammation biomarkers. Total and differential leucocyte counts were collected from inpatient electronic database. Data were analyzed by 2-Way ANOVA and Dynamic Bayesian Network (DBN) inference.
Results: Significant differences in ICU length of stay (LOS), hospital LOS, and ventilator days were observed in the NI vs. no-NI patients. Plasma IL-6, MCP-1, MIP-1α, IL-1RA, sIL-2Rα, IL-4, IL-5, IL-7, IL-13, IL-15, IL-17, GM-CSF, IFN-α, and IFN-γ were significantly elevated within the first 24 h in patients that developed NI vs. no-NI. Importantly, these biomarkers exhibited four unique dynamic patterns in NI, two of which being distinct within 24 h post-injury when compared to no-NI. NI group had higher total leucocyte and neutrophil counts, while no-NI group had higher monocyte and lymphocyte counts over days that corresponded with the mean day of diagnosis of infection. HMGB1 was significantly elevated by 4 h in patients that went on to develop infection. DBN suggested that MIG and MCP-1 induce each other through forward-feedback loops in both groups, and that MIG and MCP-1 affect IP-10 and IL-6 in NI group, while MIG alone affect IFN-α, IP-10, IL-6, and MCP-1 in no-NI group.
Conclusion: These studies suggest the presence of unique inflammation patterns and networks upon admission and over the initial 24 h post-injury that are associated with post-trauma NI, and imply an early, central role for chemokines.
THE EFFECT OF MECHANICAL VENTILATION WITH 100 % OXYGEN ON LUNG INJURY AND FUNCTION AFTER MURINE BLUNT CHEST TRAUMA
K. Wagner1, M. Gröger1, O. McCook1, A. Scheuerle1, B. Jung2, E. Calzia1, M. Georgieff1, P. Radermacher1, and F. Wagner1. 1Ulm University Hospital, Ulm, Germany, 2Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
Objectives: It is well-established, that long-term hyperoxia is associated with oxidative stress and enhanced cell death resulting in acute lung injury (1). However, in various animal models resulting from hemorrhage and sepsis short-term ventilation with 100 % oxygen attenuated inflammation and apoptosis, resulting in improved organ function and survival (2). The effect of pure oxygen after blunt chest trauma is still unknown. Therefore we tested the hypothesis whether short-term hyperoxia affects the pulmonary inflammatory response and thereby lung after murine blunt chest trauma.
Methods: In anesthetized and spontaneously breathing C57BL/6 mice, blunt chest trauma was initiated by a single blast wave, followed by instrumentation and induction of lung-protective mechanical ventilation with an FiO2 of 21 or 100 % (n=8 per group) (3). After 4 hours of ventilation and repeated measurements of lung mechanics and PaO2/FiO2 ratio, blood and lung tissue were collected for subsequent analyses of lung histology (hematoxylin-eosin staining), cytokines (multiplex ELISA), hemoxygenase-1 (HO-1), cleaved caspase-3 (immunoblot), nuclear factor-kB (NF-kB) activation (electrophoretic mobility shift assay), nitrotyrosine formation and P2X7 receptor (P2RX7) expression (immunohistochemistry).
Results: Pulmonary compliance did not differ between the two groups. In mice ventilated with pure oxygen, PaO2/FiO2 ratio increased at the end of the experiment, but was significantly reduced compared to the control group. Lung histology, cytokines as well as the activation of caspase-3 and NF-kB were comparable. But 100 % O2 attenuated the expression of P2RX7 and the formation of nitrotyrosine. This effect coincided with a significant increase of HO-1 in lung tissue.
Conclusion: Short-term ventilation with pure oxygen was not associated with a deletorious effect regarding apoptosis, local and systemic inflammation as well as lung mechanics after murine blunt chest trauma. The fact, that 100 % oxygen resulted in a reduction of radical stress, most likely resulting from a protective upregulation of HO-1, suggests to reconsider current recommendations and to leave the PaO2 up in the early phase of trauma.
Supported by the KFO 200 (DFG Ra 396/9-2)
1. Kallet RH: Respir Care. 2013;58:123-41
2. Hauser B: Crit Care Med 2009;37:2465-9
3. Wagner F: J Trauma. 2011;71:1659-67
EFFECT OF HYPEROXIA ON TISSUE CBS AND CSE EXPRESSION AFTER BLUNT CHEST TRAUMA
E. Knöller1, A. Scheuerle2, K. Wagner1, F. Wagner1, M. Gröger1, P. Radermacher1, and O. McCook1. 1University of Ulm Anesthesiology, Ulm, Germany, 2University of Ulm Pathology, Ulm, Germany
Background: Chest trauma causes alveolar hypoxia, consecutive hypoxemia and thereby tissue hypoxia [1,2]. Increased pulmonary expression of the two H2S producing enzymes CBS and CSE was shown to represent an adaptive stress response to chest trauma . Though not directly affected by the thorax trauma, the kidneys are highly sensitive to hypoxemia . In the kidneys, constitutive CBS and CSE expression is crucial for the maintenance of organ function . Since H2S is also referred to as an O2 sensing molecule , we investigated the effects of pure O2 ventilation on the pulmonary and renal tissue CBS and CSE expression after chest trauma.
Methods: After exposure to pressure wave-induced blunt chest trauma anesthetized C57Bl6J mice were randomly assigned to 4 hrs of lung-protective term mechanical ventilation with air (n=6) or 100 % O2. The left lung and kidneys were removed, formalin-fixed and embedded in paraffin. 5μm sections were deparaffinised, exposed HIER, 10% goat serum, 1° antibodies (CSE, CBS), 2°ab anti-rabbit IgG AP followed by Dako REAL chromogen and counterstained with hematoxlin. Slides were analyzed using the Axio Vision (rel. 4.8) software (Zeiss, Jena Germany) and are represented as mean densitometric sum red.
Results: Pure O2 ventilation significantly reduced both the lung CBS (p=0.029) and CSE expression (p=0.001). In contrast, hyperoxia was associated with a significantly higher kidney CBS (p=0.029) and CSE expression (p=0.005) expression.
Conclusion: We confirmed that pulmonary CBS and CSE expression is up-regulated after chest trauma, which was attenuated by therapeutic hyperoxia, thereby further supporting their role as an adaptive stress response. Surprisingly, when compared to animals that did not undergo surgery and trauma, chest trauma resulted in a reduction of the constitutive enzyme expression in the kidneys, which was restored by therapeutic hyperoxia. It remains to be elucidated whether this benefit of hyperoxia is due to a relief of whole body hypoxia and/or an attenuated stress response in the lung translating into the kidney.
Supported by the DFG KFO 200 (Ra 396/9-2) and the Perspektivförderung Innovationsfond Medizin Land Bw
1. Knöferl et al, Shock 2003;19:519-25
2. Wagner et al, J Trauma 2011;71:1659-67
3. Bos et al, J Am Soc Nephrol 2013;24:759-70
4. Olson et al, Respir Physiol Neurobiol 2011;179:103-10
IDENTIFYING A POTENTIAL MECHANISM OF VENTILATOR INDUCED LUNG INJURY USING A NOVEL ASSESSMENT OF THE LUNG MICRO-ANATOMY
M.C. Kollisch-Singule1, B. Emr1, B. Smith2, C. Ruiz3, S. Roy1, J. Satalin1, K. Snyder1, P. Andrews4, N. Habashi4, R.N. Cooney*1, G. Nieman*1, and L.A. Gatto3. 1SUNY Upstate Medical University, Syracuse, NY, 2University of Vermont, Burlington, VT, 3SUNY Cortland, Cortland, NY, 4R Adams Cowley Shock Trauma Center, Baltimore, MD
Objective: Improper mechanical ventilation settings can exacerbate acute lung damage by causing a secondary ventilator induced lung injury (VILI). Current protective ventilator strategies focus on Macro-ventilatory parameters (Macro-V). We hypothesize that the key to identifying the mechanism of VILI is to better understand the impact of the mechanical breath at the Micro-Anatomical level (Micro-V). We identified the impact of a change in a single Macro-V setting (TLow) using Airway Pressure Release Ventilation (APRV) on Micro-V.
Methods: Rats were randomized into 1 of 3 groups: 1) Control (n=2); 2) APRV75% (n=2)-TLow set to terminate at 75% of the Peak Expiratory Flow Rate (PEFR) and 3) APRV10% (n=2)-TLow set inappropriately to terminate at 10% of the PEFR. Lung injury was induced in APRV groups by Tween lavage then ventilated with their Macro-V settings. The right lung was fixed at peak inspiration for standard histology. Conducting airway (CA) and alveolar (Alv) air space areas were demarcated and air space areas quantified using digital image analysis.
Results: Control rats have the greatest Alv air space area (56.9%±0.8%; p<0.05). APRV75% (48.9%±2.5%) has greater Alv air space occupancy than APRV10% (27.3%±2.6%; p<0.05). Control rats have the least CA air space occupancy (19.5%±0.8%; p<0.05). APRV75% has less CA air space occupancy (31.0%±2.34%) as compared with APRV10% (49.3%±2.95%; p<0.05).
Conclusions: Previous studies have identified that APRV with TLow set to terminate at 75% of the PEFR protects the lung, whereas APRV10% has been shown to be less protective. APRV75% results in a more similar distribution of gas to Control than APRV10%. We postulate that optimizing gas distribution at the Micro-V level is a mechanism of lung protection seen with the application of appropriately set APRV (75%) in animals with lung injury.
THROMBIN INHIBITION AFTER POLYTRAUMA AND HEMORRHAGE IN RATS
X. Wu, D.N. Darlington, and A.P. Cap. US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: We have developed an Acute Coagulopathy of Trauma (aCOT) in rat that mimics the clinical hypocoagulation in both civilian and military patients with severe trauma and hemorrhage. This model of polytrauma and hemorrhage allows for the study of thrombin inhibitors that may, in turn, contribute to the development of coagulopathy.
Objective: To determine whether changes in thrombin inhibitors correlates with prolongation of prothrombin time, an indicator of aCOT, after polytrauma and hemorrhage.
Method: Sprague-Dawley rats (n=8) were anesthetized with Isoflurane. Polytrauma was induced by damage to the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and by fracturing the right femur, than bled to a mean arterial pressure of 40mmHg and held there until 40% of the blood volume was removed. No fluid resuscitation was given. Blood samples were taken before (time 0) and at 30, 60, 120 and 240min. Rat anti-thrombin III, thrombomodulin, thrombin anti-thrombin, α2- macroglobulin and tissue factor pathway inhibitor were measured by immunoassay.
Results: Prothrombin time significantly increased over the 4hr period thereby validating the model of coagulopathy. Plasma levels of thrombin inhibitors showed complex changes over time. Rat anti-thrombin III fell at 30, 60 and 120min, but rose by 4hrs to levels not different than control. α2- macroglobulin also fell over time (significant at 2hr) to return to levels not different from control. Plasma thrombomodulin was elevated throughout the 4hrs. Tissue factor pathway inhibitor levels did not change significantly over time.
Conclusion: Polytrauma and hemorrhage led to a complex change in inhibitors to thrombin. There was an early (30-120min) decrease in both anti-thrombin III and α2-macroglobulin suggesting an early consumption by thrombin. However, both anti-thrombin III and α2-macroglobulin returned to normal by 4hrs, suggesting that thrombin consumption was reduced by 4hrs. Since the changes in thrombin inhibitors did not correlate with PT, thrombin inhibitors are not likely to contribute to the development of aCOT in this model. This project was funded by MRMC.
NK CELLS DEPLETION INCREASES END ORGAN DAMAGE IN A MODEL HEMORRHAGIC SHOCK WITH TRAUMA
S. Chen1, 2, R. Hoffman1, L. Kohut1, P. Loughran1, and T. Billiar*1. 1Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, 2Department of Biochemistry, School of Biosciences and Technologies, Central South University, Changsha, China
NK cells play critical roles in both innate immune defense and the development of systemic inflammatory response. To study the roles of NK cells in the immunological response in a model hemorrhagic shock with tissue trauma(HS+T), NK cells were depleted by i.p. administration of 75ug NK1.1-PK136 on two consecutive days, then followed by resuscitated hemorrhagic shock 2h with pseudofracture (HS+T); injections of Isotype IgG or saline served as negative controls of NK cell depletion. The plasma levels of IL-6, MCP-1 and ALT were measured at 6hrs and the numbers of different lymphocyte populations in blood were determined by flow cytometry. Apoptotic cells in lung tissue were detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. Plasma ALT, IL-6 and MCP-1 in the mice injected with either isotype IgG (n=7), saline (n=4) or NK1.1-PK136 (n=10) were elevated followed by HS+T, however plasma ALT levels were significantly higher in the NK cell depleted group than that of the controls. The numbers of blood neutrophils, B cells, CD3T cells and Ly6G-CD11b+ cells were similar at the baseline in the three groups. The number of blood NK cells in the HS+T control groups were reduced notably compared to those at baseline but was significantly higher than that in the HS+T group with NK depletion. The numbers of circulating B cells and CD3T cells also dropped significantly while the neutrophils and Ly6G-CD11b+ cells increased markedly in the three HS+T groups compared to those at baseline. Nevertheless, there was no significant difference in these lymphocyte phenotyping between the three HS+T groups. A significant increase in apoptosis of alveolar and bronchial epithelial cells was induced in the HS+T group with NK1.1 Ab (9±2.1/low power filed, n=2) but not the control treatment(0.5±0.58/low power filed, n=2). These findings collectively suggest that NK cells play essential roles in protecting against end organ damage in the early phase of hemorrhagic shock with trauma.
CARDIOVASCULAR RESPONSIVENESS TO ALPHA1-ADRENERGIC- AND VASOPRESSIN RECEPTOR STIMULATION DURING HEMORRHAGIC SHOCK AND RESUSCITATION
Y.M. Wong1, H. LaPorte1, R.L. Gamelli*1, K.L. Byron2, and M. Majetschak*1, 2. 1Loyola University Chicago, Health Sciences Division, Burn and Shock Trauma Research Institute, Maywood, IL, 2Loyola University Chicago, Department of Molecular Pharmacology and Therapeutics, Maywood, IL
Introduction: Pressure support resuscitation during hemorrhagic shock with arginine vasopressin (aVP) has been proposed previously. Changes of the cardiovascular responsiveness to vasoactive drugs after hemorrhage (hem) and resuscitation (resus), however, are not well defined. Thus, we assessed hem/resus associated changes in cardiovascular reactivity to aVP and phenylephrine (PE), a selective α1-adrenergic receptor (AR) agonist.
Methods: Rats (355±8g) underwent hem to a MAP of 25mmHg for 15min, 60min and 60min plus 30min crystalloid resus to a MAP of 70mmHg (control=no hem; n=4-5/group). Series 1: At the end of the hem or hem/resus period, 10 increasing doses of PE (5-1000 μg) or aVP (0.1-50 ng) were injected iv in 5min intervals. MAP was monitored continuously, the area under the MAP curve for each dose calculated and dose-response curves generated. Series 2: Mesenteric arteries were harvested from normal animals and after 60min hem/resus. Vascular reactivity of arteries to PE and aVP was assessed by pressure myography. Non-linear regression analyses were calculated (GraphPad Prism). Best fit values were compared with Extra sum-of-squares F test. Data are given as mean (95% confidence interval). *: p<0.05 vs. control.
Results: Series 1: The EC50 for PE was 99(91-106) μg for control, 60(54-67)* μg after 15min hem, 95(84-107) μg after 60min hem and 172(148-200)* μg after 60min hem/resus. The Hill slopes of the PE curves were 1.6(1.4-1.8) for control and 0.96-1.01(0.86-1.1)* for all other groups. For aVP, the EC50 was 542(370-795) ng for control, 540(402-725) ng after 15min hem, 949(802-1123)* ng after 60min hem and 318(269-376)* ng after 60min hem/resus. The Hill slopes of the aVP curves were 0.67(0.49-0.84) at baseline, 1.05(0.74-1.35) after 15min hem, 1.27(1.04-1.5)* after 60min hem and 1.07(0.87-1.27)* after 60min hem/resus. Series 2: Vascular reactivity to PE and aVP was not affected by hem/resus.
Conclusions: After hem and resus, cardiovascular sensitivity to aVP is enhanced and sensitivity to PE reduced, suggesting differential regulation of α1-AR and vasopressin receptor responsiveness. This regulation depends on the integrated cardiovascular response and cannot be attributed to changes in intrinsic vascular reactivity. The results of this study may partially explain beneficial effects of vasopressin on hemodynamics during hem/resus.
DOES RESUSCITATION WITH PFC IMPROVE OUTCOME IN HEMORRHAGIC SHOCK? EVALUATING COMPETING STATISTICAL MODELS OF BENEFIT
P. Reynolds* and B.D. Spiess. Virginia Commonwealth University Medical Center, Richmond, VA
Background: Hextend (HEX) is a colloid used for primary fluid resuscitation following severe hemorrhage. Although an excellent volume expander it has limited O2 carrying capacity. Perfluorocarbon (PFC) emulsions are intravascular oxygen therapeutics that temporarily enhance tissue oxygenation, but cannot be used as primary volume expanders. We tested the hypothesis that HEX supplemented with PFC would promote greater hemodynamic support and lactate resolution compared to unaugmented HEX. Many investigators determine benefit by performing numerous multiple pairwise contrasts, between treatments at each time, and between times within each treatment. This greatly inflates the risk of false positives. However, inter-individual variation over time may obscure real differences between treatments.
Methods: Eighteen male NZW rabbits were hemorrhaged to MAP 35-40 mmHg and maintained for 1hr before random allocation to resuscitation with either HEX or HEX+PFC (15 mL/kg). MAP was monitored at 500Hz; and data summarized every 2 min. Lactate was evaluated every 15 min. Data were analysed by (1) repeated measures ANOVA (RM-ANOVA) on 7 fixed time points; and (2) longitudinal mixed model piecewise random coefficients models (LPRC), which describes change over real time.
Results: RM-ANOVA could not detect a PFC effect in either MAP or lactate (p >0.5). However, LPRC showed that both the timing and trajectory of MAP and lactate post-resuscitation differed significantly; PFC significantly depressed MAP recovery (p =0.002) and resulted in reduced rates of lactate clearance.
Conclusions: Addition of IV PFC to Hextend for resuscitation from hemorrhagic shock conferred no benefit, and may have been deleterious, as assessed by random coefficients models. However conventional ANOVA could not detect differences between groups. Random coefficients models are recommended because inter-individual variability and real time effects can be accounted for in the model. Interpretation based on multiple contrasts on fixed time points is of limited value, ignores much of the information in a time series, and may be highly misleading.
CARDIOPROTECTION BY ESTROGEN RELATED RECEPTOR (ERR)-β AND -γ AGONIST DY131 AFTER TRAUMA-HEMORRHAGE (T-H) IS VIA DY131-DERIVED RESTORATION OF CARDIAC SIRTUINS (SIRT)
S. Yang, S. Hu, B. Cam-Etoz, W.J. Hubbard, K.I. Bland, and I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
In mammals, 7 orthologs of SIRT have been identified, SIRT1-7; however the exact biological function of most of these SIRT remains partially known. Our prior results have shown that T-H induced cardiac depression, which is associated with decreased cardiac SIRT. Additionally, ERR-β and -γ agonist DY131 restored cardiac function after T-H; however, it is unclear if DY131 has any effect on cardiac SIRT after T-H. To study this, left ventricular (LV) performance and cardiac SIRT1-3 were studied in male adult Sprague-Dawley rat T-H model, treated with DY131 or other agents. Seven groups were used (n=6/group): sham, sham+DY131, T-H+vehicle, T-H+17β-estradiol (E2; 1 mg/kg), T-H+ICI 182,780 (ICI; 3mg/kg)+E2, T-H+DY131 (40μg/kg), and T-H+ICI+DY131. DY131 or E2 was given IV at the onset of resuscitation; estrogen receptor (ER) antagonist ICI was given IP 30 min before DY131 or E2 treatment. Two hr thereafter, LV performance was measured; blood and heart tissue were harvested. Cardiac SIRT1-3 were determined by Western blot. Plasma and heart tissue cytokines (i.e., TNF-α, IL-6, IL-10) were measured by ELISA. Our results showed that like E2, DY131 restored LV performance and all measured cardiac SIRT after T-H. Moreover, DY131 attenuated T-H-induced increase in plasma/cardiac TNF-α/IL-6; however, it produced further increase in IL-10. However, ICI did not alter DY131 effect on LV performance and cardiac SIRT/cytokines. Thus, it can be concluded that T-H induced cardiac depression and decreased cardiac SIRT; however, DY131 restored LV performance, which appears to be ER-independent and via DY131-derived restoration of cardiac SIRT after T-H (NIH R01GM39519).
AMPK ACTIVATORS REGULATE CONTRACTILE FUNCTION OF MESENTERIC ARTERIES
V.A. Locke1, 2, A.S. Miner2, 1, R.W. Barbee*1, 3, and P.H. Ratz*2, 3. 1Depts of Emergency Medicine & Physiology, Virginia Commonwealth University, Richmond, VA, 2Depts of Biochemistry & Pediatrics, Virginia Commonwealth University, Richmond, VA, 3VCURES, Richmond, VA
Vasoconstrictor tone in the splanchnic circulation redistributes blood flow during hemorrhage and resuscitation. A metabolic sensor, AMP kinase (AMPK), has been proposed to relax arteries by inhibiting myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK) activities. Because AMPK activation might be beneficial in re-establishing splanchnic blood flow during resuscitation, we sought to explore the role of AMPK activators (AICAR, A769662, berberine (BBR) and simvastatin (SIMV)) on regulation of mesenteric artery (MA) contraction. MA from male NZW rabbits were used to measure contractile tension at a fixed muscle length (Lo) or vessel diameter using, respectively, wire or pressure myographs. VSM activation state is defined by the degree of MLC phosphorylation (MLCp), and ROCK activation by the degree of ROCK regulatory protein phosphorylation (MYPT1-pT853 and MYPT1-pT696). Homogenates of fast-frozen tissues were analyzed by Western Blot for these phospho-protein indices of MA activation. AICAR caused a significant rightward shift and depression of a KCl-induced concentration-response curve (CRC) but was without effect on a PE-induced CRC. A769662 appeared to have no effect on contraction. BBR significantly reduced the sensitivity of PE-induced CRC’s but did not depress the maximum tension in both artery systems. SIMV caused a strong inhibition of PE- and KCl-induced maximum tensions. BBR (30μM) and SIMV (30μM) did not relax permeabilized (β-escin) MA contracted with 560nM Ca2+, but the ROCK inhibitor H-1152 produced strong relaxation, suggesting that the relaxation of intact tissues induced by BBR and SIMV was not due to AMPK-induced inhibition of MLC kinase. PE (10μM) caused a rapid increase in peak tension followed by a rapid decline in < 1 min. From 2 to 60 min, tension remained stable at ~1.1-fold the KCl-induced maximum value (T0). MLCp and MYPT-pT853 increased to, respectively, 4- and 2-fold the basal values at 2 min and fell to stable values of 3 and 1.5-fold basal from 30-60 min. PE did not increase MYPT-pT696. A 30 min time point for PE-induced activation was therefore chosen to examine the effect of 30μM BBR on indices of MA contraction regulation. BBR inhibited tension by ~50% and appeared to reduce both basal and PE-stimulated MCLp. These data support the hypothesis that AMPK activators play a role in activation and regulation of MA. Supported by DOD Grant W81XWH-12-1-0525.
ORGAN SPECIFIC MITOCHONDRIAL FUNCTIONS AND CELL DEATH AFTER HEMORRHAGIC SHOCK AND RESUSCITATION
P. Sharma, B. Benford, and M. Bodo. Uniformed Services University of the Health Sciences, Bethesda, MD
Background: Hemorrhagic shock due to extensive blood loss following traumatic injury is associated with mitochondrial dysfunctions and multiple organ failure. The ineffective treatment of HS is mainly due to our lack of knowledge about the severity of organ specific mitochondrial damage and effective resuscitative strategies. Our goal is to determine if and how mitochondrial electron transport chain enzymes are altered in various organs after HS and resuscitation, and their relation to organ damage. In this study, we have evaluated the effectiveness of normal saline, hypertonic saline, and Ringer’s lactate solution followed by blood infusion in ameliorating the physiological, biochemical and mitochondrial electron transport chain enzymes (ETC) in liver and kidneys following HS in rats.
Materials and Methods: Anesthetized, male Sprague-Dawley rats underwent computer-controlled HS at 40 mmHg for 30 min followed by fluid resuscitation for 60 min and then infusion with shed blood. Based on the type of resuscitative fluid, animals were randomly divided into five groups consisting of (1) sham, (2) HS without resuscitation, (3) resuscitation with normal saline, (4) resuscitation with hypertonic saline, and (5) resuscitation with Ringer’s lactate solution. Animals were monitored for physiological, hemodynamic, biochemical parameters, organ dysfunctions, and mitochondrial ETC (by Western blotting).
Results: Non-resuscitated animals were unable to survive due to hypotension, poor oxygen metabolism, and lactic acidosis. In comparison to sham, all ETC enzymes were significantly low in non- resuscitated animals.
Conclusion: The pattern of mitochondrial ETC damage is organ specific and its recovery depends on the type of fluid resuscitation.
This study was supported by funding from U.S. Army Medical Research and Materiel Command (USAMRMC) Award Number: W81XWH-10-1-0507 to PS.
PLATELET FUNCTION AFTER POLYTRAUMA AND HEMORRHAGE IN RATS
D.N. Darlington, J.D. Keesee, X. Wu, and A.P. Cap. US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: Acute Coagulopathy of Trauma (aCOT) can occur after polytrauma and hemorrhage in both the civilian and military patients and is associated with an increased mortality rate.
Objective: To determine if polytrauma and hemorrhage is associated with altered platelet function.
Method: Sprague-Dawley rats (n=9) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and by fracturing the right femur. The rats were bled to a mean arterial pressure of 40mmHg and held there until 40% of the blood volume was removed. No fluid resuscitation was given. Blood samples were taken before (time 0) and at 30, 60, 120 and 240min. Platelet aggregation was measured on a Multiplate (Diapharma) after stimulation with ADP, collagen, thrombin (PAR4) or arachidonic acid and expressed as area under the curve per 1000 platelets.
Results: Prothrombin Time significantly increased over time (18.6±0.3, 20.8±0.5, 21.6±0.5, 21.3±0.3 and 21.9±0.6sec). The ability of ADP to stimulate platelet aggregation was increased at 30 and 60min after trauma/hemorrhage, but was significantly reduced by 2 and 4 hrs. Arachidonic acid stimulation was also significantly lower 2 and 4 hrs after trauma/hemorrhage. Collagen and Thrombin (PAR4) stimulation was significantly elevated 2-4 hr after trauma/hemorrhage. The velocity at which aggregation occurred was significantly elevated for all agonist over the 4hrs, except ASPI which did not change.
Conclusion: Polytrauma and hemorrhage led to complex changes in platelet function, exemplified by both an elevation in platelet function by collagen and thrombin, and a decrease by ADP and arachidonic acid by 4hrs. This deficit in platelet function seen after ADP and arachidonic acid stimulation may contribute to the development of aCOT. This project was funded by MRMC.
INFLAMMATORY RESPONSES IN BRAIN FROM SWINE SUBJECTED TO TRAUMATIC HEMORRHAGE AND TREATED WITH A C1 INHIBITOR
M.A. Dubick*, Y. Li, D.L. Grubbs, J.L. Barr, and J.J. Dalle Lucca. US Army Institute of Surgical Research, San Antonio, TX
Introduction: Hemorrhage is a leading cause of death from potentially survivable injuries in both military and civilian trauma patients. Complement activation has been proposed as a major pro-inflammatory mediator after traumatic hemorrhage that can lead to development of multiple organ dysfunction. The present study evaluated the effects of a C1 complement inhibitor (C1 Inh) in anesthetized, instrumented swine (n=8-12/group) subjected to traumatic hemorrhage.
Methods: A 22 ml/kg controlled plus splenic uncontrolled hemorrhage and femur fracture with soft tissue injury (TH) was induced. After a 30 min shock period, the pigs were resuscitated with 50 ml of 500 U/kg of C1 inhibitor (C1 Inh) alone or with 45 ml/kg LR to a systolic pressure of 65 mmHg. Other groups received 50 ml of the C1 Inh alone or no treatment. Sham animals were instrumented but not hemorrhaged or injured. Swine were monitored for an additional 5.5 hr after injury, euthanized and sections of brain (cerebral cortex (CC), hippocampus (H) and brain stem (BrS)) were collected for histopathology by H&E staining and the remainder flash frozen and stored at -80° C until analyzed for select indices of oxidative stress and cytokines.
Results: TH resulted in a 37% and 100% increase in BrS and H IL-6 levels, respectively and 15% and 34% increased in IL-1 in these tissues, but these levels were not affected by C1 Inh treatment alone or with LR. Thiobarbituric acid reactive substances were 24% higher in BrS after TH, but were unchanged in the other tissues. C1 Inh alone or with LR reduced nitric oxide and raised NADH reducing equivalents in all tissues examined compared to TH. No significant histological changes were observed in brain tissue by TH alone or with C1Inh treatment.
Conclusion: Our initial concern that BrS and H would be more susceptible to hypoxic injury due to TH than CC was not observed. Observed changes in cytokine levels or indices of oxidative stress were minor and agree with the histological findings.
INADEQUATE PMN CHEMOKINE RECEPTOR EXPRESSION AFTER POLYMICROBIAL ABDOMINAL SEPSIS CONTRIBUTES TO DEFECTIVE PMN CHEMOTAXIS IN THE AGED
D.C. Nacionales, R. Ungaro, L.F. Gentile, M. Lopez, F. Needell, E. Vanzant, B.E. Szpila, A.L. Cuenca, F. Moore*, A. Joseph, C. Leeuwenburgh, S. Brakenridge*, H. Baker, and P.A. Efron*. University of Florida, Gainesville, FL
Objectives: Although it is known that the aged have higher morbidity and mortality compared to young adults after sepsis, the mechanism behind these poor outcomes remains unclear. We have shown that following cecal ligation and puncture (CLP), peritoneal cavity (PC) neutrophil (PMN) chemotaxis is dysfunctional while their capacity to phagocytose and produce ROS remains intact. Our genomic analysis also illustrated poor early upregulation of innate immunity genes in aged blood leuckoytes. We investigated whether PC PMN chemotaxis, chemokines or receptors play a role in the increased PC bacterial CFUs and the mortality seen in aged mice after CLP.
Methods: Using Ingenuity Pathway Analysis, we identified chemokine genes with decreased expression in murine aged (vs young) blood leukocytes that had human homologues. Subsequently, 6-10 week (young) and 20-24 month (aged) mice underwent CLP, were sacrificed at day 1 and PC lavage was performed. PC bacterial load was determined and chemokines were quantified by ELISA. Cell surface expression of chemokine receptors on PC CD11b+Ly6G+ PMNs was determined through flow cytometry.
Results: Our analyses showed that the soluble factors MIP1-a, MCP-1, MIP-2, KC, & osteoponin (OPN) as well as the cell surface receptors CCR1, CCR5, and CXCR2 had decreased genomic expression in aged murine blood leukocytes. Consistent with our previous data, the PC bacterial load was increased in aged mice 1 day after CLP. The percentage and mean fluorescent intensity (MFI) of CCR1+, CCR5+, CXCR2+ cells were also significantly lower on aged murine PC PMNs (p<0.05). In contrast, the peritoneal lavage concentrations of MIP-2, MIP-1a, MCP-1, and KC concentrations were all significantly greater in the aged compared to young mice (p<0.05), while OPN trended towards higher levels in the elderly.
Conclusion: Despite the increased levels of PC chemokines present after CLP, aged mice are unable to effectively clear PC bacteria. The reduced percentage and expression level of specific chemokine receptors (CCR1, CCR5, and CXCR2) on the PC PMNs in the context of increased ligand concentrations could explain the increased susceptibility of aged mice, and possibly elderly humans, to abdominal sepsis. A thorough understanding of this phenomenon may be key to the development of novel.
MUTATIONS AFFECTING LIPOTECHOIC ACID (LTA) AUGMENT SUSCEPTIBILITY OF ENTEROCOCCUS FAECALIS BIOFILMS TO ANTIBIOTICS
M.J. Henry-Stanley*1, G. Dunny2, D. Hess*3, and C.L. Wells*1, 3. 1University of Minnesota, Minneapolis, MN, 2University of Minnesota Department of Microbiology, Minneapolis, MN, 3University of Minnesota Department of Surgery, Minneapolis, MN
Microbial biofilms are attached to surfaces in aqueous environments encased in a matrix composed of polysaccharide, protein, DNA, and lipid. Trauma patients with implanted devices are especially susceptible to biofilm infections, and because biofilms are antibiotic-resistant, treatment may involve removal of the implanted device. Studies by our laboratory have provided a role for lipid in the antibiotic resistance of biofilm bacteria. We tested the hypothesis that mutations in LTA, a lipid-containing molecule embedded in the cell wall of gram-positive bacteria, might affect the susceptibility of developing E. faecalis biofilms to antibiotics. OG1RF is a wild-type and INY3000, 3048 are mutant strains that express shortened LTA poly(glycerol phosphate) chains and INY3000 also expresses shortened fatty acid chains. Biofilms were cultivated 16 hr on suture in medium supplemented with cell wall active antibiotics and numbers of viable bacteria were quantified from suture sonicates. The antibiotic concentrations reflected the minimum inhibitory concentration for planktonic OG1RF. Both LTA mutants showed increased susceptibility to vancomycin and oxacillin compared to corresponding data from *OG1RF, and INY3048 also showed increased susceptibility to oxacillin compared with #INY3000 (P<0.01, ANOVA, n≥7 biofilms, Fig.1). Thus, LTA may play a role in the antibiotic resistance of E. faecalis to cell wall active antibiotics and may be a target for the design of therapies to increase antibiotic efficacy.
BOTH TRAUMATIC BRAIN INJURY AND α-7 NICOTINIC ACETYLCHOLINE RECEPTOR ANTAGONISM IMPROVE SURVIVAL IN A MURINE PNEUMONIA MODEL
E.G. King1, T. Hsieh2, D.M. Stepien2, E. Duffy2, and D.G. Remick*2. 1Boston University Medical Center, South Boston, MA, 2Boston University School of Medicine Department of Pathology, Boston, MA
Introduction: Previous work has demonstrated improved survival in mice subjected to traumatic brain injury (TBI) given Pseudomonas aeruginosa (PSA) pneumonia compared to non-TBI mice. Other studies have shown improved outcomes in murine models of inflammation when animals were treated with an agonist of the α-7 nicotinic acetylcholine receptor (nAChR), GTS-21. The current study tested the hypothesis that GTS-21 will improve survival in a murine model of PSA pneumonia.
Methods: Three groups of adult female ICR mice either underwent 1) mild TBI induced by a weight drop model, 2) sham TBI, or 3) administration of GTS-21 (4mg/kg) intraperitoneally (IP) q12h. 48 hours later, PSA (5x107 CFU) was given intratracheally. Mortality was followed over 28d with serial hemodynamic parameters recorded using a cervical collar pulse-oximeter and blood draws via facial vein. A second group of mice were sacrificed at 4h post-pneumonia, blood and bronchoalveolar lavage (BAL) fluid was collected. Additionally, to examine the effect of α-7 nAChR antagonism on survival, the α-7 nAChR antagonist methyllycaconitine (MLA) (1mg/kg) was administered IP at the time of TBI and q12h for 48h prior to pneumonia. Survival was followed for 28d.
Results: TBI mice displayed a survival advantage following PSA pneumonia as compared to non-TBI mice (p<0.0001). The survival of mice that received GTS-21 (no TBI) was equivalent to the TBI group, and significantly better than non-TBI (p=0.0003). TBI mice had significantly more cells in BAL fluid (p<0.0001) and significantly less BAL colony-forming units (p=0.0004) than either the non-TBI or GTS-21 groups. Both TBI and GTS-21 resulted in deceased plasma IL-6 (p=0.0008) and MIP-2 (p=0.0001) levels. No significant difference was observed in BAL cytokines. Hemodynamic parameters and complete blood counts were similar among all three groups. Additionally, TBI provided no survival benefit in the mice that also received MLA.
Conclusion: The α-7 nAChR agonist, GTS-21 improves survival in a murine model of pneumonia and decreases levels of plasma IL-6 and MIP-2, similar to the effects following TBI. Antagonizing the α-7 nAChR abrogates the improved survival observed in TBI. These findings suggest that the cholinergic anti-inflammatory pathway may have a role in the improved survival of TBI mice after Pseudomonas pneumonia.
FIBROCYTE INDUCED T-CELL PROLIFERATION DUE TO IL-15 ASSOCIATED PRODUCTION IMPROVES MORTALITY IN SEPTIC MICE
C. Fry and J.A. Nemzek*. University of Michigan, Ann Arbor, MI
Numerous studies correlate mortality with loss of T-cells during sepsis. Consequently, treatments that preserve T-cells have proven beneficial. Previous work in our lab demonstrated increased survival after cecal ligation and puncture (CLP) for mice receiving fibrocytes (80%) compared to mice receiving saline (20%). In addition, mice receiving fibrocytes had significantly higher numbers of splenic CD4 T-cells, and CD8 T-cells after CLP, compared to mice receiving saline. To determine whether antigen presentation by fibrocytes caused the increase in T-cells, the major histocompatibility complex class II and costimulatory molecules were tested. Fibrocytes were derived from wild-type (WT) or MHC class II knockout (KO), CD40KO, CD80KO, or CD86KO mice. The KO fibrocytes were co-cultured with CFSE loaded splenic T-cells for one week. The cultures were harvested at various timepoints and flow cytometry used to detect loss of CFSE, an indicator of cell proliferation. There were no significant differences between T-cells incubated with WT or any of the KO fibrocytes. Next IL-15 was investigated. Fibrocytes were derived from normal mice and co-cultured with CFSE loaded splenic T-cells for one week with or without a neutralizing IL-15/IL-15R alpha antibody. Although fibrocyte/T cell cocultures demonstrated a significant increase in T-cell proliferation, there was no significant proliferation of CD4 T-cells in the presence of the IL-15/IL-15R alpha antibody. In addition, there was a 2-fold decrease in proliferating CD8 T-cells in the presence of antibody as compared to fibrocyte T-cell co-culture. Interestingly, when fibrocytes were co-cultured with splenic T-cells for up to 1wk, there was a significant increase in IL-15rby on T-cells compared to T-cells alone. Moreover, throughout co-culture with the T-cells, the fibrocytes have a significantly increased expression of intra-cellular IL-15R alpha compared to fibrocytes alone, suggesting that direct contact causes increases in receptors necessary for transpresentation of IL-15 on both cell types. Collectively, these data suggest that the enhanced T cell proliferation after adoptive transfer of fibrocytes is not related to antigen presentation, but may be a function of IL-15 transpresentation.
ABSENCE OF MATRIX METALLOPROTEINASE-8 (MMP8) INCREASES MORTALITY IN A PEDIATRIC SEPSIS MODEL
S.J. Atkinson1, 2, K. Harmon2, J. Kuethe1, C.C. Caldwell*1, B. Zingarelli*2, and H. Wong*2. 1University of Cincinnati, Cincinnati, OH, 2Cincinnati Childrens Hospital Medical Center, Cincinnati, OH
Introduction: We previously demonstrated that genetic ablation or pharmacologic inhibition of MMP8 improves survival in an adult murine sepsis model (Crit Care Med 2012;40:379). Since the immature host’s inflammatory response differs from the adult’s, we hypothesized that developmental age influences the role of MMP8 in sepsis.
Methods: Using a cecal slurry intraperitoneal injection sepsis model, we compared wild-type (WT, C57 BL/6) and MMP8 null pediatric-aged mice (age 12-14 days). Survival after cecal slurry (0.5 mg/kg) challenge was monitored for 7 days. Bacterial burden was measured in tissues and peritoneal fluid 18 hours after injection. Peritoneal lavage samples were analyzed for white blood cell subset numbers and inflammatory cytokine concentrations.
Results: Pediatric MMP8 null mice had a significantly higher mortality than WT mice (Figure). Bacterial burden in the peritoneal lavage was increased in the MMP8 null mice vs. WT mice. The median log-transformed colony count (IQR) for MMP8 null mice was 4.5 (4.0 - 6.4) vs. the WT median of 0.0 (0 - 4.5; p=0.03). Bacterial burden in blood and lung samples followed the same trend of a higher burden in MMP8 null mice vs. WT, but did not reach statistical significance. White blood cell numbers and cytokine concentrations in the peritoneal fluid were not significantly different between MMP8 null and WT mice.
Conclusion: In contrast to our findings in adult mice, absence of MMP8 increases mortality in a pediatric model of sepsis. The mechanism of increased mortality appears to involve a defect in MMP8-dependent bacterial clearance. (Supported by NIH T32GM008478 and R01GM096994).
SURFACTANT PROTEIN SP-D ALTERS THE EXPRESSIONS OF NF- KB AND IL-1β IN INTESTINE IN PNEUMONIA MOUSE MODEL INDUCED BY STAPHYLOCOCCUS AUREUS
A. Sharif, Q. Meng*, O. Abdel-Razek, M. Cooney, G. Wang*, J. Liu, and R.N. Cooney*. SUNY Upstate Medical University Hospital, Syracuse, NY
Introduction: The gut plays an important role in systemic inflammation and is felt to be critical in the development of multiple organ failure. Pneumonia-induced sepsis influences intestinal epithelial turnover kinetics and functional parameters. The surfactant proteins SP-A and -D are important in innate immunity in the lung and protect against a wide variety of pathogens. Using a murine model of S. aureus pneumonia we examine the effect of SP-A and -D on intestinal expression of IL-1β and NF- κB.
Methods: Three groups of mice: WT mice C57BL/6, SP-A and SP-D double knockout (SP-A/D KO), and humanized transgenic (hTG) SP-D mice (9-12 weeks) were used in this study. Mice were anesthetized by IP injection of ketamine and xylazine then S. aureus (5x107 CFU /50 μl/mouse) were delivered by intra-tracheal injection to induce pneumonia. Mice were re-anesthetized 24 h later for bronchoalveolar lavage (BAL), tissue harvest. 1000 μl of BAL fluid (BALF) and intestine were collected. Colony-forming units (CFU) from BAL fluid were counted. The expression of NF-κB, Pi-κB, MYD88, TLR2, TLR4, TNF-α and IL-1β mRNA were measured by quantitative RT-PCR. Protein levels of NF- κB p65 subunit were measured by Western blot and normalized to β-actin, IL-1β was assayed by ELISA. Data are means ± SE, n = 7 /group, statistical significance p<0.05 vs. control by Student’s t test or one-way ANOVA.
Results: Pneumonia increased the mRNA expression of NF-κB and IL-1κ and protein levels of NF-κB p65 and IL-1β in intestine from WT and SP-A/D KO mice. BAL CFUs were decreased in infected hTG SP-D mice compared to infected SP-A/D KO mice (175±41 vs. 25±6, P<0.01). Constitutive expression of SP-D in hTG mice reduced gut expression of NF- κB p65 and IL-1β mRNA and protein by 65% - 30%. In contrast, the expression of Pi-κB, MYD88, TLR2, TLR4 and TNF-α mRNA did not show significant changes in gut from WT mice, SP-A/D KO mice and hTG SP-D mice.
Conclusions: S. aureus pneumonia enhances the expression of NF- κB and IL-1β in intestine from WT and SP-A/D KO mice. SP-D attenuates S. aureus growth and intestinal inflammation via the NF- κB pathway. These findings enhance our understanding of how SP-D protects the host against infection and intestinal inflammation during S. aureus pneumonia.
SEPSIS-INDUCED CHANGES IN AMINO ACID TRANSPORTERS AND SIGNALING VIA TORC1 IN SKELETAL MUSCLE
L.J. Laufenberg, A.H. Pruznak, and C.H. Lang*. Penn State College Medicine, Hershey, PA
Sepsis not only decreases basal muscle protein synthesis but also produces a marked resistance to the normal anabolic response to essential amino acids, such as leucine (Leu). The etiology for the sepsis-induced Leu resistance has not been elucidated and is the focus of this study. CLP-induced sepsis in adult male rats decreased basal protein synthesis in gastrocnemius (30%), associated with a reduction in mTOR activation as indicated by >50% reduction of 4E-BP1 and S6K1 phosphorylation. The ability of oral Leu to increase protein synthesis and mTOR kinase was completely prevented in septic rats. Although sepsis increased 2- to 4-fold the mRNA for selected amino acid transporters, such as CAT1, LAT2 and SNAT2, CAT1 and LAT2 protein content in septic muscle was decreased 20% (with no change in SNAT2 protein). Conversely, sepsis decreased the proton-assisted amino acid transport (PAT)-2 by 70%, but without a coordinate change in PAT2 protein. The intracellular Leu concentration was increased in septic muscle, compared to basal control conditions, and Leu further increased the intracellular Leu concentration similarly in both control and septic rats. The Rag GTPase proteins A/B/C/D are also central to amino acid-stimulation of mTOR. While sepsis did not alter RagA, B and C, there was a 30% reduction in muscle RagC protein. Sepsis also decreased LAMTOR-1 (p18) and -2 (p14), which forms an essential mTOR anchoring complex. Hence, sepsis alters impairs multiple pathways responsible for cellular distribution of amino acids as well as proteins necessary to the recruitment of mTOR to the endosome-lysosomal compartment needed for full nutrient activation of muscle protein synthesis. (GM38032).
A CRITICAL ROLE OF ARGININE VASOPRESSIN RECEPTOR 2 ACTIVATION IN VASCULAR HYPERPERMEABILITY DURING SEVERE OVINE SEPSIS
E. Lopez1, O. Fujiwara1, F. Lima-Lopez1, 2, B. Enkhtaivan1, Y. Zhu1, J. Rojas1, H.K. Hawkins2, 1, R.A. Cox2, 1, and P. Enkhbaatar*1. 1The University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX
Introduction: Arginine vasopressin (AVP) is used in sepsis as an adjuvant vasopressor due to its AVP V1a receptor agonist effect. AVP expands plasma volume by activating V2 receptor (V2R), but high doses of AVP have shown detrimental effects in severe sepsis. We hypothesized that V2R activation promotes vascular hyperpermeability.
Methods: Using our ovine sepsis model, sheep were injured by cotton smoke insufflation (48 breath) and lung instillation of 3.5X 10^6 CFU Methicillin-resistant staphylococcus aureus (MRSA). Sheep were placed on a ventilator and monitored in a conscious state for 24H under analgesia. Lactated Ringer’s was titrated to hematocrit. Urine output was measured via Foley catheter. Groups: 1) C: injured, treated with vehicle; 2) D: injured treated with V2R agonist desmopressin (36.16 ng/kg/h); and 3) T, injured treated with V2R antagonist tolvaptan (416.6 ug/kg/h). Treatments began 1H post-injury (IV).
Results: 24H survival rate: 3/6 in C, 5/6 in T, 6/6 survived in D. Total fluid accumulation was lower in T vs. D at 12H (1008 vs. 2071mL, p>0.05) and 24H (983 vs 4672mL, p>0.05). T accumulated less fluid than C at 12H (1008 vs. 2294mL p>0.01). There was no difference in hematocrit and hemoglobin, suggesting similar plasma volumes. Plasma protein at 24H was lower in D vs. T (4.033 vs 4.840 g/dL, p<0.05). Lung water content was higher in D vs C (8.012 vs 5.581, p<0.05). In addition, desmopressin decreased electric impedance in cultured endothelial cells similar to VEGF (Fig. 1).
Conclusion: AVP V2R activation mediates microvascular hyperpermeability in MRSA ovine sepsis. Selective AVP V1aR agonist or combination with AVP V2R antagonist should be considered for treatment of septic patients.
DO TYPE 2 INNATE LYMPHOID CELLS HAVE A ROLE IN SEPTIC LIVER INJURY?
N. Hutchins1, 2, C. Chung*1, 2, and A. Ayala*1, 2. 1Rhode Island Hospital, Providence, RI, 2Brown University Warren Alpert School of Medicine, Providence, RI
Sepsis is the 10th leading cause of death in critically ill patients; thus, the development of novel treatments is necessary. Traditionally, therapies directed against the pro-inflammatory stage of sepsis, such as anti-IL-1β have failed in clinical trials. The recent discovery of cytokine, IL-33, an IL-1 super-family member that is a ligand for orphan receptor, ST2, suggests it may have a unique role in the pathology of sepsis. Important to this discussion, is the report that type 2 innate lymphoid cells (ILC2) which express ST2 bind IL-33, but not IL-1α and IL-1β. This induces ILC2s to produce type 2 (TH2) cytokines, such as IL-5, IL-13, IL-4, and IL-9. While most studies have documented a role of ILC2s in mucosal barrier surfaces, such as the skin, lungs, and intestine, their role in the liver and the pathology of liver disease states other than hepatic fibrosis, like sepsis, is essentially unknown. Therefore, we hypothesized that ILC2s play a role in the pathology associated with acute liver injury seen in sepsis. To test this, C57BL/6 mice were subjected to Sham or cecal ligation & puncture (CLP) to induce sepsis. Liver non-parenchymal cells (NPCs) were isolated and analyzed by flow cytometry, 24hr post-CLP. Our initial results indicate that while there were no % ILC2+ differences between Sham and CLP, there was decreased total number of ILC2s in CLP animals (Figure 1A). In addition, there is increased IL-33 secreted into the blood from CLP animals compared to Sham controls (Figure 1B). Liver ILC2 populations also expressed higher levels of the co-inhibitory molecule, programmed cell death-1 (PD-1; a molecule we have shown plays a role in mediating liver injury) in CLP animals. Together, this implies that the IL-33 receptor+ (ST2+) ILC2s may play a novel role in mediating the pathology associated with acute liver injury seen during sepsis and, thus, further investigation is warranted to determine their impact as potential therapeutics (supported by NIH T32 GM065085 & R01 GM046354).
INK128 MTOR INHIBITOR AS AN IMMUNE RESPONSE MODIFIER (IRM) TO RESTORE SEPSIS IMMUNITY
L. Wanke-Jellinek1, 2, K. Shimizu*1, S. Ishikawa1, P. Gao1, J. Keegan1, and J.A. Lederer*1. 1Brigham and Women’s Hospital, Boston, MA, 2Technical University Munich, Munich, Germany
Background: The loss of immune homeostasis in patients with traumatic injuries can predispose to opportunistic infections and sepsis. We hypothesized that early treatment with immune response modifiers (IRMs) like mTOR inhibitors can provide immune protection from infections by redirecting the immune system response to injury.
Methods: Groups of outbred mice underwent burn injury and were treated with mTOR pathway inhibitors (INK128, Torin2), 1mg/kg, 2 hours later. At 7 days after injury, mice underwent cecal ligation and puncture (CLP) to induce sepsis. Using CyTOF and conventional flow cytometry as well as multiplex assays for a systems biology approach we measured the phenotypic and functional details of how treatments with these IRMs influence immune system functions in vivo.
Results: Treating injured mice with INK128 significantly increased CLP sepsis survival. In contrast, treatment with Torin2 was not protective even though both drugs act as mTOR inhibitors. INK128 significantly increased blood monocytes, Gr-1+ monocytes, and PMNs at 7 days after injury. Also, a significant increase in B1- type B cells was observed in the spleen, lymph nodes and blood. Torin2 did not induce these changes in immune cell subsets. The effects of these related IRMs on sepsis-induced immune function were determined by measuring T cell and innate cytokine production by spleen cells. We found that INK128 increased IL-1a, -5, -6, -12p40, -17, IFNg and GM-CSF production as compared to untreated or Torin2-treated mice.
Conclusions: These findings demonstrate that INK128, but not Torin2, acts as an IRM to protect injured mice from CLP sepsis. The divergent effects of these related IRMs on the immune system highlight the protective activity of INK128 as enhancing B1-type B cells and several innate immune cell types - monocytes and PMNs.
ESTROGEN DECREASES AKT AND INCREASES NF-KB ACTIVATION IN HEPATOCYTES DURING INFLAMMATION IN VITRO
B.G. Harbrecht*, A. Gentile, I. Nweze, J.W. Smith*, J. Lakshmanan, and B. Zhang. University of Louisville, Louisville, KY
Background: Estrogen is responsible for the improved outcome for women after shock and sepsis. Estrogen (E2) protects the liver from injury following inflammation and chemical toxins and its effects are mediated through estrogen receptors that alter cellular gene expression and intracellular signaling pathways. Hepatocytes contain alpha and beta E2 receptors and the G-protein receptor that regulates downstream phosphorylation pathways. E2 alters MAPK activation, PI3K/Akt, and NF-kB in selected cells and increased Akt activation in the liver but the specific mechanism for E2 in reducing hepatic injury in inflammation is not known. E2 decreases hepatocyte nitric oxide synthase expression and regulates other metabolic pathways in the liver. We hypothesized the E2 would alter hepatocyte intracellular signaling pathways to regulate hepatocyte function.
Methods: Primary rat hepatocytes were isolated from male Sprague-Dawley rats. After overnight culture, hepatocytes were exposed to proinflammatory cytokines (IL-1beta, Interferon-gamma) and differing concentrations of E2. Cellular proteins were collected after 60 minutes for western blot analysis.
Results: In contrast to in vivo sepsis where E2 was associated with increased Akt activation, in vitro E2 administration decreased hepatocyte Akt phosphorylation (see Figure). E2 also decreased IkappaB consistent with increased NF-kB activation (Figure) but had no effect on AMPK phosphorylation (not shown).
Conclusions: E2 selectively activates intracellular signaling pathways in hepatocytes during inflammation. Improved understanding of the pathways altered by E2 will help define the cellular mechanisms responsible for the beneficial effects of E2 administration in sepsis.
THE ENDOCANNABINOID N-ARACHIDONOYL DOPAMINE (NADA) AND WIN55,212-2 MODULATE INFLAMMATORY ACTIVATION OF HUMAN ENDOTHELIAL CELLS
S. Khakpour*, K. Wilhelmsen, A. Tran, F. Xu, and J. Hellman*. University of California San Francisco, San Francisco, CA
Background: While cannabinoids, such as Δ9-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, since they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and decrease vascular barrier function. We hypothesized that endocannabinoid pathways modulate endothelial inflammatory responses.
Methods: Quantitative PCR was used to assess the expression of cannabinoid receptors and metabolic enzymes in different EC niches. To determine the effects of cannabinoids on EC cytokine production, human lung microvascular EC (HMVEC-Lu) were pre-treated with a panel of cannabinoids and then stimulated with bacterial lipopeptide, LPS, or TNF-α. Cytokines were quantified in HMVEC-Lu supernatants.
Results: Primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel TRPV1. In contrast to leukocytes, CB2R is only minimally expressed in some EC populations. Furthermore, ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNF-α.
Conclusion: These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.
PROTEOMIC ANALYSIS IDENTIFIES AGE-ASSOCIATED RELEASE OF HISTONE H4 DURING ACUTE INFLAMMATORY STRESS
M.E. Starr*1, D. Okamura1, R.L. Lin2, L.Y. Lee2, B.M. Evers1, 3, and H. Saito*1, 2. 1University of Kentucky, Department of Surgery, Lexington, KY, 2University of Kentucky, Department of Physiology, Lexington, KY, 3University of Kentucky, Markey Cancer Center, Lexington, KY
Background: Systemic inflammatory response syndrome (SIRS), caused by sepsis or trauma, is a serious clinical condition which is particularly threatening to elderly patients who suffer much higher mortality rates than the young. SIRS can lead to an array of complications resulting in vascular damage, microvascular coagulation, pulmonary dysfunction, and death. We previously reported age-dependent exaggerated pulmonary inflammation, edema, and interstitial proteinaceous debris, characteristic of acute lung injury (ALI), using a murine lipopolysaccharide (LPS)-induced endotoxemia model of SIRS. Our previous studies using this model also demonstrated significant age-associated mortality, inflammation, and thrombosis after LPS-injection to young and aged mice.
Objective: The purpose of this study was to identify and characterize proteins involved in pulmonary dysfunction which differ by aging.
Methods: Two-dimensional gel electrophoresis was performed on lung homogenates from young (4-months) and aged (24-months) mice injected with or without LPS (2.5mg/kg, i.p.). SYPRO Ruby stained 2D-gel images were analyzed with Progenesis Discovery Software and protein spots were excised and identified by MALDI TOF-MS.
Results: We selected 35 protein spots which exhibited increased levels after LPS-injection and were further augmented by aging. Among these proteins was core histone protein, histone H4. Histones (DNA packaging proteins) were recently shown to be cytotoxic to endothelial cells and mimic sepsis-associated complications upon release from dying cells (Nature Medicine 15; 2009). Age-associated increase of histone H4 in the lung from young and aged mice with LPS-injection was confirmed by one-dimensional gel electrophoresis and Western blot analysis. Additionally, detection of histone H4 in the plasma after LPS-injection confirmed release of histone H4 into the circulation during endotoxemia. We further injected young and aged mice with recombinant histones (50mg/kg, i.v.) which caused 17% versus 67% mortality in young and aged mice, respectively.
Conclusion: Release of histones, particularly histone H4, into the circulation from dying cells during acute inflammatory stress may largely contribute to the augmented endothelial cell damage and organ dysfunction associated with heightened mortality in the elderly with SIRS-related complications.
NETOSIS AND PLASMA DNA PROVIDE AN ALGORITHM FOR THE MOLECULAR NATURE OF INFLAMMATION IN SIRS AND SEPSIS
J. Angermeyer1, J. Roos1, J. Schneider1, H. Piechotka1, E. Barth3, M. Huber-Lang*2, and E. Schneider1. 1Ulm University, Sektion Experimentelle Anaesthesiologie, Ulm, Germany, 2Ulm University - Dept. Trauma Surgery, Ulm, Germany, 3Ulm University, Dept. Anaesthesiology, Ulm, Germany
Introduction: In patients with systemic inflammatory response syndromes (SIRS) cardiovascular function and plasma concentrations of C-reactive protein (CRP) constitute well accepted markers for the severity of inflammation. However, causative molecular agents causing inflammation remain to be obscure.
Methods: A diagnostic pilot study of adult patients with SIRS and sepsis was performed to question the contribution of Neutrophil Extracellular Trap Formation (NETosis) and circulating genomic DNA by quantification of “netting” granulocytes1 in blood smears corresponding to fluorometric measurements of double-stranded (ds-) and single-stranded (ss-) DNA in plasma for a defined period of time during intensive care treatment. Blood counts, C-reactive protein (CRP), and cytokines were documented in parallel with SIRS criteria.
Results: In defined episodes with SIRS and high CRP, we found a strong correlation between NETosis and the circulating dsDNA, whereas episodes with high thrombocyte counts followed elevations of ssDNA. During sepsis, high plasma concentrations of dsDNA and NETosis correlated with an infection associated biomarker, Lipopolysaccaride Binding Protein (LBP).
Conclusion: The selective determination of NETosis in granulocytes, dsDNA and ssDNA on the one hand and CRP or LBP on the other provides a new algorithm to specify the molecular nature for inflammation in patients with SIRS or sepsis. Genomic DNA appears to be the major stimulus for inflammation in both SIRS and sepsis and may be a new and relevant therapeutic target.
1Brinkmann, B. et al. Neutrophil extracellular traps: how to generate and visualize them. J. Vis. Exp. 2013
ELEVATED LEVELS OF PLASMA INTERLEUKIN-27 IN MURINE MODELS OF SYSTEMIC INFLAMMATORY RESPONSE
T.R. Langner, P. Hake, M. O’Connor, H. Wong*, and B. Zingarelli*. Cincinnati Childrens Hospital Medical Center, Cincinnati, OH
Interleukin-27 (IL-27), a heterodimeric cytokine produced by antigen presenting cells, has important modulatory functions in the innate and adaptative immune responses. Our recent clinical study suggests that circulating IL-27 may serve as a biomarker for sepsis (Wong HR et al., Shock 40:382, 2013). Here, we tested the hypothesis that systemic elevation of IL-27 is dependent on the presence of bacterial infectious pathogens in murine models of systemic inflammatory response. Male C57BL/6 mice (4-5 weeks old) were subjected to three models of systemic inflammation induced by pathogen-associated molecular pattern molecules (PAMPs): 1) peritonitis by intraperitoneal (ip) injection of zymosan of S. cerevisiae (500 mg/kg); 2) polymicrobial sepsis by cecal ligation and puncture (CLP); 3) endotoxemia by ip injection of lipopolysaccharide (LPS, 50 mg/kg) of E. coli (LPS). Plasma cytokines were detected at 6 and 24 hrs after challenge by multiplex array. In each model, mice exhibited a similar mortality rate with approximately 50% lethality at 48 hrs. Injection of LPS caused a robust elevation of IFN-γ, IL-1β and IL-17f at 6 hrs when compared to the zymosan and CLP model. IL-6 levels at 6 hrs were more pronounced in mice with endotoxemia or polymicrobial sepsis when compared to the zymosan-induced peritonitis. TNF-α levels were similarly elevated in all three models. IL-27 levels rose significantly at 6 hrs after challenge with PAMPs in the three models when compared to control baseline levels at time 0 (947±259 pg/mL) and were similar among the three groups; plasma IL-27 levels were 1754±421 pg/mL in the LPS group; 2294±857 pg/mL in the CLP group and 1875±364 pg/mL in the zymosan group. Mice exhibited a further elevation of IL-27 at 24 hrs after challenge with PAMPs. However, the zymosan-treated group exhibited a remarkable elevation of IL-27 (7585±342 pg/mL, P<0.05) when compared with the LPS (2102±342 pg/mL) or CLP (2567±1028 pg/mL) groups. Our data suggest that elevation of IL-27 represents a mechanism of innate immune defense induced by the immunogenic challenge of PAMPs. Our data have also identified a novel cytokine pattern response for fungal pathogens. (Supported by NIH R01 GM067202; R01 AG027990; R01 GM099773; R01 GM096994).
TOLL-LIKE RECEPTORS UP-REGULATE COMPLEMENT FACTOR B VIA NF-κB PATHWAY IN HUMAN KIDNEY TUBULAR CELLS
D. Li1, 2, L. Zou*1, Y. Feng1, Y. Gong1, J. Cai1, and W. Chao*1. 1Massachusetts General Hospital, Boston, MA, 2Xiangya 3rd hospital, Changsha, China
Introduction: Toll-like receptors and complements are two important components of the host innate immunity. Complement factor B (cfB) is a necessary component of the alternative pathways (AP) of complement activation. Our previous studies have shown that mice deficient of cfB were partially protected from acute kidney injury (AKI) and had improved survival during bacterial sepsis. However, how cfB synthesis is regulated in the kidney is less clear.
Methods: Human proximal tubular cells (HK-2 cells) were stimulated with different concentrations of lipopolysaccharides (LPS) and polyinosinic-polycytidylic acid (Poly(I:C)) for various periods of time. Ten μM of BAY 11-7082, a specific NF-κB inhibitor, was added 30 min before HK-2 cells treatment with 100 μg/ml of LPS or 25 μg/ml of Poly(I:C) for 24 h. HK-2 cells were also stimulated with 2 μg/ml of lipoteichoic acid (LTA), 1 μg/ml of Pam3cys or 0.25 μM of CpG oligodeoxynucleotides (CpG) for 48 h. cfB levels in cultured medium were determined by Western blot.
Results: As indicated in Fig A, Poly (I:C) and LPS induced time- and dose-dependent increase in cfB levels in HK-2 cell culture media. These effects were significantly inhibited by BAY 11-7082 (Fig B). Moreover, LTA and Pam3cys, but not CPG, also induced cfB production in HK-2 cells (Fig C).
Conclusions: These data demonstrate that activation of TLR2, TLR3 and TLR4, but probably not TLR9, induces a robust cfB production in human proximal tubular cells in vitro and this effect appears to be mediated via NF-κB signaling.
CIRCULATING C5A RECEPTOR TO MONITOR SEPSIS?
S. Denk1, D. Rittirsch1, H. Barth2, M. Perl3, P. Ward*4, and M. Huber-Lang1. 1Department of Orthopedic Trauma, Hand, Plastic, and Reconstructive Surgery, University Hospital Ulm, Ulm, Germany, 2Institute of Pharmacology and Toxicology, University of Ulm, Ulm, Germany, 3Clinic for Surgery, Trauma Surgery and Orthopedics at the BGU Murnau, Murnau, Germany, 4Department of Pathology, University of Michigan Medical School, Ann Arbor, Ann Arbor, MI
Numerous studies suggested an excessive activation of the complement system and an accumulation of the anaphylatoxin C5a in the course of sepsis. In parallel, neutrophils lost their ability to bind C5a during experimental sepsis, which was associated with severe neutrophil dysfunction. Therefore the aim of the study was to investigate the molecular regulation of the C5a receptor (C5aR) on neutrophils during human sepsis.
A prospective, clinical and experimental study was conducted, including 60 consecutive ICU patients with septic shock (according to Bone’s criteria) and 30 healthy volunteers. C5aR expression on neutrophils was measured by flow cytometry and Western blotting. For detection of circulating C5aR in serum, both immunoblotting and a novel technique of flow cytometric immuno-capturing analysis were used.
Patients with septic shock displayed significant evidence of complement activation and signs of complementopathy in contrast to healthy volunteers. Neutrophils from patients with septic shock exhibited decreased C5aR expression, which negatively correlated with survival. As C reactive protein (CRP) is known to be an activator of complement, the effect of CRP on C5aR expression was analyzed. In vitro incubation of healthy neutrophils with CRP led to a dose- and time-dependent decrease of the C5aR which was associated with a decrease of heat shock protein 70 (HSP70). Further investigations revealed that C5aR is shed from the neutrophil surface during sepsis as a so far undetected circulating C5aR (cC5aR). The appearance of this cC5aR in the serum of patients with septic shock was correlated with negative clinical outcome.
Therefore, the C5aR content on neutrophils as well as the presence of the cC5aR in serum may be useful as surrogate markers in monitoring the immune status of ICU patients.
MITOCHONDRIA REGULATE NEUTROPHILS BY PRODUCING ATP FOR PURINERGIC SIGNALING
Y. Bao, C. Ledderose, T. Seier, and W. Junger*. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
Polymorphonuclear neutrophils (PMNs) have evolved highly sophisticated strategies to detect, locate, and kill invading microbes before these invaders can multiply and cause systemic infections. Our previous work has shown that formyl peptides receptors (FPR) induce the release of cellular ATP from PMNs. Released ATP then regulates cell functions by autocrine feedback via purinergic (ATP and adenosine) receptors. Here we studied whether mitochondria produce the ATP that drives these purinergic signaling mechanisms.
Although glycolysis is the main source of intracellular ATP, inhibition of mitochondria with CCCP or KCN blocked FPR-induced ATP release. This suggests that mitochondria of PMNs are critical for ATP release and cell activation. Studying mitochondrial signaling with JC-1, dihydrorhodamine 123 (DHR 123), and Rhod-2 revealed that a small portion of mitochondria (5-10%) was activated within seconds of FPR stimulation. Blocking mitochondrial ATP production with CCCP impaired FPR-induced calcium signaling and decreased oxidative burst, degranulation, phagocytosis, and chemotaxis. These results suggest that mitochondria are required as triggers of neutrophil activation and that ATP release and calcium signaling regulate cell functions. Funded by NIH R01GM60475.
LIPOPOLYSACCHARIDES DIRECTLY INCREASE CALCIUM OSCILLATIONS AND INTRACELLULAR CALCIUM IN HUMAN CARDIOMYOCYTES
R. Wondergem, B. Graves, T.R. Ozment*, and D.L. Williams*. East Tennessee State University, Johnson City, TN
Lipopolysaccharides (LPS) are involved in endotoxin-mediated heart failure and chronic cardiac myopathies. We have shown previously that lipopolysaccharides (LPS) directly affect murine-derived HL-1 cardiac myocyte function via TLR-4 by inhibiting Ca2+ oscillations, by decreasing intracellular Ca2+ concentration, [Ca2+]i, and by impairing pacemaker “funny” current, If, (Wondergem et al. Am. J. Physiol. Cell Physiol. 299:C665-71, 2010). Here we report LPS effects on human, induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (iCell® Cardiomyocytes). In marked contrast to the inhibitory effects of LPS on HL-1 cells, LPS (S. enteriditis or E. coli) in a dose-dependent manner (0.01- 1.0 μg/ml) increased the rate of Ca2+ oscillations and the [Ca2+]i in iCell Cardiomyocytes. Ca2+-free external solution abolished this stimulation by LPS. Ultra-pure LPS (E. coli), which is considered a TLR-4 specific agonist, exerted identical effects as the impure LPS. Prior addition of the monoclonal-antibody blocking peptide, MAb hTLR4, attenuated the effect of ultra-pure LPS. Pam3Csk, a TLR-2 agonist, had no effect on Ca2+ oscillations or [Ca2+]i. iCell Cardiomyocytes also expressed TLR-4 as determined by flow cytometry. We conclude that LPS, acutely applied to human iCell Cardiomyocytes, results in TLR-4-dependent increases in Ca2+ oscillations and [Ca2+]i, which are consistent with a hyperdynamic response. This is in marked contrast to the hypodynamic response that we reported previously for acute effects of LPS on murine cardiomyocytes.
ROLE OF NECROPTOSIS IN D-GALACTOSAMINE AND LIPOPOLYSACCHARIDE-INDUCED HEPATIC FAILURE
H. Cho*, S. Kim, H. Choi, J. Shin, and S. Lee*. Sungkyunkwan University, Suwon, Republic of Korea
Necroptosis is a newly recognized and regulated form of necrotic cell death that has been shown to be a crucial control point for several inflammatory diseases. The kinases receptor interacting protein (RIP) 1 and RIP3, are known as key modulators of necroptosis. Fulminant hepatic failure (FHF) is a life-threatening clinical syndrome that results in massive inflammation and hepatocyte death. In this study, we aimed to examine the pattern changes of necroptosis and its role in molecular pathogenesis of D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced liver injury. Male C57BL/6 mice were given an intraperitoneal injection of necrostain-1 (Nec-1, RIP1 inhibitor; 1.8 mg/kg; dissolved in 2% dimethyl sulfoxide in phosphate buffered saline) 1 h before receiving GalN (800 mg/kg)/LPS (40 μg/kg). After GalN/LPS injection, the levels of serum alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and hepatic cleaved caspase-8 protein expression significantly increased. Moreover, GalN/LPS significantly increased RIP3 protein and mRNA expression, and RIP1/RIP3 necrosome complex formation in liver tissue. In GalN/LPS group, treatment with Nec-1 increased survival rate and attenuated the increase in serum ALT, TNF-α and IL-1β levels, cleaved caspase-8 protein expression, and RIP1/RIP3 complex formation. Furthermore, treatment with Nec-1 before GalN/LPS injection attenuated portal inflammation, hepatic necrosis and apoptosis evidenced by histological features of hematoxylin and eosin, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In conclusion, our findings suggest necroptosis is strongly associated with GalN/LPS-induced liver injury and may contribute to promotion of inflammatory process.
CHRONIC ALCOHOL INGESTION AND T CELL CYTOKINE PRODUCTION IN SEPSIS
L. Margoles, R. Mittal, J. Lyons, Z. Liang, M. Serbanescu, M.L. Ford, and C.M. Coopersmith*. Emory University, Atlanta, GA
Objectives: To identify alterations in T cell function associated with immune dysregulation and increased mortality from sepsis in the setting of chronic alcohol ingestion.
Methods: Mice received either alcohol (20% weight/volume) or water for 12 weeks followed by cecal ligation and puncture twice with a 30 gauge needle. Mice were sacrificed at 24, 72, or 120 hours and splenic lymphocytes harvested for intracellular cytokine staining (n=3-9/group). Intracellular production of IL2, IL17, TNF, and IFNγ by CD4+ and CD8+ T cells after ex vivo stimulation was assessed by flow cytometry.
Results: At 24h, the frequency of IFNγ-producing CD8+ T cells was increased in alcohol versus water-fed sham mice (11% vs 8.3%, p=0.006). In both the alcohol and water-fed groups, sepsis decreased CD8+ T cell IFNγ production compared to sham mice (7.6% vs 11%, p<0.0001 for alcohol; 5.3% vs 8.3%, p=0.003 for water). Alcohol-fed septic mice had higher CD8+ T cell IFNγ-production than water-fed septic mice (7.6% vs 5.3%, p=0.009). At 72h, IL2-producing CD4+ and CD8+ T cells decreased in alcohol-fed sham mice compared to water-fed sham controls (14.8% vs 29.7%, p=0.0001 for CD4+; 6.2% vs 16.7%, p=0.009 for CD8+). Alcohol-fed septic mice had decreased IL2-producing CD4+ and CD8+ T cells compared to water-fed septic mice (13.2% vs 26.7%, p<0.0001 for CD4+; 5.1% vs 16.9%, p=0.0004 for CD8+). At 72h, the frequency of IL17-producing CD4+ and CD8+ T cells was increased in alcohol-fed septic mice compared to water-fed septic mice (56.4% vs 42.3%, p=0.02 for CD4+; 53.9% vs 35.1%, p=0.02 for CD8+) without a change in the sham groups. There were no significant differences between the alcohol and water-fed septic mice at 120h.
Conclusion: The increased mortality from sepsis in the setting of chronic alcohol ingestion is associated with alterations in IFNγ at 24h and IL2 and IL17 at 72h. The differences in IFNγ and IL2 between the alcohol and water-fed sham groups suggest a functional change due to alcohol alone. While sepsis decreased IFNγ-production in both the alcohol and water-fed mice, the greater magnitude of change in the alcohol-fed group suggests an additive effect between sepsis and chronic alcohol ingestion. The elevated IL17 production in alcohol septic mice over water septic mice without a concurrent difference between the sham groups suggests the change is specific to sepsis in the setting of alcohol ingestion.
THE EFFECTS OF INTESTINE-SPECIFIC EPIDERMAL GROWTH FACTOR IN THE ABSENCE OF LYMPHOCYTES AND ITS ROLE IN MODULATING CROSSTALK BETWEEN THE GUT EPITHELIUM AND LYMPHOCYTES IN SEPSIS
Y. Ali, Z. Liang, J. Lyons, R. Mittal, and C.M. Coopersmith*. Emory University, Atlanta, GA
Background: Systemic and intestine-specific epidermal growth factor (EGF) significantly improves survival and intestinal integrity in septic immunocompetent mice, however, systemic administration of EGF, surprisingly, worsens survival in septic Rag-/- mice. The purpose of this study was to determine the effects of intestine-specific EGF on survival and intestinal integrity in septic Rag-/- mice.
Methods: Rag-/- C57BL/6 mice and transgenic Rag-/- C57BL/6 mice that overexpress intestine-specific EGF (Rag-/-EGF) were subjected to cecal ligation and puncture (CLP). These mice were either followed for 7-day survival (n=25/group) or tissue samples were collected at 24 hours post-operatively for analysis (n=6-14/group).
Results: When compared to septic Rag-/- mice, septic Rag-/-EGF mice demonstrated a 3.6-fold decrease in intestinal permeability (15,473 ng/mL vs. 4,225 ng/mL FD-4, p<0.05), a dampened local cytokine response (1.5- to 5-fold decrease in IL-1, IL-6, IL-10, TNF-α, and MCP-1, p<0.05), a dampened systemic cytokine response (1.7- to 4.5-fold decrease in IL-1, IL-6, IL-13, and MCP-1, p<0.05), a 1.5-fold decrease in apoptosis based on caspase-3 staining (17.6 vs. 11.9 apoptotic cells/100 crypts, p<0.05), a 1.9-fold decrease in apoptosis based on morphologic analysis (18.2 vs. 9.4 apoptotic cells/100 crypts, p<0.001), and decreases in pro-apoptotic proteins Bax and Tradd (2.5-fold and 10-fold, respectively, p<0.05). There was no significant difference in proliferation and villus length. There was no difference in survival between septic Rag-/- mice and septic Rag-/-EGF mice (48% and 52%, respectively, p=0.75).
Conclusion: This study demonstrates that the survival advantage of EGF seen in immunocompetent mice is dependent on the presence of a functional adaptive immune system, and that there is more to the improvement in survival in sepsis than improvement in intestinal integrity alone. Despite the inability to improve survival in septic Rag-/- mice, intestine-specific EGF is not associated with increased mortality as is seen in the systemic administration of EGF. These results reveal the importance of communication between the intestinal epithelium and the adaptive immune system in survival in the setting of sepsis.
A CXCL12 ANALOGUE INCREASES NEUTROPHIL RECRUITMENT AND FUNCTON IN CLP-INDUCED SEPSIS
S. Guan1, 2, H. Fan*1, A.J. Goodwin1, P. Halushka1, B. Zingarelli*3, and J. Cook*1. 1Medical University of South Carolina, Charleston, SC, 2Jilin University, Changchun, China, 3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Background: Previous studies demonstrated that the CXCL12 peptide analogue CTCE-0214 (CTCE) has beneficial effects in experimental sepsis induced by cecal ligation and puncture (CLP). We examined the hypothesis that CTCE recruits neutrophils (PMN) to the site of infection, enhances PMN function and improves the survival of mice with CLP-induced sepsis already treated with antibiotics. We investigated CTCE’s effect on miR-223 expression, a known regulator of PMN function.
Methods: CLP was performed and septic mice (n=15) were administrated Imipenem (25mg/kg) and CTCE (10 mg/kg) subcutaneously vs. vehicle control at designated intervals post-CLP. PMNs were quantified in the blood and peritoneal fluid by flow cytometry. Bacterial load in the blood, peritoneal cavity and lung was determined by Colony-forming units (CFU) counting. Phagocytic activity and reactive oxygen species (ROS) production by PMNs was measured in vitro by flow cytometry and CM-H2DCFDA florescence detection. Real-time PCR was used to measure PMN expression of miR-223. All comparisons are between CTCE and vehicle control groups.
Results: CTCE treatment increased PMN demarginalization in CLP-induced sepsis as evidenced by increased PMNs in the blood (2.4±0.57 fold at 18h, 2.9±0.63 fold at 24h respectively, p<0.05) and in peritoneal fluid (2.0±0.2 fold at 24h, p<0.05). CTCE treatment reduced bacterial load in peritoneal fluid (CFU decreased 78±8.5%, p<0.05), blood (CFU decreased 77±11.4% vs. vehicle, p<0.05) and lung (CFU decreased 79 ±8.3% vs. CLP vehicle, p<0.05). The improved PMN recruitment and bacterial clearance correlated with reduced mortality by CTCE (20% vs. 67% CLP vehicle, p<0.05). In vitro, CTCE augments PMN function by enhancing phagocytic activity (26.3±1.8%, in 10ng/ml, p<0.05), increasing intracellular production of ROS (31.9±3.7%, p<0.05) and improving bacterial killing (CFU decreased 26.5±3.2%, p<0.05). CTCE reduced PMN miR-223 expression (38±7.9%, p<0.05).
Conclusion: These composite findings support the hypothesis that CTCE is beneficial in experimental sepsis, possibly by augmenting PMN recruitment and function.
(Supported by NIH GM27673, GM67202).
SONIC HEDGEHOG-CONTAINING MICROPARTICLES FROM APOPTOTIC T CELLS MODULATE MACROPHAGE AND ENDOTHELIAL CELL RESPONSES IN VITRO
N. Feilen, S. Canipe, and M. Clemens*. University of North Carolina Charlotte, Charlotte, NC
Increased T cell apoptosis is a hallmark of sepsis. It is now recognized that apoptotic T cells release microparticles (MP) that may have biologic effects. Sonic Hedgehog (shh) is a gen product that is released in MP and modulates inflammation in liver fibrosis but their potential role in sepsis is not known. Therefore, we tested whether MP contain shh and whether these might affect macrophages and endothelial cells which are targets of inflammation. Apoptosis was induced in a T cell line (CEM) by sequential treatment with phytohemagglutinin followed by PMA and actinomysin D. Released MP were isolated by differential centrifugation. Western blot analysis showed that isolated MP contained shh. We then tested whether MP modulated the effect of LPS on iNOS induction in RAW 264.7 cells. RAW cells were treated with LPS (10ng/ml), various doses of MP or LPS + MP and iNOS induction was estimated by production of nitrite at 24 hours. As expected, LPS increased nitrite production. MP at doses less than 125 μg protein/ ml had no effect while 250 and 500 μg/ml significantly increased nitrite production. However, when given with LPS, 125 μg/ml increased nitrite production over that seen with LPS alone indicating an additive effect. This effect was significantly decreased by treatment with cyclopamine an inhibitor of the hedgehog pathway by inhibiting smoothened activation indicating that the effect of MP was mediated by shh. In separate experiments, the effect of MP was tested on endothelial cell wound healing using a scratch test in 2H11 endothelial cells. Wound healing was found to be inhibited by MP at concentrations above 20 μg/ml. These results indicate that apoptotic T cells shed microparticles containing sonic hedgehog and that these microparticles combine with LPS to increase macrophage activation and impair wound healing in endothelial cells. Moreover, vascular endothelial cells appear to be more sensitive targets since they were affected at lower doses of MP. Thus MP from apoptotic T cells or possibly other cell sources may be important modulators of the inflammatory response working via the sonic hedgehog pathway.
PRE-HOSPITAL HYPOTENSION IS ASSOCIATED WITH ALTERED INFLAMMATION DYNAMICS AND WORSE OUTCOMES FOLLOWING BLUNT TRAUMA IN HUMANS
K.W. Almahmoud*1, R.A. Namas*1, 2, A.M. Zaaqoq*1, 3, O.M. Abdul-Malak*1, R. Zamora*1, 2, A. Al-Khafaji4, T. Billiar*1, 2, and Y. Vodovotz*1, 2. 1University of Pittsburgh, Pittsburgh, PA, 2McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 3Texas Tech Health Sciences Center, Amarillo, TX, 4University of Pittsburgh, Critical Care Medicine, Pittsburgh, PA
Background: Hypotension-induced inflammation is frequently implicated in morbidity following traumatic injury. We sought to compare the dynamic, systemic acute inflammatory response to blunt trauma - with or without an episode of pre-hospital hypotension in humans.
Methods: From a cohort of 472 blunt trauma survivors studied following IRB approval, two stringently matched sub-cohorts were derived. 22 patients who sustained a pre-hospital hypotensive episode following blunt trauma (15 males and 7 females; age: 45±3.8; Injury Severity Score [ISS]: 20.7±1.8) were matched with 28 normotensive trauma patients (20 males and 8 females; age: 46.1±2.5; ISS: 20.8±1.3). Serial blood samples were obtained from all patients (3 samples within the first 24 h and then from days 1 to 7 post-injury). Twenty-three plasma inflammatory cytokines were assayed using Luminex™. NO2-/NO3- was measured using the nitrate reductase/Griess assay. Two-Way Analysis of Variance (ANOVA) was used to compare groups. Dynamic Bayesian Network (DBN) inference was utilized to infer causal relationships based on probabilistic measures.
Results: Statistically significant differences were observed in ICU length of stay [LOS], Total LOS, days on mechanical ventilator, and Marshall score between hypotensive and normotensive patients. Shock markers (shock Index, pH, lactate, and base deficit) were significantly altered in hypotensive patients. Plasma levels of IL-6, MCP-1, IL-7, IL-8, IL-10, IFN-γ, MIP-1α, IL-17, and NO2-/NO3- were significantly elevated within the first 24 h in hypotensive vs. normotensive patients. Moreover, MCP-1, GM-CSF, IL-10, IL-1β, IL-6, MIP-1α, IL-7, IL-8, IL-17, IP-10, and sIL-2Rα were significantly elevated in hypotensive vs. normotensive patients up to 7 days post-injury. DBN suggested that MCP-1 and MIG in hypotensive and normotensive patients respectively, affect plasma IL-6 production differentially.
Conclusion: Based on analysis of stringently matched cohorts, we suggest that an episode of pre-hospital hypotension post-trauma can lead to early reprogramming of inflammation, which is associated with worse outcomes. Network analysis suggests that chemokines play a central role in modulating the production of IL-6 early post-trauma.
A NOVEL METHOD TO DISTINGUISH MICROGLIA FROM INFILTRATING LEUKOCYTES AFTER TRAUMATIC BRAIN INJURY
D.M. Trahanas, C.M. Cuda, H. Perlman, and S.J. Schwulst*. Northwestern University, Chicago, IL
Background: Traumatic brain injury (TBI) activates microglia (MG) resulting in similar morphology and surface receptor expression as infiltrating leukocytes (IL). The current method for distinguishing between these populations is immunohistochemistry which requires preparation of fixed tissue and is prone to artifact.
Hypothesis: Immunomagnetic enrichment of brain leukocytes can reliably differentiate between MG and IL with flow cytometry after TBI.
Methods: C57BL/6 mice underwent TBI (n=8) or sham injury (n=8). Brains were collected at 24 hours post injury. Brain tissue was dissociated and myelin removed from cellular material. CD45+ cells were selectively enriched via magnetic bead separation. Multicolor flow cytometry was used to separate MG from IL. Data was analyzed with the statistical software program PRISM and are reported as the mean number of cells/mg brain tissue ± SEM.
Results: TBI results in an increase in both MG (10944±1277 vs. 5584±1081; p<0.02) and IL (1474±199 vs. 717±149; p<0.03) as compared to sham-injured mice. The distinction between MG and IL becomes more apparent with severe TBI when gross intracranial hemorrhage is present (Figure1).
Conclusions: Immunomagnetic enrichment of brain leukocytes followed by multicolor flow cytometry is a reliable method to distinguish between MG and IL after TBI. Unlike immunohistochemistry which only permits semi quantitative analysis from localized regions of the brain, this new method allows for the characterization and quantification of whole populations of cells from an entire brain or hemisphere.
HEPATIC STEATOSIS INCREASES SUSCEPTIBILITY TO LIVER I/R INJURY AND IS ASSOCIATED WITH INCREASES IN INFLAMMASOME COMPONENT LEVELS IN THE LIVER
C. Di Ciano-Oliveira and O.D. Rotstein*. St. Michael’s Hospital, Toronto, ON, Canada
Background: Obesity is a modern day epidemic and has considerable impact on health. One manifestation of obesity is that of fatty liver (steatosis). Individuals with fatty livers are known to be highly susceptible to insults to the liver, such as ischemia/reperfusion (I/R) (1). Recent reports indicate that the caspase-1 inflammasome plays a role in mediating damage in several models liver injury including I/R (e.g. ref. 2). Whether exaggerated inflammasome activation is responsible for the intensified injury in fatty liver has yet to be determined. We hypothesize that fatty livers are primed for inflammasome activation by a exposure to I/R injury.
Methods: Two murine models of fatty liver were employed: genetically obese ob/ob mice, and mice fed a choline and methionine deficient diet (MCD). The latter causes hepatic steatosis without causing overall obesity. To induce liver I/R, the portal triad was clamped, occluding blood flow to ~70% of the liver, during the ischemic period and was removed for the reperfusion period. Liver injury was monitored by serum ALT. The expression of inflammasome components, NLRP3, ASC and pro-caspase-1, in liver tissue was assessed by Western blotting. Activation of the inflammasome was determined by examining the serum levels of cleaved caspase-1.
Results: Both ob/ob mice and mice fed MCD diet had increased I/R-induced liver injury when compared to their controls. Specifically, serum ALT after I/R: WT mice, 3067 +/- 3284 U/L versus ob/ob mice, 33,624 +/- 6,188 U/L; normal diet mice, 350+/-297 U/L versus MCD fed mice 1603+/-256 U/L. The livers of ob/ob mice had increased baseline levels of NLRP3 and pro-caspase-1 as compared to WT controls. Upon I/R injury, abundant amounts of active caspase-1 was detectable in the serum of ob/ob mice. The livers of MCD diet fed mice had increased expression of pro-caspase-1 and ASC. Surprisingly, active caspase-1 was not detectable in injured MCD diet fed mice.
Significance: I/R injury to multiple organ systems occurs in patients sustaining trauma with major blood loss. An understanding of the mechanisms which lead to augmented liver I/R injury in obese patients and their translation into therapeutic strategies may suggest novel interventions in man.
1. Selzner & Clavien. (2001) Semin Liver Dis. 21(1):105-13.
2. Huang et.al. (2013) J Immunol. 191(5):2665-79.
TIME OF INJURY AFFECTS SYSTEMIC INFLAMMATION FOLLOWING SEVERE BLUNT TRAUMA
A.M. Zaaqoq*2, 1, R. Namas*1, K.W. Almahmoud*1, O.M. Abdul-Malak*1, D. Barclay1, T. Billiar*1, and Y. Vodovotz*1. 1University of Pittsburgh Medical Center, Pittsburgh, PA, 2Texas Tech Health Sciences Center, Amarillo, TX
Background: Circadian oscillations of the immune system, among other host factors, are key regulators of inflammation. We hypothesized that due to this diurnal variation, time of injury can affect the systemic inflammatory response and outcomes post-trauma, and tested this hypothesis by examining trajectories of circulating inflammatory mediators in blunt trauma patients injured during daytime vs. nighttime.
Methods: In a cohort of 472 blunt trauma survivors and after excluding patients with alcohol use and major surgical intervention, stringently-matched two sub-cohorts of severely injured patients [injury severity score (ISS) >20] were identified. Forty “daytime” patients (age 50.5 ± 2.9, male (M) /female (F) 28/12, ISS 22.9 ± 0.5) presented during the shortest daylight in Pittsburgh throughout the year (8:00 am-5:00 pm) and twenty “nighttime” patients (age 50 ± 4.4, M/F 13/7, ISS 22 ± 2) presented during the shortest night period (10:00 pm-5:00 am). Serial blood samples were obtained (3 samples within the first 24 h and daily from days 1-7) from all patients. Twenty-three plasma inflammatory mediators were assayed using Luminex™. NO2-/NO3- was measured using the nitrate reductase/Griess assay. Two-way Analysis of Variance (ANOVA) was used to compare groups. Dynamic Bayesian Network (DBN) was utilized to infer causal relationships based on probabilistic measures.
Results: The total length of stay was significantly prolonged in the patients injured during nighttime. Plasma levels of IL-17, sIL-2Rα, IL-1Ra, IL-5, NO2-/NO3-, MIP-1β, TNF-α and eotaxin were significantly elevated in the nighttime group within the first 24 h post-injury. IL-1Ra, IL-5, TNF-α, eotaxin, and GM-CSF were elevated in the nighttime group up to 7 days post-injury. DBN suggested that IP-10 affects both MIG and MCP-1 in the daytime group, whereas MIG affects plasma MCP-1, IL-6, and IP-10 in nighttime group.
Conclusion: Our results suggest that time of injury affects the clinical outcome in severely-injured patients, possibly implying a role for circadian variation or other factors in post-injury inflammation and outcomes.
IMPACT OF INJURY SEVERITY ON DYNAMIC INFLAMMATION NETWORKS FOLLOWING BLUNT TRAUMA
K.W. Almahmoud*1, R.A. Namas*1, 2, O.M. Abdul-Malak*1, A.M. Zaaqoq*1, 3, N. Azhar1, R. Zamora*1, 2, A.B. Peitzman*1, T. Billiar*1, 2, and Y. Vodovotz*1, 2. 1University of Pittsburgh, Pittsburgh, PA, 2McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 3Texas Tech Health Sciences Center, Amarillo, TX
Background: Outcomes of injury following trauma depend both on the extent of injury and the host’s response to injury, along with medical care. We hypothesized that dynamic inflammation networks manifest differently as a function of injury severity. To test this hypothesis, we analyzed an extensive time course of circulating inflammatory mediators in blunt trauma patients as function of injury severity score (ISS).
Methods: From a cohort of 472 blunt trauma survivors studied following IRB approval, three separate sub-cohorts were studied after matching for age and gender distribution: Mild ISS (49 patients [33 males, 16 females; age 42±1.9; ISS 9.5±0.4]); Moderate ISS: (49 patients [33 males, 16 females; age 42±1.9; ISS 19.9±0.4]) and Severe ISS: (49 patients [33 males, 16 females; age 42±2.5; ISS 33±1.1]). Serial blood samples were obtained from all patients (3 samples within the first 24 h and then from days 1 to 7 post-injury). Twenty-three plasma inflammatory cytokines were assayed using Luminex™. NO2-/NO3- was measured using the nitrate reductase/Griess assay. Two-Way Analysis of Variance (ANOVA) was used to compare groups. Dynamic Bayesian Network (DBN) inference was utilized to infer causal relationships based on probabilistic measures.
Results: Statistical differences were observed in ICU length of stay [LOS], Total LOS, days on mechanical ventilation, Marshall Score, prevalence of pre-hospital hypotension, and prevalence of nosocomial infection among the three groups. Moreover, Severe ISS group had elevated admission levels of lactate, and CPK when compared to Moderate and Mild ISS group. Plasma levels of IL-6, IL-7, IL-17, sIL-2Rα, GM-CSF, IP-10, MIG, and MCP-1 were significantly elevated in Severe ISS vs. Moderate and Mild ISS. DBN suggested that IL-6 production in Severe ISS was affected by MCP-1, MIG, and IP-10; by MCP-1 and MIG in Moderate ISS; and by MIG alone in Mild ISS.
Conclusion: Our results suggest that ISS correlates linearly with morbidity and prevalence of infection. Moreover, over the initial 24 h post-injury, different chemokine networks - manifesting as different dynamic pattern - affect IL-6 production as a function of injury severity.
ABERRANTLY ACTIVATED NEUTROPHILS DEPRESS MONOCYTE DERIVED DENDRITIC CELL (MDDC) FUNCTION BY INHIBITING NOTCH-2 ACTIVATION
G. Bandyopadhyay*, J. Cullen, P.E. Bankey*, and C.L. Miller-Graziano*. University of Rochester Medical Center, Rochester, NY
Some severely injured trauma patients (Pt) experience post-injury CD1a+MDDC differentiation defects and increased infection risk. CD1a+MDDC are critical to initial infection defense. These Pt also experience increased circulating neutrophil (PMN) numbers, prolonged PMN activation by trauma induced mediators (such as Thrombospondin-1 [TSP-1] & IL-17) and delayed PMN apoptosis. Activated PMN can alter MDDC function in other diseases. We found that pre-exposure to certain Pts’ activated PMN resulted in decreased control monocyte (MO) to CD1a+MDDC differentiation [58.8% compared to control PMN-treated MO]. New reports suggest that triggering of Notch2 signaling pathways plays a critical role in immunostimulatory MDDC differentiation and function as well as IL-17 induction. We hypothesized that post-injury activated PMN alter MO Notch signaling to induce CD1a+MDDC defects. Experimental Design: Control PMN were pretreated with TSP-1 mimic (TSP-PMN), or IL-17(IL-17-PMN) or IL-10(IL-10-PMN) overnight then washed. Control autologous MO were co-cultured with these pre-treated PMNs for two more days [MO: PMN = 1:10], then re-isolated using magnetic beads. These re-isolated MO were in vitro differentiated to CD1a+MDDC using IL-4 + GM-CSF. DC differentiated from cultured MO [Cnt MDDC] or MO previously cocultured with untreated PMN [CPMN-MDDC] served as controls. The CD1a+ MDDC differentiation and intracellular activation of Notch-1 and 2 were assessed by flowcytometry. Results: Exposure of MO to activated PMN resulted in decreased CD1a+ MDDC differentiation [CD1a+ MDDC percentage- 63.7± 6.9%, 53.6±0.9%, 41±11.8%, 40.1±4.1% and 40.5±5.1% in Cnt MDDC, CPMN-MDDC, TSP-PMN treated, IL10-PMN treated & IL-17-PMN-treated MDDCs respectively]. The few differentiated CD1a+MDDC from MO previously exposed to activated PMN had decreased active Notch-2 levels [Notch-2 MFI- 1041±90.1, 1230±343.9, 865.1±108.5, 703±32 and 379.3±136 in Cnt MDDC, CPMN-MDDC, TSP-PMN treated-, IL-10-PMN treated- & IL-17-PMN- treated MDDCs respectively]. Their immunostimulation of T cells was also depressed. Conclusion: Our data suggest that certain trauma mediator activated PMN can negatively affect MO differentiation to immunostimulatory CD1a+MDDC by inhibiting Notch-2 signaling.
URIC ACID DRIVES INFLAMMASOME ACTIVATION FOLLOWING RADIATION INJURY
V.M. Stoecklein1, 2, A. Osuka*1, and J.A. Lederer*1. 1Harvard Medical School/Brigham and Women, Boston, MA, 2Ludwig-Maximilians-University, Munich, Germany
Radiation exposure causes extensive damage to cells and tissues, resulting in local and systemic inflammatory responses. The inflammasome pathway is central to initiating inflammatory responses to cell death and danger-associated molecular patterns (DAMPs). We therefore hypothesized that the inflammasome may signal acute and chronic immune responses to radiation injury. We used a whole-body gamma ray mouse model of radiation and found that radiation induces dose-dependent inflammasome activation in various immune cell subsets. Time course analysis furthermore showed that inflammasome activation is delayed until 1 day after radiation injury and was sustained until day 7. Because of the delayed nature of the observed inflammasome activation we reasoned that activation of the inflammasome following radiation is secondary to sensing of a DAMP. Since the wide-spread cell death that occurs after radiation injury is known to lead to the release of the DAMP uric acid, we looked into the possibility that sensing of uric acid is the main driver of inflammasome activation following radiation injury. To test this hypothesis we treated mice with the xanthine oxidase inhibitor allopurinol before and after radiation injury. We found that inflammasome activation is blunted by allopurinol treatment. In summary, our study demonstrated that radiation injury leads to activation of the inflammasome pathway secondary to sensing of uric acid. Targeting uric acid might be a promising approach to modify the inflammatory response to radiation injury.
HEPATOCYTE-DERIVED EXOSOMES REGULATE LIVER RECOVERY AND REGENERATION AFTER ISCHEMIA/REPERFUSION IN MICE
H. Nojima, G.C. Wilson, C. Freeman, R.M. Schuster, J. Blanchard, M.J. Edwards, E. Gulbins, and A.B. Lentsch*. University of Cincinnati, Cincinnati, OH
Introduction: Exosomes are small extracellular membrane vesicles that originated from a variety of different cell types, including hepatocytes. Exosomes may play important roles in intercellular communication. Exosomes are also heterogeneous in content and exosome release is regulated by several mechanisms. Our previous work has demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after ischemia/reperfusion (I/R) injury. In the present study, we sought to determine if hepatocyte-derived exosomes mediate the CXCR1 and/or CXCR2-mediated regulation of liver recovery and regeneration after I/R injury.
Methods: Murine models of partial ischemia/reperfusion (I/R) were used to study liver Recovery and regeneration. The number of exosomes isolated from serum and cell culture media was assessed by enzyme-linked immunosorbent assay (ELISA). Hepatocyte proliferation was determined via DNA corporation, and liver regeneration was assessed by immunohistochemical staining for proliferating cell nuclear antigen (PCNA).
Results: Exosome release from hepatocytes was found to be dependent upon CXCR1 and CXCR2. CXCR1-/- hepatocytes produced fewer exosomes, whereas CXCR2-/- hepatocytes produced more exosomes compared to their wild-type controls. Exosomes from wild-type hepatocytes induced dose-dependent increases in hepatocyte proliferation in vitro. Interestingly, exosomes from CXCR1-/- hepatocytes had no effect on hepatocyte proliferation, whereas exosomes from CXCR2-/- hepatocytes had similar effects as those from wild-type cells. Mice treated with hepatocyte-derived exosomes showed a dose-dependent increase in hepatocyte proliferation after I/R.
Conclusion: Together, these data suggest that exosome release by hepatocytes is controlled by CXCR1 and CXCR2 and that hepatocyte-derived exosomes promote hepatocyte proliferation and liver regeneration after I/R injury.
AUGMENTED HEPATIC ER STRESS IS ASSOCIATED WITH INCREASED MORBIDITY AND MORTALITY
A. Abdullahi1, 2, E. Bogdanovic1, 2, L. Diao1, 2, and M.G. Jeschke*1, 2. 1Sunnybrook Research Institute, Toronto, ON, Canada, 2University of Toronto, Toronto, ON, Canada
Introduction: The liver, with its metabolic, inflammatory, and acute-phase functions plays a critical role in post-burn patient outcome and recovery. In fact it has been shown that liver impairment worsens the prognosis in patients with thermal injury. A key element in the hepatic post-burn response appears to be endoplasmic reticulum (ER) stress. The ER is a multifunctional organelle within hepatocytes that is required for protein folding and processing. We have previously shown that burns cause ER stress within the liver, impairing protein folding and leading to hepatocyte apoptosis. Here we examine whether augmented ER stress impacts survival post thermal injury.
Methods: Mice were injected intraperioneally with Tunicamycin (1μg/g body weight). After 24h, the mice were subjected to a 30% total body surface area (TBSA) scald burn. Survival of the mice post burn was evaluated in addition to ER stress signaling pathways, liver structure and function, mitochondrial integrity, mitochondrial associated membranes (MAM), and associated signaling pathways were analyzed. Genomic changes were also analyzed in the liver using GeneChip analysis.
Results: Tunicamycin administration post burn injury induced a robust hepatic ER stress response confirmed by increased expression of pancreatic ER kinase (PERK), inositol requiring transmembrane kinase/endonuclease-1 (IRE-1) and activating transcription factor-6 (ATF6). While almost no mice died from the burn or Tunicamycin groups, mice receiving both Tunicamycin and a burn had a 90% mortality. Histological and transmission electron microscopy (TEM) analysis revealed significant hepatic damage including hepatocyte apoptosis and elevated serum aminotransferase enzymes, mitochondrial dysfunction, damage of MAM and alterations in ER integrity in these mice. Additionally, mammalian target of rapamycin (mTOR) which controls MAM integrity and mitochondrial function was depleted in these mice. Genomic analysis confirmed the above data as well as indicating substantial reduction in genes involved in oxidative metabolism, cholesterol biosynthesis and transport.
Conclusion: Augmented ER stress is associated with increased mortality after burn. Thus, we propose that specific interventions focus on modulating the ER stress response post thermal injury, as it may protect burn patients from the deleterious effects on the liver and improve patient outcome.
DIVERGENT EFFECTS OF NECROTIC AND APOPTOTIC LUNG EPITHELIAL CELLS IN THE PATHOGENESIS OF CHEST TRAUMA INDUCED SEPTIC ACUTE LUNG INJURY (ALI)
C. Hohmann1, 2, P. Kellermann2, A. Schultze2, B. Klohs2, M. Huber-Lang*2, and M. Perl*3, 2. 1University of Leipzig, Leipzig, Germany, 2Dept. of Orthopedic Trauma, University of Ulm, Ulm, Germany, 3BG-Trauma Center Murnau, Murnau, Germany
Necrotic and apoptotic cell death both appear to be involved in the pathogenesis of ALI. However, the direct immunomodulatory effects of necrotic or apoptotic cells ac-cumulating in the lung during ALI still remain unclear. To study this, lung epithelial cells (MLE12) rendered either apoptotic via UV-light exposition or necrotic by repeated freeze-thaw cycles were instilled at 1x10^4 (low) or 1x10^7 (high) concentrations into lungs of C56Bl/6 mice at 4hrs after blunt chest trauma followed cecal ligation and puncture 20hrs thereafter. 12hrs later lung tissue, plasma, and bronchoalveolar lavage fluid (BALF) were harvested. Lung cytokines were quantified by sequential ELISA, lung active caspase-3 (C-3) via western blotting. TUNEL staining was performed in lung sections. Total protein in BALF and lung histology (H&E) were assessed. N=6-8/group, Two-way ANOVA, SNK, p<0.05. Instillation of 1x10^4 necrotic cells markedly increased concentrations of KC, G-CSF and MIP-2 in the BALF and of IL-6 and IL-10 in the plasma, whereas the deposit of apoptotic cells did not alter the cytokine profile when compared to saline treated ALI controls. In lung tissue, instillation of either 1x10^4 or 1x10^7 necrotic - but not apoptotic - cells resulted in significantly enhanced levels of KC and MCP-1 compared to trauma control animals. TUNEL sections and C-3 analysis revealed increased lung apoptosis following instillation of apoptotic cells, whereas application of necrotic cells did not induce secondary lung apoptosis when compared to control animals. Taken together instillation of a low number of necrotic lung epithelial cells induced inflammation and progressed lung damage in a clinically relevant double hit model of chest trauma induced septic ALI. Apoptotic cells did not exert such proinflammatory effects but induced secondary lung apoptosis.(Supported by DFG-PE 908/2-1).
AMELIORATION OF ENDOTOXEMIC CARDIAC DEPRESSION IN AGING MICE BY IL-37: A CRITICAL ROLE OF SUPPRESSION OF MYOCARDIAL PRODUCTION OF MCP-1
H. Slimani, Y. Zhai, N.G. Yousif, L. Ao, D. Fullerton, C. Dinarello, and X. Meng*. University of Colorado, Aurora, CO
Major surgeries performed in the elderly are increasing with the increase in life expectancy, and such surgeries frequently cause endotoxemia and the systemic inflammatory response. Aging hearts have exaggerated responses to injurious stimuli. Thus, it is important to protect aging hearts during major surgeries. Endotoxin activates TLR4 to induce the production of cardiodepressant cytokines, including TNF-α and IL-1β. IL-37 is an anti-inflammatory cytokine and has been shown to suppress the inflammatory responses caused by endotoxin. We tested the hypothesis that IL-37 protects the aging heart against endotoxemic cardiac depression via suppression of myocardial inflammatory responses.
Methods: WT and IL-37 transgenic (tg) mice, males of adult (4-6 months) and aging (18-20 months), were treated with endotoxin (E. coli 011:B4; 0.5 mg/kg i.v.). Left ventricular (LV) function was measured with a pressure-volume microcatheter at 6 h after injection. Plasma and myocardial tissue homogenate were prepared for analysis of MCP-1, TNF-α and IL-1β by ELISA.
Results: Endotoxin caused greater depression of LV function in WT aging mice that was associated with exaggerated myocardial production of MCP-1, TNF-α and IL-1β in comparison to WT adult mice. Aging IL-37Tg mice had improved ejection fraction and cardiac output after LPS injection that were associated with lower myocardial levels of MCP-1, TNF-α and IL-1β. To determine the role of MCP-1 in myocardial production of TNF-α and IL-1β, as well as in LV dysfunction, we treated WT aging mice with MCP-1-neutrlizing antibody and found that neutralization MCP-1 reduced myocardial TNF-α and IL-1β levels and improved LV function.
Conclusions: Endotoxemia results in worse LV functional injury in aging WT mice. MCP-1 plays an important role in mediating the production of cardiac depressant cytokines and resultant LV dysfunction. IL-37 improves LV function in aging mice during endotoxemia through suppression of myocardial production of MCP-1 and cardiodepressant cytokines.
ANALYSIS OF PRINCIPAL DRIVERS AND DYNAMIC NETWORKS OF SYSTEMIC INFLAMMATION SUGGESTS COMPLEX INTERACTION BETWEEN AGE AND INJURY SEVERITY
R.A. Namas*1, 2, O.M. Abdul-Malak*1, Q. Mi1, K.W. Almahmoud*1, A.M. Zaaqoq*1, 3, D. Barclay1, A.B. Peitzman1, T. Billiar*1, 2, and Y. Vodovotz*1, 2. 1University of Pittsburgh, Pittsburgh, PA, 2McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 3Texas Tech Health Sciences Center, Amarillo, TX
Background: Both age and injury severity are associated with dysregulation of post-trauma inflammation. Using data-driven modeling, we sought to elucidate principal drivers and dynamic networks of the post-traumatic inflammatory response across age and injury severity in blunt trauma patients.
Methods: From a cohort of 472 blunt trauma survivors, and after excluding 8 patients with incomplete datasets, we identified 3 sub-cohorts using supervised K-mean clustering (MATLAB™): Young (18-37 y), Middle-aged (38-60 y), and Old (>60 y). Their demographic characteristics were as follows: Young (146 patients: males [M]/females [F] 108/38; age 26±0.4; injury severity score [ISS] 23±1]); Middle-aged (184 patients: M/F 107/77; age 49±0.5; ISS 19±0.8) and Old (134 patients: M/F 84/50; age 72±0.7; ISS 17±0.7). 3 samples within the first 24 h and then from days 1 to 7 post-injury were obtained from all patients. Plasma inflammatory mediators were assayed using Luminex™. 2-Way ANOVA was used to compare groups. Principal Component Analysis (PCA) and Dynamic Bayesian Networks (DBN) were utilized to identify inflammatory drivers and networks, respectively.
Results: Young group had significantly higher ISS and days on ventilator when compared to Middle-aged and Old. To exclude the confounding factor of ISS among age groups, we stratified age groups based on injury severity (Mild, Moderate, and Severe ISS). There was no difference in clinical outcomes among age groups in the Mild and Moderate groups. In severe ISS, Old and Middle-aged patients had significantly longer hospital length of stay, while the Old group stayed longer on mechanical ventilation vs. Young. Plasma IL-5, IL-7, and IL-10 were elevated in Old vs. Young patients. CC and CXC chemokines were significantly elevated in Old vs. Young and Middle-aged patients. PCA suggested a strong Th2 response in Old patients, with a more robust innate response in Young and Middle-age. DBN suggested that MIG in Middle-aged and both MCP-1 and MIG in Young and Old patients affect the production of IP-10, IL-1RA, IL-6, MIP-1β, and sIL-2Rα.
Conclusion: Our results suggest a complex, dynamic interaction between age and injury severity in driving systemic inflammation post-trauma, and point to an enhanced chemotactic and anti-inflammatory response with robust adaptive immunity as a function of age.
MURINE LUNG CANCER DECREASES SEPSIS SURVIVAL AND WORSENS SEPSIS-INDUCED IMMUNE COMPROMISE
J. Lyons, R. Mittal, Z. Liang, L. Margoles, M.L. Ford, and C.M. Coopersmith*. Emory University, Atlanta, GA
Objective: Develop a novel murine model of sepsis in the setting of cancer and characterize the immune response under these conditions.
Methods: B6 mice were given subcutaneous injections of a murine lung cancer cell line (LLC-1) and followed for 3wks to allow tumor growth. Cancer mice and age-matched unmanipulated controls were then subjected to cecal ligation and puncture (CLP), and either followed for survival to 7d (n=11/group) or sacrificed at 24h (n=8-9/group). After sacrifice, peritoneal fluid (PF) and blood were analyzed for cytokine profiles (TNF-α, IFN-γ, IL-1, IL-6, IL-10, IL-13). Splenocytes were assayed by flow cytometry for immune subset frequency.
Results: Mice with cancer had worsened 7-day CLP survival vs. controls (9% vs. 82%, p=0.002). A significant decrease was seen in the frequencies of both CD4+ and CD8+ T cells (11.4%±1.8 vs. 3.9%±1.0, p=0.0003, 7.6%±0.8 vs. 3.2%±0.8, p=0.003; Figure 1). Absolute numbers of CD4+ and CD8+ cells were also decreased. PF of cancer mice showed a decrease of pro-inflammatory cytokines IL-1 and IL-6 (340±48pg/ml vs. 2134±506pg/ml, p=0.002, 3422±139pg/ml vs. 27394±6916pg/ml, p=0.017). The remaining cytokines listed above did not vary significantly in either PF or blood.
Conclusion: Pre-existing murine lung cancer profoundly worsens survival of sepsis. This decrease is potentially attributable to a worsening of sepsis-induced immune dysregulation, characterized by significant loss of splenic T cells and diminished production of pro-inflammatory mediators at the site of injury.
OBESITY IS ASSOCIATED WITH IMMUNE DYSFUNCTION AND MORTALITY FOLLOWING MURINE POLYTRAUMA
R.D. Winfield*, R.E. Southard*, A. Fuchs, I. Turnbull, S. Ghosh, C. Davis, N. Karlow, J. Hilliard, and G.V. Bochicchio. Washington University in St. Louis, St. Louis, MO
Introduction: Obesity is associated with greater complication rates following severe injury. The obese have evidence of immune dysfunction following trauma; the nature of the dysfunction and relationship to outcome remains poorly characterized. We hypothesized that obesity would be associated with cell-specific immune dysfunction after traumatic injury and that this would impact outcome.
Methods: Cohorts of male C57Bl/6J mice were raised to 20-22 weeks of age. Obesity was induced in one cohort utilizing a diet consisting of 60% kcal fat while control animals were raised on a diet with 10% kcal fat. Upon reaching the appropriate age, animals were subjected to polytrauma consisting of hepatic laceration, pseudofracture (proximal hind leg soft tissue crush injury followed by injection of isogenic bone homogenate), and 15% hemorrhage. At fixed intervals, animals were euthanized and blood collected for FACS analysis of leukocyte populations. In a separate experiment, survival was determined.
Results: Prior to injury, obese animals were significantly heavier than control (42 vs. 31 gm, p<0.001). Pre-injury analysis demonstrated no differences in neutrophil, dendritic cell, or monocyte frequency in blood samples from the two groups. At six hours post-injury, obese and control animals showed increased percentages of neutrophils (75 and 73% respectively, NS) as well as inflammatory monocytes (5 and 6%, NS) that persisted to 18 hours. At 24 hours, frequencies of these cell subsets had decreased considerably in control mice. In contrast, obese animals showed persistent elevation in the percentage of circulating neutrophils (80%; control: 39%, p<0.001) and inflammatory monocytes (5%; control: 2%, p<0.001). By 48 hours, both groups had defervesced to a similar level. In survival analysis, no animal had expired at 48 hours; however, by 96 hours, all obese animals were dead or moribund; no control animals expired.
Conclusions: Obese animals show a differential inflammatory response to polytrauma noted by prolonged neutrophilia and prolonged increase in circulating inflammatory monocytes at 24 hours. There was a significant survival difference between obese and control animals with demise in the obese by 96 hours. We speculate that early inflammatory differences seen in obese animals correlate with this survival difference and may partially explain poor outcome in obese trauma patients.
INTERACTIONS BETWEEN EXTRACELLULAR HISTONES AND THE NLRP3 INFLAMMASOME MEDIATE ACUTE LUNG INJURY
J.J. Grailer, M. Kalbitz, F. Zetoune, and P. Ward*. University of Michigan Medical School, Ann Arbor, MI
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) often results from sepsis and from hemorrhagic shock. The pathophysiologic mechanisms of ALI/ARDS are not fully understood. Inflammasomes are multi-protein intracellular innate immune pathways implicated in a myriad of inflammatory disorders. The NLRP3 inflammasome senses pathogen and danger associated molecular patterns, ultimately resulting in the caspase-1-dependent processing and secretion of interleukin (IL) -1β and IL-18. Using a standard model of ALI in mice, featuring airway instillation of E. coli lipopolysaccharide (LPS), ALI severity was significantly attenuated in NLRP3-deficient (NLRP3-/-) and caspase-1-/- mice, compared to wild type mice. IL-1β levels in bronchoalveolar lavage fluids (BALF) were severely reduced in NLRP3-/- and caspase-1-/- mice during LPS ALI. Extracellular histones also appeared in the BALF and had the ability to directly activate the NLRP3 inflammasome in vitro and during LPS-induced ALI in vivo. Antibody-mediated neutralization of histones significantly reduced IL-1β levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K+, increased [Ca+2]i, and generation of reactive oxygen species, which are known features of the NLRP3 activation pathway. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism for inflammatory propagation. Extracellular histones and IL-1β levels in BALF were elevated in other ALI models (C5a- and IgG immune complex-induced) suggesting the presence of a common inflammatory mechanism. These data indicate that interactions between extracellular histones and the NLRP3 inflammasome mediate ALI. Such findings suggest novel targets for the treatment of ALI, for which there is currently no FDA-approved efficacious drug.
A NEW CECAL SLURRY PREPARATION PROTOCOL WITH IMPROVED LONG-TERM REPRODUCIBILITY AS AN ANIMAL MODEL OF SEPSIS
H. Saito*1, 2, B.J. Hacker1, A.M. Steele2, M. Saito1, and M.E. Starr*1. 1University of Kentucky, Department of Surgery, Lexington, KY, 2University of Kentucky, Department of Physiology, Lexington, KY
Background: While cecal-ligation and puncture (CLP) is considered the gold standard model for sepsis research, this model may not be preferable for certain experiments where animals of unusually different sizes are compared or when animals are under different dietary regiments and/or have cecums of exceptionally different sizes. Intraperitoneal injection of cecal slurry (CS) is an alternative method for inducing polymicrobial sepsis in animals. The currently accepted protocol for CS preparation with 5% dextrose solution does not allow for long-term storage, thus the CS has to be prepared each time an experiment is to be performed.
Objective: The objective of this study was to develop an improved protocol for CS-induced sepsis that allows for long-term storage with reproducible results.
Methods: Cecal contents were collected from ten C57BL/6 mice, weighed, passed through a sterile mesh and diluted in one of five different buffer solutions: PBS, 5% dextrose/water, 5% glycerol/PBS, 10% glycerol/PBS, or 15% glycerol/PBS. The CS stocks were dispensed into aliquots, and stored at either 4, −20, or −80°C. Viability of bacteria was assessed by the colony formation unit calculated after plating samples on agar plates. To test sepsis-inducing efficacy, various amounts of the CS stocks kept in glycerol/PBS at −80°C were injected to young (4-mo-old), middle-aged (13-mo-old), and aged (25-mo-old) male C57BL/6 mice, and mortality rates compared.
Results: Nearly 100% of bacterial viability was maintained for 6-weeks or longer if the CS was prepared in 5%, 10%, or 15% glycerol/PBS and kept at −80°C. Bacterial viability was significantly lower if stored in 5% dextrose or PBS alone. Storage at 4 or −20°C, as compared to −80°C, resulted in much lower bacterial viability regardless of type of buffer used. Mouse survival studies clearly showed dose- and age-dependent mortality with high reproducibility when using the same stock for over a 6-month period.
Conclusions: The cecal slurry injection model of sepsis is useful, particularly in cases when the CLP model is not preferable. This new cecal slurry preparation protocol using glycerol instead of dextrose is durable for preservation by freezing at −80°C without significant loss of bacterial viability for several months, allowing for experiments to be performed more conveniently and with higher reproducibility.
LIPOXIN A4 PROMOTES BLOOD BACTERIA CLEARANCE IN SEPSIS BY INCREASING NEUTROPHIL PHAGOCYTOSIS AND APOPTOSIS
B. Wu, J. Walker, B. Spur, A. Rodriguez, and K. Yin*. Rowan School of Medicine, Stratford, NJ
In sepsis, hyperactivation of neutrophils can lead to tissue injury while neutrophil dysregulation with reduced migration, decreased free radical production and phagocytosis may impair the host’s ability to clear infection. Lipoxin A4 (LXA4) is a pro-resolution lipid mediator which inhibits neutrophil function. As LXA4 reduces neutrophil migration and free radical generation, the role of LXA4 in regulating neutrophil function for bacterial clearance is unclear. Using the cecal ligation and puncture (CLP) rat model of sepsis we show that LXA4 (7 μg/kg, i.v.) administered 1h after surgery, reduced blood bacterial load compared to CLP rats given vehicle saline. In these animals, LXA4 increased peripheral blood neutrophil apoptosis, decreased free radical production by 2-fold and augmented phagocytic ability by 10-fold. Furthermore, the results indicated that LXA4 -induced apoptosis by a free radical-independent mechanism. To investigate if LXA4 acted directly on neutrophils, peripheral blood leukocytes were taken from CLP rats 1h after surgery. LXA4 (300 nM) decreased free radical production without affecting either apoptosis or phagocytosis in peripheral blood neutrophils. Taken together, LXA4 increased bacterial clearance in peripheral blood by enhancing neutrophil phagocytic ability and apoptosis without excessive free radical generation. This neutrophil phenotype is most likely the most efficient at clearing pathogen without excessive activation leading to tissue injury. As LXA4 affected neutrophil function by both direct and indirect mechanisms the data suggest that LXA4 promotes neutrophil mediated bacterial clearance by multiple mechanisms.
CILOSTAZOL INHIBITS HMGB1 RELEASE IN LPS-ACTIVATED RAW 264.7 CELLS THROUGH AMPK- AND P38 MAPK-DEPENDENT HO-1 INDUCTION AND INCREASES THE SURVIVAL RATE OF SEPTIC MICE
Y. Ko, H. Kim, and K. Chang*. Gyeongsang National University, Jinju, Republic of Korea
High-mobility group box 1 (HMGB1) has been recognized as an important cytokine in the late phase of sepsis. We investigated whether cilostazol, antiplatelet agent, protects mice against cecal ligation and puncture (CLP)-induced sepsis and shows anti-inflammatory action in LPS-treated RAW264.7 cells via HMGB1 regulation. Cilostazol treatment increased AMPK phosphorylation and heme oxygenase-1 (HO-1) expression in a time- and concentration-dependent manner in RAW 264.7 cells. In these cells, the expression of HO-1, but not p-AMPK, was inhibited by SB203580, a p38 MAPK inhibitor. In addition, cilostazol significantly reduced LPS-activated HMGB1 release, which was reversed by both SB203580 and the silencing of HO-1 or AMPK. Interestingly, the increased expression of HO-1 in response to cilostazol was significantly reduced by silencing AMPK, whereas the increase in p-AMPK levels induced by cilostazol was not reduced in cells transfected with siHO-1-RNA, indicating that the induction of HO-1 depends on AMPK activity. The inhibition of HMGB1 by cilostazol was antagonized by either compound C or ZnPPIX. LPS-stimulated NF-κB luciferase activity was significantly inhibited by cilostazol but antagonized by SB203580. Finally, the administration of cilostazol increased the survival rate and reduced the serum HMGB1 levels and lung injury of CLP-septic mice, and these effects were antagonized by ZnPPIX. We concluded that cilostazol reduces the levels of HMGB1 in LPS-activated RAW 264.7 cells and increases the survival rate of mice with CLP-induced sepsis, in which p38 MAPK and AMPK play pivotal roles in the induction of HO-1.
MIR-223 IS ESSENTIAL FOR MESENCHYMAL STEM CELL-ELICITED CARDIO-PROTECTION IN POLYMICROBIAL SEPSIS
X. Wang1, W. Huang1, K. Essandoh1, G. Liu1, Y. Yang1, Y. Wang1, C.C. Caldwell1, B. Zingarelli*2, and G. Fan*1. 1University of Cincinnati, Cincinnati, OH, 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in polymicrobial sepsis induced by cecal ligation and puncture (CLP). However, the underlying mechanisms remain obscure. Recently, we reported that loss of pre-miR-223 aggravated cardiac dysfunction and mortality in polymicrobial sepsis. Thus, we asked if miR-223 plays any role in MSC-mediated cardio-protection in sepsis. To address this issue, MSCs harvested from the bone marrow of pre-miR-223 deficient (miR-223-/-) and wild-type (miR-223+/+) mice were injected to male wild-type mice via the tail vein (1×10^6 cells/mouse) 1 h after CLP (a single through-and-through puncture with an 18-gauge needle). Lung fibroblasts and PBS were used as controls. The lethality of mice receiving fibroblasts or PBS was not different (83% and 88%, respectively, at 32h after CLP). The treatment of mice with miR-223+/+ MSCs exhibited 8% mortality, whereas the mice receiving miR-223-/- MSCs showed 71% mortality at 32h post-CLP. Next, cardiac function was measured using echocardiography in these MSC-treated mice at 12 h post-CLP. We observed that the left ventricular ejection fraction (EF%) and fractional shortening (FS%) both were significantly improved in miR-223+/+MSC-injected mice, but not in miR-223-/-MSC-treated mice, compared with controls (n=8). Furthermore, the results of ELISA analysis for serum cytokine levels showed that CLP-induced elevation of IL-6 and TNF-α in controls was greatly reduced in miR-223+/+MSC-delivered mice, but not in miR-223-/- MSC-treated mice. Using a luciferase reporter assay, we identified that miR-223-5p interacted with the 3-UTR of TNFR1. Western-blotting analysis also showed that the levels of TNFR1 were dramatically increased in miR-223-/- MSCs compared to miR-223+/+MSCs. Given that TNF-α/TNFR1 are well known to induce MSC apoptosis, we therefore added TNF-α (100 pg/ml) to the cultured MSCs for 3 h, and apoptosis was measured using a TUNEL-staining and a DNA fragmentation-detection kit. The results from both methods consistently indicated that the degree of apoptosis was much higher in miR-223-/- MSCs than miR-223+/+MSCs. Taken together, our results suggest that loss of pre-miR-223 in MSCs up-regulates TNFR1 expression, leading to transplanted miR-223-/-MSC death in CLP mice. Thus, miR-223 might be required for MSC-mediated cardio-protection in sepsis.
SERIAL ANALYSIS OF TRAUMA PATIENT LEUKOCYTE MRNAS ENCODING PROTEINS INVOLVED IN COAGULATION
R.R. Meyer1, M. Bhattacharjee1, N. Varghese1, M. Sharma1, A.E. Pusateri*2, and B.H. Brownstein*1. 1Washington University School Medicine, St Louis, MO, 2US Army Medical Research and Material Command, Fort Detrick, MD
Introduction: In a study of leukocyte mRNA taken longitudinally from trauma patients in Glue Grant studies, there was differential expression of some mRNAs for coagulation proteins. We have currently explored changes in mRNA abundance for all known coagulation, anticoagulant and complement proteins, all of which play a role in coagulation, and use systems biology to understand coagulopathy after injury.
Objective: To monitor the changing mRNA abundance for proteins involved in coagulation, and to characterize their pattern of expression over time from injury. We focus on three classes of mRNAs; coagulation, anticoagulant and complement.
Methods: Blood from 167 trauma patients at specific intervals from <12h post-trauma out to 28 d. Leukocytes were purified, RNA was extracted, labeled, and hybridized against an Affymetrix GeneChip®. We examined mRNAs specifying proteins for coagulation, anticoagulation, and complement. Multivariate analysis was applied to determine differential expression from uninjured controls, and network analyses were used for visualizing molecular interactions.
Results: For the first 24h, 8, 3h bins representing intervals of time from injury revealed that 8 of 19 coagulation protein mRNAs increased, and 2 of 19 decreased. For anticoagulant mRNAs, 13 of 41 were increased, and 5 of 41 were decreased. For complement mRNAs, 19 of 59 were increased, while 5 of 59 were decreased. Levels of differential expression ranged from 1.2 fold to 11 fold, and all 3 classes of mRNAs were variably expressed. In a previous Glue Grant study, human volunteers were given endotoxin, and their leukocyte mRNAs were analyzed. We re-analyzed those data, and scored for the 3 classes of mRNAs, and found that for coagulation, 13/19 were common to trauma and endotoxin and 3/19 of the mRNAs responded to endotoxin alone. The anticoagulant group showed 14/41 mRNAs were common to both conditions, but 17/41 were seen in the endotoxin data.
Discussion: Why do leukocytes express protein mRNAs as a consequence of trauma? Since all 3 protein mRNA classes are expressed in trauma, a panel of biomarkers could be diagnostic for coagulopathy. It has been hypothesized that the first 24-36h post injury are the most important times for understanding coagulopathy. That there are sustained levels of regulated genes for all 3 protein classes beyond 24h suggests a chronic phase of coagulopathy.
HUMAN STORED RED BLOOD CELL SUPERNATANT LEADS TO SUPPRESSION OF PRO-INFLAMMATORY CYTOKINES AND INHIBITS LEUKOCYTE PHAGOCYTOSIS AFTER TRAUMA IN A MURINE MODEL
K.R. Zettel*, K. Hart, T. Billiar*, and M.D. Neal*. University of Pittsburgh, Pittsburgh, PA
Background: Blood transfusion in trauma patients is associated with an increased risk of nosocomial infection, and this risk has been shown to increase with the transfusion of packed red blood cells (PRBCs) of advanced storage age. The mechanism by which transfusion of older blood leads to increased infectious complications is poorly understood. We hypothesize that soluble mediators in human PRBCs may suppress microbial clearance and that increased storage duration would decrease leukocyte phagocytosis.
Methods: C57BL/6 mice were subjected to trauma and 25% blood volume hemorrhagic shock (THS) for 2hrs, followed by resuscitation with either lactated ringers (LR), or human PRBC supernatant from either 5 or 42 days of storage. Mice were sacrificed 48hrs after THS and splenocytes were cultured with concanavalin A for 48hrs and then analyzed by ELISA. PRBC supernatant effect on pathogen clearance was assessed by co-culturing peritoneal leukocytes with splenocytes in either media, 20% 5d PRBC or 42d PRBC supernatant for 48hrs. Cells were resuspended and co-cultured with 1.4x10^3 Colony forming units (CFU) Candida albicans (CA). CA CFU were determined by plating serial dilutions into sabouraud agar plates and counting after overnight incubation.
Results: Splenocytes from mice resuscitated with 5d PRBC supernatant secreted less IFN-γ (475±84) than those resuscitated with LR (630±0). Mice resuscitated with 42d PRBC supernatant secreted less IFN-γ (288±95) than either LR or 5d PRBC supernatant (p<0.05). To assess the effect on phagocytosis in the presence of 5d or 42d PRBC supernatant, peritoneal leukocytes cultured with splenocytes for 48hrs were subjected to a pathogen burden of CA. Splenocyte/leukocyte mixture in the presence of 5d PRBC supernatant had decreased clearance of CA compared to media alone (405±47 vs 352±43 CFU). Strikingly, the clearance of CA was markedly decreased in the presence of 42d PRBC supernatant (427±49, p<0.05), indicating that exposure to supernatant from PRBCs of advanced storage age decreases microbial clearance by leukocytes.
Conclusion: Stored PRBC supernatant suppresses the pro-inflammatory response after trauma and suppresses leukocyte phagocytosis. These data may provide insight into the mechanisms by which transfusion of stored PRBCs is associated with nosocomial infection.
INDUCED NEGATIVE REGULATOR EXPRESSION TO THE MAP KINASE CASCADE CONTRIBUTE TO EPINEPHRINE INDUCED MONOCYTE SUPPRESSION
N. Shubin and G.E. O’Keefe. University of Washington, Seattle, WA
Background: Post-traumatic immune-suppression is considered a major contributor to nosocomial infection and is responsible for a considerable proportion of the 200,000 annual sepsis-related deaths in the US. Monocytes and macrophages are thought to lose innate inflammatory function following severe trauma. Additionally, catecholamines are chronically induced 5-10 fold after severe injury. Previously, it was demonstrated that catecholamines exhibit an anti-inflammatory effect on monocytes by decreasing pro-inflammatory cytokine secretion. However, the direct mechanisms for this have not been fully elucidated. Therefore, we hypothesized that the trauma-induced catecholamine, epinephrine, stimulates monocyte counter-inflammatory molecule expression, thus leading to decreased monocyte pro-inflammatory cytokine secretion.
Methods: A pilot study evaluating human primary monocyte genome-wide expression changes following epinephrine and/or LPS exposure (n=3 for each group) was conducted to broadly determine which counter-inflammatory molecules were induced. SiRNA studies were then executed in THP-1 monocytes to evaluate the capacity for one of these negative-regulator candidates, Map kinase phosphatase-1 (MKP-1), to diminish epinephrine-induced monocyte TNF-α gene expression.
Results: Following monocyte epinephrine stimulation, we observed a marked increase in expression of two known negative regulators of the MAP kinase cascade, MKP-1 and NURR1 (27.3 and 28.2 fold-increase compared with non-stimulated cells, respectively). Additionally, following epinephrine and LPS dual exposure, MKP-1 and NURR1 expression levels were further induced. These results were validated in THP-1 cells. Moreover, these MKP-1and NURR1 expression increases coincided with significantly diminished TNF-α expression levels following epinephrine and LPS exposure, when compared with LPS exposure alone. MKP-1 mRNA knockdown, via MKP-1 siRNA, partially rescued this epinephrine-induced TNF-α gene expression loss.
Conclusions: These data demonstrate that epinephrine-induced MKP-1 expression suppresses monocyte pro-inflammatory cytokine expression following LPS stimulation. Further, these data suggest that MKP-1 and NURR1 may play novel mechanistic roles in trauma-induced monocyte suppression.
LACTOBACILLUS RHAMNOSUS GG AND L-THREONINE TREATMENT ENHANCES MUC-2 MRNA EXPRESSION AND INCREASES CELL SURVIVAL IN HEAT STRESSED INTESTINAL EPITHELIAL CELLS
C.H. Baird, L. Khailova, and P. Wischmeyer*. University of Colorado, Aurora, CO
Background: Mucin-2 (MUC2) production preserves gut barrier function, and has recently been shown to regulate gut homeostasis. MUC2 deficiency is associated with intestinal disease as spontaneous colitis develops in MUC2 knockout mice. Preservation of MUC2 could have protective clinical implications for a multitude of inflammatory states. Both probiotics such as Lactobacillus Rhamnosus GG (LGG) and specific amino acids have been shown to be protective during intestinal inflammation but therapeutic agents have yet to be optimized. Although dietary l-threonine (THR) deprivation in rats has been linked to decreased mucin production in vivo, no current literature exists on THR’s effect on intestinal cell MUC2. Our laboratory has shown that both the amino acid, L-Threonine (THR), and probiotic administration can protect the intestine however the mechanism is unknown.
Purpose: The purpose of this study was to investigate the effects of LGG and THR on cell survival and MUC2 production in heat stressed (HS) intestinal epithelial (IEC-18) cells.
Methods: IEC-18 cells were treated for 30 min +/- 20 mM THR, +/- 1X105 CFU/mL LGG and exposed to heat-stress. LGG was removed and cells were washed in PBS. Either 0mM (CT)or 20mM THR was re-added and cells recovered for 24hrs. Survival was evaluated via MTS assay (all groups were normalized to unstressed internal controls). mRNA was analyzed in a separate set of experiments to determine if THR, LGG, or a combination of both were associated with changes in MUC2 gene expression.
Results: THR increased survival fourfold vs. HS CTs (p<0.001). Interestingly, cells treated with LGG also showed significant protection vs. HS CTs (P<0.001). MUC2 mRNA increased in non-stressed cells with THR and with LGG and was highest in cells treated with both (p<0.05 vs. non treated control cells). HS cells also showed an increase in MUC2 mRNA with both treatments (p<0.05 vs. HS CTs).
Conclusion: This is the first reported indication that THR and LGG treatment can affect MUC2 mRNA levels in IEC-18 cells in both control and stressed conditions. Both treatments also increased intestinal cell survival during lethal mechanical injury, which could potentially lead to clinical interventions to prevent or treat a multitude of intestinal inflammatory diseases.
FLUORESCENCE IMAGING OF ATP IN HUMAN NEUTROPHILS WITH ORGANELLE-LOCALIZABLE FLUORESCENT CHEMOSENSORS IN SEPSIS PATIENTS
K. Sueyoshi1, Y. Sumi*1, Y. Inoue1, Y. Kuroda1, K. Ishii3, H. Nakayama4, K. Iwabuchi4, Y. Kurishita2, I. Hamachi2, and H. Tanaka1. 1Juntendo University, Urayasu Hospital, Urayasu, Japan, 2Graduate School of Engineering, Kyoto University, Kyoto, Japan, 3Riken Advanced Science Institute, Wako, Japan, 4Institute for Environmental and Gender Specific Medicine, Juntendo University, Urayasu, Japan
Introduction: Adenosine triphosphate (ATP) is involved in both intracellular and extracellular signaling. We have shown that ATP release and feedback via ATP receptors are essential for neutrophils (PMNs) activation. Meanwhile, two new fluorescent chemosensors (PMAP-1, MitoAP-1), which can detect ATP in specific region of living cells, were developed. In this study, we established the method of staining human PMNs with these chemosensors and evaluated the fluorescence intensity in sepsis patients.
Objective: To evaluate the ATP localization using these chemosensors in PMNs of sepsis patients.
Methods: We enrolled 5 sepsis patients (age:73±6 yrs old, APACHEII:21.3±7.0, SOFA:6.4±5.1, 3 male, 2 female) admitted to our hospital between October 2013 and January 2014. PMNs were isolated from the whole blood of these sepsis patients and healthy controls (HC) (n=8). Live PMNs with these chemosensors were observed by a confocal microscopy. Mean fluorescence intensity (MFI) on plasma membrane(PMAP-1) and in mitochondria(MitoAP-1) of PMNs were evaluated using flow cytometry. CD11b expression on PMNs was evaluated.
Results: These chemosensors localized on plasma membrane and in mitochondria of PMNs. MFI of PMAP-1, MitoAP-1 and CD11b in sepsis patients were significantly higher than HC (PMAP-1: 162.7±34.4 vs 121.6±21.6, MitoAP-1: 511.0±139.6 vs 282.0±54.7, CD11b: 397.0±117.9 vs 288.6±83.2;p<0.05).
Conclusion: The higher MFI of PMAP-1 and MitoAP-1 in sepsis patients suggests the pivotal role of ATP for the activation of PMNs. This study would contribute to understanding the dynamics of ATP in PMNs of sepsis patients.
HYPERTONIC SALINE INHIBITS IRF1 INDUCED RANTES SYNTHESIS IN SMALL AIRWAYS CELLS
S. Mitra, F. Gamboni, C.C. Silliman*, and A. Banerjee*. University of Colorado Denver, Aurora, CO
Airway epithelium is the first line of defense against inhaled pathogens, which provides a physical barrier to airborne insults and serves an important immune function. It is also the source of several inflammatory mediators including TNF-α, RANTES and IL-8.The expression of these molecules is a complex multistep process requiring one or more transcriptional regulators.
Objective: The purpose of this study was to elucidate the mechanism by which HTS inhibits IRF1 induced expression of RANTES in Small Airway Epithelial Cells (SAECS) stimulated with Cytomix (TNF-α 10ng/mL, IL-1β 10ng/mL, IFN-γ 10ng/mL).
Methods: Small airway epithelial cells (SAECS) were transfected with luciferase reporter constructs and stimulated with Cytomix +/- HTS. Changes in the activity of 45 different transcription factors were assayed using the Cignal Finder multi pathway dual luciferase reporter array. Gene expression of the IRF1-induced proinflammatory chemokine RANTES (CCL5) was evaluated at 2 hours and 4 hours via quantitative RT-PCR and ELISA. Nuclear levels of IRF1 were evaluated by immuno-blotting and immuno-fluorescent microscopy.
Results: SAECS stimulated with Cytomix show an increase in IRF1 activity via both increases in the levels of IRF1 driven luciferase expression and RANTES. HTS inhibited this increased expression of RANTES.
Conclusion: HTS does not inhibit RANTES expression by preventing nuclear entry of IRF1 suggesting that this inhibition may be post nuclear following IRF1 nuclear entry.
VITAMIN E SUPPLEMENTATION DECREASES OXIDATIVE STATUS IN PEDIATRIC BURNED PATIENTS
L.E. Sousse*1, 2, D.L. Traber*1, 2, M.G. Traber3, J.O. Lee1, 2, E. Watts3, S.W. Leonard3, C.R. Andersen1, K.R. Jennings1, L. Sidossis1, 2, H.K. Hawkins*1, 2, R.P. Mlcak1, 2, O.E. Suman1, 2, R.A. Cox*1, 2, P. Enkhbaatar*1, C.C. Finnerty*1, 2, and D. Herndon*1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals for Children, Galveston, TX, 3Oregon State University, Corvallis, OR
Our recent findings demonstrate that burn injury significantly depleted long-term stores of vitamin E (alpha-tocopherol) of children by nearly 50% within one month of injury.
Objective: Our central hypothesis is that the administration of high doses of alpha-tocopherol will prevent or restore levels of vitamin E and reverse the oxidative state present in burned patients.
Methods: Using a reverse crossover study, pediatric burned subjects (n=49, 47 ± 18% total body surface area burn, 11 ± 4 years) were randomly assigned into one of two groups: an early treatment group who received vitamin E for days 1-15 of the study (n=23), and a delayed treatment group who received vitamin E for days 16-30 of the study (n=26). Both groups were studied for thirty days, and analyses were also compared to unsupplemented controls.
Results: Plasma alpha-tocopherol significantly increases by 34% between days 1-15 and days 16-30 of the delayed treatment group (95% CI 10-64%, p<0.02), as well as a 58% increase between days 16-30 of the early treated group and days 1-15 of the delayed treated group (95% CI 16-114%, p<0.02). Malondialdehyde, which is a marker of lipid peroxidation, significantly decreases between the delayed treated groups compared to unsupplemented controls (p<0.02).
Conclusions: Our long-term goal is to improve the quality of life of the burn patient by preventing pathophysiologic abnormalities that may be aggravated by inadequate vitamin E. Supplementation of vitamin E is a novel concept that may mitigate the complications of burns, including lung injury, fatty liver disease, and peripheral neuropathy.
INHIBITION OF POLY(ADP-RIBOSE) POLYMERASE ACTIVITY BY 5-AIQ MODULATES AUTOPHAGY INDUCED BY EXPERIMENTAL SPINAL CORD TRAUMA
E. Esposito, I. Paterniti, D. Impellizzeri, R. Di Paola, G. Bruschetta, M. Campolo, M. Cordaro, R. Siracusa, and S. Cuzzocrea*. University of Messina, Messina, Italy
Introduction and Aims: Although it is known that spinal cord injury (SCI) induces necrosis and apoptosis, its relationship to autophagy is uncertain. Autophagy is implicated in various pathological states in the nervous system, such as neurodegenerative diseases, cerebral ischemia, and traumatic brain injury. However, the contribution of autophagy to neural tissue damage after SCI has not been extensively investigated. Moreover, Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the tissue injury associated with stroke and neurotrauma. Recently, several studies have broadened the role of poly(ADP-ribosyl)ation in cell killing showing that PARP-1 activation also occurs during apoptosis, and inhibition of PAR formation impairs activation of the apoptotic machinery leading to PAR-dependent mitochondrial dysfunction and rapid release of apoptosis-inducing factor. Despite its pathogenetic relevance, the involvement of PARP-1 in autophagy and the regulatory mechanisms underlying energetic derangement during PARP-1 hyperactivation associated to SCI, are still elusive. The aim of the present study was to investigate the involvement of autophagy after SCI and the role of PARP-1 in the cell’s decision to commit to autophagy following DNA damage.
Material and Methods: SCI was induced by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy, and 5-AIQ (3 mg/kg), a new synthetic inhibitor of PARP, was administered at 30min, 1hour and 6 hours after injury. In the present study the markers of autophagy expression were evidenced by transmission electronic microscope (TEM), western blot analysis for Beclin-1, microtubule-associated protein light chain 3 (LC3), p-AKT and mTOR in neural tissue after SCI and after treatment with 5AIQ.
Results: Taken together our results clearly detected elevated levels of Beclin-1, LC3 and pAKT as well as AIF and Bax after SCI, whereas treatment with 5-AIQ significantly reduced autophagy activation.
Conclusion: PARP-1 inhibition could produce its protective effects by affecting Akt, that can produce significant cytoprotective effects by phosphorylating such apoptosis-regulatory proteins, and modulating the autophagic response.
MODULATION OF CANNABINOID 2 RECEPTOR ACTIVATION IN EXPERIMENTAL ENDOTOXEMIA
J. Sardinha, M. Kelly, J. Zhou, and C. Lehmann*. Dalhousie University, Halifax, NS, Canada
Introduction: Sepsis results from a dysregulated immune response to an infection. Current medical treatments are of limited efficacy with regard to improvement of survival. The endocannabinoid system (ECS) opens a novel avenue in sepsis therapy due to its unique characteristics. Cannabinoid 2 (CB2) receptors are expressed on the surface of all immune cells, providing a direct approach to the modulation of the immune system. The present study explored the effects of CB2 receptor modulation in a mouse model of acute sepsis (endotoxemia).
Methods: Endotoxemia was induced by intravenous administration of lipopolysaccharide (LPS, 5 mg/kg; from E. coli, serotype: O26:B6). Six groups of mice were used to assess leukocyte activation within the intestinal microcirculation as well as functional capillary density (FCD) by intravital microscopy. The treatment compounds tested were: a specific CB2 receptor agonist, HU308, a CB2 receptor antagonist/inverse agonist, AM630, and the cannabinoid degradation enzyme inhibitors URB597 (fatty acid amide hydrolase inhibitor), and JZL184 (monoacylglycerol lipase inhibitor).
Results: Administration of the CB2 receptor agonist, HU308, reduced significantly the number of adhering leukocytes in submucosal venules, but did not restore muscular and mucosal villi FCD in endotoxemic mice. The CB2 receptor antagonist, AM630, did not exacerbate leukocyte activation within the intestinal microcirculation, but further reduced muscular and mucosal FCD of the intestinal wall. The cannabinoid degradation enzyme inhibitors URB597 and JZL184 both significantly reduced the number of adhering leukocytes in submucosal venules. Furthermore, JZL184 administration completely restored muscular FCD.
Conclusions: CB2 receptor activation by a specific agonist or cannabinoid degradation enzyme inhibition was effective in reduction of leukocyte activation within the intestinal microcirculation. The CB2 receptor pathway seems to be involved in the inflammatory cascade elicited early during sepsis. Therefore, modulation of the CB2 receptor pathway might offer new therapeutic options for the manipulation of the immune system in sepsis.
ADOPTIVE TRANSFER OF FIBROCYTES PRIOR TO CLP PROMOTES DECREASED BACTERIAL COUNTS
J.A. Nemzek*, and C. Fry. University of Michigan, Ann Arbor, MI
Various studies have correlated increased survival with decreased bacterial growth in sepsis. Previous work in our lab showed adoptive transfer of tissue derived fibrocytes prior to CLP improved survival in mice. In addition, mice that received fibrocytes demonstrated a significant decrease in bacterial load in the peritoneum, spleen, and blood within 24hours of CLP. To account for this, lung and spleen derived fibrocytes were compared to fibroblasts and peritoneal macrophages for the ability to perform phagocytosis of E.coli bioparticles (Molecular Probes, Life Technologies). The cells were cultured with bioparticles for 2 hours followed by assessment by flow cytometry for fluorescence, indicative of a pH change in the active phagosome. Both lung and spleen fibrocytes demonstrated significantly more phagocytosis than the fibroblasts and equivalent phagocytosis compared to peritoneal macrophages. Prior stimulation of fibrocytes with LPS had no impact on phagocytosis. To determine whether fibrocytes from the septic environment were also able to phagocytize bioparticles, mice expressing GFP on the collagen promoter underwent CLP or Sham surgery. In these mice, only fibroblasts and fibrocytes express GFP and can further be distinguished by the presence of CD45 on fibrocytes. Mice were sacrificed 24 hours post CLP. Afterwards, peritoneal and spleens cells were cultured with bioparticles and examined for phagocytosis by identifying positive cells for CD45, GFP, and bioparticles. Professional phagocytes represented by the CD45+, GFP- population in the peritoneum demonstrated an increase in phagocytosis when Sham (35%) was compared to CLP (73%). The resident fibrocytes (CD45+ GFP+) showed a 2 fold increase in phagocytosis in the septic mice, but overall were significantly less active than the other phagocytes and the fibrocytes propagated in culture. Splenic fibrocytes directly from GFP+ mice showed no difference in bioparticle expression between Sham and CLP mice. Taken together, these data suggest that the cultured fibrocytes used in adoptive transfer have a greater ability to engulf bacteria than their resident counter parts. This may explain, in part, the mechanism for improved survival in sepsis after the adoptive transfer of tissue derived fibrocytes.
THE EFFECT OF LOW-DOSE HYDROCORTISONE AFTER SEVERE BURN INJURY: A MICROARRAY LONGITUDINAL STUDY
J. Plassais1, M. Cazalis1, F. Venet2, G. Monneret2, A. Pachot1, and S. Tissot3. 1Joint Unit Hospices Civils de Lyon/bioMérieux, Hôpital Edouard Herriot, Lyon, France, 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Immunology Lab, Lyon, France, 3Hospices Civils de Lyon, Hôpital Edouard Herriot, Intensive Care Unit, Lyon, France
The systemic effect of inflammatory mediators release after severe burn is a severe cardiovascular dysfunction called burn shock. Patients need vasopressor infusion to maintain adequate delivery of oxygen but it could have deleterious effects on skin perfusion and worsen the burn depth. This shock could result from the interplay of the initial hypovolemia and the release of multiple inflammatory mediators. It has been shown that a low-dose of hydrocortisone could reduce the shock duration but the mechanisms involved remain unclear. We investigated the systemic genomic response after severe burn injuries and determine whether patterns of gene expression could be associated with low-dose of glucocorticoids.
Thirty burn patients with over 30% of total body surface area were enrolled into a randomized double-blind clinical study. 15 patients were treated with low-dose of hydrocortisone and 15 patients were treated with placebo. Whole blood samples were collected after shock onset (S1) before any treatment, one day after treatment beginning (S2) and 120h and 168h after the burn injury (S3/S4). Moreover blood samples of 13 healthy volunteers were collected. Pangenomic expression was evaluated with Affymetrix HG-U133plus 2.0 microarrays. Moderated t-tests and F-test were used to compare burn patients to controls and then gene expression profiles between the 2 groups (B-H correction, p<0.05).
Severe burn injury induced the deregulation of a considerable number of genes (n>2200 at S1) in comparison with controls with an increased number of deregulated genes over time. Within burn patients, more than 300 genes were deregulated by hydrocortisone over time. The treatment had a rapid effect on gene expression, 339 and 627 genes were differentially expressed at S2 and S3 respectively. However the number of these genes decreased drastically at S4 (only 24 genes significant). The genes identified at S2 were mostly related to the decrease of growth, development and quantity of leukocytes but these biological processes were not found significant at S3 indicating that the action of glucocorticoid in the response to burn injury is short-lived and time dependent.
This study is an informative overview of the genomic responses after burn injuries. More importantly it is the first study providing information about mechanism involved in glucocorticoid’s reduced shock duration after burn.
THE IMMUNE RESPONSE AFTER TRAUMA - IS IT DIFFERENT IN THE ELDERLY?
H. Vester1, M.S. Huber-Lang*2, Q. Kida2, F. Gebhard*2, A.K. Nussler4, C. Seeliger1, M. van Griensven*1, and M. Perl*3. 1Klinikum rechts der Isar, Technical University, Munich, Germany, 2Department of Traumatology, Ulm, Germany, 3BG-Trauma Center, Murnau, Germany, 4BGU Tübingen, Tübingen, Germany
Proximal femur fractures of the elderly are associated with a high postoperative complication (26%) and in house mortality (6%) rate. In a multi-center prospective clinical study we have investigated differences in the posttraumatic immune response of elderly patients. Upon clinical admission and within 6 hours after surgery blood was collected from young patients (<50 y) with a long bone fracture (YF), elderly patients (>70 y) with a proximal femur fracture (OF) and two healthy control groups with matching age (YH & OH). CD15+ magnetic bead-isolated neutrophils (PMN) were TUNEL stained. Serum TRAIL- and cytokine concentrations were analyzed via cytometric bead array, Fas-Ligand and TNF-Receptor-I via ELISA. N=20-26/group, One Way ANOVA and SNK Test, p<0,05.
YH: age 29±3 y, m:f=15:11; OH: age 78±6 y, m:f =15:11; OF: age 86±7 y, m:f=7:14; YF: age 31±9 y, m:f =12:8. IL-6 was significantly increased only in OF after trauma and surgery whereas a significant decrease of TNF-alpha was seen only in YF after trauma. Interestingly only YF showed a significant increase of GM-CSF serum levels whereas OF exhibited a decrease of IFN-gamma after trauma and after surgery. PMN apoptosis was significantly reduced only in YF.
TRAIL serum levels were diminished in OF after trauma and even more after surgery and in YF only after surgery. Fas-L concentrations were reduced only in YF after surgery or trauma. Taken together our data suggest, that the posttraumatic immune response is differently regulated in old and young operatively treated trauma patients. Decreased activation of PMNs and phagocytes along with dysregulation of the posttraumatic inflammatory response might contribute to the high perioperative mortality rate of the elderly suffering from a proximal femoral fracture. (Supported by EK-Fresenius 2009_A116).
THE EFFECT OF DAPHNETIN ON TLR4/NF-κB SIGNALING PATHWAY IN SEVERE ACUTE PANCREATITIS INDUCED BY SODIUM TAUROCHOLATE IN RATS
Z. Liu1, 2, J. Liu1, 2, Q. Shi2, G. Wang*1, and W. Wang2. 1SUNY Upstate Medical University, Syracuse, NY, 2Renmin Hospital of Wuhan University, Wuhan, China
Introduction: Severe acute pancreatitis (SAP) is the sudden onset of inflammation of the pancreas, often results in subsequent multiple-organ dysfunction syndrome with a unacceptably high mortality rates. Daphnetin, a coumarin extracted from Daphne odora, has been reported to possess properties of analgesic and anti-inflammatory effects.
Objective: The objective of this study was to investigate the effect and potential mechanism of daphnetin on severe acute pancreatitis induced by sodium taurocholate in rats.
Methods: Female Wistar rats were pretreated with daphnetin 30 min before retrograde infusion of 5% Sodium Taurocholate (0.1mL/kg) into the bile-pancreatic duct. Daphnetin (4 mg/kg) was administered intraperitoneally. Twelve hours after sodium taurocholate administration, histological grading, serum amylase and lipase levels was evaluated. The myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in pancreatitis tissues were determined. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the serum were measured by enzyme-linked immunosorbent assay (ELISA). The level of phosphorylation of nuclear factor of inhibitory kappa B alpha (IκB-α), the expression of Toll-like receptor-4 (TLR4) were detected by western blot. DNA-binding activity of nuclear factor kappa-B (NF-κB) was assessed as an index of NF-κB activation by electrophoretic mobility shift assay (EMSA).
Results: We found: 1) daphnetin markedly attenuated the histological alterations (p<0.05) in the pancreatitis; 2) daphnetin remarktable reduced levels of serum amylase and lipase (p<0.05); 3) daphnetin obviously decreased MPO activity and MDA content (p<0.05), but increased SOD activity (p<0.05) in pancreatic tissues; 4) daphnetin significantly down-regulated the levels of pro-inflammatory mediators, including TNF-α, IL-1b and IL-6 (p<0.05); 5) daphnetin can inhibited the phosphorylation of IκB-a, NF-κB activation and the expression of TLR4 (p<0.05).
Conclusion: Our results suggest that anti-inflammatory effects of daphnetin against severe acute pancreatitis may be due to its ability of zinhibition TLR4 mediated NF-κB signaling pathways. Daphnetin may be a promising potential therapeutic reagent for severe acute pancreatitis.
DEVELOPMENT OF FLUORESCENTLY-LABELLED PSEUDOMONAS AERUGINOSA FOR IN VIVO IMAGING IN MICE
L. Kartchner, C. Gode, D. Moore, R. Maile, and B. Cairns. University of North Carolina at Chapel Hill, Chapel Hill, NC
A common cause of increased morbidity and mortality among long-term hospital patients is the incidence of nosocomial bacterial infections. Patients develop infections that are difficult to treat due to antibiotic resistance. Currently there are no techniques that allow researchers to study the kinetics of these infections. Recent improvement of in vivo imaging has facilitated the development of multiple near-infrared fluorescent proteins to label cells for detection in vivo. In this study we developed a fluorescently-labelled Pseudomonas aeruginosa strain for in vivo detection in a developed model of mouse infection. DNA containing the sequence for four near-infrared fluorescent proteins (IRFP 682nm, IRFP 713nm, IRFP 720nm, and Katushka) was purchased and sequences were amplified using PCR. These sequences were then inserted into a Tn7 vector for incorporation and expression in the P. aeruginosa genome. Upon successful electroporation of the plasmid into Pseudomonas aeruginosa, bacteria were analyzed for fluorescent signal using flow cytometry as well as imaging with IVIS (in vitro imaging system) kinetic imaging equipment. Finally, signal detection in both C57BL/6 and BALB/c mouse strains were characterized. We believe this technique will allow us to effectively study the in vivo kinetics and colonization rates of bacteria during a relevant model of traumatic injury.
PERIPHERAL LETHAL INFLAMMATION INDUCES BRAIN REGION-SPECIFIC ALTERATIONS IN THE EXPRESSION OF CYTOKINES, IMMUNE CELL MARKERS AND CHOLINERGIC SYSTEM COMPONENTS
H. Silverman1, 2, M. Dancho1, A. Regnier-Golanov3, M. Ochani*1, P.S. Olofsson1, M. Nasim4, S.S. Chavan*1, E. Golanov5, C.N. Metz*1, 2, K.J. Tracey*1, 2, and V.A. Pavlov*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Hofstra North Shore-LIJ School of Medicine at Hofstra University, Hempstead, NY, 3Pediatrics-Neurology, Baylor College of Medicine, Houston, TX, 4Neuropathology-Anatomic Pathology, North Shore-LIJ Health System, Lake Success, NY, 5The Houston Methodist Research Institute, Houston, TX
Peripheral inflammation alters CNS immune responses and neuronal function. We and others have indicated a role for brain cholinergic signaling in the neural control of peripheral cytokine release (Nat Rev Endocrinol, 2012, 8:743). However, the impact of lethal peripheral inflammation on brain cytokines and the brain cholinergic system is not well understood. To provide insight, we studied the expression of IL-1β,IL-6, immune cell markers and brain cholinergic system components by qPCR 4 h post LPS (8 mg/kg, ip) or saline administration in mice. Seven brain areas: cortex; brainstem; cerebellum; hippocampus; hypothalamus, thalamus and striatum were evaluated. Brain Il1b and Il6 mRNA expression were differentially increased, with the highest magnitude in cortex (144-fold) and cerebellum (173-fold) for IL1b and Il6, respectively in endotoxemic mice. Gfap (astrocytes) gene expression was increased in cortex, cerebellum, brainstem, hippocampus and thalamus. Iba1 (microglia) mRNA expression was differentially decreased while morphological assessment (by IBA1 immunostaining) indicated microglia activation. Ache (acetylcholne degrading enzyme) mRNA expression was decreased in the cortex and increased in the hippocampus; Chat (biosynthesizing enzyme) mRNA expression was decreased in striatum; Chrm1 (M1 muscarinic receptor) mRNA expression was decreased in cortex and brainstem. In conclusion, our results reveal previously unrecognized brain region specificity for Il1b and Il6 mRNA expression during lethal inflammation, associated with differential immune cell marker expression and microglia morphology. In addition, our data indicate that the brain cholinergic system with a role in regulating peripheral cytokine release is altered in settings of acute lethal inflammation. This study was funded in part by NIH/NIGMS.
PHYSIOLOGICAL AND IMMUNE-BIOLOGICAL CHARACTERIZATION OF A LONG-TERM MURINE MODEL OF BLUNT CHEST TRAUMA
S. Hafner1, M. Wepler1, 2, J. Matallo1, M. Gröger1, K. Wagner1, O. McCook1, M. Huber-Lang3, P. Dietl4, M. Georgieff1, E. Calzia1, P. Radermacher1, and F. Wagner1. 1Sektion APV, Klinik für Anästhesiologie, Universitätsklinikum Ulm, Ulm, Germany, 2Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA, 3Klinik für Unfall-, Hand-, Plastische- und Wiederherstellungschirurgie, Universitätsklinikum Ulm, Ulm, Germany, 4Institut für Allgemeine Physiologie, Universität Ulm, Ulm, Germany
Background: Murine blunt chest trauma is associated with a pulmonary and systemic inflammatory response . The long term course of the immune response has not yet been studied together with analysis of pulmonary function. Furthermore, it is a matter of debate, however, whether the inflammatory response is associated with ongoing impairment of lung function. Therefore, we investigated parameters of pulmonary and systemic immune biology together with lung mechanics and gas exchange over 24 hours after murine blunt chest trauma.
Methods: Anesthetized, spontaneously breathing male C57BL/6J mice were subjected to a blast wave-induced blunt chest trauma  or sham procedure. 12 or 24 hours later, blood gas and lung mechanics were measured together with the analysis of blood, bronchoalveolar lavage (BAL), and tissue cytokine concentrations (multiplex cytokine kit), heme oxygenase-1 (HO-1), IκBα, activated Caspase-3, Bcl-xL, and Bax expression (western blotting), nuclear factor-κB activation (electrophoretic mobility shift assay), nitrotyrosine formation, and tissue P2X7 and P2X4 receptor expression (immunohistochemistry). Histological damage was assessed after HE and PAS staining. Mitochondrial respiration in permeabilized diaphragm muscle biopsies was quantified by high-resolution respirometry.
Results: Chest trauma significantly increased tissue and BAL cytokine levels at 12 hours, followed by a decrease at 24 hours, which was associated with a significant increase of HO-1, P2X7 receptor expression, and tissue nitrotyrosine formation. In contrast, neither histological damage and apoptotic cell death nor lung mechanics or gas exchange showed any significant difference between sham and trauma groups.
Conclusion: Our findings suggest that activation of the immune response is still present at later time points after murine blunt chest trauma. Discordance of the increased local immune response and preserved pulmonary function in our study may be explained by a dissociation of inflammatory response and lung function, such as previously described after CLP .
Supported by the Boehringer Ingelheim Ulm University BIU Center
1. Knöferl et al, Shock 2003;19:519-25
2. Wagner et al, J Trauma 2011;71:1659-67
3. Uematsu et al, Crit Care Med 2013;41:151-8
LACTOBACILLUS RHAMNOSUS GG IMPROVES HOMEOSTASIS IN THE COLON AND SPLEEN OF MICE WITH PSEUDOMONAS AERUGINOSA INDUCED PNEUMONIA
A. Rush, L. Khailova, C.H. Baird, and P. Wischmeyer*. University of Colorado Anschutz Medical Campus, Aurora, CO
Introduction: Pneumonia continues to be a common cause of mortality in American ICUs, resulting in over 50,000 deaths each year. Although pneumonia injury is initiated in the lungs, the disease often develops into a multi-organ syndrome. Studies have shown that preventing sepsis-induced intestinal epithelial apoptosis is associated with increased survival in a murine model of pneumonia. Probiotics, such as Lactobacillus rhamnosus GG (LGG) have been shown to decrease intestinal apoptosis in other diseases. Our laboratory has previously shown that LGG treatment significantly increases survival in mouse models of sepsis although the mechanism is unknown.
Objective: To evaluate the effects of probiotic treatment on apoptosis and proliferation in the colon and spleen of mice with Pseudomonas aeruginosa induced pneumonia.
Methods: Colonic and spleen tissue was collected from 6-week old FVB/N mice 12h after intratracheal injection of P. aeruginosa (4x108CFU) or saline with immediate administration of either LGG (1x109CFU/ml) or vehicle. Tissue was fixed overnight in 10% formalin, parrafin-embedded, and sectioned. Sections were stained with either hematoxylin and eosin (H&E), cleaved caspase 3 (CC3), or proliferating cell nuclear antigen (PCNA). Positive cells were quantified by a blinded evaluator in 10 random high-power fields per section.
Results: LGG mice had significantly lower numbers of CC3 positive cells in the colon and spleen (p<0.05) and decreased apoptosis in H&E stained colonic and spleen cells (p<0.0001) compared to pneumonia control group. PCNA positive proliferative cells in the colon and spleen were significantly higher in LGG treated mice than pneumonia control group (p<0.001)
Conclusions: LGG treatment decreases apoptosis levels and increases proliferation in the colon and spleen of mice with P. aeruginosa induced pneumonia which may be associated with improvement of survival in the LGG treated animals. Therefore, LGG may be useful as a therapy for pneumonia induced sepsis.
SUBSTANCE P REGULATES IMMUNE PRIMING FOLLOWING TRAUMATIC BRAIN INJURY
T. Hsieh, E.G. King, D.M. Stepien, E. Duffy, and D.G. Remick*. Boston University School of Medicine, Boston, MA
Introduction: Substance P is a neuropeptide released after traumatic brain injury with widespread effects, including modulating the immune system through neurokinin receptors. Prior results in a murine TBI model have shown that TBI improves survival, neutrophil recruitment into the lung and bacterial killing in Pseudomonas aeruginosa (PSA) pneumonia. This study tests the hypothesis that substance P serves as a neuro-immunomodulatory link that primes the immune system for bacterial killing.
Methods: TBI was induced using a weight-drop model. 48 hours after TBI, PSA (1.25-5x107 CFU) was intracheally instilled into adult female ICR mice. The neurokinin-1 receptor (NK-1R) antagonist (CJ-12,255, 25mg/kg) was given 1h prior to both TBI and pneumonia, and repeated every 12h. The NK-1R agonist (GR-73632, 0.5mg/kg) was given every 12h instead of TBI to attempt to replicate the post-TBI substance P signaling. Bronchoalevolar lavage (BAL) was performed 4h post-pneumonia (52h post-TBI) for cell counts and bacterial load (CFU). Mortality was followed for 14d.
Results: Blocking substance P signaling with CJ-12,255 abolished the survival advantage in mice subjected to TBI (p<0.05). BAL fluid data support the hypothesis that NK-1R activation primes the immune system resulting. NK-1R blockade inhibits TBI-induced recruitment of neutrophils to the lung space with resulting increases in pulmonary bacterial burden (Table 1). The BAL results from the TBI mice treated with the NK-1R antagonist were similar to non-TBI mice. Although NK-1R agonist treatment was unable to replicate the survival advantage of TBI, it increased bacterial clearance and neutrophil recruitment into the lungs to similar levels of TBI mice (Table 1).
Conclusion: TBI+PSA challenged mice treated with the NK-1R antagonist had increased mortality and increased bacterial loads. NK-1R agonist treatment alone increased bacterial killing but could not replicate the increased survival following TBI. Neutrophil recruitment to the lungs exhibited an inverse relationship with bacterial clearance. Our data support the hypothesis that substance P is essential for immune priming following TBI, but cannot be the sole contributor for increased survival.
ESTROGEN RECEPTOR IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD): GENDER AND AGING DIFFERENCES
R. Crupi, D. Impellizzeri, R. Di Paola, E. Esposito*, and S. Cuzzocrea*. University of Messina, Messina, Italy
Sex differences in the biology of different organ systems and the influence of sex hormones in modulating health and disease are increasingly relevant in clinical and research areas. Gender influences not only physiological differences, but also the social, economic, and cultural context in which men and women coexist. There is increasing clinical evidence for sex differences in incidence, morbidity, and mortality of lung diseases including allergic diseases (such as asthma), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, lung cancer, as well as pulmonary hypertension. The prevalence, morbidity, and mortality of COPD in women are increasing in the United States. Statistics say in fact that there are more women than men with COPD, a disease characterized by all-or-nothing progressive, presently irremediable, alveolar loss: primary or idiopathic pulmonary hypertension (PPH) is well known as a disease of women in the childbearing years, while asthma is increasing in prevalence and more rapidly in women. Based on experimental evidences, the idea of our study starts from sex-specific differences, which lie mainly in hormonal deficiency that occurs in women during climateric period or in a more advanced stages of life. With this aim in mind we investigated the role of estrogen receptors (ERs) in inflammatory lung injury and assessed the role of gender and aging in estrogens action. C57BL/6 mice were treated with bleomycin (BLEO, 1 mg/kg) for one week; after this treatment, measurement of fluid and collagen content in the lung, BALF analysis, myeloperoxidase (MPO) activity assay, lung histology and IHC were performed. Our results demonstrate that activation of ER in lung inflammatory cells might provide beneficial effects in adult, but not in aged mice of both genders, suggesting a key role of the estrogens signaling pathway in the physiology and therapeutic opportunities of lung inflammation.
PORCINE TRAUMA MODEL OF CHEST, ABDOMINAL AND EXTREMITY TRAUMA WITH HEMORRHAGIC SHOCK - IS SYSTEMIC INFLAMMATION TRIGGERED BY TRAUMA LOCALIZATION?
K. Horst1, D. Eschbach2, M. Sassen2, R. Pfeifer1, S. Ruchholtz*2, H. Pape*1, and F. Hildebrand*1. 1RWTH Aachen University Hospital, Aachen, Germany, 2University Hospital Giessen/Marburg, Marburg, Germany
Background: Small animal models have given evidence that there is a significant interaction of the local and systemic response (e.g. bone healing, inflammation) after severe trauma with long bone fractures. In this context, systemic inflammation was associated with delayed bone healing. Therefore, we examined the association between local inflammation in fracture hematoma and the systemic inflammatory response in a long-term, porcine trauma model.
Material and Methods: Trauma group consisted of 15 animals, SHAM group of 5 animals. The combined trauma model included tibial fracture, lung contusion, liver lazeration and controlled hemorrhage of 45% of total blood volume. After 90 minutes volume loss was substituted by a standardized crystalloid infusion protocol. After trauma, animals were mechanically ventilated and under ICU-monitoring for 48.5h. Blood samples as well as samples from fracture hematoma were taken before trauma, when shock period resp. reperfusion period ended and after 14,5h, 24h and 48h. Biopsy of traumatized muscle at the fracture site was also obtained. Local and systemic levels of IL-6, IL-8, IL-10 and diverse alarmins (HSP70 and HMGB1) were determined by ELISA.
Results: Compared to the SHAM group, a significant increase of systemic cytokines and alarmins was observed. Locally levels in fracture hematoma showed an earlier and more intense increase (14.5 h, 24h and 48h) compared to the systemic inflammatory response. Partially cytokine levels (IL-6, IL-8) already decreased over time while systemic response increased.
Conclusion: This clinically-relevant large animal model describes for the first time the course of fracture-associated local inflammation. Combined trauma resulted in a time-dependent activation of the local and systemic inflammatory response. In further studies it has to be further clarified how the local inflammation at the fracture site contributes to systemic inflammation and in how far systemic inflammation might result in a delayed process of early bone healing.
TROPONIN I AND MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF): A DIRECT CORRELATION IN PATIENTS WITH SEPSIS
L. Pawar1, A. Iftikhar1, K. Ochani1, S. Kamran1, K. Talati1, N. Chaudhry1, N. Kohn1, and E.J. Miller*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Hofstra North Shore-LIJ Medical School, Hempstead, NY
Introduction: Cardiac troponin I and the cleaved brain natiuretic peptide molecule (proBNP) are released by cardiomyocytes under stress, and have been suggested as indicators of myocardial depression in patients with sepsis. MIF is a pro-inflammatory cytokine which plays an important role in potentiating sepsis, and is also a cardio-depressant factor. We have shown previously that, in our mouse model, MIF is released from the lungs in sepsis, inducing cardio-circulatory depression. In this study we examined the relationship of MIF with troponin I and proBNP in newly diagnosed patients with sepsis.
Methods: 49 individuals (male/female 26/23; median age 76yrs), admitted through the emergency room at NSLIJ Forest Hills Hospital, with the diagnosis of sepsis (n=5), severe sepsis (n=6) or septic shock (n=38), were assessed. Blood was drawn within the first 48 hrs after admission. Cardiac troponin I, proBNP, and MIF were analyzed in a blinded manner. The Spearman correlation coefficient was used to examine the degree of correlation between MIF and each of troponin I and proBNP. For purposes of these analyses, levels of troponin I that were below the detectable limit of 0.04 were set to 0.02 (the mean of 0 and 0.04). As all analyses were carried out using non-parametric methods, the choice of the numerical value for levels below detectable limits did not affect the results, since these methods use ranks.
Results: The mean concentration of MIF (pg/ml +SD) in each group was sepsis (1037+1232), severe sepsis (1819+1011) and septic shock (2739+3149). The mean concentration of MIF in patients with normal left ventricular function (LVF n=36) was 1903+1806 pg/ml whereas those with depressed LVF(n=10) the MIF was 4038+5047 pg/ml. The patient MIF concentration (mean 2452+2864 pg/ml) was correlated with Troponin I (r =0.442, p=0.0015) but not proBNP or other parameters such as age of the individual or lactate concentration.
Conclusions: The data suggest a correlation between MIF, troponin I and myocardial depression in early sepsis. Interestingly there was no such correlation between MIF and proBNP. This may be a function of when the blood samples were drawn, and further studies are required to elucidate this. Nevertheless, the data suggest that therapeutic inhibition of MIF in sepsis may target and prevent sepsis-associated myocardial dysfunction. (L.P. and A.I. contributed equally)
HUMORAL ELEMENTS IN LETHAL/NON-LETHAL SEPSIS PRIME FOR SURVIVAL OR DEATH
E.L. Chiswick and D.G. Remick*. Boston University, Boston, MA
Introduction: Within 6 hours post-Cecal Ligation and Puncture (CLP) induced sepsis, mice may be stratified as predicted to Live (Live-P) or die (Die-P) based on plasma IL-6. At 6 and 24 h, Die-P mice have increased pro and anti-inflammatory mediators in their peritoneum and plasma, as compared to Live-P. Within 6 hours, Die-P mice also demonstrate decreased bacterial killing, phagocytosis, and reactive oxygen species burst (ROS). We tested the hypothesis that the increased surge in pro and anti-inflammatory factors mediates the immunosuppression observed in Die-P phagocytes.
Methods: A recently developed flow cytometry assay was used, where bacteria were simultaneously labeled with a reactive oxygen sensor (DCF), a pH sensor (pHrodo), and a ph/ROS insensitive fluorophore (Alexafluor-350). DCF and pHrodo fluorescence was normalized to Alexa-350 fluorescence to differentiate total phagocytosis from intra-phagosomal responses. Trypan Blue was used to quench non-phagocytized bacteria.
To recapitulate the early phase of peritonitis, naïve peritoneal cells (B-cells and Macrophages) were mixed with naïve neutrophils (PMN) isolated from bone marrow. Cells were incubated at 37°C in plasma or peritoneal fluid harvested 6 or 24hrs post-CLP. After 45 minutes, cells were incubated with labeled bacteria for 30 minutes. Murine fluid samples were stratified as Live-P or Die-P based on plasma Il-6 levels. Incubations on ice served as controls.
Results: Naïve phagocytes incubated with Die-P peritoneal fluid show decreased function, compared to Live-P. Although a similar percentage of cells phagocytize the bacteria, Live-P peritoneal fluid results in more bacterial phagocytosis per cell, with significantly greater phagosomal ROS and acidification.
Interestingly, Live-P plasma incubation results in fewer cells phagocytizing, but the cells that do phagocytize take in more bacteria than cells in Die-P plasma. This may be due to DNA NETs ejected by Live-P primed PMN, which were absent in Die-P primed PMN. Surprisingly, Die-P plasma results in significantly greater phagosomal ROS.
Conclusions: Mice predicted to die from CLP-sepsis generate biochemical factors that impair bacterial clearance mechanisms in naïve cells, relative to mice predicted to live. This presents an enticing area of study to decipher what soluble factors from Die-P mice drive immunosuppression.
AUTOPHAGY-RELATED ATG16L1 POLYMORPHISM WAS NOT ASSOCIATED WITH SUSCEPTIBILITY TO NOR OUTCOME OF SEVERE SEPSIS IN THE JAPANESE INTENSIVE CARE UNITS
E. Watanabe*1, T. Kimura1, Y. Sato1, O. Takasu2, T. Ikeda3, J. Kotani*4, and S. Oda*1. 1Chiba University, Chiba, Japan, 2Kurume University, Kurume City, Japan, 3Tokyo Medical University Hachioji Medical Center, Tokyo, Japan, 4Hyogo College of Medicine, Nishinomiya City, Japan
Background: ATG16L1 belongs to a family of genes involved in autophagy, a biological process, which play a role in many other pathways essential to the regulation of inflammation. Recently, pathophysiology of severe sepsis has been implicated to be associated with autophagy, i.e. type II programmed cell death. In this study, we examined the association between ATG16L1 genetic polymorphism and susceptibility to and/or outcome of severe sepsis.
Patients and Methods: The study included 253 patients who were admitted to the intensive care unit (ICU) of Chiba University Hospital between October 2001 and September 2008 (discovery cohort) and 767 patients who were admitted to ICUs of five Japanese institutions including Chiba University Hospital between October 2008 and September 2012 (multi-center validation cohort). Genotyping was performed for a single-nucleotide polymorphism (SNP) within the coding region of ATG16L1 (rs2241880).
Results and Discussion: Among the discovery cohort, ATG16L1 was significantly associated with susceptibility to severe sepsis/septic shock (SS) (n=246, P=0.036 in an additive model, P=0.014 in a dominant model, with the correlation/trend test). To confirm the result, we selected 254 ICU patients from validation cohort by propensity score matching to equalize the severity of both cohorts. In the validation cohort, however, ATG16L1 SNP was not associated with the susceptibility. As for ICU mortality of SS patients, furthermore, no significant difference was observed among ATG16L1 genotypes in both discovery (n=125 SS patients) and validation (n=271 SS patients) cohort. Recently, another autophagy-related SNP, an IRGM SNP (rs10065172) was reported to be associated with outcome of severe sepsis. However, the present study implicates that the autophagy pathway via ATG16L1 is not associated with pathophysiology of severe sepsis. Mitochondrial dysfunction via decreased expression of IRGM might be of more importance in deterioration of severe sepsis in relation to autophagy mechanisms.
Conclusions: The data suggest that the ATG16L1, an autophagy-related polymorphic locus, does not influence outcome in severely septic patients, nor susceptibility to severe sepsis in the Japanese ICU patients.
ELEVATED MYOGENIN EXPRESSION IN SKELETAL MUSCLE AFTER BURN WITH IGF-1 TREATMENT
M. Saeman, J. Song*, K. Despain, M. Liu, J. Minei*, and S.E. Wolf*. University of Texas Southwestern, Dallas, TX
Introduction: Severe burn causes decreased lean body mass and muscle loss. Even though myogenesis is activated after burn, we speculate that it is insufficient to maintain muscle homeostasis. IGF-1 has been shown to decrease loss of muscle protein in burn patients, but little is known about the effect of IGF-1 on myogenic regulation after burn. The aim of the study is to investigate whether skeletal myogenesis is affected by IGF-1 treatment after burn.
Methods: Fourteen C57BL/6 adult male mice received 20% TBSA scald burn. Immediately following injury, seven animals received 5mg/kg IGF-1 (sc, q 24h) for 3 doses; others were injected with 0.9% saline as vehicle control. In situ isometric force test of the gastrocnemius muscle was performed on day 3 after injury; muscle samples were harvested for RNA extraction and examined by real time qPCR. Student’s t-test was applied for statistical analysis.
Results: The reduction of body weight was lower in mice with IGF-1 treatment; this also corresponded with greater gastrocnemius muscle mass (p<0.05). No difference was observed in the tetanic (Po) and twitch force (Pt) of mouse gastrocnemius muscle between IGF-1 and vehicle treatment groups. Specific Pt (normalized to muscle tissue weight) was significantly decreased in the IGF-1 treatment group (p=0.013). Real time qPCR data showed that myogenic regulatory factor myogenin expression significantly increased in gastrocnemius muscle of the IGF-1 treated group (p<0.05).
Conclusions: IGF-1 treatment increased myogenin expression in the gastrocnemius muscle relative to saline treatment. This finding correlated with muscle physiological change: increased muscle weight and altered muscle function. The decrease in specific Pt seen in the IGF-1 treatment group is likely a reflection of relatively increased muscle mass that did not affect the twitch force. IGF-1 also decreased overall mouse body weight loss. These findings suggest IGF-1 protects homeostasis of muscle tissue with increased myogenesis in burn injury.
TREATMENT OF INTERLEUKIN (IL)-15 SUPERAGONIST IN VIVO: IS IT SAFE FOR USE IN MICE, AND THEN HUMAN?
Y. Guo and E. Sherwood*. Vanderbilt University, Nashville, TN
IL-15 is a cytokine that plays an essential role in the development of natural killer (NK), NKT and memory CD8 (mCD8) T cells, as evidenced by the deficiency of these cells in IL-15-/- mice. Thus, this cell commitment gives IL-15 the possibility to be superior to other cytokines in cancer and anti-viral therapy. However, the safety of IL-15 administration in vivo is not clearly elucidated and thus its suitability as a therapeutic agent is unknown. This study was designed to evaluate the safety of IL-15/IL-15Rα complex (IL-15 superagonist, SA) used in C57BL/6 mice. In a dose escalation study, treatment with IL-15 SA at a dose of 2 μg/mouse for 4 days was toxic in mice as indicated by hypothermia, weight loss and even death (Fig. A), whereas doses of less than 2 μg were non-toxic. With toxic dose of IL-15 SA treatment, splenic and hepatic NK cells were markedly expanded and activated in mice, with a preponderance of newly-generated pro-inflammatory (CD27+CD11b+) and cytotoxic (CD27-CD11b+) subsets, when compared to vehicle-treated mice. To identify whether the expansion of activated NK cells contributes to IL-15 SA toxicity, C57BL/6 mice received intraperitoneal administration of anti-asialoGM1 antibody to deplete NK cells prior to the IL-15 SA treatment described above. Body temperature, body weight and survival were significantly higher in anti-asialoGM1-treated mice than in IgG control mice, indicating less toxicity is induced (Fig. B). Anti-asialoGM1 treatment markedly diminished IL-15 SA-induced expansion of splenic NK cells, primarily the pro-inflammatory and cytotoxic NK subsets. Thus, the proposed study suggests that toxicity induced by IL-15 SA is due primarily to the expansion of activated NK cells. Of note, NKT and mCD8 T cells were also expanded in toxic mice. However, antibody-induced CD8 T cell depletion didn’t reverse IL-15 SA toxicity. More studies will be done to define the role of NKT cells in IL-15 SA toxicity.
CANNABINOID RECEPTOR ACTIVATION REGULATES P38 AND ERK THAT ARE ASSOCIATED WITH A PROTECTIVE ROLE IN A MURINE MODEL OF SEPSIS
J.W. Kuethe, B.L. Johnson, E.F. Midura, and C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Background: Sepsis remains a complex clinical condition resulting from a number of infectious etiologies. The immune response and subsequent inflammation can lead to significant tissue injury, organ failure and death. Previous reports have shown that activation of the Cannabinoid 2 Receptor (CB2R) is capable of modulating the immune response. The mechanism by which the CB2R may exert this effect during sepsis has yet to be elucidated.
Materials and Methods: Taconic C57blk/6 mice underwent cecal ligation and puncture (CLP) to produce a murine model of sepsis. Peritoneal lavage cells were collected for ex vivo cell signaling studies. The lavage cells were stimulated with LPS and in conjunction with AM630 (a selective CB2R antagonist) or GP1a (a selective CB2R agonist) or a vehicle control in an ex-vivo manner. Flow cytometry was utilized for cell analysis. Pharmacological agents were purchased from Tocris Bioscience.
Results: Our data illustrate that CB2R signaling in sepsis provides a protective role. CB2R-deficient mice experienced increased mortality, inflammation, bacterial burden and more severe lung injury after CLP. In contrast, treating mice with GP1a (a selective CB2R agonist) decreased mortality. The mice also experienced less bacterial burden and diminished CLP-induced increases in serum cytokines as well as decreased organ injury. In ex-vivo studies to further elucidate the cellular mechanism, antagonism of the CB2R resulted in a significant increase in phosphorylation of both p38 and ERK in myeloid cells. Activation of the CB2R with GP1a resulted in minimal effect in either pathway.
Conclusion: CB2R can modulate myeloid cells through activation of the MAP-kinases, p38 and ERK. We speculate that the CB2R exerts a protective effect during a murine model of sepsis through p38 and ERK activation.
ADMINISTRATION OF MITOQ PRIOR TO HEMORRHAGE INCREASES HEPATIC NECROSIS IN A RAT MODEL OF HEMORRHAGIC SHOCK AND RESUSCITATION
R.D. Powell*, I.H. Mckillop, T.T. Huynh*, and S.L. Evans*. Carolinas Medical Center, Charlotte, NC
Introduction: Hypoxic injury and oxidative stress associated with hemorrhagic shock and resuscitation (HSR) leads to cell damage and multiple organ dysfunction. Reactive oxygen species (ROS) contribute to mitochondrial damage and organ failure. We sought to determine if MitoQ (MQ), a mitochondrial-targeted antioxidant, improves outcomes in a rat model of HSR and identify potential mechanisms by which it does so.
Methods: HSR was achieved in male rats by arterial blood withdrawal to a MAP of 25±2.0 mmHg for 1-hr prior to resuscitation. MQ (5mg/kg) or saline (0.9%) was administered (iv) 15-mins prior to hemorrhage, followed by ip administration (MQ; 20mg/kg) immediately after resuscitation (n=5 per group). Morbidity was assessed based on cumulative markers of animal distress (0-10 scale), and rats sacrificed 2-hrs after procedure completion. Tissue (liver and kidney) was collected and processed for blind-scoring (0-5 scale) following histology (H&E) or immunohistochemistry using antibodies specific against TNF-α and IL-6.
Results: Morbidity at 2-hrs was increased in both HSR and MQ animals vs Sham (7.0±0.3; HSR vs 8.2±0.6; MQ vs 0.6±0.3; Sham; p<0.05). Liver tissue exhibited more necrosis in MQ vs HSR (2.5±0.08 vs 2.0±0.15; p<0.05), and IL-6 expression (2.5±0.14 vs 1.7±0.14; p<0.05). Conversely, TNF-α expression in MQ was reduced compared to HSR (2.7±0.15 vs 3.2±0.08; p<0.05). In kidneys no significant difference was detected in levels of necrosis, TNF-α or IL-6 expression.
Conclusions: Our data demonstrate that MQ treatment prior to hemorrhage increases morbidity and exerts effects preferentially in the liver over the kidney.
THE DEVELOPMENT OF ACUTE LUNG INJURY AFTER POLYTRAUMA AND HEMORRHAGE IN RATS
X. Wu, D.N. Darlington, M.G. Schwacha, and A.P. Cap. US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: We have developed an Acute Coagulopathy of Trauma (aCOT) in rat that mimics the clinical hypocoagulation in both civilian and military patients with severe trauma and hemorrhage.
Objective: Determine if acute lung injury develops to polytrauma and hemorrhage, with no resuscitation.
Methods: Sprague-Dawley rats (n=8) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, right and medial lobes of the liver, the right leg skeletal muscle, and by fracturing the right femur. The rats were then bled to a mean arterial pressure of 40mmHg until 40% of the blood volume was removed. No fluid resuscitation was given. Lung tissue was taken at 240min and from rats who had received trauma/hemorrhage and from sham control rats (no trauma/hemorrhage). Lung injury was assessed by gross histology, leukocyte and platelet infiltration, and tissue cytokine levels.
Results: There was a significant elevation in the pro-inflammatory cytokines IL-1α, IL-1β and IL-6, anti-inflammatory cytokines IL-4, IL-10 and IL-13, and chemokines MIP-1α and GRO KC (CXCL1) in lung after polytrauma and hemorrhage. There was also a significant rise in growth factors GM-CSF and M-CSF. Polytrauma and hemorrhage led to an increase in the percentage of circulating monocytes and granulocytes, and an increase in the number of granulocyte-platelet interactions. There was also an increase in the thickness of the alveolar wall, and an increased the number of neutrophils and monocytes (CD11b positive stained cells), macrophages (CD68) and platelets (CD61) in lung tissue by 240 min as compared to sham controls.
Conclusion: Acute lung injury develops after polytrauma and hemorrhage in the rat, without resuscitation, and without direct damage to the lung. This project was funded by MRMC.
ATB-346, A HYDROGEN SULFIDE-RELEASING CYCLOOXYGENASE INHIBITOR, ENHANCES MOTOR FUNCTION AND REDUCES CORTICAL LESION VOLUME FOLLOWING TRAUMATIC BRAIN INJURY IN MICE
I. Paterniti*1, M. Campolo1, R. Siracusa1, R. Di Paola1, G. Bruschetta1, M. Cordaro1, E. Esposito*1, J. Wallace*3, and S. Cuzzocrea*1, 2. 1University of Messina, Messina, Italy, 2University of Manchester, Manchester, United Kingdom, 3University of Calgary, Calgary, AB, Canada
Traumatic brain injury (TBI) is a major cause of death and disability in young people. A plethora of pharmacologic interventions, that recognized initiators and propagators of pathology, are being investigated in an attempt to ameliorate secondary injury processes that follow traumatic brain injury. In recent years, physiological roles of hydrogen sulfide (H2S) have been recognized, and there is emerging evidence that this endogenous gaseous substance can modulate inflammatory processes. Therefore, in order to evaluate the effect of Hydrogen Sulfide-Releasing Cyclooxygenase Inhibitor such as ATB-346, a controlled cortical impact (CCI) was performed in adult mice, acting as a model of TBI. Thus, our results indicated that ATB-346 treated mice showed a significant improvement in motor dysfunction and reduced brain lesion volume as evidenced by decreased 2,3,5-triphenyltetrazolium chloride staining across the brain sections. Moreover, ATB-346 significantly reduced the severity of inflammation decreasing pro-inflammatory cytokines levels such as TNF-α and IL-1β; reducing iNOS and COX-2 expression and neurotrophic factors production such as GDNF and NGF, that characterized the secondary events of TBI. These data demonstrate that ATB-346 can be efficacious in a TBI animal model by enhancing the secondary inflammation process and tissue injury that follow TBI. Therefore, ATB-346 could represent an interesting approach for the management of secondary damage following CNS diseases counteracting behavioral changes and inflammatory process.
THE EFFECT OF H2 GAS INHALATION FOR MASSIVE HEMORRHAGE SHOCK IN A RAT MODEL
Y. Kobayashi, K. Hayashida, M. Suzuki, and S. Hori. Keio University School of Medicine, Tokyo, Japan
Objectives: Hydrogen (H2) gas has potential as an antioxidant. We aimed to assess the early resuscitative effects of H2 inhalation in a rat massive hemorrhage shock (HS) model.
Methods and Results: Controlled HS was induced over a period of 15 min through the arterial catheter to induce hypotension with MAP < 50 mmHg in rats. HS was observed without fluid resuscitation for 60 mins. At the end of shock, animals were resuscitated with equal their shed blood (SB) volume of hydroxyethyl starch 130/0.4 (HES) with or without 1.3% H2 inhalation. All animals were mechanically ventilated under normoxia (FiO2 ≤ 0.26) and normothermia with monitoring for 2 h after induced HS. The survival rate at 2 h after HS was improved in the H2 inhalation group (87.5%) compared to the control group (50.0%). In rats with survival for 2 h, serum IL-6 level was increased in the control group compared with sham (mean ± SEM, 20982.3 ± 12571.5 vs. 81.9 ± 74.9 pg/mL, P<0.05), but this increase was prevented in the H2 inhalation group (13107.8 ± 2178.6 pg/mL).
Conclusions: Inhalation of H2 gas is a favorable strategy to mitigate mortality in a rat HS model, probably by inhibiting the release of pro-inflammatory cytokines.
MITOCHONDRIAL ACTIVITY IS IMPORTANT FOR T-CELL ACTIVATION AND HOST DEFENSE IN SEPSIS
C. Ledderose1, Y. Bao1, X. Li1, M. Lidicky1, N.I. Shapiro2, and W. Junger*1. 1Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 2Department of Emergency Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
Patients suffering from severe sepsis and septic shock often develop profound immunosuppression and a state of “T cell exhaustion” that is characterized by poor effector functions. We have previously reported that ATP release and autocrine purinergic signaling via P2X receptors are required for T cell activation. Here we show that mitochondria provide the ATP for this process and that defective mitochondria in sepsis patients cause T cell dysfunction.
We found that CD3/CD28 stimulation of primary human CD4+ T cells rapidly activated mitochondrial ATP production. Inhibition of mitochondrial function with CCCP or rotenone blocked the release of ATP in response to cell stimulation and impaired calcium signaling and IL-2 production. Using dihydrorhodamine 123 (DHR) as a readout of mitochondrial activity, we found that the function of mitochondria was reduced in lymphocytes of sepsis patients when compared to control patients and healthy subjects. This inhibition was paralleled by impaired calcium signaling. These findings suggest that mitochondrial ATP production and ATP release are essential for T cell activation and that impaired mitochondrial function diminishes host defenses against infections. Funded by NIH R01GM51477.
EFFECTS OF COMBINED SILENCING OF CASPASE-3, FADD AND FAS ON THE PATHOGENESIS OF CHEST TRAUMA INDUCED SEPTIC ACUTE LUNG INJURY (ALI)
C. Hohmann1, 2, P. Kellermann2, S. Weber2, F. Schaupp2, M. Huber-Lang*2, and M. Perl*3, 2. 1University of Leipzig, Leipzig, Germany, 2Dept. of Orthopedic Trauma, University of Ulm, Ulm, Germany, 3BG-Trauma Center Murnau, Murnau, Germany
Pulmonary silencing of apoptotic molecules like Fas or Caspase-3 ameliorates ALI. Whether a combined therapeutic silencing approach addressing several apoptotic molecules simultaneously would be of additional benefit remains unstudied. To address this, a small interfering (si)RNA mix (300μg/100μl) against procaspase-3, FADD and FAS or control (nt)RNA was intratracheally instilled into C57Bl/6 mice 24 hrs before and 12 hrs after chest trauma followed by cecal ligation & puncture 12 hrs there-after. 12 or 24 hrs later, lung tissue & bronchoalveolar lavage fluid (BALF) were harvested. Lung cytokines were quantified by ELISA, lung active caspase-3 (C-3), FADD and FAS by western blotting. Total protein in BALF and lung histology (H&E) were assessed. N=6-8/group, Two-way ANOVA, SNK, p<0.05. IL-1b at 12 and 24 hrs and MIP-2 at 24 hrs were significantly decreased in the lung whereas TNF-α in BALF was increased 24 hrs following trauma and siRNA treatment. FAS, FADD and active Cas-pase-3 in lung tissue at 12 hrs showed slightly reduced levels after siRNA treatment. BALF total protein was markedly increased after siRNA and ntRNA administration at 12 hrs after trauma and sepsis. However, at 24hrs ntRNA treated animals still showed markedly increased BALF protein whereas siRNA treated mice did not differ from sham. Our data indicates, that simultaneous silencing of up- and downstream apoptotic molecules in the lung in a clinical relevant double hit model of trauma induced septic ALI does not necessarily augment the beneficial effects seen after single target silencing. In this regard potential dose dependent proinflammatory effects of double stranded siRNA and the potential compensatory upregulation of alternative death pathways need to be considered in further studies. (Support DFG-PE 908/2-1).
AICAR BLUNTS HYPERGLYCEMIA DURING HEMORRHAGIC SHOCK
Y. Huang1, V.A. Locke1, A.S. Miner3, 1, B. Sharma4, P.H. Ratz*3, 5, and R.W. Barbee*2, 5. 1Dept. of Emergency Medicine, Virginia Commonwealth University, Richmond, VA, 2Depts. of Emergency Medicine and Physiology, Virginia Commonwealth University, Richmond, VA, 3Depts. of Biochemistry and Pediatrics, Virginia Commonwealth University, Richmond, VA, 4Dept. of Biochemistry, Virginia Commonwealth University, Richmond, VA, 5VCURES, Richmond, VA
Previous literature suggests that AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and hyperglycemia both promote survival during hemorrhagic shock. However, AICAR might suppress glucose appearance via AMPK (adenosine monophosphate-activated kinase) activation. We therefore sought to examine mechanisms of AICAR action both in vitro (rabbit femoral - FA and superficial inferior epigastric - SIEA arteries) and in vivo (rabbit pressure-controlled hemorrhage). FA and SIEA were sampled for in vitro contraction-response curves to the alpha adrenergic agonist phenylephrine (with inhibition of cGMP and NO) in the presence of AICAR (2 mM) or vehicle (0.3 % DMSO) or analyzed for AMPK phosphorylation (AMPKp-T172) by Western blotting. Anesthetized (ketamine sedation followed by Alfaxan infusion) male New Zealand rabbits (3.0 - 3.4 kg) were instrumented for MAP (mean arterial blood pressure), CO (cardiac output) and FA blood flow and infused with AICAR (bolus of 7.5 mg/kg, followed by infusion of 2 mg/kg/min) or vehicle (Ringer’s solution; bolus of 0.3 ml/kg followed by 0.03 ml/kg/min) for 2 hours. Animals were then hemorrhaged to a MAP of 30 - 35 mm Hg, and followed for approximately one hour. AICAR (2 mM) significantly increased by >½ log the EC50 of phenylephrine and increased AMPKp-T172 (~ 3.0 fold compared to basal/DMSO) within FA, but not SIEA. Blood glucose levels dropped during AICAR infusion compared to vehicle (P = 0.04). Just prior to hemorrhage, AICAR lowered CO (P = 0.003), FA blood flow (P = 0.001) and increased blood lactate (P = 0.006). However, the subsequent hemorrhage induced fall in CO (P = 0.018) and rise in blood glucose (P=0.056) was blunted in AICAR compared to vehicle infused animals, without significant changes in total peripheral resistance or heart rate. Appearance of lactate was slightly lower, with a tendency toward lower whole-body oxygen extraction and oxygen deficit. In conclusion, AICAR relaxes large muscular but not smaller muscular cutaneous arteries, possibly via AMPKp-T172 in vitro. However, vasodilation is overridden in vivo. Sympathetically mediated hyperglycemia is blunted, while lactate output is similar (independent of oxygen deficit), possibly due to enhanced glycolysis. Activation of AMPK might be less beneficial in hemorrhage compared to resuscitation. Supported by DOD Grant #W81XWH-12-1-0525.
SEVERE ACIDOSIS FOLLOWING INJURY IS ASSOCIATED WITH ALTERED GENOMIC INFLAMMATORY SIGNATURE AND INCREASED MORBIDITY
R.E. Plevin1, M.J. Delano1, L. Moldawer*2, H. Baker2, M. Lopez2, G.E. O’Keefe*1, R.V. Maier*1, and J. Cuschieri*1. 1University of Washington, Seattle, WA, 2University of Florida, Gainesville, FL
Background: Elevated base deficit (BD) is an indicator of ongoing hypoperfusion & physiologic shock which is associated with worse outcomes in trauma patients. Little is known about the genomic changes that result from acidosis. We investigated whether the degree of hypoperfusion correlates with genomic alterations in pathways related to infection and organ failure in the immune cells of trauma patients.
Methods: We performed a retrospective review of prospectively collected data from the Inflammation and the Host Response to Injury trauma database. Inclusion criteria were adult blunt trauma patients who had a BD recorded within 12 hours of admission and survived for 28 days post-injury. Patients were classified into 3 groups: 1) BD ≤ 6; 2) 6 < BD ≤ 10; and 3) BD > 10. Multivariate analysis was performed to evaluate the effects of BD on clinical outcomes. Affymetrix Glue Grant Human Transcriptome Arrays™ were used to analyze gene expression in neutrophils, monocytes, and lymphocytes obtained from whole blood at 12 hours of admission and 6 time points over the next 28 days in 244 patients. The microarray data was normalized and gene expression was compared between BD groups at each time point as well as across BD groups over time.
Results: In multivariate analysis, BD > 10 correlated with increased rate of multi-organ failure (45% vs. 30%), longer time to recovery from organ dysfunction (17 days vs. 12 days), and increased mortality (23% vs. 12%). Although genomic changes were evaluated in both innate and adaptive immune cells, only changes in monocyte gene expression persisted over the 28 days post-injury. Of the 477 monocyte genes with significant expression changes, 177 fit into known signaling pathways. These included genes within the inflammatory and antigen-presentation pathways, which remained most significant in patients with a BD > 10.
Conclusions: Increased base deficit is associated with more organ failure, longer time to recovery from organ failure, and higher mortality. These poor outcomes correlate with persistent changes in the genomic signature of monocytes, but not of neutrophils or lymphocytes. Thus, genetic alterations in monocyte antigen presentation and inflammatory pathways may represent an “early genomic hit” which helps explain increased rates of infection and organ failure in the most severely injured trauma patients.
CASPASE-1 DEPENDENT ACTIVATION OF MMP9 VIA THE TLR4/MAP KINASE PATHWAY IN CARDIAC MYOCYTES
D.L. Carlson*, and S.E. Wolf*. University of Texas Southwestern, Dallas, TX
Caspases are a family of genes important for maintaining homeostasis through regulating cell death and inflammation. Caspase 1 (Cas1) is an important modulator of cytokine signaling in response to stress. The primary role of Cas1 is the activation of IL-1β and IL-18 from inactive precursors; it also induces production of IL-1α, IL-6, TNF, and metallomatrix proteases (MMP). MMP9, in turn, contributes to cytokine processing, a crucial upstream signal for induction of the inflammatory response in the heart. Previously, we have demonstrated an improvement in cardiac function during sepsis in the absence of Cas1 in rodents. To delineate the mechanism through which Cas1 affects the heart we have examined the relationship between inflammation and Cas1 directly on murine myocytes. Neonatal myocytes were isolated from Cas1 deficient and control mice. Cells were cultured and stimulated with either saline or 100 ng/ml of LPS. Cells and supernatants were collected at 1, 2, 4, and 8 hr post stimulation. Control cells consistently showed an elevation in MMP9 to Cas1 deficient cells post stimulation (246±64, 219±74, 216±34, 165±38, vs 84 ±26 ng/ml p<0.05) as measured by ELISA . MMP9 expression was confirmed by qPCR (63.3±1.3, 10.2 ±4, 6.5±2.5 p<.001, compared to 1±0.65 normalized fold expression to control) and zymography. IL-1β, TNF, and IL-10 from both the media and cell lysates were compared and found to be consistently depressed in the Cas1 deficient group.
As IL-1β directly stimulates MMP9 expression via the TLR4/MAP kinase pathway, we examined phosphorylation of ERK, JNK and p38. Stimulated controls showed a 200, 278 and 576 % increase in phosphorylation of ERK as compared to non-stimulated cells. Similar increases in phosphorylation were noted for JNK and p38 in LPS stimulated control cells. In comparison, Cas1 deficient cells demonstrated significantly less phosphorylation for all genes post stimulation (25, 10, 18 % increase over non-stimulated for ERK).
We conclude that in the absence of Cas1 cardiac myocytes demonstrate a depressed inflammatory response specifically through inhibition of the TLR4/MAP kinase pathway. We propose that Cas1 might inhibit IL-1β specific upstream signaling molecules such as the metallomatrix proteases, which are required for ERK activation, thus limiting cytokine and MMP9 expression, suggesting a cardioprotective role for Cas1 during sepsis.
CHOP REGULATES EXPRESSION OF INFLAMMATORY MEDIATORS DURING SEPSIS
M. Ferlito, Q. Wang, W.B. Fulton, P.M. Colombani, and C. Steenbergen. The Johns Hopkins University School of Medicine, Division of Cardiology, Baltimore, MD
We investigated hydrogen sulfide (H2S) as a new therapeutic agent for sepsis and found that H2S decreases the inflammatory response induced by cecal ligation and puncture (CLP) and significantly improves survival of septic mice. We found that the benefit of H2S is dependent, at least in part, on the inhibition of the transcription factor C/EBP homologous protein (CHOP). CHOP is part of an intricate cytoplasmic and nuclear signaling pathway, the “unfolded-protein response” (UPR), which is activated upon endoplasmic reticulum (ER) stress. Furthermore, a new role for ER stress and UPR signaling in the regulation of inflammation and innate immunity is emerging. We have recently demonstrated, for the first time, that genetic deletion of Chop results in increased survival after CLP or LPS injection in mice (Ferlito M. et al., J Immunol, 2014). Here we investigated whether during sepsis CHOP regulates the production of specific inflammatory mediators. Sepsis was induced by CLP in male wild-type (WT) and CHOP knock out (KO) mice, and after 18 hours, blood was collected. We used a protein array to detect expression of over 40 cytokines and chemokines. We found that accumulation of 16 mediators was decreased (30% to 70%) in septic CHOP KO compared to septic WT mice. The most down-regulated mediators in the CHOP KO group include IL-1β, IL-10, IL-6, CCL4, CXCL10, CXCL9 and CCL5. Among the remaining 24 mediators, some were undetectable, others such as CCL3 and CXCL2 had similar expression, and a few, including CXCL1 were slightly (about 20%) increased in KO compared to WT mice during sepsis. Our results highlight the concept that genetic deletion of Chop does not equally affect the expression of all mediators; rather it appears to decrease mediators that may be harmful to the host while maintaining others that can be beneficial. Thus, our data strongly suggest that CHOP may differentially regulate the inflammatory response during sepsis, by both inhibiting and enhancing transcription of target genes.
CONTINUOUS CARDIAC OUTPUT ARE CORRELATED WITH LEUKOCYTES AND NEUTROPHILS COUNTS IN A CANINE MODEL OF SEPTIC SHOCK
Y. Zhang1, 2, R. Yang1, 2, S. Liu2, and L. Zhao2. 1Dalian Medical University, Dalian, China, 2Dalian Municipal Central Hospital, Dalian, China
Introduction: Septic shock is a life-threatening complication of infection and injury that occurs up to 19 million cases worldwide per year, and presenting 10% of all ICU admissions. To date, it is known that leukocytes play an important role in defensing the pathogens and initiating the immune response. While a prolonged and vigorous response can cause organ failure. The leucocytes counts changing slowly, but the hemodynamic parameters, e.g. cardiac output (CO), cardiac index (CI) continuously measured by Picco which usually used in septic shock patients. This study aimed to find whether they are correlated or not.
Materials and Methods: Length of protocol was 24hrs. Twelve male adult mongrel dogs were randomized into sham group (n=6), and septic shock group (n=6) which intravenous infusion E coli for 2hrs with a MAP of 35±5mmHg declined for1.5hrs, restored hemodynamic by fluid resuscitation rapidly, and began pumping of Amoxicillin after 6 hrs. We used some invasive and non-invasive devices, e.g. Picco catheters detected hemodynamic, and peripheral blood samples were collected at set time points (baseline, 0hr, 6hr, 12hr, 18hr, 24hr). Differences were considered significantly when P<0.05 by t-test or ANOVA.
Results: Both sham and septic shock dogs survived. The CO and CI significantly differed at the same time points of the septic shock dogs and sham ones, and increased before 6hrs then decreased (P<0.01;P <0.05),as well as MABP (P<0.05) ; SVR changed to the contrary after septic shock (P>0.05). The leukocytes and neutrophils counts in septic shock group increased sharply than the sham group after 6hrs (P<0.05; P<0.05). In septic shock group, of all the parameters, CO and CI were significantly correlated with the leukocytes (r=0.565, p<0.01; r=0.588, p<0.01), and neutrophils (r=0.556, p<0.01; r=0.592, p<0.01); however, in the sham group, they were not.
Conclusions: In summary we illustrated that CO and CI of hemodynamic parameters are correlated with leukocytes and neutrophils counts in our hyperdynamic septic shock models. It is feasible that the leukocytes counts can be relatively predicted by hemodynamic device continuously. Timing and proper intervention before overwhelming leukocytes induced organ injury will be studied in the future.
TRANEXAMIC ACID-INHIBITED DIFFERENTIAL THROMBELASTOGRAPHY IS A MORE SENSITIVE AND RAPID PREDICTOR OF HYPERFIBRINOLYTIC HEMORRHAGE IN TRAUMA THAN CONVENTIONAL VISCOELASTIC HEMOSTATIC ASSAYS
M.P. Chapman1, 4, E.E. Moore*2, 1, H.B. Moore1, 2, E. Gonzalez1, 2, T.L. Chin1, 2, C.C. Silliman3, 1, and A. Banerjee1. 1University of Colorado, Denver, CO, 2Denver Health and Hospital Authority, Denver, CO, 3Bonfils Blood Center, Denver, CO, 4Georgia Regents University, Augusta, GA
Background: Hyperfibrinolysis is an uncommon but highly lethal component of trauma induced coagulopathy (TIC). Treatment with antifibrinolytics must be administered as early as possible after injury to be effective. Unfortunately, conventional laboratory tests for hyperfibrinolysis are slow and have poor sensitivity for hyperfibrinolytic bleeding. Therefore, we developed a novel thrombelastogram (TEG)-based assay to detect tranexamic acid (TXA)-reversible fibrinolysis. We hypothesized that this assay would achieve more rapid and accurate detection of clinically significant hyperfibrinolysis in trauma patients.
Methods: Our novel TEG assay, differential TXA-inhibited functional fibrinogen TEG (DIFF-TEG), consists of two channels of platelet-inhibited whole blood, one run in the presence of TXA, the other without. The differential in clot strength between these two channels is used to calculate the degree of TXA-reversible fibrinolysis in the patient’s blood in real time. Blood was collected in the field from 89 consecutive trauma patients, and tested with DIFF-TEG and conventional TEG. Death from hemorrhage or a requirement of 10 or more units of PRBCs were considered clinically significant hemorrhage.
Results: Of 89 patients, 10 had clinically significant hemorrhage. Of these, 8 had TXA-reversible fibrinolysis on DIFF-TEG. Overall, DIFF-TEG was 80% sensitive and 94% specific for significant hemorrhage. Traditional, kaolin TEG was only 30% sensitive, but 99% specific for hemorrhage. Moreover, DIFF-TEG yielded a result 30 minutes faster than kaolin TEG, as the result is calculated at the maximum clot strength, not at the later lysis-at-30-minutes (LY30) time point as in traditional TEG.
Conclusion: The novel DIFF-TEG assay is a 2.5x more sensitive predictor of massive hemorrhage in trauma than conventional assays. It has strong predictive value for TXA-reversible hyperfibrinolytic bleeding in samples drawn immediately after injury. Additionally, this assay yields results 30 minutes faster than conventional TEG. Thus, DIFF-TEG represents a powerful new tool to guide the use of TXA in severely injured trauma patients.
OXYGEN BUT NOT GLUCOSE DEPRIVATION ACTIVATES AMPK IN RABBIT LEG ARTERIES
T. Makinde1, A.D. Latham4, A.S. Miner4, 2, Y. Huang1, V.A. Locke1, B. Sharma4, G. Chen4, R.W. Barbee*2, 5, and P.H. Ratz*3, 5. 1Dept. of Emergency Medicine, Virginia Commonwealth University, Richmond, VA, 2Depts. of Emergency Medicine and Physiology, Virginia Commonwealth University, Richmond, VA, 3Depts. of Biochemistry and Pediatrics, Virginia Commonwealth University, Richmond, VA, 4Dept. of Biochemistry, Virginia Commonwealth University, Richmond, VA, 5VCURES, Richmond, VA
AMP-activated protein kinase (AMPK) is a primary metabolic stress sensor, while Akt activity is a measure of the fed-state. Glucose and O2 levels change dramatically in hemorrhage and other pathological conditions. Femoral and saphenous arteries were isolated from male NZW rabbits (2.9 -3.5kg). Blood vessels were divided into one of four treatment groups: control (CON; 11.2mM glucose, pO2 ~150mmHg), zero glucose (GLC-0), hypoxia (HYP; <10mmHg), or hypoxia plus zero glucose (STARVE). Tissues at 37°C were quickly frozen in an acetone/dry ice slurry at 30, 60, and 120 minutes, thawed in 6% trichloroacetic acid, 10mM dithiothreitol and 30mM NaF, and processed. Homogenates were subjected to Western Blot to measure levels of activated AMPK (AMPK-pT172), Akt (Akt-pS473 and Akt-pT308), ACC (ACC-pS79) and Raptor (Raptor-pS792). ACC-pS79 and Raptor-pS792 were evaluated as downstream activation targets of AMPK. Responses from tissues in the GLC-0, HYP and STARVE groups were reported as fold-change from CON responses. Tissues from the GLC-0 group displayed no change in any of the phospho-proteins assessed at 30, 60 and 120 min. At 30 and 60 min, tissues from the HYP group displayed ~4-fold increases in AMPK-pT172 and ACC-pS79, and a 1.5-fold increase in Raptor-pS792. Akt-pS473 and Akt-pT308 were reduced by ~50% at 30 min. Tissues from the STARVE group displayed a strong 8-fold increase in AMPK-pT172, 10-fold increase in ACC-pS79 and 2-fold increase in Raptor-pS792. Raptor-pS792 declined to the CON level at 60 min whereas AMPK-pT172 and ACC-pS79 remained at high levels until 120 min, at which time AMPK-pT172 and ACC-pS79 declined to the CON level. Although AMPK phosphorylation fell to the CON level at 120 min, Akt phosphorylation did not increase (return) to the CON level at 60 or 120 min. These data show that rabbit muscular arteries respond to metabolic stress with increases in AMPK signaling and reductions in Akt signaling pathways. Notably, glucose deprivation alone was insufficient to induce metabolic stress, suggesting that these arteries may use an alternate energy substrate in the absence of exogenous glucose. However, in the absence of exogenous glucose, hypoxia induces a more dramatic metabolic stress than did hypoxia alone. The lack of sustained stressor responses and lack of increase in Akt at 120 min suggests the transition to irreversible damage. Support: DOD Grant #W81XWH-12-1-0525.
CONTROLLING PELVIC HEMORRHAGE USING A NOVEL PRESSURE GARMENT
M.H. Tiba*1, 2, G.T. Draucker1, 2, B.M. McCracken1, 2, and K.R. Ward*1, 2. 1University of Michigan, Ann Arbor, MI, 2Michigan Center for Integrative Research in Critical Care, Ann Arbor, MI
Objectives: Hemorrhage is the leading cause of death for both civilian and battlefield injury. Wounds to the groin as well as intra-cavitary wounds of the abdomen are of special concern and have been considered non-responsive to external pressure (non-compressible hemorrhage or NCH). We tested a new prototype garment for its ability to reduce or halt blood flow in a model of pelvic arterial injury.
Methods: Nine male swine, 40(1.6) kg were anesthetized, instrumented then randomized into two groups (treatment and control). Animals underwent laparotomy with placement of a 4-0 stainless steel monofilament suture through the Right iliac artery. The laparotomy was closed and the iliac suture was exteriorized. The left femoral artery was isolated and cannulated for blood pressure monitoring distal to the injury. Both groups were subjected to uncontrolled hemorrhage by pulling the suture through the iliac artery and tearing it. In the treatment group, hemorrhage was controlled by a specially designed pressure garment that was applied over the lower abdomen for 60 minutes followed by garment release and monitoring for 30 minutes or until the animal expired. The control group did not have the pressure garment applied. Hetastarch (500mL) was infused immediately after hemorrhage.
Results: All treatment animals survived for 60 minutes during pressure garment application. Average survival time for the control group was 8(8.5) min. Following pressure release, treatment animals survived an average of 17(12.1) min. Kaplan-Meier survival curve, log-rank (Mantel-Cox), and hazard ratio (log-rank) tests were used to compare the groups. There was a significant difference in survival time between treatment and control groups (p < 0.003). Log-rank hazard ratio between the two groups was (4.9) indicating that the rate of death in the control group was 4.9 greater than that in the treatment group. Lactate levels at the end of 60 minutes in the treatment group were 1.7(0.5) meq/L.
Conclusion: The pressure garment was successful in improving survival for 60 minutes after an otherwise lethal vascular injury. Such a device may be helpful as bridge to newer endovascular methods of hemorrhage control.
COMPARISONS OF NORMAL SALINE AND LACTATED RINGER’S IN NORMAL AND HYPOTENSIVE RESUSCITATION IN PIGS AFTER HEMORRHAGIC SHOCK
W.Z. Martini, K.K. Chung, and M.A. Dubick*. US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: Despite decades of use, questions continue to arise regarding the different physiologic effects of normal saline (NS) and lactated Ringer’s (LR). Recent studies have demonstrated that NS results in hyperchloremic metabolic acidosis and resultant acute kidney injury when used as a primary resuscitative fluid for various conditions. For the military these effects are of particular interest as NS is the most commonly used resuscitation fluid on the battlefield. This study compared hemodynamic, metabolic and biochemical effects of LR and NS in normotensive (E1) and hypotensive resuscitation (E2) swine models.
Methods: In E1, anesthetized pigs were randomized into control (n=6), LR (n=7), and NS (n=7) groups. Hemorrhage of 60% estimated total blood volume was induced, followed by resuscitation with either LR at 3 times the bled volume or the volume of NS to reach the same MAP as in LR group. In E2, pigs were subjected to a controlled hemorrhage of 20 ml/kg over 5 min. After 30 min, hypotensive resuscitation with LR (n=9) or NS (n=7) to reach and maintain a SBP of 80 mmHg was started along with a second hemorrhage of 8 ml/kg. Hemodynamics was measured continuously and blood samples for measurements of blood gases, base excess, lactate and electrolytes were taken at baseline (BL) and at times up to 3h.
Results: In E1, MAP was decreased by hemorrhage but returned to BL within 1h after resuscitation with LR (119±7 ml/kg) or NS (183±9 ml/kg). In E2, hemorrhage reduced MAP from 85±5 to 37±4 mmHg and target SBP on resuscitation was achieved with equal volumes of LR or NS resuscitation. Base deficit (BD) increased by hemorrhage; resuscitation with LR normalized BD but not with NS in E1; in E2, BD was higher in NS than LR. In E1, blood lactate concentrations were higher in the LR than NS group, but in E2, an opposite trend was observed, although the differences were not statistically significant. Plasma Na+ and Cl- concentrations were increased by NS in both E1 and E2, with no differences in K+. Serum creatinine and blood urea nitrogen increased after hemorrhage and resuscitation, with no differences between LR and NS.
Conclusions: NS results in a significantly different biochemical profile than LR when used as a resuscitative fluid after hemorrhage. Further studies are needed to determine the clinical relevance of these findings.
EFFICACY OF A LYOPHILIZED EXTRACT OF THE LARVAE OF THE GREENBOTTLE FLY LUCILIA SERICATA IN THE MANAGEMENT OF CHRONIC WOUNDS
E. Boelke1, P. Gerber1, B. Buhren1, N. Hoff1, H. Mehlhorn1, M. van Griensven*2, H. Schrumpf1, C. Matuschek1, T. Jansen1, A. Baran1, S. Braun1, and B. Homey1. 1Heinrich Heine University, Dusseldorf, Germany, 2Technical University, Munich, Germany
Background: Chronic wounds, are a common problem of the growing elderly population. The so-called bio-debridement or maggot-therapy with the use of living larvae of the greenbottle fly Lucilia sericata is an effective therapy to debride chronic wounds and stimulate the generation of fresh granulation tissue, finally leading to wound closure.
Objectives: Recently, we have generated a lyophilized extract of the larva of the greenbottle fly Lucilia sericata (Larveel®, Alpha-Biocare GmbH, Duesseldorf, Germany).
Methods: We present the results of the first four patients with venous leg ulcers treated with Larveel®. Moreover, we analyzed the effect of Larveel® on wound healing in an organotypic 3D-skin model (Phenion®).
Results: Four patients with venous leg ulcers that did not respond sufficiently to standard wound management strategies were treated with Larveel®. In three patients a significant improvement of the wound status was achieved, either resulting in complete wound closure or allowing surgical wound closure by mashed skin-graft transplantation. In those three patients we did not observe any relevant adverse effects of Larveel®-therapy. In one patient, Larveel®-therapy resulted in an increased wound colonization with Pseudomonas aeruginosa and progression of the ulcer. Interestingly, it has been reported that Pseudomonas aeruginosa may significantly impair the growth of Lucilia sericata, eventually killing the living larvae. Accordingly, larval therapy is particularly effective in the management of wounds infected with Gram-positive bacteria, like Staphylococcus aureus, but less so for wounds colonized or infected with Gram-negative bacteria, especially Proteus spp. and Pseudomonas spp. strains.
Analysis of an organotypic skin model (Phenion®) demonstrated that Larveel® promoted the healing of standardized artificial wounds in vitro in a dose- and time-dependent manner.
Conclusions: Larveel® may be a promising new option for the treatment of chronic wounds. Use of the extract may result in complete wound closure or may significantly improve the wound condition, enabling surgical wound closure by skin-graft transplantation. Prior to therapy microbial swab analysis should be performed to allow a concomitant therapy of the identified bacterial strains according to their resistogram.
DECREASED PRODUCTION OF ADIPONECTIN IS ASSOCIATED WITH POSTOPERATIVE COMPLICATIONS IN UPPER GASTROINTESTINAL CANCER SURGERY
A. Matsuda*1, M. Miyashita*1, T. Matsutani*2, H. Wakabayashi2, G. Takahashi1, and E. Uchida2. 1Department of Surgery, Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan, 2Department of Surgery, Nippon Medical School, Tokyo, Japan
Rationale: Postoperative complications (POCs) are responsible for longer hospital stays, increased medical costs, and poor survival. Adiponectin is an adipokine produced from adipose tissues and has an anti-inflammatory property via inhibition of NF-kB activation. The purpose of this study is to clarify the association between adiponectin and POCs in upper gastrointestinal cancer surgery.
Methods: Peripheral blood samples were collected from 46 upper gastrointestinal cancer patients (20 esophageal and 26 gastric cancer) before surgery and on postoperative days 1, 3, 5, and 7. Patients were divided into non-POC (n=30) and POC (n=16) groups. Plasma adiponectin levels were measured by ELISA.
Results: In both groups, the postoperative plasma adiponectin levels decreased transiently and then gradually recovered. The POC group had significantly lower adiponectin levels throughout the perioperative period than the non-POC group. The preoperative decreased adiponectin level was associated with increased postoperative inflammatory host responses (WBC and CRP).
Conclusions: Decreased adiponectin production is associated with POCs through an inhibition of the anti-inflammatory property.
SONIC HEDGEHOG SIGNALING MEDIATES HEMATOPOIETIC PRECURSOR CELL ACTIVATION DURING THE GRANULOPOIETIC RESPONSE TO E. COLI BACTEREMIA
X. Shi, W. Zhang, Y. Lin, Y. Chen, and P. Zhang*. MSU College of Human Medicine, East Lansing, MI
During bacterial infection, hematopoietic activity is shifted to enhance granulocyte production. This granulopoietic response is critical for reinforcing host defence. Our recent studies have revealed that activation of hematopoietic stem/progenitor cells constitutes a key component of the granulopoietic response. Hedgehog signaling has been reported to regulate stem/progenitor cell function during embryogenesis and in adulthood. In this study, we determined the role of Sonic hedgehog (SHH) signaling in mediating activation of hematopoietic precursor cells during the granulopoietic response to bacteremia. E. coli (E11775, ATCC, 5 x 10^7 CFUs/mouse) or saline was given to mice via penile vein injection. In a subset of experiments, intravenous BrdU (1 mg/mouse) was administered to animals 24 h before the termination of experiment. In response to E. coli bacteremia, SHH expression by bone marrow cells (including both differentiated lineage positive cells and undifferentiated lineage negative precursors) were markedly enhanced. This increase in SHH expression was regulated at the transcriptional level. Bone marrow SHH mRNA expression was significantly up-regulated at both 12 and 24 h after initiation of bacteremia. TLR4-ERK1/2 signaling mediated up-regulation of SHH expression by marrow cells. Inhibition of ERK activation with specific MEK1/2 inhibitor PD98059 (25 μM) blocked LPS (20 ng/ml)-induced up-regulation of SHH expression by marrow cells in the in vitro culture system. Hematopoietic stem cells and myeloid progenitor cells expressed high levels of Gli1 (the key component of the hedgehog pathway). In association with enhancement of SHH expression in the bone marrow, the expression of Gli1 by hematopoietic precursor cells was significantly up-regulated at both mRNA and protein levels following E. coli infection. Furthermore, the up-regulation of Gli1 expression correlated with the increase in the proliferative activity (as reflected by BrdU incorporation) of marrow hematopoietic precursor cells. Activation of cyclin D1 signaling appeared to be involved in Gli1-mediated hematopoietic precursor cell proliferation. These data suggest that SHH signaling may play an important role in activation of hematopoietic precursor cells during the granulopoietic response to systemic bacterial infection. (Support by NIH grant AA019676).
INTRATHORACIC PRESSURE REGULATION AUGMENTS STROKE VOLUME IN HUMAN HEMORRHAGE
N. Patel*1, R. Branson2, G. Kramer*1, M. Salter1, S. Henkel1, R. Seeton1, M. Khan1, A. Koutrouvelis1, D. Solanki1, H. Li1, A. Indrikovs1, and M. Kinsky*1. 1University of Texas Medical Branch, Galveston, TX, 2University of Cincinnati, Cincinnati, OH
Background: Early resuscitative measures e.g., establishment of IV access and infusion of vital fluids is the cornerstone in treating hemorrhagic shock. These measures require expertise, supplies, and personnel, which can be delayed or even absent. Alternative interventions that buy time for definitive care are needed. Intrathoracic pressure regulation [ITPR] is an emerging technology used to treat hypovolemia and cardiac arrest, when IV access is either limited or delayed. Preclinical studies demonstrate that ITPR increases venous return and thereby restores perfusion. We compared the impact of ITPR to placebo in humans undergoing hemorrhage under general anesthesia.
Methods: Healthy volunteers (n=7, age 21 - 35 yrs), underwent a paired study [received ITPR or placebo] on different study dates. After IRB informed consent was obtained, subjects were anesthetized, intubated and mechanically ventilated (TIVA propofol). Then a 10-mL/kg hemorrhage was performed. Twenty min after hemorrhage, ITPR (-10 mmHg vacuum) or placebo (device but no vacuum) was placed between endotracheal tube and ventilator for 60 min. Fluid (0.9% NaCl, 250 mL boluses) was administered if systolic BP < 85 mmHg. Hemodynamics e.g., stroke volume (SV), mean arterial pressure [MAP], systolic and diastolic function by echocardiography, and fluid balance were compared.
Results: Data shows [Δ Mean ± SEM] before and after ITPR/placebo intervention. There were no differences in MAP [ITPR (2.1 ± 3 mmHg); placebo (-0.7 ± 3 mmHg)] or fluid in [ITPR (17.4 ±4 mL/kg; placebo (18.6±5 mL/kg). Other volumetric indices such as urinary output and plasma volume were similar. On the other hand, ITPR augmented SV after hemorrhage (ΔSV:ITPR = 22 ± 5 mL vs. placebo = 6 ± 4 mL, p <0.05). Diastolic function was enhanced by ITPR (ΔE/e’: ITPR = -0.43 ± 0.48 vs. placebo = +0.81 ± 0.59), p <0.05).
Conclusion: ITPR did not improve mean arterial pressure; however, stroke volume was significantly improved. The increase in stroke volume could be due to better ventricular compliance, as other loading conditions e.g., preload and afterload were similar.
DEVELOPMENT OF POST-TRAUMATIC ACUTE LUNG INJURY MODEL: A PRELIMINARY STUDY
R. Pfeifer*1, 2, K. Horst1, 2, H. Andruszkow1, 2, H. Pape1, 2, and F. Hildebrand*1, 2. 1RWTH University Aachen, Aachen, Germany, 2RWTH Aachen University, Aachen, Germany
Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common complications associated with severe trauma and fat embolism. Moreover, lung is the first organ to fail and it is known to trigger the development of multiple organ failure (MOF). The main aim of this study is the development of a ventilated murin lung injury model. This allows the analysis of the dynamic of lung and multiple organ failure.
Methods: Lung injury was induced by two standardized models: hemorrhagic shock (HS) and oleic acid (OA). 30 μg of oleic acid diluted in 270 μl of serum was intravenously administered prior to the injection of pressure controlled HS (MAP of 35 ± 5 mmHg) for 90 minutes. In the end of the shock period mice were resuscitated with shed blood and saline fluid. After tracheotomy, mice were ventilated using the mouse ventilator (HSE Apparatus) (RR 130/ min. and tidal volume 200μl). Blood gas analysis (arterial oxygenation pO2 and Horivitz Index) and histology were performed to assess the pulmonary dysfunction.
Results: During the course mice developed pulmonary edema with macroscopic alterations of the lung (hemorrhagic areas, atelectasis, and edema). Histological analysis confirmed diffuse alveolar congestion and mild to severe lung edema. We observed reduced systemic oxygenation (pO2 55 mmHg) and Horovitz Index of (261 mmHg).
Conclusion: A sterile post-traumatic acute lung injury model could be established with marked macroscopic and microscopic changes within the lung. Reduced oxygenation and Horovitz Index indicate the clinical relevance of this model.
GENETIC VARIATION IN THE C-REACTIVE PROTEIN (CRP) GENE AFFECT SERUM ACUTE PHASE CRP LEVELS IN TRAUMA PATIENTS
S.D. West*1, M. Krencicki1, and C. Mold2. 1Department of Surgery University of New Mexico School of Medicine, Albuquerque, NM, 2Department of Molecular Genetics and Microbiology University of New Mexico School of Medicine, Albuquerque, NM
C-reactive protein is a serum innate immune protein used clinically to detect acute infections and to assess progression and response to treatment in chronic inflammatory diseases. Its plasma concentration may increase 1000-fold from baseline within 48 h of injury. CRP has well known host defense activities, but its role in the immune response to trauma is unknown. Highly elevated CRP levels are seen following trauma, and maintenance of these levels have been associated with a poor prognosis. Little is known about the influence of genetics on acute-phase CRP, particularly in trauma patients. Genetic variants at the CRP locus influence the baseline levels of serum C-reactive protein as well as the magnitude of acute-phase response. We sought to determine the effects of common variation at the CRP locus on serum CRP levels in trauma patients. We used previously published genotype data on 6 common single nucleotide polymorphisms in the CRP gene to categorize patient genotypes into 7 possible haplotypes. These haplotypes have been previously associated with high or low baseline serum levels of CRP. We enrolled 57 patients and collected blood at enrollment and again at 48 and 72 hrs. We measured serum CRP levels at all three time points. Additionally, DNA was analyzed for the 6 SNPs in the CRP gene to determine individual patient haplotypes. We found that CRP levels in trauma patients were significantly higher in individuals with one or more high haplotypes compared to individuals with low haplotypes at the 48 hr (high-81.5 +/- 5.4, low- 49.6 +/- 7.7, p<0.05) and 72 hr (high- 80.1 +/- 5.7, low- 39.4 +/- 3.9, p<0.05) time points. In conclusion, genetic variation in CRP haplotypes influence the magnitude of acute phase response in trauma patients.
COMBINATORIAL NETWORK ANALYSIS REVEALS NOVEL RACE SPECIFIC MOLECULAR ACTIVATION IN THE SETTING OF COMMUNITY-ACQUIRED PNEUMONIA
O.M. Peck Palmer*1, 3, G.L. Rogers2, G. Clermont3, and M. Langston2. 1Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 2Department of Electrical Engineering & Computer Science, University of Tennessee, Knoxville, TN, 3The Clinical Research, Investigation, and Systems Modeling of Acute Illness (CRISMA) Laboratory, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA
Background: In the US, the epidemiology of community-acquired pneumonia (CAP) differs among racial groups. CAP incidence is higher among black than white Americans. Black compared to white adults are on average younger and have a higher mortality rate. Social and economic factors do not fully explain this disparity.
Objective: The purpose of this study was to identify novel racial differences, if any, in molecular network activation in adults with CAP.
Methods: A nested case-control analysis was conducted on a multicenter inception cohort consisting of consented self-identified black (n=22) and white patients (n=22) matched 1:1. Patients were admitted to the emergency department (ED) with radiological diagnosis of CAP. Whole blood transcriptomic data (BeadArray Technology, Illumina - containing 24,500 gene transcripts) obtained at ED admission were employed to construct co-expression graphs for each racial group. Pearson correlation coefficients were used to weight edges. Spectral thresholding was applied to ascribe significance. Innovative graph theoretical methods were then invoked to detect densely connected subgraphs (molecular networks) and provide differential structural analysis.
Results: Novel race differences in molecular network activation were observed between the two groups despite similarities observed at ED admission in age, symptoms duration and illness severity. Three networks comprised of 10, 11, and 14 genes, respectively were identified that exhibited perfect connection only within the white group. Similarly, one network comprised of 16 genes was identified that exhibited perfect connection only within the black group. In the black group, we further identified five novel networks comprised of genes involved in innate/adaptive immunity that exhibited less than perfect connection but were significant for the black group. Interestingly, the genes within these networks were distributed across 22 novel networks significant for the white group.
Conclusions: Differential race-specific molecular network activation appears to occur during CAP. Racial disparity in CAP may thus, in part, be a functional consequence of molecular mechanisms by which diverse racial groups respond differently to infection. Graph theoretical analysis of genome-scale data is a powerful tool for the interrogation of differences in immune response.
IMMUNE-MEDIATED ACTIVATION OF COAGULATION IN PATIENTS WITH STEVENS-JOHNSON SYNDROME / TOXIC EPIDERMAL NECROLYSIS
O. Iqbal, D. Syed, J. Malalis, C. Mata, A. Lin, M. Mosier*, E. Campbell, S. Abro, D. Hoppensteadt, J. Fareed, and C. Bouchard. Loyola University Health System, Maywood, IL
Background: Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) are specific drug hypersensitivity reactions initiated by cytotoxic T-lymphocytes. Major Histocompatibility Complex-restricted presentations of the culprit drug triggers T-cell activation that causes expression of cytokines including TNF-α, interferon-γ, and granzyme B from NK cells.
Materials & Methods: Following an Institutional Review Board approved protocol blood samples were obtained from subjects suspected of SJS/TEN and normal healthy volunteers (n=2). Confirmatory biopsies for SJS/TEN were positive in 4 and negative in 7 subjects. Immunohistochemistry was performed on the skin biopsy sections using antibodies against granulysin, FasL, TNF-α and granzyme B. Cytokine levels were measured using the Cytokine High Sensitivity Array biochip from Randox Laboratories Limited. Thrombin-antithrombin complexes (Dade), fibrinopeptide A (F1.2, Dade), Plasminogen activator inhibitor-1, and platelet microparticle activity (Hyphen® Biomed) were measured using ELISA methods. Antithrombin was measured using a chromogenic method (Stago); Protein C levels using ELISA and Antithrombin using a chromogenic method (Stago).
Results: Compared to Normal Human Plasma (NHP), IL-6 (p=0.0138) and MCP-1 (p=0.0166) levels were increased significantly in biopsy-confirmed SJS-TEN patients. Other parameters measured including IL-2, IL-4, INF-γ, IL-1α, IL-1β and Epidermal Growth Factor (EGF) were not significantly increased. A marked increase in the TAT complexes (6.3±5.9 μ/ml), F1.2 (430.4±202.4 pmol/L), platelet microparticles (13.1±9.3 nM) and protein C levels (90.5±63.4%) with a corresponding decrease in PAI-1 (53.3±18.8 ng/ml) and antithrombin levels (80.7±42.4%) were also observed. Biopsy negative SJS/TEN subjects with less pronounced inflammatory stimulus demonstrated mild elevation in cytokine levels of IL-6 (29.81±25.18 pg/ml), TNF-α (7.20±5.04 pg/ml) and MCP-1 (265.10±159.09 pg/ml). Immunohistochemical studies revealed expression of TNF-α, granulysin, granzyme B and FasL.
Conclusions: Immune-mediated activation of coagulation resulted in increase in TAT, MCP-1, F1.2 and platelet microparticles and corresponding decrease of protein C, antithrombin, and PAI–1 which may progress to sepsis-associated coagulopathy and overt disseminated intravascular coagulation.
IMMEDIATE SUPPRESSION OF OVERALL INFLAMMATORY RESPONSE CAPACITY AFTER HEMORRHAGE AND RESUSCITATION
A. Bahrami, M. Jafarmadar, H. Redl*, and S. Bahrami*. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
Introduction: The current consensus has been that trauma patients with hemorrhagic shock (HS) are resuscitated into a state of an early systemic inflammatory response (SIRS). While an immediate and excessive SIRS is believed to lead to an acute organ failure, an excessive counter-regulatory anti-inflammatory response (CARS) is considered to cause a prolonged immunosuppressed state that is deleterious to the host at later stage. This assumption is based mainly on the detection of pro-inflammatory mediators early after HS.
Aim: We aimed to evaluate the influence of hemorrhagic shock upon the early inflammatory response capacity of the host by comparing overall TNF-α release capacity versus that of circulating leukocytes.
In vivo Experiments: rats subjected to HS (MAP of 30-35 mmHg for 90 min followed by resuscitation over 50 min) were challenged with Lipopolysaccharide (LPS, intravenously) at the end of resuscitation (HSR-LPS group) or 24 hours post-shock (HSR-LPS24 group). Control animals were injected with LPS without prior HS (LPS group). Plasma TNF-α was measured at 90 min after the LPS challenge. Ex vivo Experiments: whole blood (WB) was obtained either from healthy controls (CON) or from HS rats prior to LPS challenge at the end of resuscitation (HSR=140 min) and at 24h post-shock (HSR24). WB was incubated with LPS for 2h at 37 °C. TNF-α concentration and LPS binding capacity (LBC) was determined.
Results: Compared to controls receiving LPS only, HS followed by LPS challenge resulted in suppression of plasma TNF-α response in HSR-LPS and HSR-LPS24. Ex vivo: Compared to CON the LPS-induced TNF-α release capacity of circulating leukocytes was strongly declined both at the end of resuscitation (HSR) and 24h (HSR24). The LBC in WB was similar between CON and HSR and only moderately enhanced in HSR24.
Conclusion: Our data strongly suggest that the overall inflammatory response capacity is decreased immediately after HS/resuscitation. The down regulation of inflammatory response persists up to 24h and is independent of endotoxin binding capacity in whole blood.
CANDIDEMIA IN ELDERLY PATIENTS: THE EPIDEMIOLOGY, ANTIFUNGAL EXPOSURE AND RELATED OUTCOME
H. Wang, H. Han, J. Yue, F. Zhang, M. Yin, and D. Wu. Qilu Hospital of Shandong University, Jinan, China
The elderly patients affected by candidemia are growing in proportion to inpatients, but available data on epidemiology, antifungal exposure and outcome are limited. This retrospective study included 63 elderly (≥ 65 years) and 91 younger patients (16-64 years) at 4 tertiary hospitals. Elderly patients had higher APACHE II scores, increased 30-day mortality rate (31.7% vs. 17.6%, p < 0.05) and 90-day mortality rate (58.7% vs. 33.0%, p < 0.05) (Fig. 1A). Host-related (e.g., underlying solid tumour, diabetes mellitus and chronic renal failure) and hospital-related (e.g., prior ICU stay, mechanical ventilation, central vascular and urethral catheters placement) risk factors were identified more commonly in elderly patients. Resistance to fluconazole of non-Candida species in elderly patients was nearly double that of younger patients. Elderly patients less often received triazoles, and especially, they were less likely to receive antifungal therapies. In elderly patients, echinocandins usage led to a 20% lower 90-day mortality rate compared to triazoles (Fig. 1B); similar trend was not observed in younger patients (Fig. 1C). Conclusively, elderly patients with candidemia had poor prognosis characterized by certain host and hospital-related risk factors and special pathogen resistance features. Absence of antifungal therapies should be avoided, and echinocandins could be an attractive choice for early antifungal therapy.
LACTOBACILLUS RHAMNOSUS LGG TREATMENT IMPROVES INTESTINAL PERMEABILITY IN EXPERIMENTAL PSEUDOMONAS AERUGINOSA INDUCED PNEUMONIA: ROLE OF MUCINS AND CLAUDIN-2
L. Khailova, C.H. Baird, and P. Wischmeyer*. University of Colorado Anschutz Medical Campus, Aurora, CO
Background: Pneumonia is a common cause of mortality in the USA with more than 50,000 deaths each year. Clinical trials demonstrate probiotics such as Lactobacillus rhamnosus LGG (LGG) may reduce nosocomial infections, VAP and improve clinical outcome in critical illness. We have recently shown that LGG significantly reduces mortality in mouse model of P. aeruginosa induced pneumonia. LGG has been shown to induce mucins in other diseases and experimental models but the effect in sepsis is unknown.
Objective: To evaluate the effects of LGG treatment on intestinal permeability in mouse model of P. aeruginosa induced pneumonia and the role of mucins and Claudin-2.
Methods: 6-week old FVB/N mice were treated (o.g.) with or without LGG (1x109CFU/ml), and intratracheally injected with 4x108CFU P. aeruginosa or saline. Intestinal permeability was analyzed by fluorescein isothiocyanate-conjugated dextran at 24h. Colons were evaluated for mucins by AB-PAS and Muc-2 staining at 24h. Gene expression of Muc-2 was analyzed by real-time PCR at 12h and 24h. Claudin-2 expression was analyzed by Western Blot, immunofluorescent staining and real-time PCR at 24h.
Results: Intestinal permeability was significantly higher in pneumonia control mice compared to shams (P<0.05) and permeability of pneumonia mice treated with LGG was significantly reduced (P<0.05). mRNA levels of Muc-2 were significantly increased in pneumonia mice treated with LGG compared to pneumonia control mice (P<0.05) at 12h. Muc-2 staining revealed increased Muc-2 secretion in mice treated with LGG compared to control pneumonia mice. AB-PAS staining showed increased production of mucins in the LGG group. Gene and protein expression of Claudin-2 was significantly higher in control pneumonia group compared to shams (P<0.03) and significantly decreased in the LGG treated pneumonia group (P<0.03). There was an increase of Claudin-2 in the crypts of colon of control pneumonia group compared to the sham-like expression in the LGG treated group.
Conclusions: LGG treatment significantly reduces intestinal permeability in experimental P. aeruginosa induced pneumonia which may be due to increased secretion of mucins, especially Muc-2 and normalized Claudin-2 expression in the colon. Thus, LGG may a novel therapy for prevention of pneumonia.
INCREASED NERVE GROWTH FACTOR EXPRESSION AND INCREASED SYMPATHETIC NERVE FIBER GROWTH IN THE SPLEEN DURING SEPSIS
D.B. Hoover, A.M. Downs, C.E. Bond, T.R. Ozment*, and D.L. Williams*. East Tennessee State University, Johnson City, TN
Sympathetic nerve fibers are the major effector for neural modulation of immune cells in the spleen and at other sites in the immune system. Activation of these noradrenergic nerve fibers causes local release of norepinephrine, which stimulates β2-adrenergic receptors on immune cells to affect their phenotype and function. Nerve growth factor (NGF), secreted from target tissues, is essential for the maintenance of sympathetic nerves in adults, and altered supply of NGF can lead to remodeling of noradrenergic nerves under pathophysiological conditions. Since the spleen undergoes substantial cellular and biochemical changes during sepsis, we evaluated the hypothesis that these changes would affect sympathetic innervation. Sepsis was induced in male C57BL/6 mice by cecal ligation and puncture (CLP). Spleens were obtained from non-operated mice and from operated mice at 8 and 16 h after sham or CLP surgery and bisected on the short axis. Half was used for RNA extraction. NGF mRNA expression was determined by qPCR. The other half was used for histology. Paraffin sections were immunostained for the noradrenergic marker, tyrosine hydroxylase (TH) using the ABC method. Digital images of stained sections were collected, and TH nerve density was quantified using ImageJ software (NIH). At 8 h sepsis increased the expression of NGF mRNA by two-fold (sepsis: 11804±1052 transcripts per 50 ng RNA; control: 4625±235; sham: 5680±397; n=8 per group, P<0.0001), and there was already a trend for increased TH nerve density in the white pulp. At 16 h post-CLP, noradrenergic nerve density had increased over two-fold in spleens from septic mice compared to those from control and sham mice. Increased TH nerve density occurred in white pulp (sepsis: 0.87±0.03 % area; control: 0.34±0.00; sham: 0.37±0.09; n=3 per group, P<0.001) and red pulp (sepsis: 0.91±0.07 % area; control: 0.31±0.02; sham: 0.40±0.06; P<0.001). These findings provide the first evidence for upregulation of NGF in the spleen during sepsis and consequent rapid growth of noradrenergic nerve fibers. Increased nerve density would amplify neural modulation of immune function. In addition to causing nerve growth, NGF could modulate immune function directly by stimulating NGF receptors on splenocytes. Assuming activated splenocytes are the source of NGF, then effects of NGF could be widespread in sepsis.
THE FEMALE TRANSCRIPTOMIC RESPONSE AFTER SEVERE TRAUMA IS DIFFERENT FROM THAT OF MALES
M. Lopez, P.A. Efron*, F. Needell, E. Vanzant, B.E. Szpila, L.F. Gentile, A. Rountree, D.C. Nacionales, R. Ungaro, A.M. Mohr, F. Moore*, L. Moldawer*, S. Brakenridge*, and H. Baker. University of Florida, Gainesville, FL
Objective: Pre-menopausal women have better outcomes after trauma, and age is a known risk factor for increased morbidity/mortality with injury. We performed a genomic analysis of the Inflammation and Host Response to Injury Large Scale Collaborative Research Program database to determine if gender, as well as age, has a unique genomic expression pattern that could explain these phenomena.
Methods: Male(M) and female(F) patients who underwent microarray analysis were identified as young (<55 yrs(Y)) and aged (≥55 yrs(O)) patients from a prospective, multi-institutional cohort of severely injured blunt trauma patients in hemorrhagic shock. Genome-wide expression was evaluated from blood neutrophil(PMN) microarray data on days 0.5, 1, 4, 7, 14, 21 and 28 days after injury. Subsequent analysis consisted of identifying differences in gene expression (FDR<.001), individual fold gene changes (vs control,p<.05), as well as functional pathways among MY, MO, FY, FO and healthy subjects.
Results: Of the 202 trauma patients, there were 53 females (39<55yo) and 149 males (110<55yo). There were also 17 healthy controls (6 females, 11 males). 493 probe sets (491 genes) were significant among the groups at FDR<0.001; the differences were primarily based on the gender of the patient. Interestingly, <3% of these genes were sex chromosome linked directly. The distance from reference (DFR;single metric evaluating the overall aberration in all gene expression) graphically reflected these differences between gender. OF trended towards increased difficulty returning to baseline expression profiles. Individual gene expression usually peaked at day 7 in OF (ex. upregulation of innate immunity).
Conclusion: There is a unique F PMN genomic response to trauma and it may play a greater role in determining overall expression patterns than age. In addition, OF’s recovery to trauma appears to differ from M’s. Thus, improving F outcomes after injury may require distinct therapies from M.
ALCOHOL POTENTIATES POST BURN INTESTINAL ISCHEMIA THROUGH SHIFTS IN FLUID COMPARTMENTS
M.M. Chen, J.A. Ippolito, L. Ramirez, M. Choudhry, and E.J. Kovacs. Loyola University Chicago, Chicago, IL
Of the 450,000 burn patients each year, 50% have a positive blood alcohol content and this predisposes them to worsened clinical outcomes compared to non-intoxicated counterparts. Despite high prevalence and established consequences, the mechanisms responsible for alcohol-mediated complications of post burn remote organ damage are currently unknown. Animal studies have demonstrated that relative to either insult alone, intoxication prior to burn results in elevated pulmonary inflammation in part caused by hepatic mediators produced in response to intestinal barrier breakdown. This gut-liver interaction is known to regulate a wide array of human diseases, though how alcohol exacerbates intestinal injury after burn remains to be elucidated. To this end, mice received a single dose of alcohol (1.12 g/kg) or water by oral gavage and were subjected to a 15% total body surface area burn. Animals with a burn alone lost ~7% of their total body weight in 24 hours whereas intoxicated and injured mice lost only 1% body weight (p<0.05) despite a 20% increase in hematocrit (p<0.05) and a 17% increase in serum creatinine (p<0.05) over burn injury alone. This retention of water weight despite increased dehydration suggests that intoxication at the time of a burn causes a shift in fluid compartments that may exacerbate end organ ischemia and damage in the intestines. Catecholamine-mediated vasoconstriction of the splanchnic vasculature makes the intestines particularly susceptible to hypovolemia, made worse by third spacing of fluid as evidenced by edema in the liver and lungs. A 35% increase in liver weight to body weight ratio was found in intoxicated burn mice over burn alone (p<0.05) and alveolar wall thickness was 47% greater after burns preceded by alcohol (p<0.05). Meanwhile, the terminal ileum of mice receiving the combined injury revealed increased villus blunting and a 12-fold increase in bacterial translocation to the mesenteric lymph nodes (p<0.05) implying a breakdown of intestinal barrier function. These findings are of significant clinical concern as early gut ischemia-reperfusion injury may be the inciting event that initiates the aberrant inflammatory response thought to be responsible for remote organ damage in this setting. This work was supported in part by NIH AA012034 (EJK), T32 AA013527 (EJK), F30 AA022856 (MMC) and the Falk Foundation.
PLATELETS: POTENTIAL MODULATORS OF T REGULATORY CELL ACTIVITY AFTER TRAUMA?
C.B. Bergmann, P. Biberthaler, M. van Griensven*, and M. Hanschen*. Klinikum rechts der Isar, Technical University Munich, Munich, Germany
Severe injury causes rapid activation of CD4+ T regulatory cells (Tregs). Tregs have been shown to be key players of the anti-inflammatory CARS (compensatory anti-inflammatory response syndrome) following trauma. The mechanisms regulating the activation of Tregs following injury remain unclear. Recent evidence suggests that platelets play a protective role after trauma. The objective of this study was to examine whether CD4+ Tregs and platelets interact following trauma. The focus was to reveal whether reciprocal regulation processes take place or not.
The burn injury model in mice was employed. C57/Bl6 mice were either treated with a platelet-depletion antibody or with an isotype-control-antibody. Two hours following burn injury or sham-treatment, draining lymph nodes (LN) and spleen (SPL) were harvested. Measurement of Protein kinase C-theta (PKC-θ) expression and phosphorylation via phospho-flow cytometry was used to show the activation of CD4+ Treg cells and non-Treg cells following trauma.
Our first results display that burn injury induces activation in Tregs more than in non-Tregs, derived from LNs. The phosphorylation of PKC-θ tends to be increased following burn injury as compared to sham treatment in wild-type mice treated with an isotype control Ab. Interestingly, pilot studies suggest platelet depletion seems to induce an augmented activation of Tregs following burn injury. In Tregs derived from LNs of platelet depleted mice burn injury tends to induce higher phosphorylation of PKC-θ. Of note, Tregs derived from the SPL did not show increased expression or phosphorylation of PKC-θ following burn injury.
In conclusion, our observations indicate that rapid activation of CD4+ Tregs, but not of CD4+ non-Tregs, takes place following burn injury in mice. The activation of Tregs is limited to the draining LNs and cannot be observed in cells derived from the SPL. Our data suggests a potential difference in Treg activation following injury when platelets are absent. Platelets might be potential regulators of Treg activation in the early phase after trauma.
SEPSIS-INDUCED CARDIAC INFLAMMATION THROUGH MITOCHONDRIAL DNA AND RAGE
Q.S. Zang*, X. Yao, L. Ma, D. Maass, S.E. Wolf*, and J. Minei. University of Texas Southwestern Medical Center, Dallas, TX
Objective: We previously showed that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS-specific antioxidant, limited myocardial cytokines and improved heart function in a rat pneumonia-related sepsis model. This study was designed to investigate the mechanism of mtROS-dependent cardiac inflammation after sepsis.
Methods: Sepsis was produced in SD rats by infection with S. pneumoniae (4x1000000 CFU/rat). Mito-Vit-E (21.5 micro moles/kg) or control vehicle was given 30 minutes post-inoculation, and heart tissue was harvested 24 hours later. Intact mtDNA content was quantified by long PCR. Cytosolic mtDNA fragments were measured by real time PCR. Activation of NF-kappa B and caspase 1 was determined by Western blot. Levels of RAGE (receptor for advanced glycation end products) and inflammasome component ASC were examined by immunohistochemistry.
Results: Significant decrease in intact mtDNA, increase in cytosolic mtDNA fragments, activation of NF-kappa B and caspase 1, and elevation in the expression of RAGE and ASC were evident in septic rats but not in sham and Mito-Vit-E treated rats.
Conclusion: Our data suggest that, in septic hearts, mtROS are causative to damage mtDNA integrity. Released from damaged mitochondria, mtDNA fragments function as danger signals to up-regulate both NF-kappa B and inflammasome pathways, leading to promoted production of cytokines. Because certain immunogenic responses of mtDNA are RAGE-dependent, our data also suggest that RAGE-mediated signaling is involved in the mtROS-stimulated cardiac inflammation after sepsis.
PMX-DHP (POLYMYXIN B-DIRECT HEMOPERFUSION) THERAPY COULD IMPROVE MAP AND HIGH EAA (ENDOTOXIN ACTIVITY ASSAY) VALUES IN SEVERE SEPSIS AND SEPTIC SHOCK
K. Shibahara, T. Harada, N. Saito, M. Namiki, M. Takeda, and A. Yaguchi*. Tokyo Women’s Medical University, Tokyo, Japan
Introduction: Since 1994, PMX-DHP (Polymyxin B-direct hemoperfusion) (Toraymyxin®, Toray medical Co., Tokyo) has been an approved therapy for patients with severe sepsis or septic shock due to Gram-negative infection in Japan. However, its indication still is controversial. Recently, randomized controlled studies are being conducted in other countries. The purpose of this study is to determine if EAA (Endotoxin Activity Assay)(EAA™; Spectral Diagnostics Inc., Toronto, Canada) is a useful marker for the indication of PMX-DHP therapy.
Methods: From September 2012 to January 2014, adult patients treated twice with PMX-DHP every 24 hours and had EAA performed were included in this preliminary study. Patients’ clinical, microbiological and PAC data were collected from medical archives. EAA values were compared between immediately before and after each PMX-DHP therapy. Values were expressed as mean ± SD. Data were analyzed by Wilcoxon signed-ranks test. A p < .05 was considered as statistically significant.
Results: Sixteen patients (11 men, 5 women; age mean 65.3 ± 17.0) were studied. The mortality rate was 18.8.0 % at 28 days at 90days after PMX-DHP. APACHE II score and SOFA score on the first day of PMX-DHP therapy were 21.2 ± 7.7 and 6.9 ± 3.2, respectively. EAA values indicated no statistically significant differences between immediately before and after each PMX-DHP therapy (0.61 ±0.16 vs. 0.63 ± 0.22 vs.0.60 ± 0.23 vs. 0.54 ± 0.12, p>0.05). Mean arterial pressure (MAP) (mmHg) was significantly increased after the second PMX-DHP therapy (72.4 ± 8.8 vs. 86.1 ± 21.1, p=0.013). WBC (/mm3) and CRP (mg/dl) were not statistically different before and after PMX-DHP therapy. EAA values significantly decreased after the second PMX-DHP therapy compared before the first and second PMX-DHP treatment in patients with high values of EAA (>0.60) (0.53 ± 0.14 vs. 0.72 ± 0.12, 0.69 ± 0.23, p<0.05).
Conclusions: This pilot study has limitations because of small numbers for analysis. But MAP significantly improved after PMX-DHP therapy. Although WBC, CRP and EAA values were not changed after PMX-DHP therapy, EAA values were significantly decreased after PMX-DHP therapy in cases with high EAA values before PMX-DHP. PMX-DHP therapy may have efficacy for patients with high EAA values.
EXTRACELLULAR SIGNAL-REGULATED KINASE 5, A CENTRAL MEDIATOR OF ACUTE INFLAMMATORY SIGNALING
K. Wilhelmsen, A. Tran, F. Xu, K. Farrar*, S. Khakpour*, S. Sundar, and J. Hellman*. University of California, San Francisco, San Francisco, CA
Background: Acute inflammatory responses cause organ injury and failure through shared innate immune receptors and signaling pathways, dysregulated inflammation, coagulopathy, and microvascular dysfunction, in which endothelial cells (ECs) play a central role. We recently made the novel discovery that extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, mediates the activation of monocytes and ECs by Toll-like receptor 2 (TLR2) agonists, and promotes the TLR2-dependent expression of plasminogen activator inhibitor 1 (PAI-1), a coagulation pathway intermediary. Subsequently we found that ERK5 mediates TLR4-dependent inflammatory responses. We tested the hypothesis that ERK5 broadly mediates endothelial activation by bacterial and endogenous inflammatory agonists, and further analyzed the effects of ERK5 inhibition on inflammation and coagulopathy in vivo in endotoxemic mice.
Methods: HUVEC or human lung microvascular ECs (HMVEC-Lu) were pre-incubated with highly specific ERK5 inhibitors (XMD8-92; XMD17-109), an inhibitor of MEK5 (BIX02189), the only known upstream activator of ERK5, or ERK5 expression was reduced via siRNA knockdown. ECs were stimulated with Pam3Cys, FSL-1, LPS, IL-1β, or TNF-α. Neutrophil adhesion to activated EC monolayers was visualized by microscopy and quantified using a fluorescent plate reader. ERK5 activation in EC lysates was evaluated by Western blot. Mice were given ERK5 inhibitor and then challenged IV with LPS. Cytokines were quantified in EC supernatants and mouse plasmas. Lung digests were assessed by flow cytometry.
Results: All inflammatory agonists induced ERK5-dependent EC activation in vitro, including cytokine production (p<0.05), neutrophil adhesion (p<0.05) and ERK5 phosphorylation. Treatment with ERK5 inhibitor reduced mortality in endotoxemic mice (p<0.05), and attenuated the induction of cytokines, chemokines, and PAI-1 (p<0.05). Furthermore, ERK5 treated mice had fewer cellular infiltrates and reduced monocyte activation in their lungs.
Conclusion: Our results highlight critical, heretofore overlooked functions for ERK5 in regulating infectious and non-infectious acute inflammatory signaling, and suggest that ERK5 may be a novel drug target for diverse disorders, including sepsis, for which there are currently no effective treatments.
B LYMPHOCYTES PLAY A PROTECTIVE ROLE AGAINST ORGAN DAMAGE IN TRAUMA + HEMORRHAGIC SHOCK
M.H. Ramadan, R. Hoffman, and T. Billiar*. University of Pittsburgh, Pittsburgh, PA
Background: There is increasing knowledge about the innate functions of B lymphocytes however their role in the early inflammatory response after trauma haven’t been characterized. The dysregulation of the innate immune system after major trauma has been known to be one of the major causes of morbidity along with consequences of bleeding and neurotrauma. This immune dysregulation manifests early as an excessive escalation of the innate immune system, measured by the production of inflammatory mediators, leukocyte activation and immune mediated end organ damage, and late as immune suppression and increased susceptibility to infection. We hypothesized that B cells have protective properties against organ damage; based on the facts that anti-inflammatory cytokines are some of the major products of the innate reacting B cells.
Aim: Is to characterize the role of B lymphocytes in the early immune dysregulation after trauma.
Methods: We utilized our mouse trauma/hemorrhagic shock model (PF/HS). Anesthetized mice were subjected to bilateral femoral pseudo fracture and 2 hours of hemorrhagic shock. Mice were then resuscitated with lactated Ringer’s solution X3 the volume of shed blood. Mice were then sacrificed at 6 hour time point. We studied wild-type C57BL/6 compared to B-cell deficient mice (μMT strain); focusing cytokine levels, end organ damage and leukocyte phenotype in different compartments using flow cytometric analysis.
Results: We have demonstrated that there were no baseline differences in the studied cytokines and liver function tests between the wild-type and the μMT naive un-injured mice. We also confirmed the absence of B-cells from the circulation of μMT mice. We demonstrated a marked change in the circulating lymphocyte phenotype with a dramatic drop in the circulating B-cells. The amount of circulating B-cells in the wild type mice after PF/HS dropped from 35±5% to 7.5±1%, P=0.001. There was a significantly higher liver damage measured by higher LFT elevation in the μMT group (ALT 581±97 & 1401±336, P=0.04; AST 638±45 & 928±100, p=0.02). There was a nearly significant trend towards lower IL-10 levels in the μMT group and higher IL-6 and MCP-1 levels.
Conclusion: With the available data B-cells have a protective role against immune mediated organ damage from trauma in mice.