THE GENOMIC RESPONSE AFTER BURN INJURY PREDICTS ORGAN DYSFUNCTION AND MORTALITY
M. Lopez1, C.C. Finnerty2, J. Zhang1, P.A. Efron*1, J. Cuschieri*3, R.V. Maier*3, F. Moore1, H. Baker1, L. Moldawer*1, D. Herndon*2, S. Brakenridge*1. 1University of Florida, Gainesville, FL, 2University of Texas Medical Branch - Galveston, Galveston, TX, 3University of Washington, Seattle, WA
Objective: Genome wide expression analysis of blood leukocytes from severely injured trauma patients has revealed a “genomic storm” of both pro and anti-inflammatory genes (Xiao J Exp Med, 2011). This response has been associated with persistent multiple organ failure and adverse clinical outcomes. The genomic response to burn injury has been shown to be remarkably similar to that of severe trauma (Seok PNAS, 2013). It is not known whether this “genomic storm” after burn injury is associated with prolonged organ dysfunction and poor clinical outcomes.
Methods: 118 patients in whom multiple blood sampling was obtained at regular intervals early after burn injury were enrolled in a multi-center, prospective cohort study. Genome-wide expression analysis was performed on isolated blood leukocyte populations from blood collected throughout the inpatient hospital course. Subsequent analysis consisted of identifying differences in gene expression (FDR<0.001), individual fold gene changes (>2), on days 0.5, 1, 4, 7, 14, 21 and 28 days after burn injury. Epidemiologic data and clinical outcomes were tracked prospectively allowing comparison of survivor and non-survivor cohorts.
Results: The overall mortality rate in this burn injury cohort was 17 percent. Non-survivors were significantly more likely to have early multiple organ failure, pneumonia, and sepsis (p<0.0001). Genome wide expression analysis revealed 2,548 probe sets, representing 1,965 unique genes, that exhibited significant expression differences from reference (DFR) between survivors and non-survivors (P<0.001). Aberrant expression patterns peaked between 4 to 7 days and revealed persistent, significant DFR over the 28 day sample period. A subset panel of 63 genes previously shown to predict persistent organ failure and complicated outcomes in severely injured trauma patients (Xiao J. Exp. Med. 2011, Gentile Crit Care Med 2014) showed significant difference in expression between burn survivors and non-survivors. This “Sweet 63” genomic screening panel was significantly predictive of burn mortality at all time points, including as early as 0.5 days (p<0.0001), and remained an independent predictor of mortality after controlling for burn total body surface area (p=0.004). Burn mortality in this population was primarily secondary to multiple organ failure (27%) and sepsis (25%).
Conclusion: Severe burn injury triggers a “genomic storm” of persistent, aberrant gene expression similar to that previously shown in severely injured trauma patients in hemorrhagic shock, which is strongly associated with post-burn mortality. Genomic screening panels of specific burn-responsive genes may allow for early prediction of those at high risk of mortality, potentially targeting these individuals for early interventional trials to improve burn mortality from sepsis and multiple organ failure.
CHARACTERIZATION OF MICROBIOME IN CRITICALLY ILL PATIENTS AND RELATIONSHIP TO CLINICAL OUTCOME: THE ICU MICROBIOME MULTI-CENTER TRIAL PROJECT
D. McDonald2, G. Ackerman3, L. Khailova*1, C.H. Baird*1, R. Knight4, P. Wischmeyer*5. 1University of Colorado Anschutz Medical Campus, Aurora, CO, 21 BioFrontiers Institute, University of Colorado at Boulder, Boulder, CO, 3Computer Science Dept., University of Colorado, Boulder, Boulder, CO, 4University of California, San Diego, San Diego, CA, 5Dept. of Anesthesiology, University of Colorado School of Medicine, Aurora, CO
Background: Alterations in gut and oral bacterial flora is thought to be associated with infectious complications and mortality in ICU patients. However to date, evaluations of microbial ecology in ICU patients has been restricted to small culture-based studies. With the evolution of 16s rRNA microbiome techniques large-scale, prospective trials of the ICU microbiome are needed.
Objective: Conduct multi-center prospective trial of effect of critical illness on gut, oropharynx, and skin microbiome via 16s sequencing and metagenomics versus healthy controls.
Methods: Fecal, oral, and skin samples were collected from 116 mixed ICU across 4 centers in U.S. and Canada at two timepoints: within 48 h of ICU admission and discharge or day 10 (which ever came first). Sample collection and processing performed in accordance with Earth Microbiome Project protocols. Results compared to samples from self-reported healthy (with no antibiotic use in last year) individuals (n=1242 pts, (American Gut Registry)). Diverse clinical outcomes including mortality, antibiotic use, and all nutrition delivery data were collected.
Results: Regardless of timepoint, fecal ICU samples are significantly different from the American Gut healthy fecal samples within principal coordinates space; a separation predominantly explained by decreased ratio of firmicute abundance (see figure: light colored spheres= low firmicute abundance, dark colored spheres=high firmicute abundance). Correlations were observed with changes in microbiome signatures from oral, fecal, and skin sites and adverse outcomes including mortality, ARDS, and length of stay, Many operational taxonomic units significantly depleted in ICU patients have previously been identified as “optimal health-promoting species” in studies of other diseases states (ie IBD).
Conclusions: These initial data suggest significant changes in the gut, oral, and skin microbiome occur in ICU patients. Characterizing these changes opens the door to asking focused questions to understand why these differences arise, and form the basis for the development of diagnostic and therapeutic interventions utilizing microbiome signatures.
INTERLEUKIN (IL)-15 FACILITATES THE PATHOGENESIS OF CLP-INDUCED SEPTIC SHOCK BY ACTIVATION OF NK, NKT AND MEMORY CD8+ T LYMPHOCYTES
Y. Guo*, L. Luan*, N.K. Patil*, J. Bohannon*, E. Sherwood*. Vanderbilt University, Nashville, TN
Background: Sepsis and septic shock remain one of the leading causes of death in critically ill patients in the USA. However, limited therapeutic interventions are available due to the lack of understanding of critical immunological mechanisms underlying the pathogenesis of sepsis and septic shock. IL-15 is a cytokine that is essential for the generation, function and survival of natural killer (NK), natural killer T (NKT) and memory CD8+ T cells, as indicated by the deficit of these cells in IL-15 knockout (KO) mice. Previous studies by our lab, and others, have described a role for NK, NKT and CD8+ T cells in the pathogenesis of sepsis. Therefore, we hypothesized that IL-15KO mice will be resistant to septic shock.
Methods: To propose the hypothesis, wild type (WT) and IL-15KO mice underwent a lethal model of cecal ligation and puncture (CLP). Core temperature, cytokine production and survival were assessed. In further studies, IL-15KO mice were treated with 0.1µg IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) to re-establish NK, NKT and memory CD8+ T cell populations or vehicle control prior to the initiation of septic shock by CLP. A 7-day survival study was performed. IL-15KO mice were continuously given the same dose of IL-15 SA daily to maintain the regenerated NK, NKT and memory CD8+ T cells. Vehicle-treated WT mice serve as control.
Results: IL-15KO mice were resistant to septic shock compared to WT mice. IL-15KO mice displayed less sepsis-induced hypothermia (32.7 ± 1.2 vs. 25.4 ± 0.6 oC, p< 0.001) and attenuated IL-6 production in plasma (14577 ± 6926 vs. 40519 ± 9424 pg/ml, p< 0.02) and peritoneal lavage fluid (19359 ± 9069 vs. 40231± 5016 pg/ml, p< 0.02) compared to WT mice. Also, there is a significant survival advantage in IL-15KO mice over WT control in response to septic shock (40% vs. 100% mortality). Treatment of IL-15 SA induced regeneration of NK, NKT and mCD8+ T cells in IL-15KO mice and re-establishes mortality (87.5% mortality) in response to septic shock compared to vehicle-treated IL-15KO mice (40% mortality).
Conclusions: IL-15 in vivo appears to be an important mediator of inflammatory responses during septic shock and contributes to the lethality of septic mice, possibly by activation of NK, NKT and memory CD8+ T cells.
ACTIVATION OF SIRTUIN 1/3 PROTECTS AGAINST HEMORRHAGIC SHOCK INJURY BY RETAINING MITOCHONDRIAL FUNCTION
R. Song*1, K. Zhao*1, P. Li1, X. Huang1, X. Meng*2. 1Southern Medical University, Guangzhou, China, 2University of Colorado Denver, Aurora, CO
Refractory hypotension following vascular hyporeactivity is one of the major causes of mortality from severe shock in the clinic. Depressed contractile vasoresponsiveness and persistent hypotension might not only result from insufficient microcirculatory delivery of nutrients and oxygen but also from cytopathic hypoxia with mitochondrial injury. We have found that the mitochondrial permeability transition (MPT) pore opening in arteriolar smooth muscle cells (ASMCs) is involved in the pathogenesis of vascular hyporeactivity in severe shock. However, the molecular mechanism underlying mitochondria injury in ASMCs during shock is not well understood. We hypothesized that an epigenetic mechanism involving activation of Sirtuin1/3 histone deacetylases protectively modulates vasoreactivity and refractory hypotension through amelioration of MPT pore opening and mitochondria injury in ASMCs.
Methods and results: A model of severe hemorrhagic shock and reinfusion was prepared in adult Wistar rats. Human ASMCs exposed to hypoxia and reoxygenation (HR) were used to model in vitro correlates of cytopathic hypoxia. Sirtuin 1/3 (SIRT1/3) protein levels and deacetylase activities were assessed at prebleeding, 60min and 120 min postbleeding, and 0, 60 and 120 min after reinfusion, respectively. Immunoblotting revealed SIRT1/3 protein levels in ASMCs were decreased by hemorrhagic shock and reinfusion. The deacetylase activities of both SIRT1 and SIRT3 were reduced. Immunofluorescence staining revealed that the accumulation of SIRT1 in nucleus and SIRT3 in mitochondria were reduced. In vitro lentiviral overexpression of SIRT1 preserved SIRT3 deacetylase activity in human ASMCs exposed to hypoxia and reoxygenation (HR). Co-immunoprecipitation revealed that acetylation of cyclophilin D (CyPD), a component of the MPT pore, was inhibited by overexpression of SIRT1 during HR. Knockdown of SIRT3 abrogated deacetylation of CyPD induced by overexpression of SIRT1. Resveratrol or SRT1720, SIRT1 activators, suppressed MPT pore opening in ASMCs after shock, resulting in amelioration of mitochondrial swelling, mitochondrial membrane potential and intracellular ATP levels. Importantly, after 120 min postbleeding, administration of the SIRT1 activator improved vasoresponsiveness to noradrenaline and refractory hypotension during shock in vivo.
Conclusions: The mitochondrial injury of ASMCs during severe shock is associated with attenuated deacetylase activity of SIRT1/3. Negative regulation of MPT by SIRT1/SIRT3- mediated deacetylation of CyPD is necessary to improve low vasoreactivity and refractory hypotension. These novel findings indicate SIRT1/3 is a promising therapeutic candidate in refractory hypotension following severe shock and in other diseases connected with cytopathic hypoxia.
CYTOSOLIC HMGB1 IS LIVER PROTECTIVE BY ACTIVATING CASPASE-1 AFTER HEMORRHAGIC SHOCK IN MICE
Q. Sun, T. Billiar*, M. Scott*. University of Pittsburgh, Pittsburgh, PA
Introduction: The production of reactive oxygen species (ROS) and the release of damage-associated molecular patterns (DAMP) after hemorrhagic shock (HS) lead to the maturation of caspase-1 in immune and non-immune cells. Rather than leading to the maturation of cytokines, caspase-1 activation in mouse hepatocytes (HC) was previously shown by us to play a protective role by up-regulating mitochondrial autophagy and decreasing mitochondrial ROS after redox stress. Nuclear protein HMGB1 can be released into cytoplasm in HCs in response to redox stress. The translocation of HMGB1 from the nucleus to the cytoplasm has been shown to up-regulate autophagy and protect against mitochondrial dysfunction. Therefore, we hypothesize that cytosolic HMGB1 regulates caspase-1 activation in HCs after HS.
Methods: C57BL/6 (WT) and HC-HMGB1-/- (hepatocyte-specific HMGB1 knockout) mice underwent HS (1.5h hemorrhagic shock + 4.5h resuscitation). Whole liver lysates were immunoblotted for caspase-1. ALT levels were measured. HCs isolated from WT and HC-HMGB1-/- mice were treated with 6h of hypoxia (1% oxygen) and reoxygenation. Cell death was determined by Annexin V/PI staining. Levels of autophagic flux were assessed by increase in the number of GFP-LC3 puncta in HCs after bafilomycin treatment. Mitochondrial and cytosolic specific ROS production was measured by HyPer-Mito and HyPer-Cyto.
Results: Caspase-1 was activated after HS in WT mice, as shown by increased cleaved caspase-1 in the liver, but not in HC-HMGB1-/- liver (Fig), suggesting a role of HMGB1 in regulating caspase-1 activation in HCs. HC-HMGB1-/- mice had significantly higher levels of ALT compared with WT mice after HS (976±131 IU/L in WT vs 1971±442 IU/L in HC-HMGB1-/-, P<0.05), confirming a protective role of HMGB1 in HCs. Consistently, HC-HMGB1 -/- HCs had decreased caspase-1 activity and increased apoptosis and necrosis in comparison with WT after hypoxia/reoxygenation. Consistent with our previous findings in caspase-1-/- HCs, HC-HMGB1-/- HCs had decreased autophagic flux and increased mitochondrial ROS after hypoxia/reoxygenation.
Conclusion: Our data suggested that HMGB1 plays a protective role in HCs by activating caspase-1 and upregulating mitochondrial autophagy after HS. Our study links HMGB1 with caspase-1 and stress-induced autophagy, which improves our understanding of how adaptive responses are regulated after trauma and hemorrhage.
ROLE OF TLR-3/TRIF IN MYD88-INDEPENDENT SIGNALING FOLLOWING LUNG CONTUSION
S.V. Madathilparambil, B. Thomas, M. Sherman, V. Dolgachev, M. Deshmukh, S. Ramakrishnan, N. Talarico, D.A. Machado-Aranda, K. Raghavendran. University of Michigan, Ann Arbor, MI
Objective: Lung contusion (LC) is a major risk factor for the development of acute respiratory distress syndrome. Previous studies have suggested TLR-4/MyD88 is involved in the pathogenesis of the acute inflammatory response in LC. We have previously shown that TLR-3 is functionally important in the development of progressive lung injury and inflammation following LC. TLR-3 functions through a TIR-domain-containing adaptor-inducing interferon-β (TRIF), a MyD88-independent pathway.
Hypothesis: To elucidate the in vivo mechanism of the TLR-3 pathway and downstream signaling following activation by LC.
Methods: Non-lethal, closed-chest unilateral LC was induced in TLR-3 (-/-), TRIF (-/-), and wild type (WT) mice. The mice were sacrificed at 5, 24, 48, and 72 hours. The extent of injury was assessed by measuring bronchoalveolar lavage (BAL), cells and apoptotic indices (flow cytometry and cytospin), albumin (permeability injury), cytokines (inflammation), and pulmonary respiratory mechanics (flexiVent). Lung lysates were analyzed with Western blot using various antibodies. To elucidate the in vivo mechanism, a TLR-3/double-stranded (ds) RNA complex inhibitor was injected into WT mice prior to LC. Mice were sacrificed at 24 and 48 hours, and BAL cells and intracellular caspase activity were measured by flow cytometry (ApoStat).
Results: Consistent with our previous findings in TLR-3 mice, pulmonary compliance and albumin levels in TRIF mice showed a significant reduction in lung injury following LC compared to WT. There was also a significant decrease in release and production of pro-inflammatory cytokines IL-1β, IL-6, MIP-2, KC, TNF-α, and MCP-5 as well as a reduction in recruitment of alveolar macrophages and neutrophil infiltration in TRIF mice compared to WT. Alveolar macrophages obtained from TRIF mice showed a lower early apoptotic index and had lower caspase-3 activation compared to WT. Histological evaluation of WT mice revealed significantly more injury at all time points compared to TRIF. Following LC, TLR-3 and TRIF mice showed significant reductions in TRAF6, NLRP-3, activator protein-1 (AP-1), as well as p38 MAPK and ERK1/2 phosphorylated expression compared to WT mice. Phosphorylated NF-kB activation was not detected in WT, TLR-3, or TRIF mice after LC. TLR-3/dsRNA ligation inhibitor pretreatment significantly reduced injury (albumin), inflammation (IL-1β and MCP-1), and macrophage apoptosis 24 and 48 hours after LC.
Conclusion: TRIF appears to be functionally important in the development of progressive lung injury and inflammation following LC. The TLR-3/TRIF signaling pathway is independent of NF-kB but results in significant phosphorylation of p38 MAPK and ERK1/2. Finally, the TLR-3/dsRNA complex inhibitor effectively reduces injury and inflammation, suggesting TLR-3 inhibition as a potential new therapeutic strategy for lung injury following LC.
INTRAOPERATIVE HYPEROXIC REPERFUSION OF THE BRAIN AND ICU DELIRIUM
M. Lopez, P. Pandharipande, M. Pretorius, A. Shaw, F.T. Billings*. Dept. of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN
Introduction: Delirium affects 20%-30% of patients following cardiac surgery and is associated with adverse outcomes including prolonged cognitive impairment. Hyperoxic reperfusion has been associated with brain injury and long-term morbidity after cardiac arrest, but its effects in patients undergoing major surgery are not known. We hypothesize that hyperoxic cerebral reperfusion following ischemia during cardiac surgery increases postoperative delirium.
Methods: We measured cerebral tissue oxygen saturation with bilateral oximetry probes (INVOS, Covidien) in 310 subjects as part of a prospective cardiac surgery cohort. Cerebral ischemia was defined as a cerebral oxygen saturation decrease of 20% below baseline for five minutes, and hyperoxic reperfusion was quantified as the area under the oxygenation:time curve (AUC) above baseline after any ischemic period. We estimated the extent of acute functional brain injury using the well-validated CAM-ICU assessment tool for delirium, measured twice daily while patients were in the ICU. Delirium rates and duration were compared among ischemia and reperfusion groups using chi-squared and Mann Whitney U tests, respectively, and multivariable regression was performed to isolate the association between reperfusion and delirium from the effects of delirium risk factors age, baseline mini mental status exam (MMSE) scores, congestive heart failure (CHF), and use of cardiopulmonary bypass (CPB).
Results: Fifty-one percent (158/310) of subjects experienced cerebral ischemia during surgery, and 56.3% of these (89/158) had hyperoxic reperfusion. Delirium was diagnosed in 24.1% (38/152) of subjects without ischemia for a mean duration of 2.7±2.5 exams, 23.2% (16/69) of subjects with ischemia but without hyperoxic reperfusion (duration 1.8±1.2 exams), and 40.4% (36/89) of subjects with hyperoxic reperfusion following ischemia (duration 2.2±1.6 exams, P=0.04 delirium incidence and P=0.44 duration). Adjusted for age, MMSE score, CHF, and use of CPB, a 10 unit AUC increase in hyperoxic reperfusion remained independently associated with a 15.1% (95% CI 1.2 – 31.0%, P=0.03) increase in the odds of delirium (Table) and a 0.07 increase in delirium positive exams (0.02 – 0.11, P=0.003).
Conclusions: Cerebral hyperoxic reperfusion after ischemia may be a modifiable risk factor for postoperative delirium, and future studies that limit cerebral hyperoxic reperfusion are warranted.
COLD-INDUCIBLE RNA-BINDING PROTEIN PROMOTES SPLENIC T CELL ACTIVATION IN SEPSIS
A. Cerutti1, A. Sharma1, 2, W. Yang*1, 2, J. Nicastro1, G. Coppa1, P. Wang*1, 2. 1Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2Center for Translational Research, The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Cold-inducible RNA-binding protein (CIRP) is a novel inflammatory mediator that stimulates the release of proinflammatory cytokines from macrophages in sepsis. Given the immune dysregulation that characterizes sepsis, the effect of CIRP on other immune cells is an area of increasing interest that has not yet been studied. We hypothesized that extracellular CIRP promotes activation of splenic T lymphocytes during sepsis.
Methods: C57BL/6 wild type (WT) mice (20-25g) were subjected to sepsis by cecal ligation and puncture (CLP). Another set of WT and toll-like receptor 4-deficient (TLR4-/-) mice received intravenous injection of recombinant murine CIRP (rmCIRP, 5 mg/kg BW) or PBS (vehicle). At 20 h after CLP or rmCIRP injection, splenocytes were isolated, labeled, and analyzed by flow cytometry. Protein levels of CIRP in the spleen were determined by Western blotting. For in vitro studies, splenocytes isolated from healthy WT mice were cultured with rmCIRP (1 µg/ml) for 24 h, or with culture medium (CM) from primary mouse peritoneal macrophages stimulated with rmCIRP (1.5 µg/ml) for 5 h, followed by flow cytometry.
Results: Protein levels of CIRP in the spleen were increased 1.5-, 2- and 3-fold compared to sham at 6, 10, and 20 h post-CLP, respectively. At 20 h after CLP, the percentage of CD4+ and CD8+ T cells expressing the early activation marker CD69 was increased compared to sham-operated controls (Table). Similarly, after rmCIRP injection, CD69 expression on T cells was increased compared to PBS (Table). However, there was no change in CD69+ CD4+ T cell population in TLR4-/- mice after rmCIRP injection (15% in both groups). In vitro, incubation with rmCIRP in the setting of CD3/CD28 co-stimulation increased the percentage of CD69+ T cells compared to control (CD69+ CD4+: 82% vs. 79% and CD69+ CD8+: 73% vs. 63%, P < 0.05). Additionally, incubation with CM from macrophages stimulated with rmCIRP increased CD69 expression on T cells compared to CM from macrophages without rmCIRP stimulation, even in the absence of CD3/CD28 co-stimulation (CD69+ CD4+: 12% vs. 9% and CD69+ CD8+: 26% vs. 17%, P < 0.05).
Conclusions: Extracellular CIRP is able to activate splenic T lymphocytes as is observed in murine sepsis, and this activation requires TLR4. CIRP, directly with CD3/CD28 co-stimulation and indirectly via macrophage stimulation, increases T cell CD69 expression. Our findings indicate that CIRP plays an important role in provoking inflammatory responses via the activation of cellular immunity during sepsis.
DIFFERENTIAL SUSCEPTIBILITY OF HUMAN SP-B GENETIC VARIANTS ON LUNG INJURY CAUSED BY BACTERIAL PNEUMONIA AND THE EFFECT OF CHEMICALLY MODIFIED CURCUMIN
Y. Xu, O. Abdel-Razek, G. Nieman*, R.N. Cooney*, G. Wang*. SUNY Upstate Medical University, Syracuse, NY
Introduction:Staph. aureus is a common cause of nosocomial bacterial pneumonia, frequently causing acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The surfactant protein B (SP-B) gene expresses two proteins involved in lowering surface tension and host defense, respectively. Genotyping studies demonstrate significant association between SP-B genetic variants and ALI/ARDS. Chemically modified curcumins, like CMC2.24, are potent anti-inflammatory compounds which attenuate the host effects of inflammation and inhibit MMP activity in various sepsis models.
Objective: The current study investigates: 1) functional differences of human SP-B genetic variants in a murine model of bacterial pneumonia; 2) effects of CMC2.24 on lung injury caused by pneumonia.
Methods: Humanized transgenic SP-B (hTG SP-B) mice expressing either the T or C allele of human SP-B variants without mouse SP-B were used in this study. Bioluminescent labeled S. aureus Xen 36 (50µl) was injected intra-tracheally (5x108 CFU/mouse) to cause pneumonia. To study the effects of CMC2.24, infected mice were administered a daily dose of CMC2.24 (50 mg/kg) or vehicle (control) by gavage. Dynamic bacterial growth was monitored using in vivo imaging system. Lung tissue and bronchoalveolar lavage fluids (BALF) were harvested 48h after exposure to S. aureus or saline. Histological, cellular and molecular changes in the lung with or without CMC2.24 treatment were studied using bacterial culture, TUNEL, ELISA, MMP zymography and Western blot analysis. p<0.05 by t-test or ANOVA are statistically significant.
Results: Infected hTG SP-B-C mice (human SP-B C allele) showed higher mortality with increased lung apoptosis and phosphorated p38 MAPK and NF-κB expression (p<0.05) compared to infected hTG SP-B-T mice (human SP-B T allele). IL-6 and TNF-α levels were higher in BALF from infected hTG SP-B-C mice (p<0.01) compared to infected hTG SP-B-T mice. Infected hTG SP-B-C mice had higher surface tension in BALF than infected hTG SP-B-T mice (p<0.01) although the surface tension in non-infected hTG SP-B-C and -T mice was similar. In vivo imaging analysis revealed that total flux was higher in the lung of infected hTG SP-B-C mice compared to infected hTG SP-B-T mice (p<0.05), and the timing of bacterial growth peak in the lung differs between male and female mice. Treatment of infected hTG mice with CMC2.24 improves mortality and bacterial clearance compared to vehicle control (p<0.05). Lung tissue treated with CMC2.24 exhibits fewer apoptotic cells, decreased phosphorated p38 MAPK and NF-κB expression (p<0.05), and reduced MMPs-2, 9 activities (p<0.05) when compared to control.
Conclusions: Mice expressing human SP-B C allele are more susceptible to S. aureus pneumonia and CMC2.24 improves mortality and attenuates lung injury in this model.
SIRTUIN 1 ACTIVATOR SRT1720 PROTECTS AGAINST ORGAN INJURY INDUCED BY INTESTINAL ISCHEMIA-REPERFUSION
L.W. Hansen1, A. Khader1, W. Yang*1, 2, J.M. Prince1, 2, J. Nicastro1, G. Coppa1, P. Wang*1, 2. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Intestinal ischemia-reperfusion (I/R) occurs in various clinical situations including abdominal aortic aneurysm repairs, mesenteric artery occlusion, and strangulated hernias. Not only resulting in local damage, intestinal I/R also causes remote organ injury, especially in the lungs, leading to significant morbidity and mortality. Maintenance of mitochondrial biogenesis is a key factor for cell survival, which can be regulated by sirtuin 1, an energy-sensing enzyme. We hypothesized that sirtuin 1 activation with SRT1720 would reduce local and remote organ injury after intestinal I/R.
Methods: Intestinal I/R was induced by applying a microvascular clip across the base of the superior mesenteric artery of adult male C57BL/6 mice (20-25g) for 45 min, followed by reperfusion. SRT1720 (a small molecule compound, 20 mg/kg BW) or vehicle (10% DMSO in saline) was injected via the internal jugular vein at the time of reperfusion. After 4 h of reperfusion, blood, small intestine, and lung tissues were collected for analysis. The levels of mRNA and protein were determined by qPCR and Western blotting, respectively.
Results: After intestinal I/R, the elevated serum organ injury marker LDH was significantly reduced by 42% with SRT1720 treatment (Table). The mRNA levels of mitochondrial transcription factor A (TFAM), a marker for mitochondrial biogenesis, and protein levels of succinate dehydrogenase (SDH), an index of mitochondrial mass, were increased by 1.6- and 1.9-fold, respectively, in the gut of the SRT1720-treated mice compared to vehicle (Table). SRT1720 decreased intestinal mRNA levels of TNF-α and inducible nitric oxide synthase (iNOS) after I/R (Table). In the lungs, the elevated mRNA levels of keratinocyte chemoattractant (KC) were also reduced after SRT1720 treatment (Table). The microscopic architectures of both the gut and lungs were improved in the SRT1720-treated I/R mice, based on H&E staining.
Conclusion: SRT1720 preserves mitochondrial biogenesis and mass, leading to inhibition of inflammation and oxidative stress, thereby protecting against intestinal I/R-induced injury. Thus, the sirtuin 1-mediated pathway is a promising target for treatment of intestinal I/R injury.
HEMODYNAMIC EFFECTS OF BLOOD PRESSURE-TARGETED STEPWISE RESUSCITATION IN SWINE HEMORRHAGIC SHOCK MODEL
J. Hwang*, J. Lee*, K. Kim, Y. Jo, J. Lee, J. Kim, H. Chung, M. Lee, M. Yu. Seoul National University Bundang Hospital, Seongnam, Republic of Korea
Objectives: Prolonged hemorrhagic shock causes global ischemic injury and subsequent resuscitation after bleeding control frequently results in hemodynamic compromise. Previously it has been reported that abrupt reperfusion compared with slow reperfusion in ischemic organs or animals increase the organ injury. Thus, this study was designed to investigate the hemodynamic effect of stepwise increase of blood pressure during initial resuscitation in swine hemorrhagic shock model.
Methods: Fourteen anesthetized male pigs (35±5 kg) were intubated and connected to mechanical ventilators. Intravascular catheters were inserted in both femoral arteries and left femoral vein. Hemorrhagic shock was induced by losing 40% of estimated blood volume (EBV) over 40 minutes and 10% of EBV over 20 minutes through arterial catheter (1 hour) and maintained at mean arterial pressure (MAP) of 30±3 mmHg for 2.5 hours. After hemorrhagic shock for 3.5 hours, pigs were allocated to one of two groups (rapid resuscitation (RR) group, n = 7; pressure-targeted stepwise resuscitation (PSR) group, n = 7). The resuscitation of the RR group was targeted to reach MAP of 70±5mmHg immediately by transfusion of shed blood via femoral artery. The resuscitation of the PSR group was targeted to increase MAP by 10mmHg for every 10 minutes up to 70mmHg and then the MAP was maintained at 70±5mmHg until transfusion of whole shed blood. Hemodynamic parameters including cardiac outputs were monitored during hemorrhagic shock and 2 hours of resuscitation.
Results: All pigs were survived during intervention and resuscitation. During initial resuscitation period of 30 minutes, the MAP of the RR group was significantly higher than that of PSR group (p<0.05) and the heart rate (HR) was significantly lower in the RR group than in the PSR group (p<0.05). Similarly, mixed venous oxygen saturation (SmvO2) was significantly higher in the RR group than in the PSR group during 30 minutes of initial resuscitation (p<0.05). However, after 30 minutes of resuscitation, there were no differences in MAP, HR and SmvO2 between groups. Pulmonary capillary wedge pressure was not significantly different between groups during entire experimental period. However, at 2 hours of resuscitation, cardiac output and stroke volume was significantly higher in the PSR group than in the RR group (p<0.05) and the systemic vascular resistance was significantly lower in the PSR group than in the RR group (p<0.05). There were no differences in serum lactate level between the groups.
Conclusions: During initial resuscitation of hemorrhagic shock, stepwise increase of blood pressure compared with rapid normalization improves hemodynamic parameters including cardiac output, stroke volume, and systemic vascular resistance in swine hemorrhagic shock model.
FIVE-YEAR OUTCOMES AFTER LONG-TERM OXANDROLONE ADMINISTRATION IN SEVERELY BURNED CHILDREN: A RANDOMIZED CLINICAL TRIAL OF SAFETY AND EFFICACY
P. Reeves*1, 2, D. Herndon*1, 2, J. Tanksley*1, A. Ali*1, N. Crites1, K.R. Jennings2, R.P. Mlcak*1, J.O. Lee*1, O.E. Suman*1, C.C. Finnerty*1. 1Shriner’s Hospital for Children, Galveston, TX, 2University of Texas Medical Branch, Galveston, TX
Background: The administration of Oxandrolone, a testosterone analog, to severely burned patients for 4-12 months has beneficial effects on patient outcomes. We have previously demonstrated that severely burned pediatric patients have improved height, bone mineral content, cardiac work, and muscle strength. We now determine whether administering Oxandrolone for greater than one year results in additional effects on long-term outcomes. Patients were followed prospectively from the acute setting to final disposition for 5 years post discharge in a single-center setting.
Study Design: Patients between the ages of 0 and 18, with ≥30% of total body surface area burned were consented to an IRB approved protocol and randomized to receive either placebo (n=84) or Oxandrolone, 0.1 mg/kg twice daily for greater than 12 months (n=35). At discharge, patients from both groups were further stratified to a 12-week exercise program or to standard of care. Resting energy expenditure (REE), cardiac work, height, weight, body composition, muscle strength, glucose kinetics and organ function were evaluated at annual intervals through 5 years post injury.
Results: Long-term administration of Oxandrolone substantially increased bone mineral density (BMD), bone mineral content (BMC), and lean body mass (LBM) (p<0.0001, p< 0.02, and p<0.04 respectively). Oxandrolone played a role in decreasing REE for the first 6 months post-burn. However, data does not suggest any significant long-term effect on REE through Oxandrolone treatment. Those patients manifested a short-term decrease in heart rate compared to those of control profile, but this effect did not last. There was not a significant effect on stroke volume, free T4 or free fatty acids. No negative side effects were attributed to long-term administration of Oxandrolone.
Conclusion: When compared to populations receiving placebo or populations receiving Oxandrolone for less than 12 months (short-term), severely burned children who received Oxandrolone for greater than 12 months improved bone mineral density, bone mineral content, lean body mass, and short term reduction in REE. Thus, administration of Oxandrolone for greater than 12 months yields additional effects for positive outcomes. Administration of this drug in a long-term capacity should be considered for future treatment protocols.
DYSBIOSIS OF INTESTINAL MICROBIOME AFTER SEVERE BURN INJURY
Z.M. Earley1, S. Akhtar1, S. Green2, A. Naqib2, O. Khan1, A. Cannon*1, A. Hammer1, N. Morris1, X. Li1, J. Eberhardt1, R.L. Gamelli*1, R.H. Kennedy*1, M.A. Choudhry*1. 1Loyola University Chicago Health Sciences Division, Maywood, IL, 2University of Illinois at Chicago, Chicago, IL
Introduction: Sepsis remains the leading cause of death in patients that sustain severe burn injury. It is hypothesized that sepsis stems from bacterial infections, toxins, or metabolic products that activate pattern recognition receptors and lead to a systemic inflammatory response in immunocompromised individuals. In addition, studies suggest that Gram-negative bacterial infections play an important role in potentiating sepsis. Since the gastrointestinal tract is a reservoir of trillions of bacteria, we asked whether severe burn injury upsets the homeostatic epithelial barrier of the gut allowing for an overgrowth of Gram-negative aerobic bacteria and their translocation to extra-intestinal sites.
Methods: 16s rRNA gene amplicon sequencing was performed on fecal samples from 4 burn patients sustaining 22%, 32%, 44%, and 57% total body surface area (TBSA) burns. Patients with physiologically insignificant burns (i.e., less than 10% TBSA) were considered as controls. In addition, feces from the ileum and colon of a ∼20% TBSA mouse burn model were also collected 1 and 3 days after injury. Microbial 16S rRNA gene amplicon sequences were generated using an Illumina MiSeq sequencer. FISH staining was performed using fluorescently labeled oligonucleotide probes. For intestinal permeability, mice were gavaged with FITC-dextran and concentration of the dye was quantified in plasma. For bacterial translocation, MLN were aseptically removed, homogenized, and plated on MacConkey agar and cultured aerobically for 24 hours to detect the presence of bacteria from the family Enterobacteriaceae.
Results: Burn injury produced a dramatic and significant effect on the fecal microbial communities of burn injured patients relative to controls. The microbiome of the burn injured patients with 32%, 44%, and 57% TBSA clustered together and diverged from the 22% TBSA patient and the controls. The patients with 32%, 44%, and 57% TBSA died from sepsis, while the patient with 22% TBSA survived. The average abundance of bacteria from the family Enterobacteriaceae was higher in burn patients relative to controls (31.9% to 0.5%). A similar significant change in the microbiome was also noted in fecal samples from mice after burn injury compared to shams. Additionally, FISH staining of mouse intestinal tissue revealed these bacteria to be mucosal associated and potentially adhering to the small intestinal villi. In addition, we observed an increase in intestinal permeability and translocation of bacteria of Enterobacteriaceae family to MLNs in mice one day after burn compared to shams.
Conclusion: Burn injury alters intestinal microbiome favoring overgrowth of Gram-negative aerobic Enterobacteriaceae and translocation of these bacteria. Such alterations in the intestinal microbiome may be a source of peripheral Gram-negative infections in burn patients and a potential cause of sepsis.
DECLINE IN INTESTINAL ANTIGEN PRESENTING CELLS AND IL-10 PRODUCTION IN A MOUSE MODEL OF TOTAL PARENTERAL NUTRITION
T. Ochi1, Y. Feng1, F.R. Demehri1, N. Kamada2, D.H. Teitelbaum*1. 1Section of Pediatric Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, 2Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI
Background: Total parenteral nutrition (TPN) leads to serious adverse events, including systemic infections. Using a mouse TPN model, we previously showed that TPN leads to a perturbation of intestinal bacteria, and an up-regulation of pro-inflammatory mucosal cytokines, such as TNF-α, which drive loss of intestinal barrier function and mucosal atrophy. However, the mechanisms by which TPN leads to such changes remain unknown. IL-10, a major immune-regulatory cytokine, maintains mucosal homeostasis via suppressing excessive immune response against intestinal bacteria. We hypothesized that TPN decreases mucosal IL-10 expression, leading to a dysregulated inflammatory responses.
Methods: Wild-type (WT) C57BL/6 mice underwent jugular vein cannulation, and received TPN vs. Sham (enteral chow). After 7 days, we harvested small intestine, isolated lamina propria mononuclear cells (LPMCs). LPMCs cytokine production and cell populations were analyzed by ELISA and flow cytometry.
Results: LPMCs IL-10 production was significantly blunted in TPN mice (91.0±5.00 vs. 54.0±8.00, Sham vs. TPN respectively, P=0.05). Consistent with the impaired IL-10 production, pro-inflammatory cytokine, TNF-α, production by LPMCs was elevated in with TPN (88.0±3.00 vs. 102.0±0.0, Sham vs. TPN respectively, P<0.05). Flow cytometric analysis showed that CD45+MHC-II+ antigen presenting cells (APCs) significantly declined in TPN mice (Figure). Notably, all four major subsets of intestinal APCs (CD11b+CD11c-F4/80+, CD11b+CD11c+F4/80+, CD11b+CD11c+CD103+, and CD11b-CD11c+CD103+) significantly decreased. Lastly, we examined the impact of TPN on mice deficient in IL-10 (IL-10 KO). Expression of pro-inflammatory cytokines was higher in IL-10 KO mice compared to WT mice, even in Sham group (TNF-α: 443.0±0.0 vs. 321.0±36.0, Sham vs. TPN respectively, P=0.077). In contrast to WT mice, TPN did not cause a loss of intestinal APCs in IL-10 KO mice (Figure).
Conclusions: TPN led to a decline in mucosal IL-10 production and numbers of intestinal APCs, a strong source of IL-10. The loss of IL-10-mediated immune regulation with TPN may contribute to the compromised mucosal homeostasis, such as enhanced TNF-α production.
FIBRINOGEN GAMMA-CHAIN PEPTIDE-COATED, ADP-ENCAPSULATED LIPOSOMES RESCUE MICE FROM LETHAL BLAST LUNG INJURY.
K. Hagisawa, M. Kinoshita, H. Miyawaki, H. Miyazaki. National Defense Medical College, Tokorozawa, Japan
Background: Fibrinogen γ-chain (HHLGGAKQAGDV, H12)-coated, adenosine-diphosphate (ADP)-encapsulated liposomes (H12-ADP-liposomes) that accumulate at bleeding sites via interaction with activated platelets via GPIIb/IIIa and release ADP.
Objective: To elucidate the effect of H12-ADP-liposomes and its mechanisms on the lethal pulmonary hemorrhage following blast injury.
Methods: Mice were pretreated with H12-ADP-liposomes (n=14), ADP-liposomes (n=10), PBS-liposomes (n=10) or normal saline (n=14). A single shot of laser induced shock wave (LISW, 1.8 J/cm2) caused lethal diffuse alveolar hemorrhage in mice. Furthermore, to examine the adenosine effect of H12-ADP-liposomes, adenosine A2A receptor antagonist (ZM241385, n=6) or adenosine A2B receptor antagonist (PSB 1115, n=6) was administered 1 hr before the H12-ADP-liposomes + LISW procedures. Among them, the acute prognosis (survival rates) and pathological injury score (blending area) were compared. The levels of albumin and MIP-2 in the bronchoalveolar lavage fluid (BALF) were measured after LISW in each group.
Results: H12-ADP-liposomes significantly improved mouse survival and reduced the pathological injury score than normal saline (35 vs 40, p=0.004, n=5). H12-ADP-liposomes reduced the albumin leakage (0.8 vs 1.3 mg/ml, p=0.03, n=6) and MIP-2 levels in the BALF (74 vs 355 pg/ml, p<0.01, n=6) than normal saline. Adenosine receptor (especially A2B) antagonists prevented from rescuing and reducing the BALF albumin and MIP-2 leakage by the H12-ADP-liposomes treatment.
Conclusions: H12-ADP-liposomes may be effective for acute blast lung injury, functioning hemostasis and organ protection via anti-inflammatory purinergic signaling.
INTRAOPERATIVE NORMOXIA, OXIDATIVE DAMAGE, AND ORGAN INJURY FOLLOWING CARDIAC SURGERY
F.T. Billings*. Vanderbilt University, Nashville, TN
Introduction: Anesthesiologists ventilate patients with concentrations of oxygen well above those required to saturate hemoglobin (Hb). Hyperoxia increases oxidative damage in vitro, and in prior cardiac surgery studies, increased concentrations of F2-isoprostanes, robust indicators of oxidative damage in vivo, independently predicted acute kidney injury (AKI). We hypothesize that intraoperative normoxia decreases oxidative damage and organ injury after cardiac surgery.
Methods: Patients administered normoxia, defined as maintenance of a median arterial pO2 between 70-120 mmHg throughout surgery, were identified in a 486-subject cohort. We risk-matched this group with patients administered hyperoxia, defined as a median pO2 >200 mmHg, based on oxidative stress risk factors. Inspired oxygen (FIO2), Hb O2 sat, and pO2 were measured to assess oxygen administration, F2-isoprostanes and isofurans to assess oxidative damage, and AKI (AKIN criteria), delirium (any positive CAM-ICU exam), and new-onset atrial fibrillation to assess organ injury.
Results: Ten subjects were administered normoxia throughout surgery. Risk factors for intraoperative oxidative stress, including smoking (incidence 18%), body mass index (median 27.6 kg/m2), age (67 years), and cardiopulmonary bypass (73%) were similar between this group and 279 risk-matched hyperoxic patients. FIO2 (p<0.001) and pO2 (p<0.001) were 75% less in normoxic patients, but Hb O2 sat and arterial lactate concentrations, safety markers of ischemia, were similar (Figure). Peak F2-isoprostanes were 41% less (95% CI 19-63) in the normoxic group (P=0.01), and neither F2-isoprostanes nor isofurans increased above baseline in normoxic patients, while F2-isoprostanes rose 47% and isofurans 43% in hyperoxic patients (Figure). Clinically, no normoxic subjects developed AKI (20.0% in hyperoxic group, P=0.04) or delirium (23.8% in hyperoxic group, P=0.02), and 20% developed new-onset atrial fibrillation (28.8% in hyperoxic group, P=0.53).
Conclusions: In this pilot study, normoxia during cardiac surgery was associated with reduced intraoperative oxidative damage and decreased incidences of postoperative AKI and ICU delirium. A randomized clinical trial is warranted to determine if intraoperative normoxia decreases oxidative damage and organ injury, compared to the hyperoxia anesthesiologists typically provide.
PLATELET DERIVED HMGB1 REGULATES THROMBOSIS AND ORGAN INJURY FOLLOWING TRAUMA
Q. Chen, S. Vogel, P. Loughran, B.S. Zuckerbraun, T. Billiar, M.D. Neal. University of Pittsburgh, Pittsburgh, PA
Introduction: Trauma is characterized by microvascular thrombosis which is thought to contribute to organ injury, although the mechanisms of thrombosis remain largely unknown. Recently, innate immune signaling on platelets has been shown to regulate both inflammation and hemostasis. High-mobility group protein 1 (HMGB1) has been shown to be released from platelets and represents a potentially potent inflammatory signal. However, the specific functions of platelet derived HMGB1 in sterile injury and hemostasis remain unexplored. We now hypothesize that platelets release HMGB1 which regulates hemostasis and organ injury following trauma.
Methods: Mice specifically lacking HMGB1 on platelets (HMGB1Pf4) were generated using cre/loxp technology and subjected to a validated model of trauma and hemorrhagic shock (THS) with tissue harvest for analysis of injury and platelet aggregometry. Ferric chloride (FeCl3) induced thrombosis was used to study the specific role of platelet HMGB1 in thrombosis, and bleeding times were measured. To assess potential clinical relevance, platelets were obtained from human trauma patients and analyzed for HMGB1 release using ELISA and FACS.
Results: Human trauma patients had a marked upregulation of HMGB1 expression compared to healthy controls (FACS MFI 18.5±1.2 v 5.3±2.2 p<0.01). Murine trauma also resulted in a significant increase in HMGB1 expression as well as platelet hyper-aggregation and sequestration into lung and liver in WT mice. Strikingly, selective deletion of HMGB1 from murine platelets (HMGB1Pf4) resulted in reduced platelet aggregation, prolonged bleeding time (190±21sec v 98±7sec, p<0.001), and reduced clot burden following FeCl3 thrombosis compared to WT. Following THS, HMGB1Pf4 mice had reduced liver injury as measured by serum AST. Additionally, HMGB1Pf4 mice were protected from lung and liver injury as measured by histologic scoring, with markedly reduced microvascular thrombosis vs WT (21.5±1.4 v 3.4±0.9 thrombi/high power field, p<0.05).
Conclusions: HMGB1 released by platelets regulates platelet aggregation and thrombus formation following trauma and hemorrhage. Expression of HMGB1 is upregulated in both murine and human platelets. Importantly, selective deletion of HMGB1 specifically from platelets nearly eliminated microvascular thrombosis and attenuated organ injury following trauma. Targeting HMGB1 release from platelets may represent a novel therapeutic intervention for regulating microvascular thrombosis and preventing subsequent organ injury after trauma.
ANGIOPOIETIN-2 (ANG-2) IN VASCULAR INSTABILITY FOLLOWING GRAM-POSITIVE PULMONARY CHALLENGE
J. Lee2, 3, H.M. Linge1, K. Ochani1, K. Lin1, E.J. Miller*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2The Elmezzi Graduate School of Molecular Medicine, Manhasset, NY, 3University of South Alabama College of Medicine, Mobile, AL
Acute Respiratory Distress Syndrome (ARDS), occurs annually in approximately 190,000 individuals in the U.S. and remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that uncontrolled excessive secretion of Ang-2 is responsible for pulmonary microvascular permeability following pulmonary challenge. Since Gram-positive bacteria are important infectious agents associated with ARDS, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN). We then examined the mechanism of release, and the effects of Ang-2.
Methods: Male Balb/c, 8-10 wks old (n=8/group) were pre-treated i.p. with an NSF inhibitor TAT-NSF700 (0.5mg/kg) or saline. After 30min, LTA (150μg) and PGN (500μg) or saline alone were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Mice were euthanized, the lungs lavaged and blood and organs were collected. In vitro studies involved pulmonary endothelial cells cultured in the presence or absence of LTA/PGN, antibody to TLR2 (αTLR2, the LTA/PGN receptor) and inhibitors of specific pathways including TAT-NSF700 (SNARE-NSF); EGTA (calcium requiring); H-89 (PKA); L-NAME (nitric oxide); cycloheximide (protein synthesis) and IBMX (phosphodiesterase).
Results: Pulmonary challenge with LTA-PGN induced both systemic and airspace inflammation and decreased oxygen saturation. TAT-NSF700 pre-treatment reduced changes in lung tissue Ang-2; improved post challenge oxygen saturation (82% vs 96%, p=0.008); and decreased lung wet-to-dry ratios (p=0.04). In vitro, there was a time-dependent, increase in the accumulation of Ang-2 in the culture medium of endothelial cells. This change was significantly decreased in the presence of cycloheximide, EGTA, H-89 and L-NAME, and increased by IBMX. However, TAT-NSF700 had no effect on constitutive accumulation of Ang-2. LTA/PGN induced a rapid (<2 min) release of Ang-2, which was significantly attenuated by αTLR2 or TAT-NSF700. Furthermore, Western blot of cell lysates of the cells showed that TAT-NSF700 reduced LTA/PGN-induced myosin light chain (MLC) phosphorylation.
Conclusion: Blocking Ang-2 release, in vivo, prevented lung edema and improved oxygen saturation following pulmonary challenge. In vitro, TAT-NSF700 blocked LTA/PGN-induced release of Ang-2 and MLC phosphorylation an important component of endothelial cell retraction. The data suggest that inhibition of Ang-2 release in pneumonia and sepsis may be beneficial to prevent the pathogenesis of ARDS, particularly the pulmonary microvascular permeability and hemodynamic instability.
CONTRIBUTION OF FREE HEME TO IMPAIRED MICROCIRCULATION AND HEPATIC INJURY IN SEPSIS
F.A. Schleser1, R.A. Seidel1, 2, A.T. Press1, S.H. Heinemann3, M. Clemens*4, M. Bauer1. 1Clinic for Anesthesiology and Intensive Care Medicine / Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany, 2Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University, Jena, Germany, 3Institute of Biochemistry and Biophysics, Friedrich Schiller University & Jena University Hospital, Jena, Germany, 4Department of Biological Sciences, University of North Carolina, Charlotte, NC
Background: Release and degradation of heme in humans is tightly controlled. However, in pathophysiological conditions, like sepsis or malaria, patients suffer from hemolysis leading to a significant release of heme into the circulation. Recent studies suggest a correlation between hemolysis and decreased heme-binding proteins coinciding with unfavorable survival in critically ill patients. In murine sepsis the level of free heme is highly elevated. The liver is a key player in both heme metabolism and host response to infection. Therefore we investigate the impact of free heme on the hepatic microcirculation and hepatocellular integrity in the stress-exposed liver.
Methods: Livers of male Wistar rats were isolated and perfused with Krebs Henseleit buffer containing hemin. Portal pressure and liver weight were recorded. Pre-experimental treatments were: (a) no intervention (control), (b) Peritoneal Contamination and Infection (PCI, i.p. for 24h), (c) Lipopolysaccharide (LPS, 1mg/kg BW for 2h). Furthermore, one group of rats was pre-treated with hemin i.p. for 24 hours. Intravital microscopy (IVM) was performed to identify changes in liver sinusoids during heme perfusion. In vitro assays were performed using the human immortalized hepatic stellate cell (HSC) cell line LX-2. Cells were cultured and incubated with hemin ± TNF-α ± albumin. Cytotoxicity was determined via lactate dehydrogenase (LDH) release. Signaling response was determined using Ca2+-imaging with Indo-1 AM.
Results: Hemin perfusion resulted in a dose-dependent increase in portal pressure over baseline of approx. 6mmHg (1 µM: 1.67±0.3; 4 µM: 1.83±0.3, 10 µM: 2.79±0.2 mmHg). This effect was mediated by contraction of HSCs that caused an increase in tortuosity and constriction of the sinusoids as indicated by IVM and liver weight change. Moreover, LX-2 stimulation with 10 µM hemin led to a strong increase in intracellular Ca2+ further supporting the role of HSCs. There was no difference in the portal pressure response between healthy, PCI and LPS animals. No increase in LDH levels in outflow was observed in control and LPS animals with hemin, while the LDH release was 4.2 fold higher in PCI with hemin than without. In cell culture experiments a cytotoxicity of 42% was measured in LX-2 after 24 hour incubation with 40 µM hemin. Addition of TNF-α showed no supra-additive cytotoxic effect in LX-2 cells. The cytotoxic and vasoconstrictive effects were abrogated by addition of 4% human serum albumin.
Conclusion: Our results demonstrate that free heme contributes to both impaired microcirculation via contraction of HSCs and direct cytotoxicity in sepsis. Albumin at normal physiological levels protects the liver by preventing both direct cytotoxicity and perfusion failure. The change in intracellular Ca2+ levels suggests that calcium is one possible mediator of heme signaling in HSCs.
BLOCKAGE OF ARGININE VASOPRESSIN RECEPTOR 2 REDUCES INCREASE IN PULMONARY VASCULAR RESISTANCE IN OVINE SEPSIS
E. Lopez*1, O. Fujiwara1, B. Enkhtaivan1, J. Rojas1, R.A. Cox*2, 1, H.K. Hawkins2, 1, D. Herndon*2, 1, D. Prough*1, P. Enkhbaatar*1. 1The University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX
Introduction: Increased pulmonary vascular resistance augments the severity of acute lung injury during sepsis. Previously, we have reported that blocking arginine vasopressin (AVP) V2 receptor (V2R) from endogenous antidiuretic AVP significantly reduced sepsis-induced microvascular hyper-permeability. The aim of the present study was to test the hypothesis that blockade of V2R alleviates pulmonary vascular resistance and attenuates pulmonary edema, using our well-characterized highly translational ovine Methicillin-resistant Staphylococcus aureus (MRSA) sepsis model.
Methods: Sepsis was induced in 25 surgically instrumented sheep by insufflation of cooled cotton smoke (48 breaths) and instillation of MRSA (3.5X10^11CFU) into the lungs under anesthesia and analgesia. After the injury sheep were randomly allocated into 4 groups—SHAM: no injury, no treatment, n=6; CTRL: Injured, no treatment, n=7; TLVP: injured, treated with intravenous administration of tolvaptan (416.7 ug/kg/h), a V2R antagonist, n=6; and DDAVP: injured, treated with intravenous administration of desmopressin (36.2 ng/kg/h), a V2R agonist, n=6. All sheep were placed on mechanical ventilation, fluid resuscitated and monitored for 24 hrs.
Results: TLVP significantly decreased pulmonary artery (PAP, p<0.05 at 24 h) and capillary (Pc, p<0.05 at 12 and 24 h) pressures and prevented increase of left ventricular stroke work index (LVSWI, p<0.05 at 15 and 21 h) vs. CTRL group. Circulating brain natriuretic peptide (BNP), a marker of myocardial stretch stress was also reduced by TLVP (p<0.01 at 12 and 24 h). DDAVP did not affect these changes. Lung water content (by wet-to-dry ratio) was significantly increased in CTRL and DDAVP vs. SHAM. No difference was found between TLVP and SHAM.
Conclusion: The results indicate that V2R antagonist improved left heart ventricle performance, thus alleviating congestion and resistance in pulmonary circulation in MRSA sepsis. TLVP should be considered as an adjuvant therapy to vasopressors for treatment of patients with septic shock. Support: GM097480, SHC84050.
HUMAN MYELOID DERIVED SUPPRESSOR CELLS INDUCE IMMUNE SUPPRESSION AFTER SEVERE SEPSIS
R. Ungaro, D.C. Nacionales, A.L. Delmas, M. Lopez, F. Needell, J. Zhang, B.E. Szpila, B. Mathias, A.L. Cuenca, A.M. Mohr*, A. Bihorac*, C. Leeuwenburgh*, F. Moore*, S. Brakenridge*, H. Baker*, L. Moldawer*, P.A. Efron*. University of Florida, Gainesville, FL
Objectives: While myeloid derived suppressor cells (MDSCs) were first described in cancer, they have more recently been suggested to play a key role in the immune responses of persistent infectious diseases, autoimmune diseases, and chronic inflammation. Although we have previously identified a significant relative and absolute increase in circulating leukocytes that phenotypically resemble MDSCs after severe infection and injury, little is known about their precise role after the onset of severe human sepsis or trauma. We hypothesized that these cells not only have a prolonged presence after severe inflammation, but contribute at the genomic to cellular level to an environment of persistent inflammation and immunosuppression.
Methods: Blood was obtained from 83 septic and 11 healthy control subjects for phenotyping (n=71 septic), functional analysis and genotyping (n=12 septic) of enriched MDSC populations. Patient recruitment was within 12 hours following the onset of severe sepsis/septic shock. Blood samples were drawn <12 hours and 1, 2, 4, 7, 14, 21 and 28 days after sepsis. MDSCs were enriched as CD33+CD11b+HLA-DR-/low by cell sorting. RNA was extracted and genome-wide expression analysis was performed on samples from days 7 and 14 (p<0.001 f-test). Ingenuity Pathway Analysis (IPA) and distance from reference (DFR) were calculated. Also, Gene Ontology and Biocarta analysis were performed. T-cell suppression assays by MDCSs were also conducted.
Results: Sepsis was associated with dramatic increases in the numbers of MDSCs, peaking at 12 hours but remaining elevated for 28 days (p<0.001). Neither the number of MDSCs nor their function differed at days 7 and 14; therefore, they were combined for analysis. Individual gene analysis of enriched cells from septic patients was consistent with MDSCs, with HLA gene expression (HLA-DOA, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRB1, HLA-DRB3 and HLA-DRB4) being suppressed and arginase 1 (ARG1) being upregulated (p<0.001). Canonical pathway and causal network analysis further supported these pathway alterations as well as a pattern of simultaneous low grade inflammation with immune suppression (p<0.001). Gene ontology revealed significant increases (DFR, p<0.05) in the “NO biosynthesis” and the “regulation of ROS production” pathways, consistent with MDSCs, as well as decreased “detection of bacterium.” T cell suppression assays by MDSCs from septic patients revealed >90% (mean and median) suppression index with MDSCs from septic patients.
Conclusion: Human severe sepsis/septic shock are associated with an increase in the circulating MDSCs, and this increase contributes to both the persistent inflammation and immunosuppression. Further analysis of these cells and their role after severe infection may allow for interventions that could improve patient morbidity and mortality after sepsis or trauma.
TREATMENT WITH IL-7 ENHANCES THE RECOVERY AND FUNCTIONALITY OF NAÏVE T CELLS IN A MURINE MODEL OF SEPSIS
T. Rice, D.H. Hildeman, C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Background: During sepsis, the initial depletion and incomplete restoration of T cells are key aspects of persistent immune suppression. Importantly, clinical trials have shown that critically ill patients with reduced T cell diversity have increased mortality and susceptibility to nosocomial infections. It has previously been demonstrated that IL-7 treatment during sepsis can reduce T cell loss. However, it has not been shown whether IL-7 treatment will accelerate T cell replenishment and / or conserve functionality during sepsis. Specifically, we hypothesized that IL-7 treatment will enhance recovery of naïve T cells, an indicator of T cell diversity, and their antigen responsiveness.
Methods: A sublethal cecal ligation and puncture (CLP) was performed on C57BL/6 mice. In some cohorts of mice, twenty-four hours after CLP, the necrotic cecum was excised, the peritoneum washed and antibiotics administered (CLP-E). Mice were treated with IL-7 or saline, intraperitoneally. Splenic T cells were isolated, enumerated, and characterized by flow cytometry.
Results: We observed that naïve T cell numbers were reduced three days following CLP or CLP-E. Subsequently, naïve CD4 T cell populations failed to return to baseline levels 7 days after CLP-E (sham 9.54 vs CLP-E 5.5, p=0.01). Treatment with IL-7 lead to an increase in naïve T cell numbers when compared to untreated CLP-E mice (7.7 vs 5.5, p =0.04). Further, there is also a significant increase in non-responsive naïve T cells 7 days following CLP-E injury (sham 10.8% vs CLP-E 22.8%, p = 0.002). Mice treated with IL-7 demonstrated a decrease in these non-responsive naïve T cells when compared to CLP-E mice (16.5% vs 22.8%, p =0.02).
Conclusion: From these data, we conclude that IL-7 administration can accelerate the recovery of naïve T cell population diversity. In addition, IL-7 treatment increases the antigen responsiveness, or functionality, of the naïve T cell population. Altogether, this pre-clinical study demonstrates that IL-7 treatment during sepsis will likely reduce mortality and nosocomial infections.
INCREASED PD-1 EXPRESSION AND ALTERED T CELL REPERTOIRE DIVERSITY IN SEPTIC SHOCK PATIENTS
N. Takeyama, A. Tomino, M. Hashiba, M. Ando, T. Gocho, H. Miyabe, H. Kano, M. Tsuda, T. Hattori, A. Hirakawa. Dept of Emergency and Acute Intensive Care Medicine, Fujita Health University, Totoake, Japan
Sepsis is a complex medical condition that exerts a variety of consequences on the immune suppression, often manifested as an increased susceptibility to nosocomial infection. We have investigated the impact of sepsis on the quantity and quality of lymphocytes. Lymphocytes functions were monitored by changes in T cell receptor (TCR) diversity and expression of the inhibitory receptor programmed death 1 (PD-1).
Blood was obtained from 13 septic shock patients and 4 healthy volunteers. Mortality in the septic shock patients was 30.8% and the most frequent cause of sepsis was digestive tract origin. TCR diversity and PD-1 expression were analyzed in peripheral blood mononuclear cells (PBMCs) and whole blood, respectively, isolated from septic shock patients at 3 time points (Day 1, 3 and 7 after diagnosis of septic shock). TCR diversity was measured in genomic DNA isolated from PBMCs using Human Immun TraCkeRb test (ImmunID Technologies, Grenoble, France). Multi-N-plex PCR was performed using a primer specific to a V genes family and several primers specific to J segments. The signal is measured as a function of the fluorescence intensity of the reference marker. Rearrangements validation and maps generation were detected and analyzed using the Constel’ID software (ImmunID Technologies). PD-1 expression on CD4 T cell and HLA-DR expression on CD14 cells were measured by flow cytometry.
TCR diversity was markedly decreased in septic patients at day 1 compared to the healthy volunteer. A recovery of TCR diversity was observed at days 3 and 7 except for dead patient. PD-1 expression was significantly increased in septic patients at day 1. HLA-DR expression was significantly decreased in septic patients at day 1. Total lymphocyte count reduced in septic patients at day 1, but lymphocyte count recovered at days 3 and 7 except for dead patient.
We observed a lower TCR diversity, increased PD-1 expression and lymphopenia in early stage of septic shock. A quickly recovery of TCR diversity and lymphocyte count were observed in live patients. On the other hand, dead patient stayed on the lower level over the course of the study. These results suggest that impaired antigen presenting capacity and lymphopenia might participate in increased mortality.
THE EFFECTS OF AICAR ON METABOLIC PATHWAYS IN THE LUNG FOLLOWING MURINE HEMORRHAGIC SHOCK
L.R. Klingbeil1, 2, G. Piraino2, P. Hake2, J.R. Ledford2, B. Zingarelli*2. 1University of Cincinnati, Cincinnati, OH, 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Background: Despite advances in the resuscitation of trauma victims, mortality from hemorrhagic shock and the ensuing multiple organ dysfunction syndrome (MODS) remains high. Mitochondrial dysfunction has been suggested in severely injured trauma patients who develop MODS early in the course of resuscitation despite early goal-oriented maximization of oxygen delivery. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls autophagy and metabolic recovery through mitochondrial biogenesis. Following phosphorylation, AMPK regulates mitochondrial biogenesis via peroxisome proliferator-activated receptor γ co-activator α (PGC1-α), which can also be regulated by sirtuin 1(Sirt-1). 5-amino-4-imidazole carboxamide riboside (AICAR) is an adenosine analog that activates AMPK. We hypothesize that treatment with AICAR during murine hemorrhagic shock ameliorates lung injury by increasing AMPK-dependent pathways of cellular autophagy and mitochondrial biogenesis.
Methods: Hemorrhagic shock was induced in anesthetized young male mice (2-4 months old) by withdrawing blood from the femoral artery to a mean arterial pressure (MAP) of 30 mmHg for 90 minutes. At the end of the shock period, mice were either injected with 100mg/kg AICAR or 200µL normal saline intraperitoneally. The mice were then resuscitated with the shed blood plus two times that amount in Lactated Ringer’s solution. Mice were sacrificed 3 hours after resuscitation and lungs were harvested for biochemical assays and histological analysis.
Results: Following resuscitation, the AICAR-treated mice had a significantly higher MAP compared to the saline-treated mice (65.8±1.1 versus 56.7±1.0 mmHg, p<0.05). After hemorrhagic shock, there was significantly higher neutrophil infiltration, as evaluated by myeloperoxidase (MPO) assay, in the lung of the saline-treated mice when compared to the mice treated with AICAR (138.9±7.9 versus 109.6±13.0 U/100mg tissue, p<0.05). The lung protective effect of AICAR was confirmed at histological examination as the AICAR-treated group had a lower median lung injury score than the saline group (6.0 versus 7.8, p<0.05). At Western blot analysis, there was a significant increase in nuclear PGC1-α in the lung of AICAR-treated mice compared to the saline group (1.3±0.08 versus 0.9±0.09 relative intensity, p<0.05). Cytosolic Sirt-1 was also significantly upregulated in the lung of AICAR-treated mice compared to the saline-treated mice (0.81±0.04 versus 0.64 ±0.04 relative intensity, p<0.05). Additionally, cytosolic pAMPK and AMPK levels were increased in the AICAR-treated mice which indicates an improved ability to mount a metabolic response to hemorrhage.
Conclusion: Our data suggests that treatment with AICAR during murine hemorrhagic shock decreases inflammation and activates metabolic repair mechanisms in the lung via AMPK-dependent pathways.
CORTICOSTEROID THERAPY BENEFITS SEPTIC MICE WITH ADRENAL INSUFFICIENCY BUT HARMS SEPTIC MICE WITHOUT ADRENAL INSUFFICIENCY
X. Li*. University of Kentucky, Lexington, KY
Objectives: Corticosteroid (CS) therapy is frequently used in septic patients given the rationale that there is an increased demand for CS in sepsis and up to 60% of severe septic patients experience adrenal insufficiency. However, the efficacy of CS therapy and whether the therapy should be based on the results of adrenal function testing are highly controversial. The heterogeneity of septic patients, the technical difficulties in identifying adrenal insufficiency and a lack of adrenal insufficiency animal model present significant barriers to address this longstanding clinical issue.
Subjects: Scavenger receptor BI (SR-BI) null and adrenal-specific SR-BI null mice.
Interventions: Sepsis was induced by cecal ligation and puncture (CLP).
Measurements and Main Results: Using SR-BI-/- mice as the first relative adrenal insufficiency animal model we found that CS therapy significantly improved the survival in CLP-treated SR-BI-/- mice but causes more septic death in wild type controls. We identified a cocktail of CS that provides better protection than an individual CS, and importantly, the CS cocktail still provided effective protection 18h post CLP; using adrenal-specific SR-BI-/- mice as an inducible CS (iCS) deficient animal model, we found that iCS specifically suppresses IL-6 production without affecting TNF-α, nitric oxide and IL-10 production. We further found that iCS does not induce peripheral lymphocyte apoptosis but promotes phagocytosis of macrophages and neutrophils.
Conclusions: This study provides a proof-of-concept that CS treatment is an effective therapy for septic patients with adrenal insufficiency but it harms septic patients without adrenal insufficiency. This study also reveals that iCS functions through specifically regulating IL-6 production and promoting phagocytosis of immune effector cells. Our findings indicate that iCS has both immunosuppressive and immunopermissive properties, which contradicts the concept that CS is a potent immunosuppressive molecule.
PPARα NULL MICE HAVE DECREASED HEART FUNCTION AND MYOCARDIAL FATTY ACID OXIDATION IN SEPSIS
S.W. Standage*1,2, B.G. Bennion1, D.R. Ledee3, W.C. Liles1, J.K. McGuire1,2, A.K. Olson3,2. 1University of Washington School of Medicine, Seattle, WA, 2Seattle Children’s Hospital, Seattle, WA, 3Seattle Children’s Research Institute, Seattle, WA
Objective: Children with severe septic shock have downregulated peroxisome-proliferator activated receptor alpha (PPARα) signaling. We have recently shown that PPARα null (KO) mice have a considerable survival disadvantage in sepsis compared to wild type (WT) mice. KO mice demonstrate biochemical and histological signs of cardiac injury. Cardiac expression of genes related to fatty acid oxidation are more downregulated in KO hearts than in WT hearts. We hypothesized that KO mice have worse cardiac function in sepsis than WT mice and that they utilize less fatty acid substrate for myocardial oxidative phosphorylation.
Methods: Sepsis was induced via cecal ligation and puncture (CLP). Echocardiography was performed pre-op (baseline), 8 hours, and 24 hours after CLP. Twenty four hours after CLP, cardiac function and substrate fractional contributions to the citric acid cycle were measured using ex vivo working heart perfusions with 13C labeled mixed fatty acid, lactate, ketones, and unlabeled glucose. Hearts from non-operated mice were perfused as controls.
Results: Baseline cardiac function was similar between KO and WT mice. At 8 and 24 hours, KO fractional shortening was significantly lower than WT (26.8 vs. 30.9%, p=0.05, at 8 hours and 24.5 vs. 33.3%, p=0.0015, at 24 hours respectively). In the ex vivo perfusions, cardiac function was similar in non-operated KO and WT hearts. In septic mice, the KO hearts had decreased systolic pressure (63.7 vs. 77.4 mm Hg, p=0.002) and lower cardiac output (5.7 vs. 8.3 mL/min, p=0.016) compared to WT. The calculated power of the KO hearts is lower than that of the WT hearts in sepsis (543 vs. 927 mm Hg mL/min, p=0.007). Myocardial oxygen consumption is higher in WT hearts at both baseline and septic conditions (5.8 vs. 6.9 μmol/g/min, p=0.012, and 6.2 vs. 9.1 μmol/g/min, p=0.005 respectively). Metabolic analysis showed that hearts from septic KO mice had reduced fractional contribution of fatty acids to the citric acid cycle versus hearts from septic WT mice (15.0 vs. 28.4%, p=0.014).
Conclusions: KO mouse hearts have an impaired functional response to sepsis. Thus, PPARα appears to be necessary for the heart’s adaptation to the hemodynamic derangements during sepsis. KO hearts also had impaired fatty acid utilization for the citric acid cycle during sepsis, which suggest a potential mechanism for the observed cardiac dysfunction.
AGE-DEPENDENT CHANGES OF METABOLIC PATHWAYS IN THE MYOCARDIUM FOLLOWING MURINE HEMORRHAGIC SHOCK
D. Matsiukevich, L.R. Klingbeil, G. Piraino, P. Hake, B. Zingarelli*. Cincinnati Children’s Hospital, Cincinnati, OH
Background: Mortality of trauma patients suffering from hemorrhagic shock with the ensuing multiple organ dysfunction syndrome (MODS) remains high. However, incidence of MODS in pediatric patients, including myocardial injury is lower than in adults. An inability to use oxygen at the cellular level, energetic failure and mitochondria dysfunction has been suggested in the development of MODS in critical illness. However, the molecular mechanisms that link age to susceptibility to MODS are not known. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls autophagy (disposal of damaged organelles) and metabolic recovery through mitochondrial biogenesis. Following activation by phosphorylation, AMPK regulates mitochondrial biogenesis through PGC1-α.
Hypothesis: We hypothesize that these metabolic signaling pathways are altered in the heart during hemorrhagic shock and are age dependent.
Methods: Hemorrhagic shock was induced in anesthetized young male (2-4 months old) and mature mice (9-10 months old) by withdrawing blood from the femoral artery to a mean arterial pressure of 30mmHg for 90 min. The mice were then resuscitated with the shed blood plus two times that amount in Lactate Ringer’s solution. Mice were sacrificed at 3 hours after resuscitation and hearts were harvested for biochemical assays.
Results: Because of the central role of AMPK in mitochondrial biogenesis and autophagy, we measured the activity of the catalytic α1-subunit of AMPK. There was an increase of nuclear pAMPK 1α in young mice by 2.8 folds after hemorrhagic shock when compared to age-matched controls (p=0.05). However, there was pAMPKA 1α down regulation in mature mice after hemorrhagic shock by 2.02 folds (p=0.05) when compared to age-matched controls. Similarly, after hemorrhagic shock PGC1-α nuclear expression dramatically increased in the young group by 1.9 folds (p=0.008) in comparison to age-matched controls. On the contrary, PGC1-α expression significantly decreased by 3.5 times in mature mice (p=0.009) after hemorrhagic shock when compared to age-matched controls.
Conclusion: Our data suggest that AMPK-dependent pathways are activated during hemorrhagic shock only in young animals. However, activation of these metabolic pathways are impaired with aging.
SELECTIVE ROLE FOR NEUTROPHIL-DEPENDENT HIF1A IN ATTENUATING POST-ISCHEMIC MYOCARDIAL INFLAMMATION
M. Koeppen1, T. Eckle2, H. Eltzschig2. 1Ludwig-Maximilians-University, Munich, Germany, 2University of Colorado, Aurora, CO
Objectives: Myocardial ischemia increases morbidity and mortality in critical ill patients. Conventional therapeutic strategies aim for an early reperfusion of the ischemic myocardium, but the injured tissue elicits a response termed reperfusion injury, which increases myocardial damage. The transcription factor Hypoxia-inducible factor 1a HIF1a coordinates cellular processes, which can reduce ischemia-elicited organ damage. In this study we investigated the tissue-specific role of HIF1a in myocardial ischemia-reperfusion injury.
Methods: The Institutional animal care and use committee (IACUC) at the University of Colorado - Anschutz Medical Campus approved the animal experiments. To circumvent the embryonic lethality of global HIF1a-deficiency, we used an inducible Cre-LoxP-System to generated whole-body HIF1a-deficient mice (UbcCre/Hif1αloxP). Using a similar approach, we also generated mice with a HIF1a tissue-specific deletion in cardiomyozytes (MyosinCre/Hif1αloxP), endothelial cells (VEcadherinCre/Hif1αloxP) and bone-marrow derived leucocytes (LysMCre/Hif1αloxP). Then, we submitted these mice an in vivo-model for acute myocardial ischemia-reperfusion injury. Animals from the experimental group matched control animals in gender, age and weight. In a subset of experiments we isolated neutrophils by negative-selection and transferred these into neutrophil-depleted mice (adoptive transfer). In vitro we used special cell culture dishes to measure the oxygen consumption of isolated neutrophils (OxoDish®). Statistical differences were determined by t-Test or ANOVA were applicable; p<0.05 was considered significant.
Results: First, we submitted UbcCre/Hif1αloxP to myocardial ischemia-reperfusion injury and found 24% larger infarct sizes than control animals (p<0.05, n=5). Next, we compared the infarct sizes of MyosinCre/Hif1αloxP, VEcadherinCre/Hif1αloxP and LysMCre/Hif1αloxP with the respective control animals. Only the LysMCre/Hif1αloxP animal produced infarct sizes comparable to the whole-body HIF1a-deficient mice. Since neutrophils are the most common bone-marrow derived leucocytes in the post-ischemic myocardium, we characterized the influence of neutrophil-based HIF1a to the cardioprotective properties. For this purpose we performed an adoptive transfer of HIF1a-expressing and HIF1a-deficient neutrophils into neutrophil-depleted mice. When neutrophils did not express HIF1a, infarct sizes increased by 21% (p<0.05; n=6). Subsequent in vitro studies revealed that HIF1a reduced the inflammatory neutrophil functions (measured by a reduction in oxygen burst by 17%; p<0.05, n=6).
Conclusion: Our study indicates a strong cardioprotective role of neutrophil-expressed HIF1a in myocardial ischemia-reperfusion. HIF1a elicits its protective effects through a reduction of neutrophil-depedendent reperfusion injury.
TOLL-LIKE RECEPTOR 3 STIMULATES CARDIOMYOCYTE PROLIFERATION AND CARDIAC REGENERATION AFTER MYOCARDIAL INFARCTION: CORRELATION WITH THE WARBURG EFFECT
X. Wang, T. Ha, H. Ma, J. Kalbfleisch, R. Kao, D.L. Williams*, C. Li*. East Tennessee State University, Johnson City, TN
Heart failure is a leading cause of death globally. Heart failure can result in a loss of cardiomyocytes. The adult heart cannot regenerate cardiomyocytes and replace damaged heart tissue. In the adult heart >90% of energy requirements are supplied by mitochondrial oxidative phosphorylation. In contrast, the neonatal heart can regenerate cardiomyocytes after injury, but this ability is lost when cardiomyocyte metabolism changes from glycolysis to oxidative phosphorylation seven days after birth. Recent evidence shows that activation of immune cells results in a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This phenomenon is known as the Warburg effect. Interestingly, stimulation of immune cells with TLR ligands can induce the Warburg effect. This study investigated whether TLR ligands will induce cardiomyocyte proliferation and whether this correlates with the Warburg effect. First, we examined the role of TLR3 in cardiac regeneration in neonatal hearts. TLR3-/- (n=8) and WT (n=8) neonatal mouse hearts (one day postpartum) were subjected to myocardial infarction (MI) for 21 days. Cardiac function was measured by echocardiography. Cardiomyocyte proliferation and regeneration were examined by histone 3 phosphorylation (phospho-histone H3) and EdU incorporation into the cardiomyocytes. Cardiac function in WT neonatal hearts completely recovered after injury. In addition, the infarct size was smaller, and more p-histone H3 and EdU were found in cardiomyocytes. In contrast, TLR3-/- neonatal hearts showed impaired cardiac function, larger infarct size and less phospho-histone H3 and EdU incorporation. This data indicates that TLR3 is required for neonatal mouse heart regeneration after MI. Next, we examined whether a TLR3 ligand (poly I:C) will induce cardiomyocyte proliferation after myocardial ischemia/reperfusion injury. WT or TLR3-/- mice (n=6/group), treated with or without Poly I:C, were subjected to ischemia (45 min) followed by reperfusion up to 14 days. Poly I:C administration significantly attenuated I/R-induced cardiac dysfunction and increased cardiomyocyte proliferation in WT mice. In contrast, poly I:C was not effective in TLR3-/- mice. We also examined the effect of poly I:C on metabolic reprogramming in cardiomyocytes. Cardiac myocytes were isolated from adult hearts and treated with or without Poly I:C (n=6). We observed that poly I:C treatment increased lactate and NAD+/NADH. Akt and GSK-3β phosphorylation were also increased, when compared with control. The data indicate that treatment of adult mice with a TLR3 ligand stimulates cardiomyocyte proliferation and induces metabolic reprogramming to glycolysis. We conclude that TLR3 plays a major role in promoting cardiac repair after ischemic injury and this correlates with the Warburg effect in cardiomyocytes.
METABOLIC REPROGRAMMING OF MACROPHAGES WITH TOLL-LIKE RECEPTOR LIGANDS
B. Fensterheim*, I. Trenary*, J. Young, E. Sherwood*. Vanderbilt University, Nashville, TN
Background: Toll-like receptor (TLR) ligands can endow short-term, non-specific protection against infection when administered prophylactically, a process known as trained immunity. However, the mechanism of protection remains undefined. Protective TLR ligands can either prime or inhibit the cytokine response to infection, and thus cytokine responses during infection do not correlate with protection. Alternatively, both macrophages and phosphoinositide 3-kinase (PI3K) signaling have been independently shown to be essential for protection. PI3K signaling plays a critical role in immune cell metabolic reprogramming, which consists of Warburg-like aerobic glycolysis. Here, we probe how TLR ligand priming alters the metabolic response of macrophages to lipopolysaccharide (LPS) challenge.
Methods: We treated wild type (WT), MyD88 knockout (KO), and TRIF KO murine bone marrow-derived macrophages with LPS, monophosphoryl lipid A (MPLA), CpG DNA, or polyinosinic:polycytidylic acid (poly(I:C)), and, after a washout period, measured their response to LPS challenge. Specific endpoints included lactate production, IL-6 production, and protein kinase B (Akt) and IκB kinase α/β (IKKα/β) phosphorylation.
Results: We found that all MyD88-associated TLR ligands, including LPS, MPLA, and CpG DNA, primed macrophages to have an augmented lactate response to LPS challenge, whereas TRIF-associated poly(I:C) did not. Mechanistically, MyD88 KO macrophages did not produce lactate in response to TLR priming or LPS challenge, whereas lactate production in TRIF KO macrophages was identical to that observed in WT. PI3K-related Akt phosphorylation was promoted by all MyD88-associated TLR ligands. Cytokine responses to LPS challenge were variable: priming with CpG DNA augmented LPS-induced IL-6 production whereas priming with LPS, MPLA or poly(I:C) was attenuating. IKKα/β phosphorylation paralleled cytokine responses.
Conclusions: MyD88-associated TLR ligands appear to universally prime the macrophage metabolic response to LPS challenge, while having variable effects on cytokine production. Furthermore, macrophages from MyD88 KO mice do not develop metabolic reprogramming in response to TLR ligands. We hypothesize that MyD88 and PI3K -dependent metabolic priming gives macrophages a critical ‘head-start’ in coordinating the host response to infection. Therefore, metabolic reprogramming, rather than altered cytokine responses, may be a primary mechanism of trained immunity.
ALUM AS A PREVENTATIVE THERAPY ABOLISHES MORTALITY IN MURINE NEONATAL SEPSIS BY IMPROVING EFFECTOR CELL FUNCTION AND BACTERIAL CLEARANCE
A.L. Cuenca, A. Cuenca, D.C. Nacionales, P.A. Efron*, R. Ungaro, L. Moldawer*, 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 (Wynn et al. Blood 2008). 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. We have previously shown that alum works independent of caspase-1 and we hypothesize that its action on effector cells improves survival (Cuenca et al. Shock Suppl 2014).
Methods: Wild-type C57BL/6 (WT) neonates (4-7 days old) mice underwent a cecal slurry (CS) model of intra-abdominal sepsis. Neonates received either, no pretreatment (control), or 20 mg alum (IP) 24 hrs prior to CS administration. Peritoneal washes were harvested at 2, 6, 18, 24, and 36 hours post sepsis and analyzed by flow cytometry and Luminex™ to determine absolute numbers for cell recruitment and cytokine concentrations. Phagocytic function, ROS production of macrophages and neutrophils, and bacterial clearance in peritoneal wash was analyzed at 18, 24, and 36 hours.
Results: Mice pretreated with alum had significant expansion of hematopoietic stem cells 36 hours after sepsis in both spleen and bone marrow (p<0.0001). When examining the number of cells in the peritoneum, there was a significant increase in the absolute number of monocytes, macrophages, dendritic, and natural killer cells 24 hours after sepsis in the treated animals compared to the untreated (p<0.05). The treated animals also had improved neutrophil phagocytosis at 18 and 24 hours after sepsis (p=0.002). At 24 and 36 hours after sepsis, alum treated animals had significantly greater bacterial clearance in the peritoneum compared to untreated animals ( p< 0.0001; p=0.002). Mice pretreated with alum also had 100% survival compared to groups receiving CS alone and s.c. administration of alum also improved survival (p<0.001).
Conclusions: Alum stimulates hematopoiesis which is associated with an increase in the numbers of macrophages, monocytes, dendritic cells and NK cells recruited to the peritoneum. Alum also improves the phagocytic function of neutrophils. When phagocytic function is combined with the increased numbers of effector cells, alum pretreatment improves bacterial clearance and results in 100% survival for neonates with sepsis.
MOLECULAR MECHANISMS OF MONOPHOSPHORYL LIPID A INDUCED NEUTROPHIL MOBILIZATION AND RECRUITMENT
A. Hernandez*, L. Luan*, J. Bohannon*, B. Fensterheim*, E. Sherwood*. Vanderbilt University Medical Center, Nashville, TN
Background: The Toll-Like Receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) is used as a vaccine adjuvant. Prior reports attribute MPLA immune adjuvant effects to TRIF-dependent signaling.
Our studies show MPLA primes innate immune responses against bacterial infections, dependent upon augmented neutrophil recruitment. Mechanisms by which MPLA enhances neutrophil mobilization and recruitment are unknown. Our aim was to identify the role of TLR4-, TRIF-, and MyD88-dependent signaling in facilitating neutrophil mobilization and recruitment upon MPLA administration.
Methods: MyD88- and TRIF-dependent signaling in bone marrow-derived macrophages in vitro and intraperitoneal (IP) leukocytes in vivo was determined by IκB kinase (IκK) and IRF-3 phosphorylation, respectively. Neutrophil numbers in blood and peritoneum were measured after IP MPLA challenge in TLR4, TRIF, and MyD88 knockout (KO) mice and wild type (WT) controls. G-CSF (expansion), KC and MIP-2 (recruitment) were measured in plasma and peritoneum at the same time points.
Results: MPLA induced phosphorylation of IκK and IRF-3 in bone marrow-derived macrophages and IP leukocytes, respectively. MPLA increased neutrophils in blood and peritoneum by 3 hours (figure), an effect absent in TLR4 KO mice. In MyD88 KO mice, blood neutrophils increased post-MPLA to levels similar to WT mice, whereas MPLA-induced IP neutrophil recruitment was ablated. TRIF KO mice showed delayed mobilization of neutrophils into blood, but peak blood neutrophil numbers in TRIF KO mice were similar to WT mice. IP neutrophil recruitment in TRIF KO mice was similar to WT. MPLA induced a significant increase of plasma G-CSF at 6 hours. MPLA increased plasma and IP MIP-2 and KC. Production of G-CSF, MIP-2 and KC were nearly ablated in MyD88 KO mice and were attenuated in TRIF KO mice after MPLA challenge.
Conclusions: MPLA activates MyD88- and TRIF-dependent signaling and induces neutrophil mobilization and recruitment. MPLA-induced neutrophil mobilization from bone marrow into blood is partially mediated by TRIF-dependent signaling and is independent of MyD88 whereas neutrophil recruitment to the site of MPLA administration is mediated by MyD88 signaling. MPLA induces G-CSF, MIP-2 and KC, which are regulated by MyD88 signaling. These findings advance knowledge of the molecular mechanisms that regulate the antimicrobial effects of MPLA.
IMAGING CELL DEATH IN VIVO DURING POLYMICROBIAL SEPSIS
L. Zou1, H.H. Chen1, D. Li1, Y. Feng1, G. Xu1, C. Chen1, L. Wang2, D.E. Sosnovik1, W. Chao*1. 1Massachusetts General Hospital, Charlestown, MA, 2Children’s Hospital Los Angeles, Los Angeles, CA
Introduction: Apoptotic cell death occurs during sepsis and may contribute to immune suppression in some subgroups of septic patients. Therefore, finding a way to detect lymphoid cell death early and noninvasively may have significant clinical implications. Annexin-V, a naturally occurring protein in human, binds with high affinity and specificity to phosphatidylserine (PS). During apoptosis, PS is translocated from the inner to outer layer of the plasma membrane. The current study is designed to evaluate the feasibility and sensitivity of detecting cell death using fluorescence-labeled Annexin V (AV-750) in a mouse model of bacterial sepsis.
Methods: Mild and severe polymicrobial sepsis was created with cecum ligation and puncture (CLP). AV-750 was administered intravenously and cell death was determined by: 1) In vivo: fluorescence reflectance imaging using an IVIS Spectrum imaging system; 2) Ex vivo imaging in isolated organs; 3) In vitro: splenocyte and thymocyte death measured with Annexin V and propidium iodide staining and flow cytometry (FACS). Caspase-3 cleavage and activity, and TUNEL staining were also used to detect apoptosis.
Results: CLP-induced polymicrobial sepsis led to apoptosis in lymphoid cell organs such as thymus, spleen and liver as demonstrated by enhanced caspase-3 cleavage and caspase-3 activity as well as marked increase in TUNEL-positive cells. Compared with severe sepsis, mice with mild sepsis had lower serum cytokines and mortality. AV-750 imaging performed at 24h after surgery revealed that fluorescent signal significantly increased in upper abdominal field in severe septic mice and the signal gradually reduced in mild septic and sham mice (Fig. A). In thoracic region, the signal significantly increased in the septic mice compared to the sham. However, there was no difference between mild and severe septic mice (Fig. A). These in vivo imaging results were validated by ex vivo imaging of spleen and thymus (Fig. B) as well as cell death detection by FACS in splenocytes and thymocytes. Importantly, there was a significant positive correlation between in vivo, ex vivo Annexin V signal and FACS in detecting cell death.
Conclusion: Noninvasive imaging of organs such as thymus, liver and spleen cell death in vivo using Annexin V during sepsis are feasible and sensitive.
MYELOID CELLS STEER MESENCHYMAL STEM CELLS INTO THE WOUND BED: A MECHANISM FOR ELDERLY’S DEFICIENT HEALING
S. Amini-Nik*1, 2, A. Abdullahi2, N. Yu2, C. Belo2, M.G. Jeschke*1, 2. 1Department of Surgery, University of Toronto, Toronto, ON, Canada, 2Sunnybrook Research Institute, Toronto, ON, Canada
Introduction: Due 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 have already shown that lack of Mesenchymal Stem Cells (MSCs) in the wound bed of old mice led to a delayed healing, partly due to deficient recruitment of these cells to the wound bed. This was accompanied with the lack of myeloid lineage cells in wound bed of aged mice. To unravel the mechanism of this deficient recruitment, we continued this line of research and hypothesized further that altered migration of myeloid lineage cells is leading to the lack of MSCs in the wound bed.
Methods: We compared skin healing characteristics of old patients (as well as aged mice) with the young ones. Young (8 weeks old) and aged mice ( >52 weeks) were subjected to 30% full thickness burn and their skin healing was monitored up to two weeks post burning.
Results: We further report here that the secretome of myeloid cells induces MSCs migration. Conditioned media of bone marrow derived macrophages (BM-Mac) of young animal rescued deficient MSCs migration in aged mice whereas media from aged animal did not affect MSCs migration. Array analysis revealed essential genes were altered in macrophages of aged mice, providing some candidates that might be used to enhance the healing of aged animal. Using flow cytometry analysis, we showed that BM-Mac of aged animal have significantly lower F4/80+ macrophages, suggesting altered differentiation capacity of myeloid lineage cells in aged animal. Interestingly, the lack of MSCs observed in aged animal was predominantly seen in CD90+ subpopulation of MSC. Moreover, MSCs of aged animal are lacking some genes which are crucial for cell migration.
Conclusions: In summary, that lack of MSCs in the wound bed of old mice is associated with the lack of myeloid lineage cells. Secretome of myeloid lineage cells of young animal contains proteins that can rescue deficient migration of MSCs, in particular CD90+ subpopulation of MSCs.
Applicability of Research to Practice: Administrating the candidate proteins that could rescue deficient migration of mesenchymal stem cells might be beneficial to the skin healing of elderly burn patients.
SPHINGOSINE-1-PHOSPATE/S1P RECEPTORS MODULATE HEMATOPOIETIC PROGENITOR CELL MOBILIZATION FOLLOWING TRAUMA/HEMORRHAGIC SHOCK
I. Alamo, K.B. Kannan, H. Ramos, P.A. Efron*, A.M. Mohr*. University of Florida, Gainesville, FL
Background: During homeostasis most hematopoietic progenitor cells (HPC) reside in the bone marrow (BM), but a small number traffic to the blood and tissues and some home back to the BM. Following traumatic injury and hemorrhagic shock there is increased mobilization of HPCs to the peripheral blood and sites of injury. The continued recruitment of HPCs following injury and stress is associated with persistent elevation of granulocyte stimulating factor, dysfunctional wound healing, and the persistent elevation of stromal derived factor-1 at the site of injury. Sphingosine-1-phosphate (S1P), a bioactive lipid, and its receptors (S1Pr1, S1Pr2, and S1Pr3) are new players in HPC trafficking. The aim of this study is to investigate the role S1P and its receptors in a unique lung injury (LC)/ hemorrhagic shock (HS)/chronic restraint stress (CS) model.
Methods: Male Sprague-Dawley rats (N=4-6/group) were randomly assigned into one of the four models: naïve, CS alone, combined LCHS, or LCHS/CS. Following LC, rats were subjected to 45 minutes of HS (MAP of 30-35). During the two hour CS period, restraint cylinders were rotated and exposed to 80-85 decibel alarms for two minutes every 30 minutes to prevent habituation. On day seven, blood and BM were harvested. S1P levels in the BM were analyzed by ELISA. BM S1Pr1, S1Pr2, and S1Pr3 were assessed by PCR. Data presented as mean±SD in each group. *p<0.05 vs. naïve and **p<0.05 vs. LCHS by t-test.
Results: There was significant S1P elevation in the BM that increased with each model of more severe injury and correlates with previously described findings of increased HPC mobilization to peripheral blood (Table). In the LCHS/CS model that has persistent HPC mobilization, there is a statistically significant increase in expression of BM S1Pr1, S1Pr2, and S1Pr3 as compared to naïve (Table). However, only the expression of S1Pr2 and S1Pr3 in the BM are significantly increased in LCHS/CS as compared to LCHS alone (Table).
Conclusion: In models of varying degrees of HPC mobilization, there is an increase in S1P levels in the BM. S1Pr1-3 all play a role in the mobilization of HPCs after LCHS/CS. S1Pr2 and S1Pr3 following LCHS/CS may play a key role in persistent HPC mobilization and the failure of HPCs to home to the BM. Alleviating the prolonged mobilization of HPCs from the BM after severe injury is a potential therapeutic target to improve BM function.
SPRAY-DRIED PLASMA AND FRESH FROZEN PLASMA ARE FUNCTIONALLY EQUIVALENT WITHIN AN IN VIVO MOUSE MODEL OF HEMORRHAGIC SHOCK
D. Potter1, G. Baimukanova1, S. Keating1, X. Deng1, S. Gibb1, M. Muench1, M. Fomin1, P. Spinella2, R. Kozar3, S. Pati1. 1Blood Systems Research Institute, San Francisco, CA, 2Washington School of Medicine in St Louis, St. Louis, MO, 3University of Maryland, Baltimore, MD
Background: In retrospective and prospective observational studies, fresh frozen plasma (FFP) has been associated with a survival benefit in massively transfused trauma patients. A dry plasma product, such as spray-dried plasma (SDP), offers logistical advantages to FFP. Recent studies on FFP have demonstrated that FFP modulates systemic vascular stability and inflammation injury. The effect of SDP on these measures has not been thoroughly examined. This study compares SDP to FFP using in vitro assays of endothelial function and in vivo assays of lung injury using a mouse model of hemorrhagic shock (HS) and trauma.
Methods: FFP, SDP and lactated ringers (LR) were compared in vitro using assays of endothelial cell (EC) permeability, cytokine production and content, gene expression and tight- and adherens-junction stability. The resuscitation products were also compared in a murine model of HS. Mean arterial pressures (MAPs) and physiological measures were assessed. Pulmonary vascular permeability was measured using tagged dextran. Lung tissues were stained for CD68, VE-cadherin and Occludin.
Results: Treatment of ECs with FFP and SDP, but not LR, preserved the integrity of EC layers in vitro and resulted in similar EC gene expression patterns and cytokine/growth factor production. FFP and SDP also reduced HS induced pulmonary vascular permeability in vivo to the same extent. In mice with HS, MAPs and base excess were corrected by both FFP and SDP to levels observed in sham treated mice. Treatment after HS with FFP and SDP but not LR reduce alveolar wall thickening, leukocyte infiltration, and the breakdown of EC junctions, as measured by staining for VE-Cadherin and Occludin.
Conclusions: Both FFP and SDP similarly modulate pulmonary vascular integrity, permeability and inflammation in vitro and in vivo in a murine model of HS and trauma.
EARLY TRAP PATHWAY PLATELET INHIBITION PREDICTS COAGULOPATHIC HEMORRHAGE IN TRAUMA
M.P. Chapman1,2, E.E. Moore*1,3, H.B. Moore1, E. Gonzalez1, A.P. Morton1, C.C. Silliman1,4, A. Sauaia1, A. Banerjee1. 1University of Colorado, Denver, CO, 2Georgia Regents University, Augusta, GA, 3Denver Health Medical Center, Denver, CO, 4Children’s Hospital of Colorado, Aurora, CO
Introduction: Platelet dysfunction correlates to severity of injury in trauma and has been shown to be a bellwether of mortality. A causal linkage between specific modes of platelet dysfunction and trauma-induced coagulopathy, however, has yet to be established. We therefore sought to determine if platelet dysfunction immediately following injury correlated to coagulopathic hemorrhage.
Methods: Blood was collected from a convenience sample of 101 trauma activation patients at the scene of injury or upon hospital arrival. Impedance aggregometry was performed using the ROTEM Platelet system using ADP or thrombin receptor activating peptide 6 (TRAP-6) as agonists. Two outcomes were assessed: (1) massive hemorrhage, defined as transfusion of ≥10 units of PRBCs or death from hemorrhage within 6 hours of injury and (2) clinical coagulopathy, defined as a 2 or 3 as scored by the attending trauma surgeon, wherein 0 = None, 1= Mild (hemostasis delayed, no intervention required), 2 = Moderate (surface bleeding continues and requires intervention), 3 = Severe (bleeding continues despite suturing and hemostatic agents and is evident in vascular suture lines). Predictive value of aggregometry parameters for these outcomes was determined using the area under the receiver operating characteristic curve (AUROC).
Results: The AUROC for prediction of massive hemorrhage was best for the area under the aggregation curve (AUC) of the citrated TRAP and ADP activated samples at 0.97 (95% CI 0.9057 to 1.026, p=0.0008) and 0.95 (95% CI 0.8786 to 1.021, p=0.003), with sensitivities and specificities of 80% and 98% for TRAP and 100% and 90% for ADP, at thresholds of < 53 and <43 Ω·min, respectively. The AUROC for prediction of clinical coagulopathy using the TRAP AUC was 0.88 (95% CI 0.7341 to 1.016, p=0.004) with sensitivity and specificity of 67% and 90% at a threshold of <65 Ω·min, but was not significant for ADP. These findings were independent of acidosis and standard coagulation metrics.
Conclusion: Inhibition of either the TRAP and ADP platelet activation pathways is predictive of massive hemorrhage following trauma. Inhibition of the TRAP pathway correlates with observable clinical coagulopathy. ADP pathway inhibition, however, lacks specificity for coagulopathy, but may correlate to physiologic decompensation associated with impending hemorrhagic shock. Conversely, TRAP pathway inhibition is specific for, and may bear a causal linkage to, clinically relevant coagulopathy in trauma.
LONG-TERM FOLLOW-UP OF PULMONARY FUNCTION IN PEDIATRIC BURNED PATIENTS
L.E. Sousse*1,2, R.P. Mlcak2, C.R. Andersen1,2, C.C. Finnerty*1,2, O.E. Suman1,2, D. Herndon*1,2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals for Children, Galveston, TX
Introduction: Inhalation injury, which is among the causes of acute lung injury and acute respiratory distress syndrome, continues to represent a significant source of mortality in burned patients. From 1986-2014, inhalation injury was diagnosed by bronchoscopy in pediatric burned patients (n=932), and pulmonary function tests were collected (n=221, 62 ± 20% total body surface area burn [TBSA], 10 ± 5 years of age) up to 7.5 years post burn.
Methods: Mixed multiple regression models related each PFT outcome to age at date of measure, TBSA, ventilation days, and days post burn. Mixed multiple regression models were also used to relate PFT outcomes from unburned subjects (n=57, 12 ± 3 years of age) to age at date of measure.
Results: Time post burn was associated with significant increases in forced vital capacity (FVC), forced expiratory volume (FEV), forced expiratory flow (FEF), maximum voluntary ventilation (MVV), total lung capacity (TLC) and diffusion capacity (DLCO) (p<0.03). The values from burned patients converged over time with the values from unburned patients, approaching values from unburned patients approximately 5 years post burn. Greater TBSA was associated with significant decreases in FVC, FEV, TLC and FEV/FVC (p<0.05). More ventilation days were associated with significant decreases in FVC, FEV, FEF, MVV and DLCO (p<0.02). Increased age at date of PFT measurements was associated with significant increases in MVV, functional residual capacity (FRC), TLC and residual volume (RV) (p<0.001).
Conclusions: Lung injury is sustained up to five years post burn in our pediatric burned patient population, which is indicated by a convergence of values between burned patients with inhalation injury and unburned subjects over time. Therapeutic targets must be identified that would enable interruption of the pathogenic sequence leading to lung injury.
CHARACTERIZATION OF A NOVEL PSYCHOLOGICAL SYMPTOM SCALE TO ASSESS INDIRECT BRAIN INJURY IN BURN PATIENTS
J. Gatson*, A. Burris*, H. Phelan*, D.L. Carlson*, J. Minei*, S.E. Wolf*. UT Southwestern Medical Center, Dallas, TX
Introduction: In previous studies of indirect brain injury (iBI) in both animals and humans, an increase in neuro-inflammation, edema, and neuronal cell death has been reported. Recently, our research group has published data demonstrating that a brain biomarker (neurofilament-H [NFL-H]) is significantly elevated in the blood after burn and correlates with the percent total body surface area (TBSA) burns. The use of neuropsychological assessments is warranted to identify which individuals are suffering from chronic iBI.
Methods: In this pilot study, we wanted to characterize a novel psychological symptom scale. Also we wanted to elucidate if NFL-H correlates with the psychological scores. To assess the psychological states of the patients (n=29), we devised the brain neuropsychological symptom scale (BNSS) to capture symptoms of confusion, anxiety, agitation, and psychosis during their hospital stay. Each symptom was scored a 1. Psychological disorders receive a score of 2 and were also captured (PTSD, depression, delirium). As documented by the attending psychologist, information pertaining to the various symptoms and disorders were collected from the patient’s chart. We conducted correlative analysis to study relationships between BNSS scores and blood levels of NFL-H in individual patients.
Results: In this study, with respect to BNSS scores collected from the 29 patients, a range of sores between 0 and 6 were collected. Interestingly, we found a positive correlation between BNSS scores and NFL-H levels on day 14 after burn (p < 0.01; Figure 1).
Conclusion: In this novel study we found that NFL-H predicts psychological symptoms/disorders and the BNSS may be useful in elucidating which patients may suffer from chronic iBI.
HYPOXIA INDUCES PROLONGED PLATELET ACTIVATION FOLLOWING TRAUMATIC BRAIN INJURY
M. Goodman*, E.F. Midura, A.T. Makley*, R.A. Veile, L.W. Friend, C.C. Caldwell*. University of Cincinnati, Cincinnati, OH
Background: Hypoxia following traumatic brain injury (TBI) can exacerbate secondary brain injury, contributing to increased morbidity and poor clinical outcomes. The physiologic mechanisms by which hypoxia may adversely affect the injured brain include inflammation, endothelial dysfunction, and microvascular thrombosis. Platelets and platelet-derived microparticles (PMPs) may also exacerbate secondary brain injury, but the effects of hypoxia on these inflammatory mediators following TBI are unknown. We hypothesized that hypoxia may augment post-traumatic secondary brain injury by inducing platelet activation.
Methods: C57Bl/6 male mice were anesthetized and moderate traumatic brain injury was induced by a 400g weight drop from 1 cm height. Sham mice underwent anesthesia without head injury. Ten minutes after injury, mice were exposed to hypoxia (FiO2=15%) or normoxia (FiO2=21%) for 30 minutes. Blood was collected at 24 and 72 hours after injury (n=5 per group). Soluble P-selectin was measured by ELISA as a marker of platelet activation. Microparticles and CD41+ PMPs were characterized by Nanoparticle Tracking Analysis.
Results: Following TBI, soluble P-selectin was significantly increased at 24 hours compared to sham (226.1±35.1 ng/mL vs. 135.1±4.1 ng/mL, TBI vs. Sham, p<0.05) with normalization of levels by 72 hours. Hypoxia alone without head injury did not increase soluble P-selectin. In animals exposed to hypoxia following TBI, soluble P-selectin levels remained elevated at 72hrs after injury (414.2±67.9 ng/mL vs. 150.1±18.6 ng/mL, TBI+hypoxia vs. TBI+normoxia, p<0.01). Mice subjected to TBI and hypoxia demonstrated similar total microparticle counts at 24 hrs, but significantly lower total microparticle counts at 72 hrs (1.97±0.26x10^8 microparticles/mL vs. 5.29±0.37x10^8 microparticles/mL, TBI+hypoxia vs. TBI+normoxia, p<0.001). By contrast, the increased number of PMPs observed at 72 hrs after TBI was sustained, even in hypoxic head-injured mice. At 72 hrs after injury, PMPs comprised 86% of all microparticles in the TBI+hypoxia group compared to 45% of all microparticles in the TBI+normoxia group.
Conclusions: Post-traumatic hypoxia leads to prolonged platelet activation and PMP generation following head injury. Sustained platelet activation and microparticle production may exacerbate secondary brain injury by contributing to microvascular thrombosis and tissue compromise in the traumatically injured brain.
SYNDECAN-1 IS CRITICALLY IMPORTANT TO VASCULAR BARRIER INTEGRITY IN THERMAL INJURY
C. Nelson1, 2, A. Smith2, J. Lee2, E. Lopez*2, D. Herndon*1, D. Prough*2, P. Enkhbaatar*1, 2. 1Shriner’s Hospital for Children, Galveston, TX, 2University of Texas Medical Branch Galveston, TX
Background: Vascular hyperpermeability is a detrimental complication of various pathological conditions, such as a thermal injury. Endothelial glycocalyx plays a critical role in vascular wall integrity.
Study objectives were to elucidate the role of syndecan-1 (Sdc1), a major constituent of endothelial glycocalyx in vascular hyper-permeability after burn and smoke inhalation injury. We hypothesized that Sdc1 KO mice display more severe vascular leakage following injury.
Method: We tested our hypothesis using two cohorts of mice, a BALB/c ‘wild-type’ (WT) and a Sdc1 ‘knock-out’ (KO) mice subjected to cotton smoke inhalation and a 40% 3rd degree cutaneous flame burn. Mice were euthanized at different time points (0, 3, 6, 9, 12, 18, and 24h) after injury, and vascular permeability was determined in different organs i.e., lung, kidney, and brain, by Evans Blue (EB) dye injection. EB was administered just before euthanasia, and allowed to circulate prior to study end point. Preceding organ tissue extraction, blood with circulating dye in vasculature was flushed out with cold saline perfusion. Tissues were removed, and EB concentration was determined in their homogenates by spectrophotometer with wavelengths 620nm and 740nm. Quantity of EB dye represented concentration of albumin leaked through endothelium.
Results: No mortality observed within 18 h study period in both WT and KO mice. Following combined injury, the pulmonary vascular permeability was significantly increased in Sdc1 KO mice vs. sham mice at 18h; there were no significant changes observed in WT mice. EB concentration was significantly higher in the lung of KO vs. WT mice at 18h. Kidneys showed hyper-permeability in the KO group as well, with significant increase compared to both sham and WT at 6h. In addition, EB was significantly higher in brain tissue homogenate in KO compared to WT mice at 6h and 9h (Table).
Conclusion: Results indicate that endothelial glycocalyx, specifically syndecan-1, plays a critical role in vascular hyper-permeability of vital organs following cutaneous burn and smoke inhalation injury. Future studies should focus on therapeutic approaches to restore vascular endothelial barrier function during burn trauma.
MUSCLE MICRORNA PROFILE ALTERATION FOLLOWING SEVERE BURN
J. Song*1, M.R. Saeman1, L.A. Baer2, K. Despain1, C. Wade*2, S.E. Wolf*1. 1University of Texas Southwestern Medical Center, Dallas, TX, 2University of Texas Health Science Center-Houston, Houston, TX
Introduction: Injury causes systemic epigenetic changes probably associated with clinical outcomes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. Muscle is significantly involved in the metabolic response after severe burn; however, the miRNA profile in this state is unknown. The aim of the study is to outline the microRNA profile in response to severe burn in an animal model.
Methods: Twelve male Sprague-Dawley rats were randomly assigned to sham and burn groups. Rats received 40% total body surface area (TBSA) scald burn or sham. On day 14, hindlimb skeletal muscles were harvested. Approximately 20mg of plantaris tissue samples from three animals were pooled in each treatment group for total RNA extraction. The microRNA profile of each biological sample was measured using Affymetrix miRNA 4.0 Arrays chips at our institutional microarray core facility. Each chip contained a total of 36, 353 small non-coding RNA probes and the chip reproducibility is greater than 0.95. The raw data signal intensity was normalized with Robust Multi-array Average (RMA). Data was analyzed with Transcriptome Analysis Console (TAC 2.0) software.
Results: A total of 36,222 miRNAs were detected in each grouped sample, of these 1,218 rattus norvegicus miRNAs probes were identified. The highest probe signal intensity (binary log ratio) was 16 for miRNA-206-3p in both groups. There were 74.5% transcripts in the sham group and 73.9% in the burn group, with a signal intensity less than 2. We identified 703 (57.7%) up-regulated miRNAs and 515 (42.3%) down-regulated miRNAs in the burn group compared to sham. Among the up-regulated profiles, 8 miRNAs were increased over 5 fold. All of the down-regulated miRNAs were within a -3 fold change (Table 1). MiR-182 was the most up-regulated miRNA following burn. It increased 12.81 linear fold (log ratio 6.14 in BA, 2.46 in SA). It inhibits Col1a2 gene (for type I collagen) and promotes NEU2 gene (for Sialidase-2). MiR-409a-3p was the most down-regulated miRNA in response to burn (-2.95 fold change). Interestingly, it functions with miRNA-182 to inhibit muscle Col1a2 gene expression after burn.
Conclusion: In summary, we describe the miRNA profile in muscle 14 days after burn. Up-regulation predominated in response to burn. The interaction network between epigenetics and genomic profile confirmed with biological response is under investigation.
TIMING OF SURGICAL INTERVENTIONS FOLLOWING BLUNT TRAUMA IS ASSOCIATED WITH DIFFERENTIAL DYNAMIC INFLAMMATORY NETWORKS AND ADVERSE CLINICAL OUTCOMES
O.M. Abdul-Malak*2, R.A. Namas*2, A.M. Zaaqoq*1, K.W. Almahmoud*2, T. Billiar*2, Y. Vodovotz*2. 1Texas Tech University, Amarillo, TX, 2University of Pittsburgh, Pittsburgh, PA
Background: Surgical interventions are often a necessary component of post-traumatic care. However, while at times lifesaving interventions may pose a secondary insult to the immune system post-injury. We hypothesized that the timing of interventions post-trauma could be associated with a distinct and altered inflammatory response and differential clinical outcomes. Accordingly, we carried out an extensive time course analysis of circulating inflammatory mediators based on the timing of initial surgical interventions (Early [≤24h], Delayed [1-7 days], and No major interventions [NMI]) in a subset of blunt trauma patients.
Methods: Surgical intervention data were retrospectively collected for a cohort of 472 blunt trauma survivors. The cohort was subsequently stratified into 194 patients with Early surgical interventions [age 44.6±1.4, M/F 135/59, ISS 22.4±0.9, Glasgow coma scale (GCS) 13.78±0.25], 61 patients with Delayed surgical interventions [age 46.7±2.3, M/F 50/11, ISS 22.3±1.3, GCS 14.1±0.4], and, after matching for age and ISS, 145 patients with NMI [age 46.7±2.3, M/F 99/46, ISS 19.32±0.6, GCS 13.5±0.16]. Plasma was sampled 3 times within the first 24 h and then daily up to day 7 post-injury and assayed for multiple inflammation mediators using Luminex™. Two-Way ANOVA and area under the curve (AUC) analysis were used to determine statistical significance (p<0.05) coupled with Dynamic Bayesian Network (DBN) analysis to infer network connectivity.
Results: ICU length of stay (LOS), total LOS, and days on mechanical ventilation were statistically significantly prolonged in the Early and Delayed groups vs. the NMI group. Moreover, the Early and Delayed groups had a higher degree of organ dysfunction from days 1 through 7 post-injury vs. the NMI group suggested by Marshall Multiple Organ Dysfunction Score. Within the first 24 h and up to day 7, levels of IL-5, IL-10, IL-7, IL-8, MCP-1, sIL-2Rα, IL-6, IL-1RA, MIP-1β, and MIG were statistically elevated with a >1.5 fold difference in the Early intervention group vs. the NMI group. The Delayed intervention group exhibited higher levels of IL-7, IL-17A, IL-15, MIP-1α, IL-1β, IL-4, sIL-2Rα, IL-1RA and IFN-γ with a >1.5 fold change from days 1 through 7 post-injury vs. the NMI group. DBN inference in the first 24 h post-injury suggested a core chemokine-based network involving MIG and MCP-1 upstream of IP-10 and IL-6 in the Early group. In contrast to the Early group, MIG was the core chemokine upstream of IP-10 and MCP-1 in the Delayed group. In the NMI group, MIG was the core chemokine upstream of IP-10, IFN-α, MCP-1, and IL-1RA.
Conclusion: Our results suggest that the timing of surgical interventions can elicit a temporally distinct, dynamic inflammatory response post-trauma, which is associated with differential clinical outcomes.
NLPR12 HELPS TO LIMIT PROGENITOR CELL DEATH FOLLOWING RADIATION-THERMAL COMBINED INJURY
B. Linz, J. Dunn, L. Kartchner, J. Brickey, J. Ting, B. Cairns*, R. Maile. University of North Carolina at Chapel Hill, Chapel Hill, NC
The hematopoietic system can rapidly expand in response to tissue damage. NLRP12, expressed in leukocytes and bone marrow cells, has been implicated in regulation of immune responses and limiting inflammation. We tested the hypothesis that NLRP12 regulates immune reconstitution as a suppressor of TNFα-induction of apoptosis. To test this, C57BL/6 wild type and Nlrp12-/- female mice underwent a radiation-thermal combined injury (RCI) consisting of 20% total body surface area burn and 5Gy γ-radiation. In wild type mice, we observed elevated NLRP12 expression in target tissues after RCI compared to controls. Following RCI, Nlrp12-/- mice failed to reconstitute peripheral innate neutrophil and monocyte populations, had increased levels of serum TNFα, and TNFα-mediated apoptosis of bone marrow progenitor cells. These data suggest that NLRP12 is essential for survival after combined radiation and burn injuries though suppression of progenitor apoptosis. We have uncovered a central role for NLRP12 in regulating myelopoiesis following immune ablation. Accordingly, we propose that NLRP12 acts as an important regulator of cell death during events that trigger myelopoiesis, suggesting a novel role of NLRP12 in immune reconstitution following injury.
ROLE OF MAST CELLS AND THEIR INFLAMMATORY MEDIATORS IN BURNS AND WOUND HEALING
C. Kuo1,2, A. Collins1, C.C. Caldwell*3, S.J. Ono3,2. 1Cincinnati Children’s Hospital, Cincinnati, OH, 2Shriners Hospitals for Children - Cincinnati, Cincinnati, OH, 3University of Cincinnati, Cincinnati, OH
The inflammatory process is a critical component of both wound healing and management of burn injuries. Mast cells are important mediators of allergic inflammation, but their contributions to inflammation in healing responses are poorly understood. To further explore the role of mast cells in the burn wound healing progress, we identify specific mast cell mediators that are differentially expressed during different phases after burn injuries. Scald burns are induced in CF-1 outbred mice (26 to 30g), after the mice are anesthetized and 18% of their exposed backs are immersed in 90°C water for 9 seconds. Mice were sacrificed from 1.5 hours to 6 days following the scald burn. Inflammatory phase reactions including histamine release, gene expressions and pathological changes in the skin tissue were evaluated. The results show that mast cells recruited in scald tissues from mere hours to several days following injury. Significant induction of systemic histamine release was found in 6 hours following the burn, while mast cell proteases, chemokines and cytokines were induced in scald tissues, compared with control mice, at different time points following burn injury. Mast cell proteases could induce inflammation and the histamine induction might suggest their role in epithelial permeability and adhesion molecules in scald burns. Depending upon the study, mast cells could play roles in angiogenesis, recruitment of immune cells to the wounds and cell proliferation at different phases of burn wound healing. A better understanding of identified mast cell- and tissue mediators in mediating inflammatory reactions will provide insights into mast cell function and novel treatments for burn and wound healing.
BLUNT TRAUMA RELEASES CELL FREE NUCLEIC ACIDS, BUT ITS PROGNOSTIC RELEVANCE DOES NOT SURFACE UNTIL THE FOLLOWING DAY.
D.C. Holden, A. Lewin, J. Thomas, R. Ungaro, B.E. Szpila, S. Brakenridge*, F. Moore*, P.A. Efron*, L. Moldawer*. University of Florida, Gainesville, FL
Objectives: Injuries continue to be the fifth leading cause of death overall and the leading cause of death for persons less than 45 years of age in the U.S. The purpose of this study was to determine if immediate and early measures of circulating nucleic acids could be useful in predicting early leukocyte genomic changes and clinical outcomes in trauma patients.
Methods: The Genomics Based Prognostics Study will ultimately enroll 200 severely injured blunt trauma patients and 40 healthy volunteers recruited from UF Health-Shands and UW Harborview Medical Center. Analyzed in this study are a subset of 10 trauma and 4 healthy control subjects enrolled at UF Health. Blood was collected from patients within 12 hours following trauma, and again at 24 hours. A single blood collection was performed on the healthy controls. In order to measure the levels of freely circulating DNA from the processed plasma, samples were centrifuged first at 200 x g to separate plasma, the plasma was centrifuged thrice at 5000 x g, and then divided into an unfiltered and 0.1 µm filtered group. The copies/ml of cytochrome c oxidase (mitochondrial) and rhodopsin (nuclear) DNA in the plasma were determined by qPCR. Blood leukocyte genomics were also determined by NanoString.
Results: The levels of both freely circulating mitochondrial (mt) and nuclear (nc) DNA in the plasma of trauma patients appear to be elevated at 12 and 24 hours compared to the healthy control subjects (p=0.028-0.190). 0.1 µm filtration of plasma does not affect the mt and ncDNA levels in trauma any differently than in healthy control populations. The levels of mt and ncDNA are well correlated in trauma patients at 24 hours (Spearman rank correlation, r=0.879). However, the levels of mt and ncDNA are poorly correlated in trauma patients at 12 hours (r=0.317). Although the sample size is small, there appears to be a moderate correlation between the levels of mt and ncDNA at 24, but not 12 hours, and patient recovery outcome (r= 0.592-0.395 at 24 hours). There also appears to be a moderate correlation between 24, but not 12 hours, gene expression and 24 hours mt and ncDNA copies (r= 0.648). There is no correlation between 12 hours genomic expression and mt and ncDNA copy numbers.
Conclusion: Blunt trauma results in the immediate release of nucleic acids that exist primarily in their free form (not encapsulated in microvesicles or apoptotic bodies). However, both mt and ncDNA concentrations are positively correlated with changes in leukocyte gene expression and clinical outcomes at 24 but not 12 hours suggesting that such measurements may reflect the degree of tissue injury and activation of inflammation. The findings suggest that the level of DNA release may regulate gene expression and subsequent clinical trajectory.
N-ACETYL CYSTEINE RENDERS AIRWAY BARRIER AT RISK FOR BACTERIAL PASSAGE AND SUBSEQUENT INFECTION
J.A. Friedman, L. Diebel*, D.M. Liberati*. Wayne State University, Royal Oak, MI
Background: The use of inhaled aerosols in mechanically ventilated patients is common practice in the ICU. However, a recent trial using a combination of nebulized heparin, albuterol, and the mucolytic/antioxidant N-acetylcysteine (NAC) demonstrated an increased incidence of pneumonia. The mechanism(s) may include changes in epithelial mucus barrier protection and resultant bacterial infection. Other possibilities include a decreased inflammatory response to bacterial presence. Recent work has demonstrated the importance of mucus in the respiratory airway barrier. We hypothesize that NAC will alter mucus properties, cytokine response to bacteria, and bacterial transcytosis in vitro.
Methods: Calu-3, a human bronchial epithelial cell monolayer, was grown in a 2-chamber cell culture system. Epithelial mucin and free O-linked oligosaccharide (OSC) content of Calu-3 cultures pretreated with NAC (0.3µg/mL for 15 mins), albuterol (10-6 M for 24 hrs), or control were quantified using ELISA. In a separate experiment, Klebsiella pneumonia (105 CFU/mL) was added to the apical chamber of Calu-3 monolayers pretreated with NAC, albuterol, or control. Basal cytokine response and bacterial transcytosis (at 60, 120, 240 mins) were measured.
Results: Epithelial mucin content and free OSC content of Calu-3 cultures were decreased with 15 minutes of NAC administration. In response to inoculation with K. pneumonia, Calu-3 cultures had an attenuated basal IL-6 and TNF response when pretreated with NAC. Bacterial translocation of K. pneumonia was increased in NAC pretreated cultures at 60, 120, and 240 minutes.
Conclusion: NAC adversely affected respiratory mucus chemical properties and epithelial barrier integrity. Short-term administration of NAC attenuated apical mucin content, free OSC, cytokine response to bacterial presence, and increased the translocation of bacteria across the epithelial surface. Our data supports that “routine” use of NAC in mechanically ventilated patients, including those with smoke inhalation injury should be avoided.
TREATMENT WITH A TLR4 AGONIST ENHANCES NEUTROPHIL RESPONSES TO INFECTION AFTER BURN
J. Bohannon*, L. Luan*, A. Hernandez*, E. Sherwood*. Vanderbilt University, Nashville, TN
Background: Infection is the leading cause of death in severely burned patients. Prophylactic treatment with the TLR4 agonists lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA) is known to induce resistance to subsequent bacterial challenge. MPLA is a less toxic derivative of LPS and is currently used as a human vaccine adjuvant, making it well-suited for clinical use to enhance immune responses to infection. Treatment of mice with MPLA enhances bacterial clearance, leading to improved survival in various models of sepsis including Pseudomonas aeruginosa peritonitis and burn wound infections. This study aimed to define the mechanisms responsible for improved bacterial clearance and survival in MPLA-treated burn-infected mice.
Methods: Mice underwent severe burn injury to ∼35% total body surface area, followed by systemic treatment with MPLA or vehicle control for two days. Mice were then inoculated with P. aeruginosa, topically or intraperitoneally, and responding neutrophils were measured in bone marrow, blood, burn wound and peritoneal cavity.
Results: MPLA treatment decreased neutrophil numbers in bone marrow, and decreased expression of CXCR4 on bone marrow neutrophils, a chemokine receptor important in sequestering neutrophils within the bone marrow compartment. Conversely, MPLA increased neutrophil numbers in the blood, peritoneal cavity and burn wound site. These effects were observed as early as 3 hours after MPLA treatment and remained constant after the establishment of a burn wound or intraperitoneal infection. MPLA treatment induced G-CSF, a cytokine necessary for mobilization, recruitment 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 (See figure). G-CSF neutralization blocked MPLA-induced mobilization of neutrophils out of the bone marrow and into the blood, as well as recruitment to the wound and peritoneum.
Conclusions: Our results suggest that G-CSF facilitates MPLA-induced mobilization of neutrophils from the bone marrow into systemic circulation, allowing for a rapid response to sites of infection. The ability of MPLA to enhance neutrophil-mediated antimicrobial responses after burn injury makes it an attractive therapeutic candidate for clinical use in burn patients for the prevention of deadly post-burn infections.
INTEGRATION OF INJURY-SITE IMMUNE ACTIVATION WITH SYSTEMIC CYTOKINE PRODUCTION MAY SERVE AS A CLINICAL INDEX TO PREDICT SEPSIS IN BURNED PATIENTS
P. Chen1, M. Stanojcic*1, M.G. Jeschke*1,2. 1Sunnybrook Research Institute, Toronto, ON, Canada, 2University of Toronto, Department of Surgery, Toronto, ON, Canada
Since the detection of clinical sepsis usually occurs at a stage where irreversible damage to patients has already occurred, the goal is to devise indices that can predict sepsis in advance before the infection worsens to the point of no return. Results from our study suggest that burn patients exhibiting an aberrant pro-inflammatory response and/or an early anti-inflammatory (AI) state in the acute phase of thermal injury foreshadows sepsis.
A holistic immune profile of burned patients is assessed by a 2-step approach. Firstly, abundance of burned site-derived macrophages and its production of IL-1b were assayed to assess inflammasome activation. Subsequently, blood of burned patients was collected to assess plasma cytokine concentration. Finally, the immune profiles of burned patients that eventually develop sepsis are compared to the non-septic counterparts. All patient specimens used for the study were collected within 96-hours post burn.
Surveying a total of 43 individuals (12 controls, 13 >20% TBSA non-septic patients, 18 >20% TBSA septic patients), we found that septic burned patients have a 2.8-fold less macrophages recruited to the site of injury in contrast to the non-septic group (p<0.05); however, the amount of IL-1b produced by these recruited macrophages in the septic cohort is 3.8-fold higher relative to the non-septic counterpart (p<0.05). In contrast, a significantly higher systemic AI profile is detected in the septic cohort relative to the non-septic group, shown by a 4.5- and 2.1-fold increase of plasma IL-10 and IL-1RA, respectively (p<0.05 for both).
From this clinical study, it shows that the clash of the immune functions between the wound site and the peripheral organs coupled with the scarce immune cells at the wound site foreshadows sepsis. Importantly, these findings collectively appear to have a very strong sensitivity in predicting sepsis onset a priori in thermally injured patients, making it invaluable for clinicians as it allows preemptive therapies to be administered to these “at-risk” patients to avoid sepsis all together.
PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA AGONIST IMPROVES SURVIVAL IN A MURINE MODEL OF POST-BURN INFECTION BY MODULATING THE MACROPHAGE FUNCTION
H. Miyazaki, M. Kinoshita*, S. Seki, D. Saitoh. National Defense Medical College, Tokorozawa, Japan
Background: Severe burn injury causes the Kupffer cells dysfunction and attenuates the host defense against bacterial infections. Peroxisome proliferator-activated receptor-γ (PPAR-γ) is abundantly expressed in macrophages and plays a critical role for regulation of immune responses during the inflammation. Pioglitazone, a PPAR-γ agonist, exert anti-inflammatory actions in different disease models, including septic shock. We herein investigated the role of PPAR-γ during the inflammatory response in post-burn bacterial infection by using the pioglitazone.
Material and Methods: C57BL/6 mice received a 20% full-thickness burn injury. Five days after injury, they were administered with pioglitazone (10 mg/kg, i.p.) or vehicle, followed by E. coli challenge.
Results: Burn injury did not affect the expression of PPAR-γ in hepatic mononuclear cells including Kupffer cells. Although burn injury augmented the elevation of serum TNF-α after E. coli challenge in mice, pioglitazone pretreatment significantly suppressed this elevation. Burn injury also down-regulated the phagocytic activity by Kupffer cells and impaired bacterial clearance in the liver after sublethal E. coli challenge. Interestingly, pioglitazone pretreated burn-injured mice augmented the phagocytic activity by Kupffer cells and restored bacterial clearance in the liver, thereby improving mouse survival after post-burn E. coli infection.
Conclusion: It is concluded that PPAR-γ agonist modulates inflammatory responses following post-burn infection via a mechanism dependent on suppressing proinflammatory cytokine production and enhancing phagocyte function of Kupffer cells/macrophages.
BURN-EVOKED ROS IMMEDIATELY AFTER INJURY ARE CRUCIAL TO RESTORE THE NEUTROPHIL FUNCTION AGAINST POST-BURN INFECTION IN MICE
M. Kinoshita*1, H. Miyazaki1, S. Ono*2, K. Hagisawa1, S. Seki1, D. Saitoh1. 1National Defense Medical College, Tokorozawa, Japan, 2Tokyo Medical University, Hachioji, Japan
Although reactive oxygen species (ROS) basically play beneficial roles to maintain host homeostasis against external disturbance/stress including infection, excessive ROS generation by activated neutrophils can sometimes cause organ damage. We investigated the role of burn-induced ROS generation in the injured hosts, focusing on post-burn infection. C57BL/6 mice received a 20% full thickness burn injury. In these mice, the burn-induced ROS generation was inhibited during and immediately after injury by pretreatment with superoxide dismutase (SOD) (at 1 h and immediately before injury), or the subsequent ROS production was inhibited by post-treatment with SOD (at 1 and 2 h after injury), which could not scavenge the ROS produced immediately after injury. As expected, inhibition of ROS production immediately after injury improved the burn-induced acute lung injury at 6 h, while inhibition of the subsequent ROS production did not lead to any improvements. Burn injury rendered the mice susceptible to bacterial infection at five days after injury and impaired bactericidal activity of neutrophils. Nevertheless, inhibition of the ROS production immediately after injury did not improve the burn-induced susceptibility to infection or the neutrophil dysfunction. Interestingly, inhibition of the subsequent ROS production potently restored the neutrophil functions and hematopoietic function of the bone marrow myelocytes, thereby improving the post-burn infection. Thus, although the inhibition of burn-evoked ROS generation is effective against burn-induced organ injury, it may be ineffective against post-burn infection. Preservation of the immediate burn-evoked ROS production, but the inhibition of subsequent ROS production, may be crucial to protect against post-burn infection.
MAJOR THERMAL INJURY UPREGULATES THE CELLULAR LEVEL EXPRESSION OF THE MELATONIN SYNTHESIZING ENZYME AANAT IN GUT CD117+ AND ENTEROCHROMAFFIN CELLS
A. Lala, C. Gumm, M. Kim, A. Ali, N. Fazal, A.C. Azim, W.M. Al-Ghoul*. Chicago State University, Chicago, IL
Our group has shown that the gut is a major source and target for melatonin (Al-Ghoul, et al., Int J Biol Sci 6(3):282-93, 2010 and Results in Immunol. 4: 14-22, 2014). Here, we focus on the cellular sources for melatonin in intestinal tissues and underlying gut associated lymph tissues (GALT) as assessed by immunohistochemistry for the melatonin synthesizing enzyme AANAT using fluorescent microscopy and flow cytometry. Our hypothesis is that major thermal injury is associated with changes in the activity and distribution of AANAT expressing resident and trafficking cells in the gut mucosa milieu. Towards this end, we compared cellular homogenates and histological sections of ileum and colon mucosae, as well as spleen, MLN, and liver preparations from three matched groups of adult male mice, namely, control, thermally injured (TI; hot water scald: 90° C, 9 sec.; 20-25% TBSA on shaved dorsum) and TI that received melatonin treatment (TI+Mel; 1.86 mg melatonin/kg b.wt., daily i.p. injection); all rats were sacrificed on day 3 postburn and all animal treatments were in accordance with NIH guidelines and IACUC approved protocols. Our results show high levels of AANAT immunoreactivity in ileum and colon enterochromaffin and CD117 immunopositive cells. Furthermore, gating CD117+ based on granularity to tease out mast cells, revealed relatively high percentages of AANAT and CD117 immunopositive cells in liver, spleen, ileum, and colon relative to the MLN alongside a consistent pattern of increase in this cell subpopulation in all of these tissues with thermal injury alone. Interestingly, this increase was attenuated with melatonin treatment in all tissues except spleen where it was actually increased. As such, our results suggest enterochromaffin, as well as mast cells as a source of mesenteric melatonin and that tissue variability in melatonin production may be at least partly regulated by a combination of mast cell trafficking alongside cell level changes in AANAT expression. (This research was supported by research grant Award Number SC3GM099632 to WMA from the NIH National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.)
EE-3-SO4 ADMINISTRATION FOLLOWING TRAUMATIC BRAIN INJURY IMPROVES PHYSICAL PERFORMANCE
T. Yu, B. Cam, W.J. Hubbard, K.I. Bland, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
We have previously shown that rats subjected to 60% hemorrhage, and then treated with EE-3-SO4 (1 mg/kg BW) produced 87% survival after 6 hrs. without resuscitation, where all vehicle controls die. In light of these promising results, we are now examining the efficacy of treating experimental moderate traumatic brain injury (TBI) with 17a-ethinyl estradiol-3-sulfate (EE-3-SO4). Combat and civilian TBI is often accompanied by hemorrhage, so the prospect of delivering a treatment simultaneously for both injuries is compelling. The model for experimental TBI in the rat utilizes lateral fluid percussion (LFP), delivered to the exposed brain via a 4.6 mm trephine bore at the right bregma-lambda junction. Trephination per se temporarily forestalled the steady daily weight gain seen in rats. Logic dictates that a rat must be physically active to seek and consume food and water, so we hypothesized that there was also a transient reduction in activity post-trephination and before TBI. We fabricated a device that utilized passive infrared (PIR) to detect motion. PIR detects perturbations in ambient infrared radiation, which is not apparent to the rats. We measured activity for 12 hrs. (6 pm to 6 am), since this corresponds to the rat’s nocturnal circadian activity. The data logging is automated and scored as the presence or absence of movement “events” in a 10 second interval. Data was collected before trephination (Day 1), after trephination but before TBI (Day 2), and 24 hrs. after TBI (Day 3). Rats subjected to TBI are identified by a solid bar on the graph’s right lower side. The rat groups were, from left to right: 1) normal rats (no trephination), 2) Sham (trephination but no TBI), 3) TBI+Vehicle (saline iv), 4) TBI+EE-3-SO4 high dose (5 mg/kg iv), and 5) TBI+EE-3-SO4 low dose (1 mg/kg iv). EE-3-SO4 or vehicle were given 1 hr post-TBI, in a non-resuscitative (∼140 ml) volume. Weight gain and activity are shown below, expressed as percentage of pre-trephination weight or activity. With both there is a clear pattern of reduced weight gain and activity after trephination. Furthermore, while preliminary, the trends suggest that EE-3-SO4 helps to ameliorate the detrimental effects of TBI on physical activity. In conclusion, the pattern for transiently lower activity or weight gain in craniectomized rats supports the assertion that trephination per se should be regarded as a measurable injury to be considered when interpreting LFP-induced TBI data. The results also point toward positive effects of EE-3-SO4 on physical activity post-TBI (DOD CDMRP Grant number PT075653).
INTERLEUKIN-15 TREATMENT EXPANDS SPLENIC NK AND CD8 T CELLS BUT FAILS TO IMPROVE SURVIVAL DURING BURN WOUND SEPSIS
N.K. Patil*, L. Luan*, Y. Guo*, J. Bohannon*, E. Sherwood*. Vanderbilt University Medical Center, Nashville, TN
Background: Burn injury creates exposed wounds that offer an ideal opportunity for microbial colonization and dissemination into blood and organs leading to sepsis. Studies have shown that burn injury and sepsis cause immunosuppression and predispose the host to opportunistic infections. Numerous therapeutic agents are currently being evaluated to boost patient’s immune cell response during sepsis. Interleukin-15 (IL-15) is an immunomodulatory cytokine that is predominantly involved in the development of natural killer (NK) and T lymphocytes. IL-15 has shown promising results in clinical trials for cancer patients and improves survival in a bacterial peritonitis model of sepsis. Therefore, we hypothesized that treatment with IL-15 will improve host resistance to infection and improve survival during burn wound sepsis.
Methods: We used a clinically relevant mouse model of Pseudomonas aeruginosa burn wound infection. A 35% TBSA full thickness burn was induced and the wound was infected (1 x 108 cfu) on day 4 post-burn. IL-15 (1 μg, IP) was administered on days 1, 2 and 3 post-burn. Splenic lymphocyte counts and activation were characterized by flow cytometry on day 4 (prior to wound infection). Organ injury and survival were also evaluated.
Results: IL-15 treatment caused a significant increase in the spleen weight (229±9 mg vs 87.65±4.8 mg) and marked expansion of splenic NK cells (>14 fold), NKT cells (>18 fold) and memory CD8+ T cells (>9 fold) counts in the spleen as compared to the vehicle treated mice. There was no increase in the CD4+ T lymphocyte count, but B lymphocyte count was increased (>2 folds). As compared to the vehicle treatment, IL-15 administration also lead to a significant increase in the expression of the early stimulation marker CD69 on CD4+ (22.8±0.8 vs 9.3±0.8%), CD8+ (16.7±1.3 vs 3.3±0.22%) and NK cells (32.17±3 vs 20.59±1.7%) in the spleen, implying robust immune activation. IL-15 administration also caused a significant increase in serum liver injury markers, ALT (47.8±5.7 vs 29.25±4.6 Units/L) and AST (164±31.8 vs 77±21.19 Units/L). Although IL-15 administration increased lymphocyte numbers and activation, it failed to improve survival during wound sepsis compared to vehicle-treated mice (100 % mortality on day 3 post wound infection).
Discussion: The lack of beneficial effect of IL-15 treatment on survival might be due to its effect on only specific lymphocyte populations and not a robust overall expansion of other immune cell populations. In a recent clinical trial, IL-15 treatments lead to hypotension and liver injury. Thus, the toxicity of IL-15 might also be playing a critical role in its failure to improve survival. Therefore, our results show that despite expansion of specific immune cells, IL-15 might not be a good therapeutic candidate to treat burn-associated wound infection and sepsis.
IN VIVO EVALUATION OF PLATELET FUNCTION IN RATS USING CONFOCAL INTRAVITAL VIDEOMICROSCOPY
I. Torres Filho*, L.N. Torres, C. Valdez, C. Salgado, A.P. Cap*, M.A. Dubick*. US Army Institute of Surgical Research, San Antonio, TX
Introduction: Platelet adhesion plays an important role in hemostasis and thrombosis, contributing to other pathophysiological processes including inflammation and immune-mediated responses to pathogens. We aimed to investigate the thrombus formation and platelet adhesion under in vivo, in situ conditions in a model of hemorrhage/resuscitation and in normotensive animals. We used confocal intravital videomicroscopy to study platelet interactions in vivo in the anesthetized rat microcirculation after hemorrhagic shock and different platelet storage procedures.
Methods: The system allowed the collection of images in separate channels, each with a different fluorescence or brightfield image, using a fixed-stage microscope, a Yokogawa confocal scanner head, and solid state lasers for excitation at 488 nm and 561 nm. After infusion of fluorescently-labeled platelets, an endothelial injury was induced using a pulsed N2 dye laser applied through the microscope immersion objective 63x (NA 1.0), parfocal with the focal plane. Upon induction of injury in cremaster muscle microvessels, the adhesion of platelets in the developing thrombus was detected and imaged. Multiple microvessels (n = 5-8 per rat, diameter: 16-50 μm) were targeted for injury. Blood samples were collected for analysis while systemic physiological parameters were simultaneously monitored and recorded. In hemorrhagic shock studies, animals were subjected to 40% hemorrhage and resuscitated with normal saline (30-45ml/kg) before laser endothelial damage. In platelet storage studies, animals were normovolemic.
Results: The relative concentration of labeled platelets (approx. 10% of total native platelets) decreased only by 1-3 % after 1 hour of injection. A fluorescence image of labeled platelets, a brightfield image of the thrombus, and a composite image showed co-localization of platelets with the thrombus. Thrombus area and height averaged 200 +/- 20 μm2 and 9 +/- 1 μm, respectively, in 50 microvessels. The integrated fluorescence intensity difference associated with the adherence of labeled platelets provided a quantitative index of adhesion, averaging 3.6 +/- 0.5 arbitrary units. We found that cold-stored platelets (4 oC) performed as well as those stored at room temperature. Moreover, platelet adhesion and thrombus formation after hemorrhagic shock / saline resuscitation were strongly reduced.
Conclusion: This methodology allows evaluating the functionality of platelets in vivo under real blood flow conditions and after animals and platelets have been exposed to different experimental conditions.
Supported by US Army Medical Research & Materiel Command.
TRANSFUSION RELATED SYSTEMIC FIBRINOLYSIS IS AFFECTED BY RED BLOOD CELL AGE AND BLOOD TYPE
A.P. Morton1, E.E. Moore*2,1, H.B. Moore*1, E. Gonzalez*1, A. Sauaia*1, A. Banerjee*1, C.C. Silliman*1,3. 1University of Colorado, Aurora, CO, 2Denver Health and Hospital, Denver, CO, 3Bonfils Blood Center, Denver, CO
Introduction: Systemic hyperfibrinolysis is associated with mortality in severely injured trauma patients, although this mechanism is unclear. Addition of lysed red blood cells (RBCs) to healthy volunteer blood is associated with hyperfibrinolysis. The red cell storage lesion (RCSL) occurs as stored RBCs age and includes hemolysis, changes in membrane stability, and bioactive substances in the storage media such as intracellular proteins, cytokines, and lipids, whose effects on fibrinolysis are unknown. We hypothesize that resuscitation with older RBCs will increase tPA-induced fibrinolysis due to the RCSL.
Materials and Methods: Six units of RBCs (3 A, 2 O, 1 B) were collected in AS-3 via standard protocol and stored for 42 days at 4° C. Blood samples were collected from 8 healthy volunteers on Day 1, 21, and 42. A 75ng/ml tPA-challenge TEG assay was performed on each citrated native sample after 25% volume replacement with RBCs. Fibrinolysis was measured via LY30, and data were analyzed using a mixed model to evaluate unit and time-dependent changes.
Results: There was a U-shaped relationship between age of RBCs and LY30 as demonstrated by the significant quadratic term (p=0.0031) independent of RBC unit type. Median LY30 for all units were 34.1% (day 1), 23.7% (day 21), and 27.4% (day 42). This effect was significantly modified by blood type as demonstrated by the significant quadratic term (p=0.0029). Median LY30 by RBC unit type were 34.1 (day 1), 24.2 (day 21), 26.8 (day 42) for type A, 32.3 (day 1), 21.9 (day 21), 31.4 (day 42) for type O, and 37.9 (day 1), 31.2 (day 21), 26.6 (day 42) for type B.
Conclusions: There is a U-shaped relationship between fibrinolysis and age of transfused RBCs. Fibrinolysis is elevated when fresh (Day 1) or old (Day 42) blood is transfused. This relationship is significantly modified by blood type, with type O RBCs showing the highest levels of early and late fibrinolysis, and type B RBCs showing an overall decrease in fibrinolysis as storage age increases. Early fibrinolysis was an unexpected finding. Potential mediators include early mechanical hemolysis or interactions between bioactive substances in the storage media with the plasmin/anti-plasmin system that regulates fibrinolysis.
MILD TRAUMATIC BRAIN INJURY (MTBI) PREVENTS THE COAGULOPATHY INDUCED BY BACTERIAL PNEUMONIA
D.G. Remick*, J. Seo, T. Hsieh, S. Kurosawa*, D.J. Stearns-Kurosawa*. Boston University School of Medicine, Boston, MA
Background: The consumptive coagulopathy that develops during a severe infection is a major contributor to organ failure, hemorrhage risk and death. We recently published that MTBI protects mice from the lethality of Pseudomonas aeruginosa (Psd) pneumonia. The current studies examined if MTBI would prevent this coagulopathy.
Methods: MTBI was induced with a fixed weight drop apparatus and 48 h later Psd was instilled intratracheally. Plasma was collected ∼18 hours after Psd to measure coagulation parameters.
Results: In naïve mice the clotting time was 26 ± 2 seconds which doubled 18 hours after induction of Psd pneumonia to 52 ± 4 seconds (p<.001). Fibrinogen consumption occurred with a decrease from in fibrinogen concentration from 189 ± 11 µg/ml in naïve mice to 94 ± 5 µg/ml in the Psd mice (p<.001). D-dimer levels also increased from 17 ± 4 ng/ml in naïve mice to 23 ± 4 ng/ml in Psd mice. These changes indicate accelerated coagulation since there was consumption of fibrinogen coupled with suppression of fibrinolysis (i.e. relatively low D-dimer). MTBI prior to induction of pneumonia prevented all of the Psd induced changes in the coagulation parameters. Clotting times were actually lower in the MTBI + Psd mice (11 ± 0.6 seconds) compared to the naïve or Psd mice. A portion of the faster clotting times may be due to increased fibrinogen levels which were significantly higher in the MTBI + Psd mice (674 ± 47 µg/ml). Notably, D-dimer concentrations were significantly increased in the MTBI + Psd (55 ± 10 ng/ml) compared to naïve or Psd mice (p<0.001 for all comparisons).
Conclusions: MTBI blunted the coagulopathy resulting from Pseudomonas bacterial pneumonia. The mechanism of this beneficial effect appeared to occur through augmenting fibrinolysis and accelerating the acute phase response.
HYPERTONIC SALINE PRIMES MITOCHONDRIAL METABOLIC ADAPTATIONS THAT PRECLUDE CYTOMIX-TRIGGERED STRESS
A. D’Alessandro, F. Gamboni, S. Mitra, C. Anderson, T. Nemkov, K. Hanse, A. Banerjee*. University of Colorado Anschutz Medical Campus, Aurora, CO
Introduction: Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. Recent mechanistic insights suggested an anti-inflammatory role of HTS on lung cancer cells (A549) by disrupting cytokine signaling at distinct intracellular steps (inhibition of I-κBα phosphorylation, NF-κB translocation into the nucleus, NF-κB nuclear binding in response to TNFα or IL-1β stimulation). Here we hypothesize that exposure to HTS prevents pro-inflammatory mitochondrial activation/uncoupling, induced by cytokine stimulation, in primary Small Airway Epithelial Cells (SAECs), by priming cell survival checkpoints (p53 pathway) that trigger metabolic adaptations.
Methods: SAECs were cultured with isotonic (290 mOsm) medium (controls) or 400 mOsm (HTS, NaCl) medium, either in presence or absence of cytomix (10 ng/ml IFNγ, IL1β and TNFα). Small molecule metabolites (60-900Da) from lysed cultured cells and supernatants were fractionated by UPLC, then identified and relatively quantified by Mass Spectrometry-(QExactive). Cell morphology and mitochondrial activity (JC1 fluorescence assay) were monitored by live cell microscopy. WB and RT-PCR for Tp53, p21 and TIGAR were performed on cell extracts.
Results: HTS-dependent increases in Tp53 and p21 levels were observed both at the mRNA and protein level, suggesting a role for HTS in the activation of basic stress responses. While Tp53 is increasingly recognized as metabolic modulator in cancer, its contribution to metabolic modulation in primary cells is unknown. Here, HTS-dependent activation of the Tp53-p21 axis corresponded to a dramatic metabolic modulation, by up-regulating glycolysis and pentose phosphate pathway (6-phosphogluconate FCHTS/Iso>2, P<0.001) and depressing Krebs cycle fluxes downstream to α-ketoglutarate (median TCA intermediates FCHTS/Iso<0.75, P<0.009). HTS pre-treatment prevented the cytomix-induced hyperactivation of mitochondrial metabolism and depressed the mitochondrial membrane potential (High/low membrane potential ratio: Iso+cytomix=0.256±0.075; HTS + cytomix=0.069±0.004 P<0.001). These events corresponded to the intracellular accumulation of glutamine (FCHTS/Iso>1.5, P=8.7E-06), suggestive of reduced glutaminolysis.
Conclusion: HTS protection against cytomix-induced inflammatory responses appears to involve activation of the Tp53-p21 axis, a key cell response to stress stimuli. Consistently with observation in cancer cells, priming of the Tp53-p21 axis coincided with a substantial metabolic modulation, through increased glycolysis and depressed glutaminolysis. These events forestall mitochondrial hypercatabolism and uncoupling, thereby protecting cells from cytomix-triggered pro-inflammatory cascades in HTS-pretreated cells.
PROTEOMIC ANALYSIS OF NEWLY SYNTHESIZED PROTEINS IN C2C12 MYOTUBES IN RESPONSE TO ENDOTOXIN (LPS) AND INTERFERON-γ (IFNγ)
C.S. Coleman, C.H. Lang*. Penn State College of Medicine, Hershey, PA
Selective identification of newly synthesized proteins provides insight on biological processes that depend on the continued synthesis of specific proteins either to maintain homeostasis or to remodel the exiting proteome in response to stress. Here we use tandem LC-mass spectroscopy to identify nascent polypeptide chains on isolated ribosomes to define the population of actively translated proteins. Ribosomes were isolated from C2C12 myotubes or after exposure to a combination of E. coli LPS and IFNγ for periods of either 4h or 16h. Three independent samples were obtained for each growth condition, and proteins identified were subsequently pooled to produce a data set of unique proteins for each growth condition. This approach identified from untreated C2C12 myotubes a total of 1205 proteins, representing approximately 6% of protein-encoding genes in the mouse genome. All enzymes in glycolysis and the TCA cycle were identified, suggesting that these processes require continued synthesis of their protein components. Likewise, enzymes for the activation of all 20 amino acids were present as nascent polypeptide chains. Clustering of the identified proteins within Gene Ontology terms for cellular components and biological processes provided an extensive landscape of actively translated proteins in C2C12 myotubes. We identified 1260 and 1441 proteins, respectively, for the 4h and 16h treatment groups, and for >200 of these proteins, their nascent polypeptide chains were either not detected in the untreated group or their detection becomes significantly more robust after LPS/ IFNg. Many of these induced proteins (∼20%) can be clustered within immune response and macrophage activation functions highlighting the important innate immune function played by muscle. Another prominent cluster contains proteins in the ubiquitin-proteasome pathway, among which are components in the degradation of unfolded proteins and ERAD. Additionally, transcription factors are well represented, among which included Rrn3, Pa2G4, Supt6h, NFkb2, Stat1, Stat3, Morf4l2, Cebpb, Mycn, Junb, Xbp1. (Supported GM30832).
COMPREHENSIVE ANALYSIS OF MICRORNA PROFILES IN NORMAL RODENT MESENTERIC LYMPH
W. Sakamoto1, T. Masuno*1, T. Tsukamoto1, H. Jun1, A. Takayuki1, B.T. Naing2, D. Zhao2, T. Takizawa2, H. Yokota1. 1Department of Emergency and Critical Care Medicine Nihon Medical School, Tokyo, Japan, 2 Department of Molecular Medicine and Anatomy, Tokyo, Japan
Background: MicroRNAs (miRNAs) are small noncoding RNAs that play an important role in posttranscriptional gene regulation by targeting the 3’-untranslated region (3’-UTR) of specific target mRNAs. miRNAs are secreted into extracellular fluids, such as plasma, saliva, and urine. Previous studies have suggested that lymph, especially mesenteric lymph, is a major source of the gut-derived inflammatory mediators that induce distant organ injury. However, very little is known about miRNAs in mesenteric lymph. The aim of this study was to provide the comprehensive analysis of miRNAs of normal rodent mesenteric lymph.
Methods: This study was approved by the animal ethics committee of Nippon Medical School. Mesenteric lymph was obtained from 8-10 week old male Sprague-Dawley rats (n = 6). The mesenteric lymph duct was cannulated, and mesenteric lymph was collected for 1 hour and a half. Samples were centrifuged to remove cellular debris. Plasma samples were also obtained from a femoral artery. We performed a quantitative PCR-based miRNA array analysis of lymph miRNA levels and compositions using TaqMan MicroRNA Assay Rodent Cards. The levels of miRNA expression were compared between lymph and plasma. Data on lymph and plasma miRNAs were normalized to exogenous cel-miRNAs as a “spike-in” control using a modification of the method of Mitchell et al (PNAS 105: 10513-10518, 2008). We also used the Ingenuity Pathway Analysis (IPA) tool to search targets of miRNAs.
Results: Among 375 kinds of rat miRNA probed in array cards, 220 miRNAs (59% of the preload miRNAs) and 240 miRNAs (64%) were detected in lymph and plasma, respectively. Eleven miRNAs were exclusively detected in lymph, while 31 miRNAs were only present in plasma. Comparison of miRNA expression profiles between lymph and plasma revealed that 35 miRNAs (e.g., miR-673-5p) were significantly upregulated in lymph compared to plasma (P < 0.05). in silico analysis of miRNA-mRNA interactions showed that some upregulated miRNAs had the in silico target sites in the 3’-UTR of inflammatory-related genes (e.g., PLA2, HMGB1, TLR4, and IL-1B).
Conclusion: We found significant differences in miRNA levels between lymph and plasma. Our data provide new information on mesenteric lymph miRNAs that might have a role in the generation of remote organ injury.
GAP ANALYSIS OF ANIMAL HEMORRHAGIC SHOCK MODELS THROUGH RAPID EVIDENCE ASSESSMENT STRATEGIES
P. Reynolds*. Virginia Commonwealth University Medical Center, Richmond, VA
The ethical justification for animal-based research is that data are expected to indicate potential benefits and harms of proposed therapies before humans are put at risk. To inform clinical trial conduct, animal data must be reliable, unbiased, and transparently reported. Reliable studies are randomized, blinded, and adequately powered, with a pre-defined primary outcome. Adequate reporting consists of documentation of methods in sufficient detail to allow replication. I performed a systematic rapid evidence assessment review of animal hemorrhagic shock models examining the efficacy of low volume fluid resuscitation strategies. Out of 307 papers extracted, 24 were retained, and summarized in RevMan5. Studies were assessed in three domains: risk of bias, outcome relevance, and model design QA/QC criteria (surgery, stabilization, hemorrhage). Results were disappointing. Studies were at high risk of bias. Randomization methods were reported in only 1 study, and blinding performed in 2. Most studies were grossly under-powered. Weight-based metrics were used to assess hemorrhage, but there was no control of body weight within trials, contributing to large variation. QA/QC variables were either not reported or highly variable between studies. Few clinically-relevant endpoints (mortality, end organ damage) were reported, and follow-up times were too short (< 4 hr) to capture adverse events. Protocols were unstandardized, inappropriate (proposed therapy could not target injury), and overly-reliant on p-values to interpret clinical data. To be useful, animal hemorrhage models must assess and document confounders (experimental/surgical preparation, hemorrhage model), clinically-relevant outcomes, and potential adverse effects. Research protocols must include thorough and systematic evaluation of the literature to identify gaps and need before animals are wasted in uninformative experiments.
THE PATHOPHYSIOLOGY OF POLYTRAUMA AND HEMORRHAGE IN RATS
D.N. Darlington1, 2, X. Wu1, 2, A.P. Cap*1, 2. 1US Army Institute of Surgical Research, San Antonio, TX, 2Dept Surgery, University of Texas Health Science Center, San Antonio, TX
Background: Coagulopathy can be induced in rats after polytrauma and hemorrhage, and manifests as an elevation in Prothombin Time (PT), activated Thromboplastin Time (aPTT) and fall in clot strength, and is similar to the clinical hypocoagulation seen in both civilian and military patients after trauma. The coagulopathy seen in this rat model may be due to either a deficit in thrombin generation or an elevation in fibrinolytic activity. We have therefore examined both thrombin and plasmin generation in an attempt to determine the cause of the coagulopathy.
Method: Sprague-Dawley rats (300-400g, n=8) were anesthetized with Isoflurane. Polytrauma was induced by damage to the small intestines, left and medial liver lobes, right leg skeletal muscle, and by fracturing the right femur. The rats were bled 40% of the blood volume. No fluid resuscitation was given. Blood samples were taken before (time 0) and 30, 60, 120 and 240 min after trauma. Coagulation factors were measured in plasma by standard immuno or enzymatic assay. Platelet aggregometry was measured by Multiplate (Roche).
Results: Polytrauma and hemorrhage cause both PT and aPTT to rise significantly, and plasma fibrinogen and fibrin monomer concentrations to fall significantly over the 4hrs. Plasma thrombin activity did not change over time. However, plasmin activity, D-Dimer, and tissue plasminogen activator were significantly elevated. Serine protease inhibitors, Anti-Thrombin III and a2-Macroglobulin, fell significantly by 2hrs. The thrombin to plasmin activity ratio fell steadily over the 4hr time period. The ability of platelets to aggregate after stimulation by collagen and thrombin was elevated over the 4hrs, but aggregation to ADP significantly fell by 2-4hrs.
Conclusion: The fall in the thrombin/plasmin activity ratio strongly suggests that polytrauma and hemorrhage in this rat model leads to a fibrinolytic coagulopathy. The falling fibrinogen levels and the steady thrombin activity suggest that clot formation is occurring. However the rise in PT and aPTT, and fall in clot strength suggest that the clot is not maintained, probably due to fibrinolysis as evidenced by the rise in plasmin activity. The fall in Anti-Thrombin III and a2-Macroglobulin suggests that these inhibitors are being consumed, and most likely buffering an elevation in thrombin and plasmin generation. These data suggest that the coagulopathy after trauma is fibrinolytic, and that anti-fibrinolytic drugs like tranexamic acid may be an effective treatment for coagulopathy after trauma and hemorrhage. This project was funded by MRMC.
EFFICACY AND CARDIOVASCULAR EFFECTS OF THE ABDOMINAL AORTIC AND JUNCTIONAL TOURNIQUET IN A SWINE MODEL OF HEMORRHAGIC SHOCK
J.M. Rall1, J. Cox1, T. Seifert1, M. Clemens1, 2, J. Ross1. 1Office of the Chief Scientist, 59th Medical Wing, Wilford Hall Ambulatory Surgical Center, Lackland Airforce Base, TX, 2Department of General Surgery, San Antonio Military Medical Center, Joint Base San Antonio, Fort Sam Houston, TX
Recent advancements have been made to mitigate junctional and non-compressible hemorrhage in the pre-hospital setting. The abdominal aortic and junctional tourniquet (AAJT) is a newly developed FDA-approved product designed to stop inguinal, axillary, and pelvic hemorrhage. The AAJT is a wedge-shaped bladder, secured by a strap and manually inflated to completely block blood flow. Studies have been published illustrating the efficacy of the AAJT. However, safety concerns have been raised about the use of the product including creation of obstructive shock and the danger of over-resuscitation injury in the context of tactical combat casualty care (TCCC) 2013 guidelines for fluid resuscitation. To address these concerns, we hypothesized that application of the AAJT would not result in significant reduction in central venous return or pulmonary artery pressure nor result in over-resuscitation injury in either a normotensive (toploading) model or a hypotensive model of severe controlled hemorrhage. Yorkshire swine were randomized into four groups: presence or absence of hemorrhage and AAT placement. Hemorrhage was accomplished by removal of 40% estimated blood volume over 30 minutes. Cardiovascular, cardiopulmonary, and oxygenation status values were compared amongst groups. Results show that the AAJT was effective in blocking blood flow to the femoral artery by a loss of pulse pressure and muscle oxygenation as measured by near infrared spectroscopy (NIRS). Cardiac output, mixed venous oxygen status, and central venous pressure were not different amongst AAJT-treated animals and untreated controls. No evidence of over-resuscitation injury was present as evidenced by pulmonary artery pressure and pulmonary histology. Therefore, we conclude that in this model there is no evidence suggesting apparent risk for cardiovascular collapse or over-resuscitation in response to or in conjunction with AAJT use under the 2013 revision of the TCCC guidelines. However, in light of changes to pre-hospital resuscitation in the 2014 guidelines, which include the continued fluid resuscitation until a palpable radial pulse is reached, additional study may be required.
CYCLOSPORINE A ALLEVIATES HEMORRHAGIC SHOCK INDUCED-MITOCHONDRIAL INJURY IN LYMPHATIC SMOOTH MUSCLE CELLS IN RATS
H. Wang, L. Zhang, X. Bai, Z. Zhao*, C. Niu*. Hebei North University, Zhangjiakou, China
Lymphatic pump function plays an important role on the process of hemorrhagic shock. We hypothesize that hemorrhagic shock induced-mitochondrial injury in lymphatic smooth muscle cells (LSMCs) is involved in the lymphatic contractility dysfunction. Therefore, we investigated the role of cyclosporine A (CsA), a mitochondrial protective agent, on the ultrastructure and function of mitochondrion, and lymphatic contractility following hemorrhage shock (40±2 mmHg, 3 h). Firstly, we found that CsA administration (6 mg/kg, i.m) at 1 h after hypotension alleviated the mitochondrial ultrastructure injury in LSMCs, using transmission electron microscope. Subsequently, using high pressure liquid chromatography, we found that hemorrhagic shock reduced the levels of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and total adenine nucleotides, which was abolished by CsA treatment. Furthermore, we prepared lymphatic rings for the observation of lymphatic contractility with an isolated lymphatic perfusion system (110P, DMT) at a transmural pressure of 3 cmH2O. And the results showed that CsA administration reversed the effect of hemorrhagic shock inducing decreases in contractile frequency and fractional pump flow in lymphatics. In conclusion, our findings suggested that the lymphatic contractility dysfunction was related to the mitochondrial damage and dysfunction of LSMCs following hemorrhagic shock, and CsA treatment enhanced the lymphatic contractility through alleviating the mitochondrial injury and dysfunction, which may represent a novel approach for prevention and cure of lymphatic pump dysfunction during severe hemorrhagic shock. This work was supported by the Natural Science Foundation of Hebei Province.
DIFFERENTIAL REGULATION OF THE CARDIOVASCULAR RESPONSIVENESS TO VASOPRESSIN AND α1-ADRENERGIC RECEPTOR AGONISTS AFTER BURN INJURY
P.G. Vana, A.E. Evans, H.M. LaPorte, R.H. Kennedy, M. Majetschak*. Loyola University Medical Center, Maywood, IL
Background: Adequate fluid resuscitation and vasopressor support are essential cornerstones in the treatment of burn patients. The effects of burn injury on the cardiovascular responsiveness to vasoactive drugs, however, are not well understood. Thus, we assessed and compared burn associated changes in cardiovascular reactivity to arginine vasopressin (aVP) and phenylephrine (PE), a selective α1-adrenergic receptor agonist, in a euvolemic burn model.
Methods: Anesthetized Sprague-Dawley rats were subjected to sham procedure (n=6-7/group) or 30% total body surface area dorsal scald burn, followed by crystalloid resuscitation (Parkland Formula). Series 1: At 24h, 72h, 96h and 168h post burn (n = 3-7/group) rats were re-anesthetized and resuscitated to a mean arterial blood pressure (MAP) ≥ 80mmHg. Rats received then increasing doses of aVP (500 fg/kg - 50 ng/kg) or PE (5 ng/kg - 40 μg/kg) in 5 min intervals. MAP was monitored continuously. The area under the MAP curve (AUC) for each dose was calculated and used to generate dose-response curves. Series 2: Mesenteric arteries were harvested from sham animals and animals at 24h post burn. Vascular reactivity of isolated arteries to PE and aVP were assessed ex vivo by pressure myography. Non-linear regression analyses were calculated (GraphPad Prism). Best fit values were compared with the Extra sum-of-squares F test. Data are reported as mean (95% confidence interval). A 2-tailed p < 0.05 was considered significant.
Results:Series 1: As compared with animals after sham procedure (EC50 PE: 338 (174-657) ng/kg), the potency of PE was significantly reduced at 24h post burn (EC50 PE: 1108 (487-2525) ng/kg) and normalized within 168h post burn (EC50 PE: 72h- 775 (424-1416) ng/kg; 96h- 605.3 (418-877) ng/kg; 168h- 435 (325-582) ng/kg). Efficacy of PE was not affected by burn injury. The EC50 for aVP was not altered after burn injury. However, the efficacy of aVP was significantly increased at 24h post burn (Maximal AUC: sham- 220 (192-249) mmHgs; 24h- 281 (247-316) mmHgs) and normalized thereafter. Series 2: There were no significant differences in the PE responsiveness of isolated arteries between sham treated and 24h post burn animals. In contrast, the potency of aVP was significantly increased at 24h post burn (EC50 aVP: sham- 386 (159-941) pM; 24h post burn- 107 (73-158) pM). The efficacy of aVP was not altered after burns.
Conclusions: Burn injury differentially regulates the integrated cardiovascular response to α1-adrenergic receptor and vasopressin receptor stimulation and alters intrinsic vascular function. Our findings provide a scientific rationale for the use of aVP in hemodynamically unstable burn patients and may help to develop improved resuscitation strategies.
SHOCK INDEX AND INJURY SEVERITY SCORE: PREDICTORS OF MORTALITY AFTER MULTI-SYSTEM TRAUMA
K.M. Moore*. NHWSN Emory University, Atlanta, GA
Background: Decisions in the field about triage and treatment of traumatic injuries must not be delayed. Trauma patients often have complex, multiple injuries that may not fully manifest until later in the course of treatment. In the pre-hospital setting, time and resources significantly limit providers in determining the full extent of the injuries sustained in trauma. Once the patient reached the Emergency Department (ED) additional diagnostics may be performed, but the final results may not be available for hours to days. The injury severity score is presently the best option available for distilling complex and variable patient data collected over a period of time to a single number. Unfortunately, the injury severity score is not immediately available in either the pre-hospital setting or the ED.
Purpose: The purpose of this study was to compare the ability of shock index calculated from data obtained by pre-hospital providers and data collected on arrival to the Emergency Department with the Injury Severity Scores reported in the National Trauma Data Bank in their ability to predict death from trauma within 48 hours of sustaining traumatic injuries.
Methods: A secondary analysis was done using baseline data from the 2009 National Trauma Data Bank. The data were analyzed for descriptive information and logistic regression was done to evaluate the predictive ability of pre-hospital shock index and Emergency Department shock index as compared to the predictive ability of the reported injury severity score.
Results: The sample consisted of 516,156 randomly selected trauma patients injured in 2009 in the United States who were transported to a hospital reporting to the National Trauma Data Bank. The shock index value calculated in the Emergency Department was found to be the best measure for predicting death within 48 hours of admission to the hospital after traumatic injuries. For each unit increase in the calculated ED shock index, the odds of death within 48 hours of traumatic injury increased by 17%.
Conclusion: Shock index is an independent predictor of mortality in trauma patients and may be useful as an early marker of hemodynamic instability, which merits further investigation. Shock index may provide a key opportunity to improve outcomes in trauma.
USE OF RESUSCITATIVE ENDOVASCULAR BALLOON OCCLUSION OF THE AORTA (REBOA) IN REFRACTORY SHOCK FROM PELVIC VENOUS HEMORRHAGE
C.H. Hsu*1, M.L. Brenner*1, 2, M. Hoehn*2, J. Pasley1, J.J. DuBose1, T. Scalea*1. 1R Adams Cowley Shock Trauma Center, University of Maryland School of Medicine, Baltimore, MD, 2Division of Vascular Surgery, University of Maryland School of Medicine, Baltimore, MD
Hemorrhage after pelvic fractures may arise from the venous plexus, fracture surfaces, and branches of major pelvic arteries. Often times, the non-compressible nature of pelvic hemorrhage leads to significant morbidity and mortality in these patients. We recently described the use of resuscitative endovascular balloon occlusion of the aorta (REBOA) for end stage hemorrhagic shock in trauma, demonstrating its potential role in severe pelvic fractures. However, the role of REBOA in patients with refractory hemorrhagic shock from pelvic venous bleeding is unknown. Here, we describe three cases where REBOA was utilized for patients with pelvic fractures in refractory hemorrhagic shock from presumed venous bleeding. The pelvic CT scans demonstrated moderate sized hematomas in the affected regions. The average patient age was 61.7 ± 1.5 years (mean ± SD), and all were male who sustained a variety of pelvic fractures from blunt injuries. For all three patients, a 12 French sheath was placed percutaneously into the common femoral artery, and REBOA was performed using the Cook Coda balloon (Cook Medical, Indianapolis, IN). All three patients had instantaneous improvement in their hemodynamics (mean increase in SBP was 82.3 ± 19.4 mmHg, mean decrease in HR was 29.3 ± 8.3 bpm) after inflation of the Coda balloon at the level of the aortic bifurcation. The average time of pelvic occlusion was 84 ± 69.2 minutes (mean ± SD). The balloon was deflated at the time of angiography and hemodynamic stability continued. The selective pelvic angiograms of all three patients revealed no evidence of arterial injury, and they subsequently underwent a groin exploration for device removal and repair of common femoral arteriotomy. All three patients survived to discharge without REBOA-associated complications. These cases suggest that REBOA may be an effective and safe way to control pelvic venous hemorrhage caused by fractures, thus sparing patients from pre-peritoneal pelvic packing or exploratory laparotomy. Further prospective data is necessary to evaluate the potential therapeutic use of REBOA in severe pelvic fractures with associated hemorrhage.
EFFECTS OF RAPID WOUND SEALING ON SURVIVAL AND BLOOD LOSS IN A SWINE MODEL OF LETHAL JUNCTIONAL HEMORRHAGE
A. St. John, X. Wang, E.B. Lim, D. Chien, S.A. Stern, N.J. White. University of Washington, Seattle, WA
Background: Hemorrhage is the leading cause of preventable death in trauma. The current standard for junctional hemorrhage control is wound packing using hemostatic gauze. Rapid mechanical wound sealing using a hemostatic clamp to seal the wound at the skin has been suggested as a new approach to hemorrhage control but has not been studied extensively.
Objectives: Compare wound packing to rapid wound sealing in a swine model of lethal junctional hemorrhage.
Methods: Lethal junctional hemorrhage was induced in 50 immature female swine using a 5mm femoral arteriotomy. Free bleeding was allowed for 30 secs, followed by randomization to one of seven hemostatic interventions. Three control groups consisted of: no intervention (Negative Control, NC), direct wound compression only for 3 minutes (C), and plain gauze packing only (P). Wound sealing groups consisted of wound seal by hemostatic clamp (iTClamp™) alone (S) and plain gauze packing followed by wound seal (P+S). Wound packing interventions consisted of standard of care plain gauze packing + compression (P+C) and hemostatic gauze packing (Combat Gauze™) + compression (HSP+C). All animals then received a 15 ml/kg bolus of Hextend™, followed by infusion of Lactated Ringer’s as needed for mean arterial pressure < 60 mmHg up to 100 ml/kg for up to 3 hours. Survival was compared using Kaplan-Meier log rank. Blood loss, hemostat application time, and coagulation parameters were compared using ANOVA with Bonferroni adjustment.
Results: Within the control group, survival was improved with P vs. NC (p<0.001). Within the wound sealing group, survival was improved with S vs. NC and P+S vs. P (p<0.001). Within the wound packing group survival was improved with P+C vs. NC (p<0.001). Survival was not different when comparing P+S vs. standard of care P+C or HSP+C groups (p≥0.05). In the wound sealing group, total blood loss was reduced with S vs. NC and P+S vs. P (p<0.001), whereas blood loss was not reduced with P+C vs. NC or among the packing groups (all p≥0.05). Application times were faster when substituting wound seal for compression after packing, P+S vs. P+C (p<0.001). In the wound sealing group, platelet count was higher in P+S vs. P (p=0.003), and all other coagulation parameters were similar. In the wound packing group, platelet count was lower in P+S vs. P+C (p=0.001), TEG MA was lower in P+S vs. P+C (p=0.005), and fibrinogen concentration was lower in P+S vs. P+C (p<0.001), and all other coagulation parameters were similar.
Conclusion: In this preclinical model, rapid wound sealing using a hemostatic clamp improved survival and decreased blood loss from junctional bleeding. Performance was comparable to wound packing with compression while reducing application times. Coagulation profile was worse when P was combined with wound seal vs compression; this association requires further investigation.
TOLL-LIKE RECEPTOR (TLR) STIMULATION INHIBITS COLLAGEN-INDUCED ACTIVATION OF PLATELETS
A.M. Lewis1, X. Wu*2, 1, D.N. Darlington*2, 1, A.P. Cap*2, 1, M.G. Schwacha*1, 2. 1University of Texas Health Science Center San Antonio, San Antonio, TX, 2US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: Traumatic injury is the number one cause of death for patients under 44 years of age. Of those affected by trauma, hemorrhage accounts for 50% of deaths in the first 48 hours after admission. Trauma-induced coagulopathy is associated with a rise in prothrombin time, a decrease in clot strength, as well as a decrease in the ability of platelets to aggregate. This suggests a role for platelet dysfunction in trauma-induced coagulopathy. Platelets express toll-like receptors (TLRs) which can be stimulated by Damage Associated Molecular Patterns (DAMPs), intracellular entities released after cellular damage. We propose that tissue damage causes the release of DAMPs which stimulate TLRs on platelets, impacting their ability to aggregate and affect clot strength.
Study Design: Platelets were isolated from human blood samples of healthy volunteers and suspended in Tyrode’s buffer and stimulated with collagen at varying doses (1-100ug/mL). Using a submaximal concentration of collagen, platelets were then incubated with the TLR4 ligand, lipopolysaccharide (LPS) and aggregation measured. Collagen stimulation of platelets was performed simultaneously with, and after incubation with LPS. Platelet aggregation was measured by interference spectroscopy (BioTek and Chrono-log).
Results: Collagen-stimulated platelets aggregated in a dose dependent manner. LPS at higher concentrations (8-25ug/mL) inhibited platelet aggregation when stimulated with a submaximal concentration of collagen, independent of whether LPS was added prior to or simultaneously. Platelets that were stimulated with a maximal concentration of collagen were not inhibited by LPS.
Conclusion: These results show that the LPS can inhibit collagen stimulated platelet aggregation. This suggests that TLR4 mediated activation of platelets inhibits their function, providing a potential mechanism by which DAMPs contribute to the development of trauma-induced coagulopathy.
ENTERAL BLOCKADE OF DIGESTIVE PROTEASES ATTENUATES VASOPRESSOR RESISTANCE AFTER HEMORRHAGIC SHOCK
M.H. Santamaria, F. Aletti, F. DeLano*, G. Schmid-Schonbein*. University of California San Diego, San Diego, CA
Hemorrhagic shock is followed by a reduced blood pressure response to an intravenous injection of a vasopressor agent (vasopressor resistance). However, no mechanism has been accepted that could explain the vasopressor resistance in shock. We hypothesize that pancreatic digestive enzymes leaking from the small intestine during shock may cause adrenergic receptor damage and reduced pressure response to an intravenous bolus of a vasopressor (phenylephrine). Rats were exposed to hemorrhagic shock (35 mmHg mean arterial blood pressure, 2 hrs) and blood pressure was monitored during a phenylephrine bolus injection (2 µg/kg) at baseline and multiple times following reperfusion. A group of animals received enteral injections of a serine protease inhibitor (tranexamic acid, TXA, 127 mM in 12ml uniformly distributed in the small intestine and cecum). The duration and amplitude of mean arterial pressure following the phenylephrine bolus were recorded. The pressure response (duration and amplitude) to the phenylephrine bolus at 30, 90, and 120 min were significantly reduced after hemorrhagic shock compared to baseline. Enteral blockade of digestive proteases enhanced the vasopressor response to the phenylephrine bolus so that the pressure responses at the 90 and 120 min time points were not significantly different than baseline. We conclude that placement of TXA into the lumen of the small intestine in shock attenuates the acute vasopressor resistance in hemorrhagic shock as well as the acute insulin resistance, previously reported (Shock 41:56, 2014).
THE ESTROGEN RECEPTOR (ER)-β AGONIST, DPN, IS MORE EFFECTIVE IN IMPROVING CARDIAC FUNCTION, CARDIAC ESTROGEN RECEPTOR (ERS), AND ESTROGEN RELATED RECEPTOR (ERRS) EXPRESSION THAN THE ESTROGEN RECEPTOR (ER)-ALPHA AGONIST, PPT FOLLOWING TRAUMA-HEMORRHAGE
S. Hu, S. Yang, B. Cam, W.J. Hubbard, K.I. Bland, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
Our studies have shown that trauma-hemorrhage (T-H) induces cardiac depression, which is associated with decreased cardiac estrogen receptors (ERs) and the orphan nuclear receptors, estrogen related receptors (ERRs). We have also demonstrated that administration of β-estradiol-water soluble (E2; Sigma) or the ER-β agonist DPN (10mg/kg, IV) after T-H, but not the ER-α agonist PPT restored cardiac function, cardiac ERs and ERRs. However, it remained unclear whether increasing the PPT dose would be effective in not only restoring cardiac function, as well as cardiac ER-β and ERRβ expression. To study this, male adult Sprague-Dawley rats were divided into six groups (n=6/group): sham, T-H+vehicle (TV), T-H+E2 (TE), T-H+PPT with various PPT doses of 10, 50, and 100 mg/kg BW. PPT or E2 (1mg/kg) was given at the onset of resuscitation, IV. Two hrs after resuscitation, left ventricular (LV) performance was determined; blood and heart tissue were harvested and cardiac nuclei extracted. Cardiac ER-β and cardiac nuclear ERRβ were determined by Western blot. Plasma and heart tissue cytokines were measured by ELISA. Our results indicate that E2 restored LV performance, as well as cardiac ERs and ERRs after T-H; however, PPT at a dose of 10mg/kg only partially improved LV performance and restored cardiac ER-α and ERRα. Although increasing the dose of PPT to 50mg/kg restored cardiac ER-β and ERRβ, it did not restore LV performance after T-H. However, if the dose of PPT was increased to 100mg/kg, LV performance was restored following T-H. Therefore, it can be concluded that the ER-β agonist DPN is more effective in improving cardiac function, cardiac ERs and ERRs than the ER-α agonist PPT following T-H (NIH R01 GM39519).
TRIPHENYLPHOSPHONIUM (TPP) IS NOT AN INERT MITOCHONDRIAL DELIVERY MECHANISM
R.D. Powell*, D.A. Goodenow, I.H. McKillop, S.L. Evans*. Carolinas Medical Center, Charlotte, NC
Background: Hemorrhagic shock and reperfusion (HSR) injury leads to a cascade of reactive oxygen species (ROS) production, mitochondrial dysfunction, cell death, systemic inflammation and multiple organ dysfunction (MOD). Triphenylphosphonium (TPP) is a lipophilic cation used to target therapeutics to the mitochondria. MitoQ is an antioxidant comprised of a ubiquinone moiety attached to TPP. We sought to determine if TPP is an inert vehicle for ubiquinone in vital organs using a rat model of HSR.
Methods: Male Sprague-Dawley rats were hemorrhaged to a MAP of 25±2.0mmHg for 1-hr prior to resuscitation. TPP (5mg/kg) or saline (0.9%) was administered (iv) 30-mins prior to resuscitation, followed by IP administration (TPP, 20mg/kg; saline, 0.9%) immediately after resuscitation. Rats were sacrificed by exsanguination (cardiac puncture) 2-hrs post-surgery. Heart, lung, liver, spleen and kidney were collected for immunohistochemistry to determine level of mitochondrial degradation (cytochrome c) or presence of macrophages (CD68) or neutrophils (Leder stain), or assayed for lipid peroxidation (TBARS), endogenous antioxidant activity (glutathione peroxidase; GPx, and catalase activity), cytokine expression (TNF-α and IL-6) or complex-1 activity.
Results: HSR-TPP significantly increased lipid peroxidation in the heart vs HSR (15±0.38 vs 9.6±1.2μM MDA; p<0.05; n=5) and both HSR and HSR-TPP did so compared to sham in the spleen (8.1±0.38 and 9.5±0.62 vs 4.2±0.25μM MDA; p<0.05; n=5). Additionally HSR-TPP significantly increased CD68 staining vs HSR in spleen (1.8±0.54 vs 0.20±0.13; p<0.05; n=5) and kidney (2.0±0.32 vs 0.10±0.10; p<0.05; n=5). TNF-α expression increased with TPP administration in the lung vs sham and HSR (2637±141 vs 1470±116 vs 1932±196pg/mg; p<0.05; n=5), but was decreased vs HSR (580±49 vs 920±84pg/mg; p<0.05; n=5) in the liver. In addition, HSR-TPP caused a significant decrease in IL-6 in the liver vs HSR (6700±570 vs 11000±860pg/mg; p<0.05, n=5). Hepatic and renal complex-1 activity was significantly increased in HSR-TPP animals vs sham and HSR (0.13±0.01 and 0.07±0.01 vs 0.07±0.01mOD/min [liver] and 0.10±0.01 and 0.01±0.01 vs 0.01±0.01mOD/min [kidney] p<0.05; n=5). HSR-TPP also increased complex-1 activity in the heart vs sham, though not significantly (0.57±0.12 vs 0.23±0.04mOD/min; p=0.06, n=5). No differences were measured in the lung or spleen. TPP increases lipid peroxidation in cardiac tissue. Interestingly, TPP resulted in an increase in complex-1 activity in both kidney and liver and was associated with a decrease in hepatic inflammatory mediators. Conversely, TPP was associated with an increase in TNF-α in the lungs, and elevated renal and splenic macrophage counts.
Conclusions: Our data demonstrate TPP is not an inert delivery molecule and, like the mitochondrial targets compounds to which it is commonly bound, exerts effects in a tissue specific manner.
EBOLA INACTIVATION PROTOCOLS - IMPACT ON MEASURING CYTOKINES IN HUMAN WHOLE BLOOD
E.R. Duffy*, N.S. Miller, D.G. Remick*. Boston University School of Medicine, Boston, MA
Background: Ebola virus causes hemorrhagic fever and can lead to symptoms resembling septic shock. No cure exists currently and treatment consists of fluid resuscitation. Researchers are desperate for a cure, especially in light of the outbreak in Africa. The virus is highly infectious and contracted from bodily fluids of infected persons. Due to the high infection risk, the virus is inactivated in samples taken from patients before assays are run for research purposes. While current viral inactivation protocols show little effect on the traditional clinical chemistries run on patient blood samples, very little work has been done to evaluate the effect of the inactivation protocols on the measurement cytokines.
Methods: In this study, we looked at two inactivation protocols most accessible to researchers in the Ebola field, heat or chemical inactivation. Human whole blood was stimulated to induce endogenous cytokines by drawing blood from healthy donors into heparinized tubes. Lipopolysaccharide (LPS 50ng/ml) was added and the blood incubated for 4 hours at 37°C in 5%CO2. We separated the whole blood into five groups, no inactivation, heat inactivated plasma, heat inactivated whole blood, Triton X-100 treated plasma and Triton X-100 treated whole blood. Samples in plasma groups were centrifuged immediately after stimulation with the untreated group and plasma isolated. Plasma and whole blood samples were submerged in 60°C water bath for 60 minutes for heat inactivation. For the Triton X-100 groups, 10µl of 1% solution of Triton X-100 was added to the samples, vortexed and left at room temperature for 60 minutes. After the inactivation protocols, the whole blood samples were centrifuged and plasma was obtained. Samples frozen at -20°C until analysis. Cytokines measured by standard sandwich ELISA protocols. Total protein was done by standard Bradford assay and hematology parameters were taken on whole blood samples before and after protocols.
Results: Results show heat inactivation destroys the ability to measure protein content and also causes the samples to coagulate so hematology could not be done. Inactivation via Triton X-100 showed no significant impact on either cytokine content or on the hematology parameters.
Conclusion: The Triton X-100 protocol allows accurate cytokine measurement in either the whole blood or plasma. Heat inactivation prevents cytokine measurement.
THE HEMODYNAMIC CHARACTERISTICS OF MOUSE ANAPHYLACTIC SHOCK
T. Shibamoto*1, M. Wang1, 2, Y. Kuda1, M. Tanida1, Y. Kurata1. 1Kanazawa Medical University, Uchinada, Japan, 2The Fourth Affiliated Hospital of China Medical University, Shenyang, China
Aims: Systemic anaphylaxis is life-threatening, and its pathophysiology is not fully clarified. Mice are frequently used for experimental study on anaphylaxis. However, the hemodynamic features and mechanisms of mouse anaphylactic hypotension remain unknown. Therefore, we determined mechanisms of systemic and pulmonary vascular response to anaphylactic hypotension in anesthetized BALB/c mice by using receptor antagonists of chemical mediators.
Methods: Anaphylaxis was actively induced by an intravenous injection of the ovalbumin antigen into open-chest artificially ventilated sensitized mice. Mean arterial pressure (MAP), pulmonary arterial pressure (PAP), left atrial pressure, central venous pressure, and aortic blood flow (ABF) were continuously measured.
Results: In sensitized control mice, MAP and ABF showed initial, transient increases, followed by progressive decreases after the antigen injection. Total peripheral resistance (TPR) did not decrease, while PAP initially and transiently increased to 18.5±0.5 mmHg and pulmonary vascular resistance (PVR) also significantly increased. The antigen-induced decreases in MAP and ABF were attenuated by pretreatment with either a platelet-activating factor (PAF) receptor antagonist, CV6209, or a histamine H1 receptor antagonist, diphenhydramine, and abolished by their combination. Diphenhydramine augmented the initial increases in PAP and PVR, but did not affect the decrease of the corresponding MAP fall. The antagonists of either leukotriene C4 or serotonin, alone or in combination with CV6209, exerted no significant effects.
Conclusion: Mouse anaphylactic hypotension is caused by a decrease in cardiac output but not vasodilatation, via actions of PAF and histamine. The slight increase in PAP is not involved in mouse anaphylactic hypotension.
DEPENDENT INTERLEUKIN-6 PRODUCTION BY KUPFFER CELLS DRIVES HEPATIC DAMAGE AFTER INTOXICATION AND BURN INJURY
M.M. Chen, E.B. OHalloran*, E.J. Kovacs*. Loyola University Chicago, Chicago, IL
The widespread and increasing trend of binge drinking is accompanied by a concomitant rise in the prevalence of trauma patients under the influence of alcohol at the time of their injury. Epidemiologic evidence suggests up to half of all adult burn patients are intoxicated at the time of admission and the presence of alcohol is an independent risk factor for death in the early stages post burn. Both alcohol and burn injury are known to increase portal blood lipopolysaccharide (LPS) as well as prime Kupffer cells (KCs) for TLR4 signaling, leading to KC hyperactivity. TLR4 signaling can be enhanced by alcohol through upregulation of its coreceptor CD14. Furthermore, p38 mitogen activated protein kinase (MAPK) is downstream of TLR4 and known to be promote IL-6 production in KCs after burn. As we have previously demonstrated hepatic IL-6 drives pulmonary inflammation in this combined injury, we now seek to determine the role of KC’s after intoxication and burn. To this end, mice were given ethanol (1.2g/kg) by oral gavage 30min prior to a 15% scald burn injury. KCs were isolated and analyzed for CD14 mRNA expression and p38 activity. In separate experiments antecedent depletion of KC was achieved with clodronate liposomes and p38 inhibition was achieved via intraperitoneal injection of SB203580 30min after injury. KC’s isolated from intoxicated burned mice had a 10-fold (p<0.05) increase in CD14 mRNA expression and a 3-fold (p<0.05) elevation of p38 activation, suggesting amplified TLR4 signaling. This corresponded to a 2-fold (p<0.05) increase in IL-6 production after LPS stimulation ex vivo. Antecedent KC depletion attenuated hepatic damage as seen by decreases of 53% (p<0.05) in serum ALT, 37% (p<0.05) in hepatic triglycerides, as well as a 77% reduction (p<0.05) in serum IL-6 levels compared to intoxicated burned mice receiving empty liposomes. This mitigation of hepatic damage was associated with a 34% decrease (p<0.05) in pulmonary neutrophil infiltration and attenuated alveolar wall thickening, suggesting a crucial role for KCs in both hepatic and pulmonary damage in this setting. In vivo p38 inhibition conferred nearly identical hepatic and pulmonary protection after the combined injury as mice depleted of KCs, highlighting the role of KC p38 in causing injury. Taken together these data suggest intoxication and burn injury promote increased TLR4 signaling through upregulation of CD14, leading to p38-dependent IL-6 production in KCs which may serve as a potential therapeutic target in this common clinical scenario. This work was supported by NIH R01AA012034 (EJK), F30AA022856 (MMC), and the Falk Foundation.
IL-1A, IL-8, AND IFNγ SERUM LEVELS AS PREDICTORS OF CLINICAL SEVERITY OF SJS AND TEN
A. Wester, D. Surprenant, M.J. Mosier*, D. Syed, O. Iqbal, J. Fareed. Loyola University of Chicago Stritch School of Medicine, Forest Park, IL
Aim: We sought to investigate the relationship between circulating plasma cytokine levels and the severity of illness in patients with Stevens Johnson Syndrome (SJS) or toxic epidermal necrolysis (TEN). Specifically, we sought to determine if Interleukin-1a (IL-1a), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interferon-γ (IFNγ), Tumor Necrosis Factor-α (TNFα), Vascular Endothelial Growth Factor (VEGF) and Endothelial Growth Factor (EGF) correlated with the severity of illness in SJS or TEN. In other inflammatory models, such as burn injury and sepsis, the relationship between disease severity and plasma cytokine levels has been investigated. Our study aimed to determine if such a relationship exists in SJS and TEN patients in order to better understand the pathogenesis of SJS and TEN as well as to evaluate the prognostic value of plasma cytokine levels in SJS and TEN.
Methods: Seven subjects with a biopsy-proven diagnosis of SJS or TEN were recruited over a two-year period. One citrated blood sample was obtained from each subject within three days of hospital admission. Samples were assayed for IL-1a, IL-4, IL-6, IL-8, IL-10, INFγ, TNFα, VEGF, and EGF. The relationship between serum cytokine levels and perceived severity of SJS or TEN, as measured by length of hospital stay (LOS), maximum total body surface area involvement (TBSA Max), percent increase in total body surface area involvement (%TBSA Increase), SCORTEN on hospital day one (D1 SCORTEN), and SCORTEN on hospital day three (D3 SCORTEN) was investigated using non-parametric statistical analysis including the Spearman’s Rank Order Correlation and the Mann-Whitney U Test.
Results: TEN patients had higher levels of IL-1a, IL-8, and IFNγ at the time of admission (MdIL1a =1.27, MdIL-8 =534.82, MdIFNg = 1.68, n=2) than SJS patients (MdIL1a =0.11, MdIL-8 =16.5, MdIFNg = 0.28, n=5; IL-1a: U=0.000, z=-1.936, p=0.053; IL-8: U=0.000, z=-1.936, p=0.053; IFNγ: U =0.000, z=-1.936, p=0.053). There was a strong positive correlation between IFNγ levels and TBSA Max (rs =0.821, p=0.023), as well as LOS (rs =0.818, p=0.024). Increased levels of IL-1a and IL-8 were positively associated with %TBSA increase during the hospital stay (IL1a: rs = 0.867, p=0.012; IL8: rs =0.906, p=0.005), as well as both D1 SCORTEN (IL-1a: rs = 0.896, p=0.006; IL8: rs =0.837, p=0.019) and D3 SCORTEN (IL-1a: rs=0.866, p=0.012; IL8: rs =0.866, p=0.012). There was no significant association found between serum levels of IL-4, IL-6, IL-10, TNFα, VEGF or EGF and %TBSA increase, TBSA Max, LOS or SCORTEN.
Conclusions: This is the first study evaluating the relationship between serum cytokine levels and clinical disease severity in subjects with SJS or TEN. Compared to subjects with less severe disease, subjects with a greater severity of illness demonstrated greater elevation of IL-1a, IL-8 and IFNγ.
LUNG ISCHEMIA REPERFUSION INFLAMMATION IS MODULATED BY INFLAMMASOME AND EXTRACELLULAR SIGNAL-RELATED KINASE (ERK) PATHWAYS
A. Prakash*, X. Tian, W. Kevin, J. Hellman*. University of California San Francisco, San Francisco, CA
Background: Lung ischemia reperfusion (IR) can generate inflammation resulting in lung dysfunction in a variety of clinical settings, from lung transplantation to resuscitated low flow states as seen in hemorrhagic trauma to therapeutically reversed pulmonary emboli. The consequences of lung IR are potentially devastating in critically-ill patients resulting in prolonged ventilator dependence with unclear long-term effects on lung function. In our earlier published work, we showed a role for IL-1b in IR inflammatory signaling in vitro and here we explored upstream inflammasome signaling pathways for their roles in mediating this process. Also, since we found that ERK5 also mediates inflammatory activation of endothelial cells by IL-1b, we also assessed the role of ERK5 in lung IR.
Methods: C57/B6 Wildtype and inflammasome knockout (ASCko, NLRP3ko, and NLRC4ko) mice underwent reversible left pulmonary artery occlusion for 30-60min without interruption in ventilation. ERK pathways were also investigated by pretreating wildtype C57/B6 mice with specific ERK5 inhibitors or vehicle controls prior to subjecting the mice to lung IR. Plasma cytokine levels and local lung gene mRNA induction were assayed 1h following reperfusion.
Results: The pan-inflammasome (ASC) knockout mice had a severely attenuated response to ventilated lung ischemia reperfusion as measured by lower IL-6 levels expressed compared to wildtype mice. However, neither the canonical sterile inflammasome (NLRP3), nor an infectious inflammasome (NLRC4) appeared to required for the generation of inflammation following lung IR. Our data suggest that a novel sterile inflammasome subtype is involved in the release of active IL-1b downstream of the lung IR injury. Additionally, through the use of specific inhibitors, we showed that, in the presence of the ERK5 inhibitor, IL-6 and CCL-2 levels were significantly lower following IR while PAI-1 levels were conversely significantly higher. This suggests that ERK5 contributes to the inflammatory response elicited by lung IR. Our findings suggest that inflammasome and ERK signaling pathways are potential targets for manipulating inflammation in the context of sterile and infectious inflammatory conditions affecting the lung.
ZINC FINGER PROTEIN 816 IS A REPRESSOR OF TOLL-LIKE RECEPTOR 4, ERK 1 AND 2 SIGNALING AND INFLAMMATORY MEDIATOR PRODUCTION IN HUMAN B LYMPHOCYTES
O.M. Peck Palmer*1, D. Ma2, I. Tourkova1, H. Blair1. 1University of Pittsburgh School of Medicine, Pittsburgh, PA, 2University of Pittsburgh, Pittsburgh, PA
Background: The role that zinc finger protein 816 (ZNF816), thought to be a transcription factor, plays in mediating inflammatory responses to bacterial infection. Our preliminary findings demonstrated that in a cohort of adult patients hospitalized with community-acquired pneumonia, two single nucleotide polymorphisms within the ZNF816 gene were associated with differential risk of severe sepsis and inflammatory mediator production in self-reported black but not white patients. The aim of our study was to determine the function of ZNF816 in: i). the activation of Toll-like receptor 4 (TLR-4) and phosphorylation of extracellular regulated kinases 1 and 2 (ERK 1/2); and ii). the production of the inflammatory cytokines, TNFa, IL-6 and IL-10.
Methods: B-cell lines from healthy individuals from Yoruba in Ibadan, Nigeria (n=3; Coriell Institute for Medical Research, Camden, NJ), were pretreated with/without siRNA directed to ZNF816 (60 pmol/s; 5 hours pretreatment). Subsequently, the cells were stimulated with/without lipopolysaccharide (10 µg/mL) for 24 hours. Cell lysates were analyzed for ZNF816 and phosphorylated ERK 1/2. TNFa, IL-6 and IL10 concentrations were measured in the supernatant.
Results: ZNF816 expression was significantly diminished in the siRNA pretreated cells compared to the non-treated cells (p<0.01). TLR4 and ERK 1/2 expression was significantly increased in the siRNA treated cells compared to the non-treated cells (p<0.01). LPS stimulation further increased TLR4 and ERK 1/2 phosphorylation in the siRNA treated cells (p<0.05). TNFa and IL-6 production was significantly higher in both the siRNA treated and LPS stimulated cells compared to the non-treated cells (p=0.04).
Conclusions: For the first time, we demonstrated that ZNF816 functions as a repressor of the TLR4 signaling pathway. Cellular manipulation of ZNF816 signaling may be an effective target to regulate inflammatory responses.
MITOCHONDRIAL ROS ACTIVATE “PARENCHYMAL” INFLAMMATORY RESPONSE CAUSING LIVER DAMAGE
A. Weidinger1, A. Meszaros1, A. Muellebner2, I. Miller2, J. Duvigneau*2, H. Redl*1, A.V. Kozlov*1. 1L. Boltzmann Institute Exper. Clin.Traumatology, Vienna, Austria, 2University of Veterinary Medicine, Institute for Medical Chemistry, Vienna, Austria
Reactive oxygen species (ROS) are often associated with the regulation of inflammatory pathways, for instance those mediated by NF-kB, STAT 5. The aim of this study was to dissect the impact of mitochondrial ROS (mtROS) on the expression and release of inflammatory mediators (IM) in liver tissue and in blood of rats challenged with LPS. We tested effects of two mitochondria targeted antioxidants (mtAOX), mitoTEMPO and SkQ1, on the expression of IL-6, iNOS in liver and IL-1, MCP1, TNF, IL4 and the levels of nitric oxide (NO) in plasma. The levels of tissue damage markers (TDM) LDH, AST, ALT in plasma and histological examination were used to evaluate organ damage. The sampling was performed at 0, 2, 4, 8, and 16 h. TNF, iNOS, IL6 reached a maximum already 2 h after LPS challenge, substantially dropped by 4 h and further decreased till 16 h, while MCP1, IL4 reached a maximum at 4 hours. There was a clear parallelism between IM and NO levels in plasma. In contrast, TDM did not correlate with either NO or IM levels, TDM were substantially increased only by 16 h and were accompanied by the formation of focal neuroses in liver. We assumed that the major IM determined by conventional methods in blood and liver tissue are predominantly produced by circulating and liver immune cells and only a small portion produced by hepatocytes. We assumed that just this small portion causes tissue damage. To prove this assumption we developed an EPR based method allowing simultaneous determination of NO formed in extracellular and intracellular compartments. This analysis revealed that extracellular NO levels correlate with the gene expression data and with circulating NO levels. In contrast, intracellular NO did not correlate either with blood/tissue gene expression or extracellular NO levels, but it correlated with TDM. This suggests that there are two tissue pools of IM sources, immune and parenchymal, quite differently contributing to tissue damage. The first, very large one, originates from immune cells and the second, small one, occurs inside parenchymal cells. This assumption was confirmed in experiments with mtAOX. mtAOX did not influence NO levels, gene expression and TDM at 2, 4 and 8 h, but at 16 h both mtAOX completely abolished the increase in intracellular NO levels, strongly decreased the expression of iNOS, IL6 and IL-4 and also TDM levels returned nearly to control levels and no focal necroses occurred in the liver. In summary, our data suggest that, the major pool of IM/NO generated by immune cells is not as toxic as IM/NO formed in parenchymal liver cells. Activation of such “parenchymal” inflammatory response in liver cells induces tissue damage, although the extent of this response in terms of gene expression is much weaker than that of immune cells. Our data suggest that this “parenchymal” inflammatory response in liver is regulated by mtROS.
THE ROLE OF PAD4 HISTONE 3 CITRULLINATION AND NET FORMATION IN A MURINE POLYMICROBIAL SEPSIS MODEL
B. Biron1, 2, Y. Chen2, S. Pulido1, C. Chung*2, J. Reichner1, 2, A. Ayala*1, 2. 1Brown University, Providence, RI, 2Rhode Island Hospital, Providence, RI
Sepsis refers to the presence of a serious infection that correlates with systemic and uncontrolled immune activation. It is a life-threatening condition that has no FDA-approved therapeutic treatment. Over thirty clinical trials of chemical and biological modifiers designed to control the morbidity and mortality associated with the disease have failed. Thus, a better understanding of the patho-biology of sepsis is needed if we are to identify novel therapies.
Recently a novel mechanism of microbial killing by neutrophils was identified through the formation of neutrophil extracellular traps (NETs). Posttranslational histone modification plays an important role in chromatin decondensation which is regulated by citrullination. Citrullinated H3 (H3cit) has been identified as a component of NETs which is released into the extracellular space as part of the neutrophil response to infection. The conversion of arginine to citrulline residues on histones is catalyzed by peptidylarginine deiminase 4 (PAD4). Histone citrullination and NET formation are essential elements of host defense and has been shown to be necessary in innate immunity during bacterial infection In order to determine a putative role of NETs in sepsis; it is necessary to characterize the presence of PAD4 catalyzed H3cit and NET formation during the onset of sepsis as well as the effects on the immune response when this mechanism of action is blocked.
We induced sepsis in adult C57BL/6 male mice via cecal ligation and puncture (CLP). H3cit expression was measured in plasma, white blood cells, peritoneal fluid, and peritoneal cells 24 hrs post-CLP via Western blot assay. H3cit protein expression is increased in the peritoneal cavity in CLP mice compared to sham mice suggesting that H3cit could be used as a potential biomarker of NET formation in sepsis. Mice were then treated with Cl-amidine, a chemical compound known to abrogate histone citrullination and NET formation, 1hr prior to CLP. 24 hrs post-CLP, pro-and anti-inflammatory cytokine levels were measured via ELISA in various tissues and H3cit was measured in the peritoneal cavity. Administration of Cl-amidine alters the pro-inflammatory response in kidney, liver, spleen, and lung tissues as compared to untreated CLP mice while H3cit protein expression was typically suppressed.
Conclusion: This study demonstrates for the first time: (a) H3cit could be used as a biomarker for NET formation in a murine CLP model and (b) the abrogation of PAD4 alters the early immune response to sepsis. Future studies are needed to not only elucidate the role of histone 3 modification by PAD4 in the process of NET formation/neutrophil function in the septic animal, but to establish its’ impact on the morbidity/mortality encountered in polymicrobial septic challenge.
SEPSIS SUPPRESSES T CELL FUNCTION BY IMPAIRING MITOCHONDRIAL ATP PRODUCTION AND AUTOCRINE PURINERGIC SIGNALING
C. Ledderose, Y. Bao, M. Heinisch, X. Li, A.H. Lee, J. Zhang, N.I. Shapiro, W. Junger*. Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA
T cell suppression following injury contributes to the development of sepsis. The mechanisms leading to T cell suppression are poorly defined. We have previously shown that T cell activation requires mitochondrial ATP production, ATP release, and autocrine purinergic signaling via P2X receptors. Here we show that sepsis blocks these signaling mechanisms, resulting in T cell suppression.
The mitochondrial membrane potential in CD4+ T cells of sepsis patients was significantly lower than in healthy controls or in non-septic emergency department patients and was inversely correlated with sepsis severity (SOFA score; Fig. 1). This rendered T cells of sepsis patients incapable of mounting the ATP and Ca2+ signaling responses needed for proper cell activation. Incubating CD4+ T cells of healthy subjects with plasma from sepsis patients suppressed mitochondrial function and Ca2+ signaling, indicating that soluble factors cause T cell suppression. Plasma ATP levels in sepsis patients were 3-times higher than in healthy controls, and addition of ATP to T cells of healthy subjects dose- and time-dependently reduced mitochondrial function and Ca2+ signaling and suppressed T cell proliferation.
Our findings suggest that systemic ATP levels released in response to injury and inflammation interfere with mitochondrial ATP production and autocrine purinergic signaling of T cells. We conclude that targeting these mechanisms may improve T cell function and could reduce the risk of sepsis in trauma patients.
Supported in part by grants from NIGMS, NIAID, and DFG.
PD-1:B7H1 PATHWAY GENES HAVE A ROLE IN THE MODULATION OF MACROPHAGE ACTIVATION
T. Tao1, 2, X. Huang*2, C. Chung*2, A. Ayala*2. 1Department of Anesthesiology, Changhai Hospital, Second Military Medical University, Shanghai, China, 2Division of Surgical Research, Rhode Island Hospital, Brown University, Providence, RI
Background: Previous studies demonstrated that PD-1:B7H1 pathway plays a significant role in innate immune response to sepsis. Either B7-H1 or PD-1 gene deficiency could reduce CLP induced lethality by dampening the inflammatory response. In addition, the PD-1:B7H1 pathway appears to participate in the development of macrophage dysfunction. However, how the PD-1:B7H1 pathway regulates the macrophage’s Toll like receptor response and their cellular phenotype during the innate immune response is unclear.
Methods: Bone marrow derived macrophage (BMDM) were obtained from femurs and tibias of male WT and B7H1-/- mice and cultured for 7 d in the presence of recombinant M-CSF. Cells were harvested and recultured at 5×105/ml overnight before any treatment. Thioglycollate elicited peritoneal macrophage (PM) were obtained 4 days after intraperitoneal injection of 1ml sterile 4% thioglycollate. To examine the role of PD-1:B7H1 pathway on Toll like receptors activation, both BMDM and PM were treated with various concentrations of LPS (1ng/ml or 100ng/ml) for 24 hours and the levels of IL-6, TNF-α, IL-12(p70) and IL-10 in culture supernatants were assessed. To determine the impact of PD-1:B7H1 pathway in vivo, mice were challenged with 5mg/kg LPS intra-peritoneally, blood and peritoneal lavage were collected 8 hours after treatment and samples were measured for cytokines by ELISA. Meanwhile, we examined MHCII, CD80, CD86 and CD40 expression on BMDM and PM with/without LPS or IFN-γ stimulation. All statistical tests were calculated using Graphpad software.
Results: Initially, what we found is that low dose of LPS (1ng/ml) induced a higher expression of IL-6, TNF-α, IL-12(p70) and IL-10 in both in vitro cultured BMDM and thioglycollate-elicited PM derived from PD-1-/- and B7H1-/- mice compared to WT mice, but these were lost when challenged with a higher dose of LPS (100ng/ml). Alternatively, in vivo exposure to a non-lethal dose of LPS (5mg/kg LPS intra-peritoneally) induced a rapid increase in the production of inflammatory cytokines, as both PD-1-/- and B7H1-/- mice had higher cytokine levels in serum and peritoneal lavage compared with WT mice. Furthermore, B7H1-/- BMDM expressed lower surface CD80 at baseline and LPS or IFN-γ stimulation in culture, while both PD-1-/- and B7H1-/- PM had significantly higher MHCII expression than Naïve mice.
Conclusions: PD-1 and/or B7H1 gene deficiency leads to higher cytokine production in response to LPS stimulation both in vitro as well as in vivo, but has only a modest effect on LPS induced co-stimulatory cell-surface check-point proteins expression on macrophages (lower CD80 expression). Thus, we propose that one of the salutary aspects of PD-1 and/or B7H1 gene deficiency in septic mouse macrophages could be related to the preservation of this innate immune function (cytokine release).
TOTAL PARENTERAL NUTRITION LEADS TO ALTERED COMPOSITION AND METABOLIC FUNCTION OF SMALL INTESTINAL MICROBIOTA
F.R. Demehri, M.J. Koenigsknecht, V. Krishnan, J.J. Freeman, V.B. Young, D.H. Teitelbaum*. University of Michigan, Ann Arbor, MI
Purpose: Total parenteral nutrition (TPN) is associated with altered intestinal microbiota. Interestingly, metabolomic analysis has shown an increase in TPN-derived amino acids (AAs) within the GI lumen; however, the significance of these AAs is unknown. We hypothesized that TPN leads to an increase in AA-metabolizing microbiota, and that this contributes to increased bacterial translocation.
Methods: C57BL/6 mice underwent jugular cannulation and received TPN. Controls received saline and regular diet. After 6 days, mice were sacrificed and spleen, liver, ileum, and ileal lumen contents were sterilely collected. The microbial communities from these samples were assessed by 16S rRNA analysis. Using an automated, mechanical disaggregation machine, cell suspensions from whole organs were collected. Organ suspensions and ileal contents were plated under aerobic and anaerobic conditions. Bacterial isolates were grown in vitro in a nutrient panel comprised of various carbon sources to assess growth rate, as a surrogate of nutrient metabolism.
Results: Bacteria was isolated from 50% of spleens and livers of TPN mice, while no enterally-fed mice showed bacterial translocation. Translocated bacteria were identified as E. faecalis (103 CFU/g liver; 106 CFU/g spleen) and E. faecium (106 CFU/g liver; 104 CFU/g spleen) - both commensal Firmicutes implicated in TPN-associated sepsis. A single bacterial strain was identified in the liver and spleen from each mouse with positive cultures. Ileal community analysis revealed a significant decrease in Bacteroidetes and an expansion of Firmicutes and Actinobacteria. While Proteobacteria represented <1% of sequences, a 4.5-fold increase occurred with TPN.
We next measured proliferation of bacterial isolates when exposed to a panel of potential nutrients (Figure). Interestingly, in vitro growth analysis of TPN-derived bacteria demonstrated increased capacity for AA utilization versus isolates from enterally-fed mice, which showed increased growth with carbohydrates.
Conclusions: TPN leads to a shift in the small intestinal microbiome, potentially in response to an altered nutrient environment. Strains that thrive in the TPN-dependent gut demonstrate increased capacity for growth on TPN-derived AAs. Future modulation of the nutrient composition of TPN may lead to a more beneficial host intestinal microbome.
AMP PROTECTS NEURONAL CELLS BY SUSPENDING MITOCHONDRIAL ACTIVITY AND OXYGEN DEMAND
J. Zhang, X. LI, C. Ledderose, Y. Bao, J. Lu, V. Sheen, W. Junger*. Beth Israel Deaconess Medical Center, Boston, MA
Adenosine 5’-monophosphate (AMP), a naturally occurring breakdown product of ATP induces reversible hypothermia and torpor in rodents. The molecular mechanisms behind these effects and the therapeutic potential of AMP for critical use are unknown.
AMP infusion in mice dose-dependently decreased metabolism and thermogenesis. Treatment of human and mouse neurons with AMP suppressed mitochondrial membrane potential and mitochondrial activity and increased cell viability in the absence of oxygen or glucose (Fig. 1). AMP increased intracellular AMP levels, activating AMP kinase (AMPK), and blocking mTORC1 and mTORC2 signaling. These findings suggest that AMP blocks mitochondrial function by inhibiting mTORC2 induced mitochondrial activation.
We conclude that AMP treatment may help stabilize critical care patients by reducing oxygen demand and glucose consumption, leading to a transient shutdown of metabolic rate that prevents apoptosis and cell death due to hypoxia and ischemia.
Supported in part by grants from NIGMS, NIAID, National Natural Science Foundation of China, and Deutsche Forschungsgemeinschaft.
LIMB IMMOBILIZATION EXAGGERATES DISUSE ATROPHY BY ACTIVATING AUTOPHAGY-MEDIATED PROTEIN DEGRADATION
C.H. Lang*, J. Steiner, T. Speacht, A. Krause, H. Donahue. Penn State College of Medicine, Hershey, PA
Disuse muscle atrophy is a common manifestation of extended bed rest resulting from trauma and critical illness. Non-load bearing contraction can have an anabolic effect on skeletal muscle and may in the preservation of lean body mass. The purpose of the present study was to determine whether limiting muscle contraction (via casting/immobilization) would exacerbate the atrophic response of muscle produced by hindlimb suspension (HLS). Two groups of adult male C57BL/6J mice were used; ground controls (GC), and HLS where the hindquarters of the mouse achieved a 30° elevation for 14 days. The latter group had one leg unrestrained (HLS; muscle free to contract but exert no force or bear no weight) and the contralateral limb immobilized casting (HLS/C). Gastrocnemius weight was reduced 10% in HLS and 20% in HLS/C legs, compared to GC values. Accordingly, the in vivo rate of protein synthesis was reduced in both limbs of suspended mice; however, paradoxically synthesis was 15% higher in the HLS/C compared to HLS only muscle. This difference was associated with a concomitant increase in mTOR signaling (S6K1 and 4E-BP1 phosphorylation). Because of the discordant results between the measured rate of protein synthesis and muscle mass in the HLS + casted limb, we also assessed endpoints relative to protein degradation. The mRNA content for the ubiquitin-E3 ligases atrogin-1 and MuRF1 did not differ between groups. However, compared to either GC or HLS only, muscle from the HLS/C group showed increased autophagy as evidenced by 50-100% increases in LC3-II/I, Atg7 and Atg5-12 protein content. Our data indicate that limiting muscle contraction by casting exacerbates the atrophic response to disuse, likely via activation of the autophagic-lysosomal protein degradation pathway. (Supported by NSBRI MA02802 and R01GM38032).
ALTERATIONS TO THE MICROBIOME DECREASE BUTYRATE LEVELS AND CONTRIBUTE TO IMMUNE SUPPRESSION FOLLOWING BURN
J.W. Kuethe1, E. Gulbins1, D.P. Healy1, T.A. Pritts*1, D.H. Hildeman3, C.C. Caldwell*1, 2. 1University of Cincinnati, Cincinnati, OH, 2Shriners Hospitals for Children, Cincinnati, OH, 3Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
In burn centers, sepsis and secondary infections remain the leading cause of death following thermal injury, with very high mortality rates. While the gut microbiome is affected by several factors (e.g. diet, genetics, environment, etc.) it is also becoming clear that systemic inflammatory responses can drive changes to the gut microbiome. Several factors have been shown to contribute to immune suppression following thermal injury, including: increased levels of myeloid-derived suppressor cells, immune cell apoptosis, and lipid mediators. Although it remains unclear whether some of these mechanisms have an underlying common mediator, recent data suggest that the microbiome may be an underlying cause as it plays a critical role in maintaining immune homeostasis. In this current study, we observed that thermal injury induces changes to the gut microbiome. Within one day after thermal injury, we observed a significant Firmicutes/Bacteroides imbalance. This imbalance was due to a loss of bacteria in the Order Bacteroidales and an increased abundance of bacteria in the Order Clostridiales. Further, we saw a substantial and persistent loss of aerobic and anaerobic bacteria as well as levels of the short-chain fatty acid butyrate in the cecum of burn injured mice. These microbiome changes were also associated with a reduction of acid sphingomyelinase (ASM) activity in T cells. Further, we found that butyrate increased ASM activity and that ASM activity was critical to maintain T cell numbers and function. To further investigate the role of a diminished microbiome on T cells, we analyzed stool samples from Germ Reduced (GR) mice and again noted a significant decrease in butyrate and T cells. We next administered fecal material transplants (FMT) to the GR and scald injured mice and were able to partially restored T cell populations. Together, these data support our working model that thermal injury drives a loss of butyrate producing gut bacteria that favors an immune suppressed state by failing to maintain adequate ASM activity that drives decreased T cell numbers / function. Our long-term goals are to further understand the mechanisms by which inflammatory events trigger changes to the microbiome and how these changes affect hypo- and hyper-inflammatory diseases. Such changes to the gut flora following burn-injury could be a significant component of the pernicious immune condition existing in burn injured patients.
THE PREDICTIVE IMPACT OF LYSOPHOSPHATIDYLCHOLINE LEVEL FOR POSTOPERATIVE COMPLICATIONS AFTER GASTROINTESTINAL SURGERY
A. Matsuda*1, M. Miyashita*1, M. Yamada*1, T. Matsutani*2, G. Takahashi2, and E. Uchida2. 1Department of Surgery, Nippon Medical School Chiba Hokusoh Hospital, Chiba, Japan, 2Department of Surgery, Nippon Medical School, Tokyo, Japan
Rationale: Recently, inflammatory lipid mediators derived from the metabolism of membrane phospholipids have been suggested to serve as regulators of the immune response. Lysophosphatidylcholine (LPC) has an anti-inflammatory property produced via the action of the phospholipase A2 on phosphatidylcholine. The purpose of this study is to clarify the association between LPC and postoperative complications (POC) after gastrointestinal surgery.
Methods: Peripheral blood samples were collected from 43 colorectal cancer patients before, immediately after surgery and on postoperative days (POD) 1, 3, 5, and 7. Patients were divided into non-POC (n=33) and POC (n=10) groups. Plasma LPC levels were measured by ELISA.
Results: In both groups, the postoperative plasma LPC levels decreased (bottom at POD1) and then gradually recovered. The POC group had significantly lower LPC levels throughout the perioperative period than the non-POC group. The ratio of pre- and post-operative LPC level inversely correlated with IL-6 and had predictive impact for POC (cut off; 51.2%, AUC; 0.795).
Conclusions: Decreased postoperative LPC is associated with POC through an inhibition of the anti-inflammatory property.
DISRUPTION OF TNF RECEPTOR P55 OR 75 INCREASES THE SUSCEPTIBILITY OF THE HEART TO ISCHEMIA AND REPERFUSION INJURY
Y. Zhao1, L.X. Zhang3, and T.C. Zhao1, 2. 1Roger Williams Medical Center, Lincoln, RI, 2Boston University, Boston, MA, 3Medical College of Virginia, Richmond, VA
Introduction: It has been demonstrated that p38 activation-induced myocardial preconditioning effects are associated with TNF receptors to protect the heart against ischemia and reperfusion injury. However, it remains unknown whether a specific TNF-a receptor (TNFR) would be involved in the regulation of myocardial ischemia and reperfusion injury.
Hypothesis: Our goal was to investigate whether the genetic disruption of TNF-a receptor subunit p55 and /or p75 would exacerbate the magnitude of ischemic injury following an ischemic insult in mouse.
Methods: The hearts were isolated from adult wild type and p55 or p75-/- mice, and then subjected to 30 min of stabilization and myocardial ischemia followed by 30 min reperfusion in the Langendorff perfused hearts. Left ventricular function and coronary effluent were measured, and infarct size was determined by triphenyltetrazolium chloride (TTC).
Results: As compared with the wild type mice, at the end of reperfusion, disruption of TNFR p55 and/or TNFR p75 demonstrated an elevation of left ventricular end-diastolic pressure, which was associated with the decrease in left ventricular developed pressure. In addition, ablation of TNFRp55 or TNFRp75 demonstrated a diminished recovery of rate pressure products following the reperfusion. Notably, relative to TNFR p55, disruption of TNFRp75 resulted in a marked reduction in coronary effluents. There were no significant differences in recovery in left ventricular systolic pressure and heart rates. Myocardial infarct sizes by TTC staining were increased by elimination of either TNFR p55 or TNFR p75.
Conclusion: These results indicate that genetic ablation of TNFR p55 or p75 exacerbates acute myocardial ischemia and reperfusion injury.
SMALL PEPTIDE MC2 MITIGATES LIVER INJURY IN A RAT MODEL OF PARTIAL ISCHEMIA REPERFUSION
J. Frimodig1, G. Fernandez-Botran1, P. Matheson1, 2, A. Matheson1, 2, N. Garrison1, 2, and C. McClain1, 2. 1University of Louisville, Louisville, KY, 2Louisville Veterans Administration Medical Center, Louisville, KY
Ischemia reperfusion injury consists of a period of oxygen deprivation resulting from decrease or cessation of blood flow followed by return of blood flow. It is important in organ transplantation, sepsis, respiratory failure, hemorrhage, trauma, stroke, cardiac arrest, and shock. Ischemia reperfusion injury initiates an inflammatory response that is regulated by a cascade of cytokines and chemokines, many of which interact with glycosaminoglycans (GAGs) on the surface of cells and in the extracellular matrix to promote activation and recruitment of leukocytes. We hypothesized that a therapy inhibiting the interactions of cytokines/chemokines with GAGS could be beneficial in ischemia reperfusion injury. Targeting of these interactions could help prevent the recruitment and activation of leukocytes which are the primary mediators of the later phase of ischemia reperfusion injury in the liver. Our laboratory designed a synthetic octa-peptide (MC2) that suppresses the binding of recombinant IFNgamma and several other chemokines to GAGs in vitro. This peptide has been shown to have anti-inflammatory effects in several other animal models including hemorrhagic shock, peritoneal inflammation and acute liver injury.
The effects of MC2 were tested in a liver ischemia reperfusion model utilizing Sprague-Dawley rats in which the left lobe of the liver was clamped for one hour followed by 3 hours of reperfusion. Experimental groups were sham, sham plus MC2 (50 mg/kg, i.v.), ischemia reperfusion and ischemia reperfusion plus MC2. Ischemia reperfusion significantly elevated serum alanine aminotransferase (ALT), an indicator of liver damage. Animals that received the MC2 peptide at the end of the ischemic period had significantly (p < 0.05) lowered serum ALT levels compared to the ischemia reperfusion-only group. These results suggest that inhibition of cytokine/chemokine interactions with GAGs may be a useful therapy for ischemia reperfusion injury.
PROTECTIVE EFFECT OF HSF1 ON LPS-INDUCED ACUTE LUNG INJURY INVOLVES INHIBITION OF XCL-1 PRODUCTION AND CHEMOKINE RECEPTOR XCR1 EXPRESSION ON NEUTROPHIL
S. Chen1, 2, H. Lu3, G. Chen1, and X. Xiao1. 1Dept. of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, China, 2Department of Biochemistry, School of Biosciences and Technologies, Central South University, Changsha, China, 3Center for Experimental Medical Research, Third Xiangya Hospital, Changsha, China
Aim: To explore the protective effect of HSF1 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) using HSF1 knockout (HSF1-/-) mice and to determine whether the protective mechanism involves chemokine receptor XCR1 on neutrophils and its ligand XCL-1.
Methods: ALI was induced by intraperitoneal injection of 10 mg/kg LPS (Esherichia coli 0111:B4) in WT and HSF1 knockout (HSF1-/-) mice. 4hrs after LPS treatment, pulmonary microvascular permeability and Wet/Dry ratio, lung PMN accumulation were measured. Chemotaxis of bone marrow PMNs toward LPS, serum level of XCL-1, chemokine receptor XCR1 expression on the blood PMN cell surface were examined.
Results: LPS induced ALI in mice, as evidenced with a marked increase in pulmonary microvascular permeability and tissue edema, and PMN infiltration into the lungs, which were significantly exacerbated in HSF1-/- mice. In cell studies, chemotaxis of PMNs from the HSF1-/- mice was significantly higher than that from the WT mice. LPS induced higher level of serum XCL1 and greater surface expression of XCR1 on circulating PMN in HSF1-/- mice.
Conclusion: The protective role of HSF1 in LPS-induced ALI is associated with inhibition of PMN chemotaxis by suppressing XCL1 production, XCR1 receptor expression on PMN during endotoxemia.
MFG-E8 ATTENUATES IL-17 EXPRESSION IN SEPSIS BY MODULATING STAT3 ACTIVATION
C. Cen1, M. Aziz1, 2, W. Yang*1, 2, J. Nicastro1, G. Coppa1, and P. Wang*1, 2. 1Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2Center for Translational Research, The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Sepsis is a significant health concern with high morbidity and mortality. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in the regulation of inflammation. In sepsis, interleukin-17 (IL-17) produced by T-cells acts as a proinflammatory cytokine to exaggerate systemic inflammation. Although beneficial effects of MFG-E8 have been reported, its role in regulating IL-17 remains unknown. We hypothesize that MFG-E8 attenuates IL-17 expression in sepsis.
Methods: Sepsis was induced in 8-week-old male C57BL/6 wild-type (WT) mice (n=5-8 mice/group) by cecal ligation and puncture (CLP). Recombinant murine MFG-E8 (rmMFG-E8) or PBS (vehicle) was injected intravenously (25 µg/kg body weight) at time of CLP. After 10 h, IL-17 mRNA and protein expression in spleen and lung tissue was determined by qPCR and Western blotting, respectively. Expression of IL-6 in serum and tissue was determined by ELISA and qPCR, respectively. Phosphorylated STAT3 (pSTAT3), an IL-17 specific transcription factor, was determined in spleen and lung tissue by Western blotting. For in vitro studies, splenocytes from healthy WT mice were stimulated with ionomycin and phorbol myristate acetate (PMA) for 4-6 h, followed by measurement of IL-17 expression in CD4 T-cells by flow cytometry using anti-CD4 and anti-IL-17 antibodies.
Results: At 10 h after CLP, rmMFG-E8 inhibited elevated IL-17 protein levels in serum by 31%, compared to the vehicle (p<0.05, Figure). Organ injury markers in plasma were also reduced compared to the vehicle: AST by 39% (p<0.05), ALT by 43% (p<0.05), LDH by 24%. In the spleen, rmMFG-E8 reduced IL-17 mRNA and protein levels by 81% and 51%, respectively, compared to the vehicle (p<0.05). In the lung, rmMFG-E8 revealed 63% and 45% inhibition of IL-17 mRNA and protein expression, respectively (Figure). In vitro treatment of splenocytes from healthy WT mice with rmMFG-E8 showed a trend toward downregulation in ionomycin/PMA-induced IL-17 production by CD4 T-cells. IL-6, a positive regulator of IL-17, was reduced in serum by 15% (p<0.05) and lung tissue by 61% in the septic mice treated with rmMFG-E8, compared to the vehicle. The phosphorylated level of STAT3 was also downregulated in spleen by 54% (p<0.05) and lung tissue by 14% in septic mice treated with rmMFG-E8.
Conclusion: Our findings demonstrate a beneficial role of MFG-E8 in downregulating IL-17 expression via modulating STAT3 activation, implicating its potential as a novel therapeutic agent against sepsis.
“BEAT THE SHOCK CLOCK”: AN INTERPROFESSIONAL TEAM IMPROVES PEDIATRIC SEPTIC SHOCK CARE
R.E. Tuuri, M. Gehrig, C. Busch, M. Ebeling, K. Morella, and W. Russell. Medical University of South Carolina, Charleston, SC
Background and objective: Ideal care for pediatric septic shock (SS) is difficult to deliver due to knowledge gaps, subtle presentations, and resource challenges. Few high-volume pediatric emergency departments (PED)’s have demonstrated sustained improvements in care performance for SS. This shared interprofessional quality improvement team of pediatric nurses, emergency physicians, intensivists, infectious disease physicians, oncologists, pharmacists, resident physicians, and patient care technicians aimed to improve SS care in a mid-volume PED. Specifically the project aimed to reduce time to vascular access, fluid resuscitation, and antibiotics for SS.
Methods: An interprofessional team performed root cause analyses and created intensive education tools, a team care protocol, a paper-screening tool, and an order set. A comparison cohort was identified from SS diagnosis codes from the interval January 2009-June 2011. These cases were compared to patients meeting clinical criteria for SS after intervention, June 2012-June 2013. The project was ongoing during transition from paper documentation to electronic medical record (EMR) in November 2012. Non-parametric tests compared outcome measures between pre and post-intervention periods. Statistical process control charting compared outcome measures over the same time periods. Ongoing individual performance feedback and monthly team performance feedback was given to physician and nurse providers. Continued project development includes an electronic screening tool.
Results: There were 43 cases pre-intervention and 63 cases post-intervention. Median time to vascular access decreased from 37 minutes in the pre-intervention group to 24 min. post-intervention, (p=0.05). Median time to first fluid bolus decreased from 35 to 26 min., (p=0.08). Percent of boluses delivered rapidly by pressure or push pull method increased from 21% to 74% (p<0.0001). Median time to antibiotics decreased from 92 to 55 min., (p=0.02). Median to inotrope initiation was 171 min. compared to 113 min., (p=0.4). ED length of stay was 240 min compared to 217 min, (p=0.89). ICU and Hospital length of stay decreased from 3.5 and 8 days to 2 and 4 days respectively, (p=0.006) and (p<0.0001).
Conclusions: A multi-modal, interprofessional team-based QI intervention in a mid-sized PED improved the time to critical interventions for patients with SS. Challenges included continued sustained performance for quality interventions, particularly during care transitions to EMR.
ELECTRONIC HEALTH RECORD-BASED SEPSIS PROTOCOL EFFECTIVELY LOWERS TIME TO ANTIBIOTICS AND TIME TO IV FLUIDS IN EMERGENCY DEPARTMENT PATIENTS
G.E. Hayden, R.E. Tuuri, R. Scott, J. Losek, A. Blackshaw, A. Schoenling, R. Raidt, and G. Hall. Medical University of South Carolina, Charleston, SC
Introduction: We sought to evaluate the effectiveness of an emergency department (ED) sepsis work-up and treatment (SWAT) protocol in patients admitted from the ED with sepsis. We hypothesized that an Electronic Health Record (EHR)-based triage sepsis alert combined with a standardized SWAT bundle would lower door-to-antibiotic times, door-to-fluid bolus times, and mortality rates.
Methods: This was a retrospective, cross-sectional, descriptive study of patients (≥ 18 years) admitted to the hospital with an ED diagnosis of sepsis. Patients admitted through the ED with a diagnosis of sepsis before (pre-SWAT) and after (post-SWAT) implementation of the SWAT protocol were compared. The protocol involved an Electronic Health Record (EHR)-based triage sepsis alert (based on suspicion of an underlying infection and either hypotension or >2 Systemic Inflammatory Response Syndrome (SIRS) criteria), direct triage nurse to physician communication, and a SWAT activation page. Activation resulted in initiation of a SWAT bundle: standardized order sets, broad-spectrum antibiotics, immediate fluid bolus, and mobilization of an ED nurse, ED technician, pharmacist, and radiology technician. Outcome variables included door-to-antibiotic times, door-to-fluid bolus times, hospital and ICU length of stays (LOS), and mortality rates. Proportions were compared for categorical data and t-tests were used to analyze continuous data. 95% confidence intervals were calculated for differences in proportions and means. P-values <0.05 were considered statistically significant.
Results: There were 108 patients in the pre-SWAT and 130 patients in the post-SWAT group. The mean time to antibiotics was 59 minutes less in the post-SWAT group, 81 vs 139 minutes (95% CI 44 to 74, p-value < 0.001). The mean time to IV fluid bolus was 31 minutes less in the post-SWAT group, 51 vs 82 minutes (95% CI 15 to 46, p-value < 0.001). There was no significant difference in mean hospital LOS or mortality rates.
Conclusion: A sepsis work-up and treatment (SWAT) protocol emphasizing rapid mobilization of resources, standardized order sets, and early broad-spectrum antibiotics resulted in a significant decrease in the mean door-to-antibiotic time and door-to-IV fluid bolus time in an adult ED population admitted with sepsis. As improved outcomes in septic patients are associated with rapid administration of appropriate antibiotics and IV fluids, the finding of reduced door-to-antibiotic time is important. This retrospective study supports the value of an EHR-based triage sepsis alert and SWAT bundle. Identifying significant differences in mortality rates as a result of the SWAT protocol will require a larger data set.
SEPSIS INDUCES MITOCHONDRIAL ROS-DEPENDENT MITOCHONDRIAL FISSION IN THE HEART
Q.S. Zang, X. Yao, S.E. Wolf, and J. Minei. UT Southwestern Medical Center, Dallas, TX
Objective: Severe sepsis is a leading cause of death in intensive care units. Previously, using a rodent pneumonia-related sepsis model, we have demonstrated that sepsis triggers mitochondrial damage and overproduction of mtROS, which may stimulate myocardial inflammation and indirectly cause cardiac dysfunction. Mitochondrial network is regulated by fusion and fission, in order to generate new, functional organelles and to separate old, damaged ones for degradation. This study is designed to investigate how sepsis alters mitochondrial network in the heart.
Methods: Sepsis was induced in adult Sprague Dawley rats by intratracheal injection of S. pneumoniae (4x1,000,000 CFU/rat). Mitochondria-targeted vitamin E (Mito-Vit-E) was administered at 21.5 micro moles/kg by oral gavage 30 minutes after inoculation. Heart tissue was harvested, lysates prepared for western blot analysis. In addition, neonatal cardiomyocytes were isolated and cultured. Cells were challenged by LPS (100 ng/ml) (or PBS for control) for 4 hours, treated with Mito-Vit-E (1 microM) or vehicle for 2 hours, and labeled with mitochondria-specific fluorescent dye MitoTracker green for 30 minutes. Mitochondrial network was analyzed through 3-D reconstitution of confocal imaging stacks.
Results: In the heart, sepsis triggered a significant, 50% increase in the expression of mitochondrial fission protein, dynamin-related protein 1 (DRP1), detected using Western blot. This increase was suppressed by treatment with Mito-Vit-E. Because Mito-Vit-E specifically attenuates mitochondrial ROS, this result suggests that sepsis-induced change in DRP1 expression is a mtROS-dependent response. In neonatal cardiomyocytes, LPS challenge reduced the volume of individual mitochondrion by 50% but increased the total number of mitochondria per cell by 20%, indicating a loss in total mitochondrial content but an increase in fission. Similar to the data obtained in vivo, Mito-Vit-E prevented changes in mitochondrial network in cardiomyocytes in response to LPS.
Conclusion: Both in vivo and in vitro data indicate that sepsis stimulates mtROS-dependent mitochondrial fission in myocardium, suggesting that this sepsis-associated event generates aberrant mitochondria, leading to cardiac dysfunction.
SEPSIS DECREASES THE CRYPT-TO-VILLUS MIGRATION RATE OF INTESTINAL EPITHELIAL CELLS
M. Meng, Z. Liang, N. Klingensmith, J. Lyons, L. Margoles, C. Chen, and C.M. Coopersmith. Emory University, Decatur, GA
Background: The cell surface area available to perform the critical work of the intestine is dependent on the amount of cell production and loss and the speed of migration of cells from the crypt to the villus tip. Our previous work has shown that gut apoptosis is increased while proliferation is decreased in sepsis. Our understanding of migration following sepsis, however, is extremely limited.
Objectives: The purpose of this study was to evaluate changes in intestinal epithelial cell migration after a septic insult.
Methods: C57Bl/6 mice received an intraperitoneal injection of BrdU (50mg/Kg) 90 minutes prior to cecal ligation and puncture (CLP) to measure proliferating cells in S phase. Mice were then sacrificed at 6, 24, 48, and 120 hours after CLP. Villus length, migration rate of BrdU positive cells, and migration as a fraction of villus length were assayed at each time point (n=7-8/group). Migration was measured as the distance from the crypt base to the foremost BrdU labeled enterocyte. Migration distance as a fraction of villus length was measured using the distance from crypt base to the foremost BrdU labeled enterocyte divided by the distance from the crypt base to the villus tip. Data are presented as mean±SEM. Comparisons were performed using the Student’s t-test if data were found to have a Gaussian distribution. Alternatively, if data did not have a Gaussian distribution, comparisons were performed using the Mann Whitney test.
Results: Sepsis shortened the villus length as early as 6 hours after CLP. Villus length of mice from sham groups vs. CLP groups are as follows: at 6h, 541.8±11.1µm vs. 479.5±19.5µm, P<0.05; 24h, 591.9±24.4µm vs. 463.2±10.7µm, P<0.01; 48h, 571.8±31.1µm vs. 489.5±14.9µm, P<0.05; 120h, 547.5±27.1µm vs. 391.8±22.0µm, P<0.01. Sepsis also decreases cellular migration, and these changes are first detectable at 24 hours. The migration distance of sham groups vs. CLP groups are as follows: at 6h, 15.1±3.2µm vs. 10.1±2.5µm, P>0.05; 24h, 99.6±4.0µm vs. 58.7±7.9µm, P<0.01; 48h, 252.4±16.8µm vs. 160.7±19.4µm, P<0.01. After 120 hours post-CLP, most of villi in the sham group displayed no positive BrdU cells while BrdU positive cells were detectable at this time in septic mice. Sepsis also decreased migration as a fraction of villus length beginning at 24 hours (sham groups vs. CLP): 24h, 0.2±0.0 vs. 0.1±0.0, P<0.05; 48h, 0.4±0.0 vs. 0.3±0.0, P<0.05).
Conclusions: Sepsis significantly decreases the crypt-to-villus migration of intestinal epithelial cells, as measured by both absolute distance from the crypt base, and as a fraction of overall villus length. These changes are detectable by 24 hours after CLP and remain significant for up to 120 hours following onset of sepsis. Alterations in cell migration rate may contribute to the disruption of intestinal integrity during sepsis.
PLASMA MYELOPEROXIDASE- AND NEUTROPHIL ELASTASE-DNA ASSAY FOR MEASUREMENT OF NEUTROPHIL EXTRACELLULAR TRAPS
M.A. Huq, A. Tomino, M. Hashiba, M. Ando, T. Gocho, H. Miyabe, H. Kano, M. Tsuda, T. Hattori, A. Hirakawa, N. Takeyama. Fujita Health University School of Medicine, Toyoake, Japan
Aim: Release of neutrophil extracellular traps (NETs), a web like structure composed of DNA and granular proteins, such as myeloperoxidase (MPO), neutrophil elastase (NE) and others, have been identified as the important aspect of innate immunity. So far there is no reliable method to demonstrate NETs from human sample. In this study, we developed a new specific NETs assay by measuring plasma levels of MPO and NE bounded with DNA and we investigated these parameters in plasma obtained from septic patients.
Method: Samples were collected on the day of admission in the intensive care unit. Isolated plasma was digested with 0.6 μg/ml of DNAseI. Then 25 μl plasma with 75 μl PBS was added to the 96-well ELISA microplates pretreated with either anti-MPO or anti-NE (1:2000) antibodies. After overnight incubation and three washes with 0.5% triton X, 100μl of a peroxidase-labeled anti-DNA antibody was added and incubated for one and half hour. After three washes 100 μl peroxidase substance ABTS was added and absorbance was measured at 405-nm wavelength. We also measured circulating free DNA (cf-DNA) using QUBITR 2.0 Fluorometer (Life Technologies, CA) kit.
Result: Fourteen septic patients and 3 healthy volunteers were included in this study. The plasma concentrations of MPO- and NE-DNA were 118.23 ± 153.04 and 108.07 ± 117.88 in the patients with sepsis and 11.47 ± 9.12 and 14 ± 15.71 u/liter in healthy volunteers, being significantly higher in the former group (mean ± SD, p=0.01). Plasma cf-DNA of septic patients and healthy volunteers were 3379.2 ± 2325.91 and 65 ± 26 ng/ml, respectively, being significantly higher in the former group (p=0.01).
Conclusion: cf-DNA cannot differentiate between DNA that has been released by NETosis or other forms of cell death such as necrosis and apoptosis. We demonstrated that MPO and NE bounded with DNA measured by sandwich ELISA is more sensitive and specific assay for NETs formation than cf-DNA assay.
SCAVENGER RECEPTOR CLASS A (SRA/CD204) DECREASES THE INTERNALIZATION OF BACTERIA BY NEUTROPHILS
T.R. Ozment 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 this disease, effective therapies for sepsis remain elusive resulting in an overall mortality rate of around 25%. In addition, the cellular and molecular mechanisms of sepsis remain elusive. We have demonstrated that Scavenger Receptor class A I/II (SRA aka CD204) plays a detrimental role in the pathophysiology of sepsis (Ozment et al. PLoS Pathogens 10:e1002967, 2012). In the cecal ligation and puncture model of murine sepsis, mice with genetic deletion of SRA have significantly improved survival and lower bacterial burdens. We have also discovered that SRA is expressed by neutrophils and that sepsis increases neutrophil SRA expression. In order to determine the role of neutrophil SRA in clearance of bacteria during sepsis, murine bone marrow was harvested from WT and SRA knock-out (KO) mice and cultured with pHrodo Green E. coli Bioparticles (Molecular Probes) for 30, 60, and 90 m. The cells were harvested and stained with PE labeled Ly6B.2 and analyzed by flow cytometry. Whole bone marrow and neutrophil specific pHrodo Green mean fluorescence was determined. We found that genetic depletion of SRA resulted in increased uptake of the labeled E. coli. After 30 m of incubation, the pHrodo Green mean fluorescence in the SRA KO cells was increased by 22.4% in whole bone marrow and by 23.3% in neutrophils when compared to WT cells (p<0.05). At 60 m the SRA KO whole bone marrow continued to show a significant increase in pHrodo Green mean fluorescence of 16.8% (p<0.05) when compared WT. There was no significant difference in the mean fluorescence of either cell type by 90 m. These data suggest that SRA slows the internalization of E. coli by neutrophils. These data provide an intriguing explanation for the decreased bacterial burden we have reported in septic SRA KO mice. These data also suggest that targeting neutrophil SRA may represent an effective treatment modality for sepsis in the future.
LEUKOCYTES MAY ALTER THEIR MODE OF ACETYLCHOLINE RELEASE DURING SEPSIS
A.M. Downs1, T.R. Ozment*1, C. Bond2, M. Elgazzar1, D.L. Williams*1, and D.B. Hoover1. 1East Tennessee State University, Johnson City, TN, 2Ferrum College, Ferrum, VA
Recent evidence has demonstrated that acetylcholine can modulate immune responses in animal models of systemic inflammation. Acetylcholine can stimulate alpha 7 nicotinic acetylcholine (α7nACh) receptors on a variety of leukocytes to suppress the production of pro-inflammatory cytokines, such as IL-1β and TNF-α. It has been shown that leukocytes express choline acetyltransferase (ChAT), the enzyme required for the synthesis of acetylcholine. This presents the possibility that leukocytes can use acetylcholine for autocrine and paracrine suppression of inflammatory cytokines. Because cholinergic leukocytes have demonstrated anti-inflammatory functions, we investigated whether leukocytes upregulate cholinergic enzymes during sepsis. Sepsis was induced in ChAT-EGFP reporter mice by cecal ligation and puncture (CLP). Spleens, mesenteric lymph nodes, and blood were collected from mice. ChAT and vesicular acetylcholine transporter (VAChT) expression were evaluated by immunohistochemistry, qPCR, and/or flow cytometry in the spleen and lymph nodes. ChAT expression was evaluated by flow cytometry in the blood. At 3 hrs post CLP, there was no significant change in ChAT protein expression in spleen, lymph nodes, or blood. At 16hrs post CLP, there were no detectable differences in ChAT protein or mRNA expression in the spleen. However, there was a significant increase in VAChT mRNA in the spleen at 16 hrs (CLP: 2583±360 transcripts per 50ng of RNA; Sham: 1643±115; control: 1459±219; n=7 per group, P<0.05). In the blood, ChAT is primarily expressed by monocytes and T cells, while T cells, B cells, and monocytes express ChAT in the spleen. We also found that ChAT is constitutively expressed in bone marrow cells of normal, non-septic mice. ChAT appears to be restricted to myeloid lineage cells. These findings show that ChAT is more widely expressed among leukocytes and leukocyte precursors in bone marrow, lymphoid tissues and peripheral blood than previously thought. Changes in VAChT expression during sepsis suggest that leukocytes may alter their mode of acetylcholine release during sepsis. While we were unable to detect any significant changes in ChAT expression in the very early stages of sepsis, further work is needed to evaluate changes in cholinergic leukocytes during later time points. Taken together, these results suggest that cholinergic signaling could play a role in the pathogenesis of sepsis.
SERUM CYTOKINE-VARIABILITY DURING SIMULTANEOUS RADIOCHEMOTHERAPY IN PATIENTS WITH MALIGNANCIES
C. Matuschek1, S. Doerges1, W. Budach1, M. van Griensven*2, P. Gerber1, K. Orth1, and E. Boelke*1. 1University of Duesseldorf, Duesseldorf, Germany, 2Technical University, Munich, Germany
Background: The aim of this study was to investigate the fluctuation of serum levels of the common inflammatory and anti-inflammatory cytokines interleukine-10 (IL-10), interleukin-6 (IL-6) and TNF-alpha in cancer patients with different tumor entities undergoing radiation and chemotherapy as compared to healthy individuals.
Patients and Methods: A cohort of 84 patients (m=48; w=36), there of 40 patients with abdomen-basin-entities (which included rectum-, anal-, stomach, cervix-, bladder, prostate cancer) and 44 patients with head-and-neck-entities (which included larynx-, pharynx-, nose-, mouth bottom-, and esophagus cancer), was recruited to measure serum-levels before, during, and after radiochemotherapy. Fifteen healthy volunteers served as controls.
All patients received a combination of radiation, mostly between 50 and 70Gy, and chemotherapy, usually a combination of cisplatin and 5-fluorouracil. The healthy volunteers were selected to have no cancer nor any kind of inflammatory or autoimmune disease when sampled.
Results: The results show significantly higher serum base levels of all cytokines in both groups with malignancies as compared to healthy volunteers. In the cohort there were a mean of Il-6 at 29,08 pg/ml, TNFalpha at 29,91 pg/ml and Il-10 at 55,19 pg/ml, whereas we measured Il-6 at 9,61 pg/ml, TNFalpha at 13,97 pg/ml and Il-10 at 14,01 pg/ml in the healthy controls.
Additionally, we could demonstrate significant differences in the serum levels between the abdomen-basin group and the head and neck group during the application of radiochemotherapy.
Patients with abdomen-basin malignancies seem to have lower baseline levels of the pro-inflammatory cytokines IL-6 (18,82 pg/ml) and TNF-alpha (30,18 pg/ml) that increased under therapy (peak of Il-6 at 33,79 pg/ml, peak of TNFa at 35,20 pg/ml). In contrast, the immune-modulatory Il-10 showed higher baseline-levels before (67,87 pg/ml) and a significant decrease during and after therapy (bottom at 34,62 pg/ml).
Patients with head and neck entities showed elevated levels of all of the three measured cytokines with a more constant and a less significant increase or decrease following therapy. We found nearly constant values of Il-6 from peak 38,29 pg/ml to bottom 32,21 pg/ml; TNFalpha from 29,91 pg/ml to 26,91 pg/ml and Il-10 from 42,52 pg/ml to 35,17 pg/ml.
Conclusion: Patients with malignancies in abdomen-basin and head-and-neck have significant higher base line pro-as well as anti-inflammatory serum cytokine levels as compared to healthy individuals. Furthermore, patients with cancer entities in the abdomen-basin region that undergo radiation and chemotherapy show a significantly higher fluctuation of cytokine levels as compared to patients with head-and-neck cancer.
EFFECTS OF MILD INDUCED HYPOTHERMIA ON HEMODYNAMIC AND ALBUMIN TO SEPTIC SHOCK CANINE MODEL
Y. Zhang1, 2, R. Yang2, 1, S. Liu2, H. Zhou2, S. Cui2, and K. Gao2. 1Dalian Medical University, Dalian, China, 2SICU Dalian Municipal Central Hospital, Dalian, China
Objective: Mild induced hypothermia has the potential advantages to lower energy requirement and to protect cells by several pathways. However, the effects on hemodynamic and albumin in septic shock model remain unexplored.
Methods: Twelve dogs were induced septic shock by infusion E.Coli intravenously for two hours, then randomly divided into two groups: normothermia group(NH,38°C), and mild hypothermia group(MH,34°C). Anesthesia and analgesia were continuously maintained until death. Ventilation was performed through trachea cannula. Femoral artery was cannulated to monitor hemodynamic and femoral vein was cannulated to draw blood samples. Blood were pumped by hemofiltration machine to control the body temperature. Hemodynamic parameters and albumin concentration were measured at septic shock induction 0 hr, 6 hr, 12 hr, 18 hr and 24 hr. The results are expressed as mean±SD, statistical significance was defined as P<0.05.
Results: Cardiac Index (CI) of MH were higher and less intermittent than NH, however, Systemic Vascular Resistance Index (SVRI) changed reversely. No significant differences were noted in SVRI (P=0.08) and in CI (P=0.14) among the groups. Albumin were less decreased in MH than in NH, and significant difference between the groups (P=0.01, Fig 1.).
Conclusion: Mild induced hypothermia can attenuated albumin loss, and stabilize hemodynamic in septic shock model.
BURN PLUS LPS STIMULATE LIPOLYSIS BY AUGMENTING ENDOPLASMIC RETICULUM STRESS, ACTIVATING NLRP3 INFLAMMASOME AND INCREASING APOPTOSIS IN WHITE ADIPOSE TISSUE
L. Diao. Institute of Medical Science, University of Toronto, Toronto, ON, Canada
Extensively burned patients often suffer from sepsis (especially caused by pseudomonas aeruginosa), which may prolong metabolic derangement, contribute to multiple organ failure, and increase mortality. The molecular and cellular mechanisms of such infection-related metabolic derangement and organ dysfunction are unclear. We have previously shown that there is significant endoplasmic reticulum (ER) stress and lipid infiltration in the liver which contributes to liver damage and liver dysfunction after burn injury. In search of the source of lipid infiltration into the liver, we evaluated the adipose tissues and hypothesized that increased lipolysis of white adipose tissue after major burns contribute to hyperlipidemia and lipid infiltration in the liver tissue.
A two-hit rat model was established by a 60% total body surface area scald burn, followed by intraperitoneal injection of pseudomonas aeruginosa-derived lipopolysaccharide (LPS) 3 days post-burn. One day later, animals were sacrificed and liver and epididymal white adipose tissue (WAT) samples were collected for gene expression, protein analysis and histological study of NOD-like receptor, pyrin domain containing 3 (NLRP3) inflammasome activation, ER stress, apoptosis and lipid metabolism. Plasma was collected to assess circulatory triglyceride, glycerol and free fatty acid (FFA).
Our results showed that burn injury and LPS injection augmented ER stress and induced inflammasome activation in adipose tissue. Moreover, increased apoptosis was observed in the WAT. Collectively, these augment lipolysis in WAT. Increased lipolysis in WAT positively correlated with hepatic lipid infiltration, suggesting that lipid infiltration into the liver is due to increased lipolysis in WAT.
In conclusion, inflammasome activation and ER stress augment apoptosis in adipocytes and induce lipolysis in WAT after severe burn injury.
SERUM AMYLOID A INDUCES HMGB1 RELEASE IN A TLR4/RAGE- AND PKR-DEPENDENT FASHION
W. Li*1, S. Zhu*1, J. D’Amore2, J. D’Angelo2, J. Li1, H. Yang*1, P. Wang*1, K.J. Tracey*1, A. Sama2, and H. Wang*1. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Department of Emergency Medicine, North Shore University Hospital, Manhasset, NY
Background: The high mortality of sepsis is partly mediated by innate immune cells (e.g., macrophages/monocytes) which sequentially release early (e.g., TNF and IFN-γ) and late (e.g., HMGB1) pro-inflammatory cytokines.
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 whose HMGB1 levels paralleled with clinical scores, the anti-HMGB1 IgGs that paradoxically cross-reacted with human serum amyloid A (SAA), but not SAA1, coincidently detected a 12 kDa protein in the serum of a few septic patients, suggesting a restricted expression of SAA in a subset of patients. Despite the shared high amino acid sequence homology, human SAA, but not SAA1, effectively induced HMGB1 release in macrophage and monocyte cultures. However, the co-current disruption of both TLR4 and RAGE receptors completely impaired SAA-induced up-regulation of PKR and RIP3, and consequently abolished SAA-induced HMGB1 release. Pharmacological inhibition of PKR phosphorylation partially blocked SAA-induced HMGB1 release, reinforcing a possible role of PKR kinase activities in necroptosis and HMGB1 release. In animal models of lethal endotoxemia and sepsis (induced by cecal ligation and puncture, CLP), recombinant SAA dramatically exacerbated LPS-induced animal lethality, whereas repetitive administration of SAA-neutralizing IgGs (at + 0.5 and +24 h after LPS; or +6, 24, and 48 h post CLP) significantly improved animal survival rates.
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)].
VISUALIZATION OF ATP IN SEPSIS PATIENTS’ PMNS WITH ORGANELLE-LOCALIZABLE FLUORESCENT CHEMOSENSORS
K. Sueyoshi1, Y. Sumi*1, Y. Inoue1, Y. Kuroda1, K. Ishii2, H. Nakayama3, K. Iwabuchi3, H. Shigemitsu4, I. Hamachi4, and H. Tanaka1. 1Juntendo University, Urayasu Hospital, Urayasu, Japan, 2Riken Advanced Science Institute, Wako, Japan, 3Institute for Environmental and Gender Specific Medicine, Juntendo University, Urayasu, Japan, 4Graduate School of Engineering, Kyoto University, Kyoto, 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) were developed. PMAP-1 can localize on plasma membrane surface and MitoAP-1 can localize in mitochondria.
Objective: To evaluate the ATP localization using these chemosensors in PMNs of sepsis patients.
Methods: 12 sepsis patients admitted to our hospital between October 2013 and December 2014 were enrolled (age: 70±8 yrs. old, APACHEII: 22.3±8.1, 8 male, 4 female). PMNs were isolated from the whole blood of these sepsis patients and healthy controls (HC) (n=8). Mean fluorescence intensity (MFI) of PMAP-1 and MitoAP-1 of PMNs were evaluated using flow cytometry. CD11b expression on PMNs was also evaluated.
Results: MFI of PMAP-1 and MitoAP-1 in sepsis patients were significantly higher than HC (PMAP-1: 172.8±52.9 vs. 121.6±21.6, MitoAP-1: 441.5±153.7 vs. 282.0±54.7; p<0.05). CD11b expression in sepsis patients was also higher than HC (376.5±61.6 vs. 287.0±87.3; p<0.05). MFI of PMAP-1 and CD11b expression on day 3-4 decreased significantly compared to day 0-1, whereas MitoAP-1 kept high level (PMAP-1: 172.8±52.9 vs. 118.0±23.9, CD11b: 376.5±61.6 vs. 271.8±82.0; p<0.01, MitoAP-1: 441.5±153.7 vs. 474.4±128.3; n.s.). MFI of PMAP-1 showed significant positive correlation with WBC and CRP (Fig. A&B).
Conclusion: The higher MFI of PMAP-1 and MitoAP-1 in sepsis patients suggests the pivotal role of ATP for the activation of PMNs. PMAP-1 could be an activation marker in sepsis. The difference in serial change of ATP between mitochondria and cell surface suggests the various role of ATP in these regions. This study would contribute to understanding the dynamics of ATP in PMNs of sepsis patients.
SELECTIVE POSITIVE ALLOSTERIC MODULATION OF THE M1 MUSCARINIC ACETYLCHOLINE RECEPTOR IMPROVES SURVIVAL IN LETHAL MURINE ENDOTOXEMIA AND SEVERE SEPSIS
M. Dancho1, M. Ochani*1, S.S. Chavan1, Y. Al-abed*2, N.M. Nathanson3, K.J. Tracey*1, and V.A. Pavlov*1. 1Center for Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY, 2Center for Molecular Innovation, The Feinstein Institute for Medical Research, Manhasset, NY, 3Department of Pharmacology, University of Washington, Seattle, WA
Activation of brain M1 muscarinic acetylcholine receptor (M1 mAChR)-mediated cholinergic signaling regulates peripheral inflammation through vagus nerve-based neural pathways (Nat Rev Endo, 2012, 8:743). Here, we describe a role of a very selective, centrally-acting M1 mAChR positive allosteric modulator - benzyl quinolone carboxylic acid (BQCA) in modulating inflammatory phenotype and survival in murine models of endotoxemia and sepsis. Initial testing in endotoxemic BALB/c mice defined dose-dependent suppression of serum pro-inflammatory cytokines following BQCA (5-20 mg/kg, i.p.) administration. Single injection of BQCA (20 mg/kg, i.p.) (as compared to vehicle) 1h prior to LPS (8 mg/kg, i.p.) administration, significantly suppressed serum TNF (75%, P<0.005) and increased IL-10 (1.8-fold, P<0.05) in WT mice, but failed to exert these effects in M1 mAChR KO mice. In addition, BQCA (20 mg/kg, i.p.) significantly improved survival in WT endotoxemic mice only (88.2% vs 31.2%, P<0.03). Furthermore, BQCA (10 mg/kg, i.p.) treatment (as compared to vehicle treatment) at 24h, 30h, and 46 h after the onset of cecal ligation and puncture (CLP)-induced sepsis, significantly improved survival in WT mice (85.7% vs 50%, P<0.05), and failed to alter survival in M1 mAChR KO mice. In addition, a significant difference was observed between the survival rates of WT mice as compared to M1 mAChR KO mice subjected to CLP (63.3% vs 23%, P<0.03). Our current results indicate the potential of selective positive allosteric modulation of the M1mAChR in the treatment of multifactorial inflammatory conditions, including sepsis. This study was funded in part by NIH/NIGMS.
APOE AND NKT CELLS MEDIATE BACTERIAL CLEARANCE IN THE LYMPHATIC SYSTEM
S. Kasravi, M. Lee, H. Woldesemayat, M. Tufaga, and H. Harris. University of California San Francisco, San Francisco, CA
Introduction: Apolipoprotein (apoE) E has a modulatory effect on inflammatory signaling following infection. Previous work in our laboratory has shown that apoE has a significant role in the relative populations and pro-inflammatory action of hepatic lymphocytes and monocytes, potentially through an antigen presentation relationship with Natural Killer T (NKT) cells. Meanwhile, other studies have shown that innate host defense systems in lymph nodes (LNs) consisting of lymphocytes and macrophages act as a block against the systemic spread of pathogens via the lymphatic system. Therefore we sought to investigate the influence of apoE and NKT cells in bacterial clearance in the lymph node.
Objective: To gain an understanding of the interaction between apoE and innate host defense in the lymph node.
Methods:Pseudomonas aeruginosa was injected into the hock of C57BL6 wildtype mice and mice genetically engineered to express 2-5% of wildtype levels of serum apoE and 3% of wildtype levels of NKT cells (HypoE-CD1d). Popliteal lymph nodes from injected mice were extracted and plated at various time points following injection, and colony forming units (CFUs) were counted 24 hours later. Data was compared via t test for two-tailed p-values.
Results: 15 minutes post injection with P. aeruginosa, popliteal lymph nodes of mice with reduced levels of apoE and NKT cells contained significantly more colony forming units than those of wildtype mice (1169 CFU vs. 366 CFU respectively, p = 0.02). However, bacteria levels in HypoE-CD1d mice diminished rapidly and at 2.5 hours post-injection, both strains of mice reached statistically equivalent CFUs. By 24 hours, there were no more detectable CFUs in the lymph nodes of either strain (See figure).
Conclusion: Low levels of apoE and NKT cells correlates with faster bacterial clearance from the lymph system, perhaps through enhanced antigen recognition and bacterial killing. Given that our previous survival studies have demonstrated that HypoE-CD1d mice are virtually immune to mortality following induction of sepsis via cecal ligation and puncture, it is possible that this survival advantage can be attributed to innate immunity at the lymphatic level.
COMPARISON OF SHEEP VS. HUMAN BLOOD GENOMIC RESPONSES TO TLR4 AGONISTS
P. Enkhbaatar*1, C. Nelson1, J. Salsbury1, J. Carmical2, K. Torres3, D. Herndon*1, D. Prough*1, and E. Sherwood*4. 1University of Texas Medical Branch, Galveston, TX, 2Baylor College of Medicine, Houston, TX, 3GenUs BioSystems, Northbrook, IL, 4Vanderbilt University Medical Center, Nashville, TN
Background: Adequate animal models are critically important for successful translation of basic science discoveries to the clinical practice. The goal of this study was to compare genomic responses of human and sheep blood to the TLR4 agonists lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA).
Methods: Venous blood, withdrawn from six healthy human adult volunteers (∼ 28 years old) and six healthy adult female sheep (∼3 years old), was mixed with 30 µL of PBS, LPS (1µg/mL) or MPLA (10µg/mL) and incubated in room temperature for 90 minutes on a rolling rocker. After incubation, 2.5 mL of blood was transferred to Paxgene Blood RNA tubes. The genomic analysis was performed at GenUS BioSystems. Quality and quantity of the Total RNA sample was assessed and gene expression was determined using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip. Agilent gene expression microarrays were scanned with a G2565 Microarray Scanner. Data was analyzed with GeneSpring GX software. Differentially expressed genes were identified with Paired T-test.
Results: 11,431 human and 4,992 sheep probes were detected above background. Among them 1,029 human and 175 sheep genes were differentially expressed at a stringency of 1.5-fold change (p<0.05). Out of 175 sheep genes, 54 had a known human ortholog. Among those genes, 22 had > 1.5-fold changes in human samples. Genes of major inflammatory mediators, such as IL-1, 6 and 8, TNF alpha, NF-kappaB, ETS2, PTGS2, PTX3, CXCL16, KYNU (kynureninase), and CLEC4E were similarly (>2-fold) upregulated by LPS and MPLA in both species.
Conclusion: The genomic responses of peripheral blood to LPS and MPLA in sheep are quite similar to those observed in humans. The ovine model can potentially be used for translational studies to mimic human inflammatory diseases, i.e., sepsis and septic shock.
Support: GM097480, GM104306, SHC84050
MUTATION OF ENTEROCOCCAL BINDING SUBSTANCE GENES INCREASES ENTEROCOCCUS FAECALIS INTERNALIZATION BY ENTEROCYTES
M.J. Henry-Stanley, G. Dunny, M. Sivertson, and C. Wells. University of Minnesota, Minneapolis, MN
E. faecalis is an antibiotic-resistant, opportunistic, nosocomial, and community-acquired pathogen. In ICU patients, intestinal colonization with E. faecalis may lead to bacterial translocation, sepsis, and multiple organ failure. Recent studies have provided a role for cell wall lipoteichoic acid (LTA) as the adhesin responsible for E. faecalis interactions with intestinal epithelial cells. Specifically, glycolipid moieties in LTA are thought to adhere to heparan sulfate (HS), a cell-surface heparin-like glycosaminoglycan found on epithelial cells. LTA may adhere to host cells in soluble form (acting as a molecular bridge between the bacterium and enterocyte) or directly, via cell surface LTA binding to HS. E. faecalis INY3000, INY3039, INY3048, and OG1SSp:ebsC are mutant strains and OG1SSp is wild-type. INY3000, INY3048, and INY3039 were generated by transposon mutagenesis and express LTA bearing shortened polyglycerol phosphate chains. In addition, INY3000 expresses shortened fatty-acid chains. OG1SSp:ebsC is an enterococcal binding substance gene deletion mutant. Studies indicate that LTA likely represents an important component of enterococcal binding substance. Gentamicin protection assays were performed in the presence or absence of heparin to investigate if alterations in enterococcal binding substance abolish LTA-dependent binding to enterocytes and to determine if binding substance mutations affect LTA interactions with HS. Invasion of HT-29 enterocytes by OG1SSp:ebsC and INY3000 appeared increased compared to wild-type OG1SSp (*P<0.01, ANOVA, n≥20). In addition, in contrast to wild-type OG1SSp, internalization of OG1SSp:ebsC, INY3000, and INY3048, by HT-29 cells was inhibited by heparin (#P<0.01, +P<.05, unpaired t-test, n≥19). These data indicate that mutations in enterococcal binding substance increase HS-dependent invasion of enterocytes by E. faecalis, thereby providing new insight into the mechanism behind E. faecalis-epithelial cell interactions.
MFG-E8-DERIVED PEPTIDE INHIBITS ADHESION AND MIGRATION OF NEUTROPHILS TO CYTOKINE-STIMULATED ENDOTHELIAL CELLS
Y. Hirano1, W. Yang*1, 2, M. Aziz1, F. Zhang1, B. Sherry*1, and P. Wang*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Dept of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY
Introduction: Excessive neutrophil infiltration is a hallmark of acute lung injury. Neutrophil migration into tissues involves rolling, adhesion, and extravasation from the vascular endothelial linings. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) has an anti-inflammatory activity. We have recently identified that human MFG-E8-derived short peptide 68 (MSP68) flanking its RGD motif reduces the neutrophil infiltration into tissues in septic animals. Here, we hypothesized that MSP68 could prevent neutrophils from interacting with activated endothelial cells for infiltration.
Methods: Bone marrow-derived neutrophils (BMDNs) were isolated from femoral bones of C57BL/6 mice, followed by Percoll gradient centrifugation, which reached > 90% purity as determined by flow cytometry with CD11b/Gr-1 staining. BMDNs stimulated with 10 nM fMLP were labeled with fluorescence dye Calcein-AM and subjected to cell adhesion and migration (using a transwell method) assays in the presence of MSP68 (ranging from 0 to 50 nM). The expression of activated integrin β1 protein and activation of p38 in BMDNs were determined by flow cytometry and Western blotting, respectively.
Results: MSP68 inhibited BMDN adhesion to TNF-α-stimulated human pulmonary arterial endothelial cells in a dose-dependent manner, with the maximum of 43% inhibition at a dose of 50 nM. Similarly, MSP68 reduced BMDN adhesion to endothelial VCAM-1-coated plate by 61%, while it did not affect BMDN adhesion to ICAM-1-coated plate. MSP68 directly bound to integrin α4β1, a VCAM-1 receptor, with apparent dissociation constant (Kd) of 1.53×10-7 M as determined by surface plasmon resonance analysis. The expression of activated integrin β1 on the surface of BMDNs was not affected by MSP68. BMDNs treated with MSP68 significantly inhibited their migration toward the chemoattractants, fMLP, MIP-2 and complement fragment C5a by 67%, 50% and 89%, respectively (P < 0.05). Furthermore, MSP68 inhibited the phosphorylation of p38 in BMDNs activated by fMLP in a time-course study (Figure).
Conclusions: MSP68 effectively prevents neutrophil adhesion to the activated endothelial cells by interfering with the binding of integrin α4β1 to VCAM-1 and thereby inhibits neutrophil migration toward various chemoattractants. Thus, the RGD motif in MFG-E8 accounts for its inhibition on neutrophil infiltration observed in sepsis.
MFG-E8-DERIVED PEPTIDE ATTENUATES ORGAN INJURY AND IMPROVES SURVIVAL IN SEPSIS
W. Yang*1, 2, F. Zhang2, A. Sharma2, S. Matsuo2, Z. Wang2, H. Wang*2, and P. Wang*2, 1. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Sepsis involves overwhelming inflammatory responses that can lead to multiple organ dysfunction and ultimately death. MFG-E8 is a secretory protein found to have multiple biological activities against autoimmune and inflammatory diseases. MFG-E8 contains an Arg-Gly-Asp (RGD) motif involved in cell-cell and cell-matrix interactions. In sepsis, excessive neutrophils migration through endothelial cells and matrix to sites of inflammation can result in organ damage. We hypothesized that MFG-E8-derived short peptides (MSP) flanking its RGD motif could provide protection against organ injury in sepsis.
Methods: The differentiated human neutrophil-like HL60 cells were incubated with a series of peptides flanking the RGD motif of human MFG-E8 and then added to a fibronectin-coated plate for 2 h, followed by the wash for cell adhesion assay. Male C57BL/6 mice (20-25 g) were subjected to cecum ligation and puncture (CLP). MSP68 (1 mg/kg body weight) or saline (vehicle) was injected intravenously at 2 h post-CLP. Blood and tissues were collected at 20 h after CLP for various measurements. Neutrophils (Gr1+CD11b+) in collected organs were identified by flow cytometry.
Results: After screening, peptide MSP68 (VRGDV) had the highest inhibition of HL60 cell adhesion to fibronectin by 54%. MSP68 treatment significantly decreased serum levels of organ injury marker AST and the proinflammatory cytokine IL-6 after CLP (Table). MSP68 improved the integrity of microscopic lung architectures after CLP, judged by H&E staining. The number of TUNEL-positive cells and expression of cleaved caspase-3 in the lungs of MSP68-treated mice were reduced by 64% and 18%, respectively, compared to vehicle (P < 0.05). MSP68 also significantly reduced neutrophil infiltration and MPO activity in the lungs and liver after CLP (Table). Moreover, the number of bacteria translocated to mesenteric lymph nodes was decreased by 57% with MSP68 treatment. Finally, the 10-day survival rate was increased from 26% in the vehicle group to 58% in the MSP68-treated group (P < 0.05, n=24/group).
Conclusions: MSP68 effectively inhibits excessive neutrophils infiltrating to organs, leading to attenuation of organ injury and improved survival in septic mice. Thus, MSP68 may be a potential therapeutic agent for treating sepsis.
AGE-DEPENDENT EFFECTS OF PHARMACOLOGICAL ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE (AMPK) ON SEPSIS-INDUCED CARDIAC DYSFUNCTION IN MICE
Y. Inata1, P. Hake1, G. Piraino1, M. O’Connor1, C. Schulte2, V. Moore2, J. James2, and B. Zingarelli*1. 1Division of Critical Care Medicine Cincinnati Children’s Hospital, Cincinnati, OH, 2The Heart Institute, Cincinnati Children’s Hospital, Cincinnati, OH
Age is an independent risk factor of high rate of multiple organ failure and pour outcome in septic patients. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis and mitochondrial biogenesis, which is activated in response to low energy levels during mitochondrial dysfunction. Considering the central role of AMPK in metabolic signaling, we investigated whether pharmacological activation of this kinase by AICAR (5-amino-4-imidazolecarboxamide riboside-1-β-D-ribofuranoside) might ameliorate sepsis-induced cardiac dysfunction. Polymicrobial sepsis was induced in anesthetized C57BL/6 male young mice (2-3 months old) and middle-age mice (11-13 months old) by cecal ligation and puncture (CLP). Mice were treated with vehicle or AICAR (500 mg/kg intraperitoneally) at 1h after CLP. Cardiac function by echocardiographic analysis was assessed at 18h after CLP. By echocardiographic analysis (Table), young vehicle-treated mice exhibited a significant decrease in systolic function as evidenced by lower fractional shortening and ejection fraction at 18h after sepsis, when compared to baseline values before the CLP procedure. Treatment with AICAR significantly improved both fractional shortening and ejection fraction when compared to vehicle treatment. Middle-age vehicle-treated mice exhibited a significant lower baseline systolic function before sepsis when compared to baseline values of young mice. However, there was no change in cardiac function after sepsis in vehicle or AICAR treated middle-age mice. At transmission electron microscopy, examination of cardiomyocytes of young vehicle-treated mice demonstrated alterations of mitochondria, characterized by disruption of cristae and translucent matrix. In young AICAR-treated mice, there were mitochondria with fusion/fission morphology, indicating active processes of mitochondrial repair. At Western blotting analysis in heart samples of young mice, the beneficial effects of AICAR were associated with increased nuclear AMPK phosphorylation and expression of peroxisome proliferator-activated receptor-γ co-activator α (PGC-1α), a major regulator of mitochondrial function and biogenesis. Our data suggest that AICAR improves cardiac function through activation of AMPK-dependent metabolic repair mechanisms. However, these cardio-protective effects of AMPK activation are age-dependent and compromised in middle-age animals.
THE EFFECT OF POST-TREATMENT WITH COMBINATION OF CGRP RECEPTOR ANTAGONISTAND NK1 RECEPTOR ANTAGONIST IN OVINE BURN AND SMOKE INHALATION INJURY
K. Ihara1, S. Fukuda1, E. Lopez*1, R.A. Cox1, 2, D. Herndon1, 2, D. Prough*1, P. Enkhbaatar*1, and H.K. Hawkins*1. 1The University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX
Background: Combined burn and smoke inhalation injury leads to significant airway hyperemia and obstruction, enhanced pulmonary fluid extravasation and severely impaired pulmonary function. Calcitonin gene-related peptide (CGRP) and substance P (SP) are neuropeptide mediators of the responses to the injury. Previously, we reported that pre-treatment with a CGRP antagonist (BIBN4096BS) significantly improved pulmonary function following burn and smoke inhalation injury in an ovine model of acute lung injury (ALI). In the present study, we tested the hypothesis that post-treatment with combined BIBN4096BS and NK1 receptor (activated by SP) antagonist (maropitant) effectively ameliorates the degree of ALI in the same model.
Methods: Surgically prepared sheep received a third-degree 40% total body surface area skin burn and 48 breaths of cooled cotton smoke (<40°C) inhalation under anesthesia and analgesia. After the injury, sheep were randomly allocated to two groups: 1) control, injured and treated with saline only, n=7; 2) injured and treated with the test drugs, n=6. All sheep were placed on a ventilator, fluid resuscitated with Parkland formula, and monitored for 48 h in a conscious state. Pulmonary gas exchange and transvascular fluid flux (lung lymph flow) were determined every 6 h. An intravenous bolus of BIBN4096BS (32 µg/kg) was given 1 h post-injury and was followed by a continuous infusion at a dose of 6.4 µg/kg/hour. maropitant (1 mg/kg) was subcutaneously injected 1h post-injury as well.
Results: PaO2/FiO2 ratio, an index of pulmonary gas exchange, was significantly higher in the treated group at 24 h post-injury (334±59 and 175±46). Plasma protein was also significantly higher in the treated group at 24h (4.9±0.1 and 3.9±0.3), 30 h (4.9±0.2 and 4.0±0.3), and 36 h (5.0±0.3 and 3.8±0.2) post-injury compared to untreated control animals. Lung lymph flow, an index of pulmonary transvascular fluid flux, tended to be attenuated by combined treatment, but no statistically significant difference was found between groups. Treatment with BIBN4096BS and maropitant was not associated with adverse effects.
Conclusions: Our data indicate that CGRP and substance P are critically involved in the pathophysiology of airway and pulmonary morbidity during combined burn and smoke inhalation injury. The fact that pre-treatment had more pronounced effects than delayed post-injury treatment suggests that CGRP and substance P may be important mediators only during the early phase after burn and smoke injury.
Support: SCH84050 and SHC85200
CHARACTERIZATION OF MICROVESICLES IN RAT MODEL OF TRAUMA AND HEMORRHAGE
B.K. Parida1, N. Prat2, R.K. Montgomery1, X. Wu1, D.N. Darlington1, and A.P. Cap*1. 1US Army Institute of Surgical Research, San Antonio, TX, 2French Armed Forces Institute of Biomedical Research (IRBA), Cedex, France
Introduction: Acute traumatic coagulopathy (ATC) develops after severe injury and hemorrhage and is clinically defined by elevated prothrombin time (PT). Microvesicles (MV) are implicated in the pathophysiology of ATC.
Objective: The purpose of this study was to characterize MV in an established rat model of ATC that demonstrates elevated PT after polytrauma and hemorrhage.
Methods: Polytrauma in anesthetized Sprague-Dawley rats was induced by damaging small intestines, liver skeletal muscle, and femur as per our previously published methods. Rats were hemorrhaged 40% of their estimated blood volume. No resuscitation was given. Venous blood samples were taken at baseline (before trauma), TP1 (15 min after polytrauma) and TP2 (after hemorrhage, 30 minutes after TP1 and 45min after polytrauma). MV were analyzed in citrated whole blood (WB) samples within 15 minutes of collection. WB was incubated with cocktails of fluorescently labeled antibodies against platelets (APC-CD42d) P-Selectin(PE-CD62P), endothelial cell marker VE cadherin (PerCP-Cy5.5-CD144), leukocytes (V450-CD45), red blood cells (PerCP-Cy5.5-erythroid cell marker) and phosphatidyl serine(PS) (FITC-lactadherin). Isotype matched Ig controls were used to set gate in flow data analysis. Activation status of platelets was monitored by measurement of PS and P-selectin expression on platelet surface. Phorbol myristyl acetate (PMA) was used as positive control for platelet activation. BD TruCount tubes were used to measure absolute concentration of MV and platelets. Data were acquired on a BD FACS Canto II cytometer and analyzed using BD FACS Diva software.
Results/Conclusion: Animals achieved wound hemostasis by TP1. Other than the protocol defined controlled hemorrhage, no other bleeding occurred. There was no significant difference in either the total MV concentration or any of the seven types of measured MV when we compared BL to TP1 and TP2. Also, we did not observe any significant difference in platelet count or activation as assessed by PS or P-selectin expression. These results suggest that activated platelets and prothrombotic MV may have been consumed in clot formation or cleared by the liver or spleen. Further studies will assess the effect of prolonged hemorrhagic shock on MV formation and platelet activation.
Acknowledgements: This work was funded by MRMC.
ANALYSIS OF CHARACTERISTICS AND OUTCOMES WITH PROPRANOLOL USE IN SEVERELY BURNED ADULTS
S.A. Finch, M.R. Saeman, M. Evans, A. Burris, and S.E. Wolf. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Severely burned patients experience a hypermetabolic state characterized by a hyperdynamic circulatory response with increased catabolism causing higher energy expenditures. Propranolol has been shown to mitigate these effects in pediatric burns and decrease energy requirements. However, the effects of propranolol in adult burns are unknown, and its use is not universal. Our study focuses on elucidating differences in adult burn patients who are prescribed propranolol versus those who are not.
Methods: We conducted a retrospective case-control study at a regional burn center between 2011 and 2012. Two groups were selected based on propranolol versus non-propranolol use during hospitalization. Selection criteria included: greater than 20% total body surface area (TBSA) burn, older than 18 years, and post-burn survival greater than 2 days. We compared gender, age, race, TBSA burn, burn mechanism, inhalational injury, hospital days, ventilator days, mortality, Charlson comorbidity index, and prior beta-blocker use. Statistical analysis was conducted using SigmaPlot and SPSS with chi-square, t-test, Mann-Whitney rank sum test, and Fisher exact test, where appropriate.
Results: We identified 60 subjects, 30 in each group. The groups showed no significant difference in gender, race, inhalation injury, burn mechanism, mortality, Charlson comorbidity index, or prior prescription of beta-blockers. There was a significant difference in age (p = 0.05) with the mean age of 39 (± 3 SEM) years in the propranolol group and 49 (± 4 SEM) years in the non-propranolol group. We found a significant difference in the median TBSA burn (p <0.01) with 31% (27, 60 [IQR]) and 27.5% (22, 34 [IQR]) in propranolol and non-propranolol groups, respectively. Median hospitalization of 24 days (20, 51 [IQR]) was longer in the propranolol group (p = 0.05) versus 19 days (10, 34 [IQR]) in the non-propranolol group. Median ventilator duration of 4 days (0, 10 [IQR]) was higher in the propranolol group (p = 0.04) versus 1.5 days (0, 3 [IQR]) in the non-propranolol group.
Conclusion: We found in our uncontrolled study that severely burned patients prescribed propranolol were younger with larger burns. They had longer hospitalizations and more ventilator days versus those not prescribed propranolol. We did not find a difference in mortality between groups. Identifying these trends gives insight into propranolol use patterns. Future, randomized control trials are needed to determine if propranolol administration results in decreased catabolism and better outcomes in severely burned adults.
TEMPORAL CHANGES IN GENOMIC STRUCTURAL LANDSCAPES OF WHITE BLOOD CELLS FROM A BURN PATIENT
K. Lee1, 2, T. Green2, D.G. Greenhalgh*1, 2, and K. Cho*1, 2. 1University of California, Davis, Sacramento, CA, 2Shriners Hospitals for Children, Sacramento, CA
Currently, human pathophysiology is explained in the context of gene functions within a static genome platform. Thus far, gene function/polymorphism-based investigative attempts to decode the mechanisms underlying polymorphic disease phenotypes of burn patients have been somewhat inconclusive or unsuccessful. Conventional genes comprise ∼1.2% of the human genome, and the vast majority of the residual genome is occupied by a plethora of transposable repetitive elements (TREs). TREs have the potential for dynamically shaping the genomes’ configuration through “copy and paste” and/or “cut and paste” functions. Recently, we identified the potential of injury-associated human endogenous retroviruses (HERVs), a TRE family, for modulating inflammatory cytokines, implicating HERVs as novel genetic elements which play a role in individual-specific disease phenotypes and clinical outcomes of burn patients. We hypothesize that burn injury-elicited HERV activities temporally and spatially alter the structural landscape of patients’ genomes. In this study, we examined whether genomic landscapes, with regard to the positions of HERVs, are altered in a set of 16 consecutive blood samples (collected biweekly for a period of 7 months) from a patient with a 91% total body surface area burn. Genomic DNAs isolated from buffy coats were subjected to inverse-PCR analyses using HERVs’ long terminal repeat sequences as probes for position mapping followed by identification of structural differences among the DNA samples from different time points. Interestingly, DNA samples from each post-burn time point had a unique genomic HERV landscape, indicating that there are temporal alterations of genome landscape within the 16 blood samples of a single patient. This finding regarding temporal changes in genomic landscape in a burn patient can be translated into three key questions: 1) what are the impacts of the alterations in genome structure due to burn-elicited HERV activity?, 2) which HERVs contribute to the temporal alterations in genome landscapes?, and 3) what are the molecules which modulate the post-burn temporal activity of HERVs in patients’ blood cells?
GLUCOCORTICOID RECEPTOR CHANGES IN RESPONSE TO BURN STRESS
D.G. Greenhalgh*1, K. Cho*1, S. Leventhal1, T. Green1, D. Lim1, N. Tran2, S. Sen1, and T. Palmieri*1. 1Shriners Hospitals for Children Northern California, Sacramento, CA, 2University of California, Davis Medical Center, Sacramento, CA
Introduction: There is significant variability in the host response to a major burn injury. Structural variability of the human glucocorticoid receptor (hGR) has been found to augment or inhibit the patient response to burn stress. Burn stressors may include the initial injury itself, subsequent surgery or infections. In a survey of healthy volunteers, wide variations in the structure of hGR were found to be associated with altered responses to steroids. Since glucocorticoids are required after injury, we hypothesize that the expression of hGR splice variants are determined by different stressors experienced during recovery. In this study, hGR isoforms from a burn patient were examined to determine its structure variants throughout his hospital stay.
Methods: Under IRB approval, mRNA was isolated from the blood of a 46 year old man with 91% burns every 2 weeks during his 263 day stay and 4 months after discharge. RT-PCR of various exon to exon combinations was performed to identify hGR splice variants using hGR-alpha as a reference. Changes in structure were correlated with his clinical status at the time. Cytokine expression was determined by real-time PCR.
Results: There were significant variations in the hGR structure throughout the hospital stay. At one month the patient developed Klebsiella sepsis. At this time there was a corresponding increased expression of a range of hGR splice variants (including the loss of exon 5, retained intron H and early hGR termination) with the potential for inhibition of function. At the same time, there was increased COX-2 mRNA expression. During other infections (Fusarium wound invasion, Stenotrophomonas and Chryseobacterium blood stream infections) other significant hGR splice variants were noted.
Conclusions: Genes are not only turned on and off but can induce a cascade of mRNA splice variants that lead to altered protein expression. We found that the stress of a major burn, especially infection, can lead to a myriad of hGR splice variants that have the potential to impact the patient’s response to treatment. These findings suggest that glucocorticoid activity is regulated, at least in part, by creating splice variants of the glucocorticoid receptor.
METABOLIC MARKERS OF INJURY AND MORTALITY IN COMBAT-RELATED TRAUMA
E.R. Lusczek*1, M.A. Dubick*2, and G. Beilman*1. 1University of Minnesota, Minneapolis, MN, 2US Army Institute of Surgical Research, San Antonio, TX
Objective: Hemorrhagic shock and injury causes significant early changes in metabolism which have been described using metabolomics in animal models and civilian trauma. We wished to evaluate changes associated with combat injury to identify unique features of injury due to combat operation.
Methods: Plasma was obtained at Emergency Department (ED) presentation and intervals thereafter from 78 patients injured during combat operations in Iraq and from 40 healthy volunteers. Water-soluble metabolites were detected and quantified with proton Nuclear Magnetic Resonance. Resulting metabolic profiles were analyzed with partial least squares discriminant analysis (PLS-DA). Potential biomarkers were evaluated with t-tests.
Results: Significant alterations to metabolism result from traumatic injury. Metabolic profiles of combat-injured patients differ from those of healthy controls (see figure, p=0.001), driven by increased acetone (0.042 vs. 0.003 mM) and cis-aconitate (0.012 vs 0.0002 mM) in patients (p<2×10-16). Among patients, increased levels of succinate and malonate differentiate those who survived from those who did not (0.05 vs. 0.003 mM, p=1×10-8 for succinate; 0.02 vs. 0.0007 mM, p=0.0002 for malonate). Patients with severe injuries (ISS>15) had metabolic profiles characterized by higher levels of cis-aconitate (0.017 vs. 0.007 mM, p=0.0015). Metabolic profiles also differentiate more subtle nuances among the injured such as method of injury (GSW vs. blast injury) and time course associated with recovery.
Conclusions: Combat injury is associated with a host of changes at the metabolic level driven by a gross change in metabolism compared to healthy individuals. More subtle alterations in metabolism among the injured reflect differences in mortality, injury severity, method of injury, and time course associated with recovery. Succinate and malonate are novel biomarkers of mortality; cis-aconitate and acetone are novel biomarkers of injury.
Gross changes in metabolism result from trauma as demonstrated by multidimensional analysis of plasma metabolic profiles.
HYPERCAPNIC CONDITIONS INCREASE EFFEROCYTOSIS OF ALVEOLAR MACROPHAGES AND REDUCE THE INFLAMMATORY REACTION AFTER BLUNT CHEST TRAUMA
A. Palmer, U. Niesler, M. Eichner, F. Gebhard, and M. Huber-Lang. Universitaetsklinik Ulm, Ulm, Germany
Introduction: Blunt chest trauma enhances phagocytosis of alveolar macrophages (AM), but doesn’t alter efferocytosis. These effects were detected previously by intratracheal instillation of E. coli or apoptotic PMN followed by their detection in alveolar macrophages. In present studies the rather unknown role of hypoxia and hypercapnia for the pulmonary clearance rate after blunt chest trauma was investigated.
Methods: Blunt chest trauma was induced by a single blast wave centered on the thorax of male CD rats. AM were isolated 2, 24 and 48 hours after blunt chest trauma or sham procedure and cultured with apoptotic PMN or E. coli under normoxic (21%O2,5%CO2), hypercapnic (20%CO2) and hypoxic conditions (5%O2). Macrophage efferocytosis and phagocytosis were evaluated by FACS analysis and concentrations of IL-6, IL-10, TNF, MCP-1 were determined in bronchoalveolar lavage fluids (BAL) by ELISA or multiplex assays.
Results: Hypercapnia induced significantly enhanced AM efferocytosis compared to AM exposed to normoxic or hypoxic conditions. However AM isolated 2 and 24 hours after blunt chest trauma cultured in hypercapnia revealed decreased efferocytosis compared to AM isolated after sham procedure. In comparison to normoxic and hypoxic conditions, incubation in hypercapnic conditions resulted in an additional increase of IL-10 and decrease of TNF in supernatants of AM, isolated 24 hours after blunt chest trauma and incubated with apoptotic PMN. In contrast the clearance of E. coli was significantly decreased under hypercapnic conditions. Furthermore in the supernatants of AM incubated with E. coli under hypercapnic or hypoxic conditions decreased concentrations of TNF and MCP 1 were detected. However under hypoxic conditions no alterations of AM efferocytosis and phagocytosis was found.
Conclusion: The results indicate that hypercapnia may contribute to maintenance of efferocytosis and enhancement of phagocytosis detectable in AM after blunt chest trauma. The data also suggest that hypercapnia as well as hypoxia exert some anti-inflammatory effects on the lungs after blunt chest trauma. (Supported by DFG KFO200, KN 475/5-2).
SCALDED RAT SERUM IS A PRO-INFLAMMATORY STIMULATOR OF HEPATIC STELLATE CELLS
F.J. Bohanon, X. Wang, D. Herndon, A. El Ayadi, C. Rastellini, C.C. Finnerty, J. Zhou, and R.S. Radhakrishnan. The University of Texas Medical Branch, Galveston, TX
Introduction: Liver injury after burn is a significant problem characterized by steatohepatitis, apoptosis and insulin resistance. Burn injury leads to intestinal mucosal injury and portal venous endotoxemia. Lipopolysaccharide (LPS) is a known activator of hepatic stellate cells (HSC) and results in an activated pro-inflammatory myofibroblast phenotype. Serum collected from animals following scald injury has shown significant pro-inflammatory cytokine profiles. In this study, we examined the effects of scalded rat serum (SRS) on HSC activation.
Methods: Rats underwent sham or 60% TBSA burn with liver and serum collected at 24 hours post burn. Liver was collected for Western analysis, qRT-PCR, and histologic analysis. Activated HSC cell lines (LX-2 and HSC-T6) were treated with LPS, SRS, control rat serum (CRS), and oridonin (a reported NF-κB and HSC inhibitor) and analyzed for α-smooth muscle actin (α-SMA), vimentin, cytokines, and NF-κB signaling.
Results: Hepatic α-SMA protein and mRNA levels were increased in burned rats compared to sham, suggesting HSC activation after burn. In vitro, CRS resulted in minimal activation of NF-κB translocation while SRS or LPS treatment dramatically increased secretion of IL-6 and MCP-1 as well as increased NF-κB translocation and nuclear DNA binding. Oridonin decreased LX-2 cell expression of α-SMA and vimentin. In addition, Oridonin significantly decreased levels of IL-6 and MCP-1 induced by SRS and LPS. Finally, oridonin completely blocked LPS-induced NF-κB translocation and DNA binding.
Conclusions: The use of CRS and SRS may be a viable model to study the effects of burn on liver tissue. Burn injury leads to HSC activation. Oridonin deactivates HSC in vitro. Oridonin decreases activated HSC secretion of LPS-induced pro-inflammatory cytokines via an NF-κB dependent pathway. Oridonin may act as a novel therapeutic agent for the treatment of liver injury after burn.
RECIPROCAL ACTIVATION OF PLATELETS AND CD4+ TREGS FOLLOWING TRAUMA
C.B. Bergmann, F. Hefele, M. Unger, S. Huber-Wagner, P. Biberthaler, M. van Griensven, and M. Hanschen. Klinikum rechts der Isar - Technical University Munich, Munich, Germany
Trauma induced injury is the leading cause of death up to the age of 45 years. CD4+ T regulatory cells (Tregs) have been shown to be key players of the anti-inflammatory host response following trauma. In depth knowledge about the mechanisms leading to Treg activation following injury are missing so far. Recent evidence suggests platelets to play a protective role after trauma. Here we test the hypothesis that trauma induces reciprocal activation between platelets and CD4+ Tregs.
Depletion of platelets or CD4+ Tregs was conducted in a murine burn injury model (male C57BL/6N mice). Draining lymph nodes, blood and spleen were harvested early (2h) and late (7d) following trauma. CD4+ Treg activation was measured using phospho- and conventional flow cytometry (ZAP-70, PKC-theta). Platelet activation was analyzed using thromboelastometry (ROTEM®) and flow cytometry (CD62P, CD63, TLR-9).
Trauma differentially activates CD4+ T cells, early after trauma only CD4+ Tregs, but not CD4+ non-Tregs, are activated. Following burn injury, platelets augment the activation of CD4+ Tregs. This effect could only be seen early after trauma. While CD4+ Tregs influence hemostasis as seen by ROTEM early following trauma, platelet activation markers were unchanged. Beyond their role in hemostasis, platelets are able to modulate the immune response following trauma.
In conclusion, our observations indicate for the first time that reciprocal activation of platelets and CD4+ Tregs is part of the protective immune response following trauma. Platelets modulate the immunologic host response to trauma-induced injury by augmenting the activation of CD4+ Tregs. Our data suggests, that reciprocal activation is taking place following trauma. CD4+ Tregs are capable of modulating the hemostatic function of platelets. The newly described interaction between CD4+ Tregs and platelets might be a suitable clinical target for the development of immunomodulatory drugs following trauma, but also a tool for immunosurveillance following trauma.
PRE-OPERATIVE EVOKED IMMUNE RESPONSES CORRELATE WITH RECOVERY FROM SURGICAL TRAUMA
B. Gaudilliere*1, G. Fragiadakis*1, E. Ganio*1, M. Tingle*1, H. Lancero*1, G. Nolan*1, and M. Angst*1. Stanford University, Palo Alto, CA
Introduction: Patients’ recovery from surgical trauma is highly variable. Protracted recovery causes significant societal and economic costs and is a widely recognized health care problem. However, the biology that drives recovery after surgery remains poorly understood. Furthermore, we lack strategies to predict recovery in individual patients.
We have recently applied mass cytometry (CyTOF) at the bedside to survey alterations of the immune system in patients undergoing hip arthroplasty1. Analysis of the resulting high-dimensional dataset revealed patient-specific responses that contained strong correlates of clinical recovery. Specifically, changes in STAT3, NF-kB, and CREB signaling shortly after surgery strongly correlated with recovery from fatigue, pain, and functional impairment.
The identification of immune correlates shortly after surgery is an important advancement in understanding the biology that drives recovery. However, a blood test interrogating a patient’s immune system before surgery and predicting clinical recovery after surgery would truly transform perioperative patient care. In this study an in vitro assay was developed to determine whether differences in patients’ pre-surgical immune states contained immune correlates of surgical recovery (the methods are summarized in Fig. 1).
Results and conclusions: Activation of canonical signaling pathways downstream of extracellular ligands (LPS, IL-2, IL-6, IL-10, GMCSF and IFNα) was elicited in pre-surgical patient samples. Surprisingly, the magnitude of this response varied significantly across patients, thereby defining pre-surgical and patient-specific “immune responses”. Among these responses, signaling downstream of the TLR4 receptor in CD14+ monocytes (namely phosphorylation of CREB and MAPKAP2) strongly correlated with recovery of hip function (R=0.64 and 0.69, SAM, FDR<0.01). The findings provide proof of concept and the mechanistic basis to develop a pre-operative diagnostic “immune stress test” to predict the course of surgical recovery. The approach outlined here in the context of surgery also provides a model system to identify critical immune mechanisms that drive recovery after major traumatic injury.
1. Gaudilliere et al., Science Translational Medicine 6, 255ra131 (2014).
UNDERSTANDING THE OBESITY PARADOX: A MURINE MODEL OF POLYTRAUMA, SECONDARY INFECTION, AND PROLONGED CRITICAL ILLNESS
R.D. Winfield*, I.R. Turnbull*, A. Fuchs*, S. Ghosh*, C. Davis*, S. Hughes*, and G.V. Bochicchio*. Washington University in St. Louis, St. Louis, MO
Introduction: Obesity is a major public health concern worldwide, while trauma remains the leading cause of death and disability among young adults. Obesity is associated with greater complication rates following injury when comparisons are made to similarly injured lean cohorts; however, mortality rates remain similar in a phenomenon known as the “obesity paradox”. We herein present a clinically relevant murine model of low-lethality polytrauma followed by secondary infection and prolonged illness to study the obesity paradox.
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 appropriate age, animals were subjected to polytrauma (hepatic laceration, pseudofracture [proximal hindleg soft tissue crush injury followed by injection of isogenic bone homogenate], and 15% hemorrhage). 24 hours post-injury, 1x106Pseudomonas aeruginosa was instilled intratracheally to induce pneumonia. Animals were monitored for survival, appearance, and weight after injury and infection.
Results: Prior to injury, obese animals were significantly heavier than control (28.6 vs. 39.4 gm, p<0.001). Mortality from polytrauma was approximately 5% in both cohorts (NS). Mortality from polytrauma with subsequent Pseudomonas infection was also similar (19% obese vs. 20% lean, NS). Both cohorts lost weight and displayed signs of illness following injury and infection. The lean cohort reached weight loss nadir at 85% of pre-injury weight on post-infection day 3 and returned to >90% of pre-injury weight by day 4. In contrast, obese animals continued to lose weight, with a nadir at 80% of pre-injury weight. Obese mice failed to regain lost weight and remained lethargic and unkempt for over 7 days (Figure 1).
Conclusions: Obese mice subjected to polytrauma and secondary infection show greater and more sustained weight loss and signs of illness relative to lean cohorts without an increase in mortality. This successfully recapitulates the clinical findings of the obesity paradox, and provides a framework for understanding the interplay between obesity, post-injury immune dysfunction, critical illness, and prolonged post-injury recovery.
SERUM AMYLOID A REGULATES THE ADIPOSE-LIVER CROSS TALK POST BURN INJURY BY AUTO/PARACRINE MODULATION OF ADIPOSE TISSUE LIPOLYSIS AND APOPTOSIS
A. Abdullahi1, 2, and M.G. Jeschke*1, 2. 1Sunnybrook Research Institute, Toronto, ON, Canada, 2University of Toronto, Toronto, ON, Canada
Introduction: The acute phase protein Serum Amyloid (A-SAA) is expressed in the liver following tissue damage. Recently extra-hepatic expression of SAA has been reported in obese and diabetic patients via the adipose tissue, and linked to both insulin resistance and systemic inflammation. Since burn patients are characterized by a similar etiology, we hypothesized that SAA regulates the dysfunctional inter-organ crosstalk between the liver and adipose tissue post thermal injury.
Methods: Serum and adipose tissue of burn patients admitted to the Ross Tilley Burn Center were collected and analyzed for SAA expression. In our mouse model, we inflicted wild type C57BL/6 mice with a 30%TBSA burn injury and evaluated ER stress, liver function, and SAA expression.
Results: We found that burn patients exhibit elevated serum levels of SAA weeks after injury (5 fold <0.05), and this was correlated with increased serum IL-6. Moreover, a novel finding of our study is that A-SAA is highly expressed in human adipose tissue post burn. Further consistent with auto/paracrine functions of SAA, increased lipolysis and leukocyte infiltration was observed in the adipose tissue of these patients post injury. Using a clinically relevant mouse model, we observed similar SAA expression profile in mice post burn injury as to our clinical patients. Also, in burned mice elevated hepatic SAA expression was associated with increased ER stress and hepatic dysfunction. The systemic alterations of SAA on adipose tissue might be mediated by ER stress, as treatment of SAA in adipocytes markedly increased ER stress and apoptosis. Collectively, we demonstrated that chronic SAA levels post burn injury maybe the result of adipose tissue secretion, and the elevation of SAA in serum may contribute to impaired liver-fat cross talk. Thus, improvements in systemic inflammation and insulin sensitivity with insulin and anti-inflammatory therapy in these patients may in part be mediated by a decrease in adipose tissue SAA production and secretion.
OXANDROLONE IN COMBINATION WITH EXERCISE ABROGATES BURN-INDUCED PROTEOLYSIS PATHWAYS IN THE DIAPHRAGM
A. El Ayadi1, Y. Wang1, A. Prasai1, M. Wetzel1, R. Mifflin1, D. Herndon*1, 2, and C.C. Finnerty*1. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX
Burn injury is sometimes accompanied by complications in respiration that may be due to a failure of respiratory muscles to function properly. These complications may require the use of mechanical ventilation. Among the respiratory muscles, the diaphragm plays an important role in the pathophysiological mechanisms that lead to difficulty in weaning patients from mechanical ventilation. Previous studies have shown that prolonged mechanical ventilation can induce muscle atrophy and stimulate proteolysis. In addition, patients in critical conditions such as burn or sepsis may require the use of steroids for the treatment of inflammatory and immune reactions. However, the dose of corticosteroids has been shown to be critical in promoting or protecting against mechanical ventilation induced proteolysis of the diaphragm. We recently reported that long term administration of oxandrolone in combination with exercise improves long-term recovery of severely burned children in height, bone mineral content, cardiac work, and muscle strength. In this study, we are using the rat burn model to investigate the effects of oxandrolone and exercise on signaling pathways involved in protein synthesis and breakdown in the diaphragm muscle.
Rats received a 60% TBSA burn using an established protocol and were housed in cages with free access to running wheels or standards cages. Oxandrolone was administered 2hrs post-burn and once a day for 3 days. Rats were sacrificed on day 3 and the diaphragm tissue was used to analyze the proteasome activity and the molecular targets involved in muscle metabolism.
Our Preliminary data show that the proteasome activity is increased following burn injury and this effect is abrogated by oxandrolone. Exercise also increases the proteasome activity in control animals; however no synergistic effect of exercise on the proteasome activity was seen with burn or burn plus oxandrolone. The MAP kinase protein ERK1/2 was activated by burn, exercise and oxandrolone. The effect of exercise on ERK ½ phosphorylation was more pronounced in the burn and burn plus oxandrolone groups. mTOR phopsphorylation in burn animals was reduced by exercise and oxandrolone while p70S6 K phosphorylation was increased in the burn animals that received oxandrolone plus exercise.
These data suggest that both exercise and oxandrolone are activating the molecular targets involved in the initiation of gene expression and ribosomal biogenesis to activate protein synthesis and preserve the diaphragm muscle function in burn animals that receive both oxandrolone and exercise.
ALTERNATIVE TO ECMO: DEVELOPMENT OF A NOVEL PERITONEAL MEMBRANE OXYGEN DELIVERY SYSTEM
K. Buesing*1, N. Legband2, M.R. Goede1, M.A. Borden3, and B.S. Terry2. 1University of Nebraska Medical Center, Omaha, NE, 2University of Nebraska-Lincoln, Lincoln, NE, 3University of Colorado-Boulder, Boulder, CO
Background: Acute respiratory distress syndrome (ARDS) continues to be a challenge for healthcare providers despite advances in different modes of ventilation. As a terminal effort to provide adequate oxygenation and carbon dioxide removal, extracorporeal membrane oxygenation (ECMO) has been used to completely bypass the patient’s respiratory and/or cardiac systems. Despite its successful application in a select group of patients, ECMO carries significant risk, which is especially worrisome in the multi-trauma patient population in whom ECMO is often contraindicated due to traumatic brain injury and the need for anticoagulation on the ECMO circuit. We have developed a unique peritoneal membrane oxygenation system that uses pure oxygen encapsulated by a phospholipid monolayer shell for use in a rat model of moderate to severe ARDS.
Objective: The objective of this study is to develop and test the efficacy of a closed-loop peritoneal membrane oxygenator, which provides continuous oxygen delivery via OMBs to a rat ARDS model.
Materials & Methods: OMB perfusate will be instilled into the peritoneal cavity of adult male Wistar rats via a closed-loop peritoneal infusion system. Intratracheal LPS will be used to simulate a moderate to severe model of ARDS. Clinical data and lung tissue samples will be collected over a 7-day time period.
Results: The closed-loop oxygenation system design has been completed and construction of the device is underway (Figure 1). Its features include modular components for greater reliability, non-permeable IV bag for infusate storage container, modified cage for tubing restraint and passage, and autonomous LabVIEW control, which can also monitor the intra-abdominal pressure of the rat and the temperature of the infused solution. Our ARDS model aligns with previously established models of moderate to severe ARDS.
Conclusions: Our closed-loop membrane oxygenator can be easily simulated in humans using peritoneal dialysis catheters. This system has the potential to serve as a safer alternative to ECMO in patients with severe respiratory failure.
COMPOSITION ALTERATIONS OF THE CARDIAC INFLAMMASOME FOLLOWING THERMAL INJURY COMPLICATED WITH SEPSIS AS RELATED TO INFLAMMATION
D.L. Carlson, X. Yao, and S.E. Wolf. University of Texas Southwestern Medical Center, Dallas, TX
Inflammation with burn injury is a common complication. Fluid resuscitation, antimicrobials, early excision, and grafting have improved survival; however, pneumonia-related sepsis often occurs after burn, progressing to multiple organ failure. This initial injury may prime the patient, producing an exaggerated response to a second insult, such as sepsis. One mechanism through which priming may occur is the inflammasome, a multi-protein complex comprising caspase-1, adaptor protein apoptosis-associated speck-like protein containing a caspase-activating recruitment domain protein (ASC), and the NLR sensors, NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRP12, or NLRC4. The aim of this study was to examine the response of the inflammasome to burn complicated with sepsis and the associated NLR factors in the heart.
C57/BL6 mice were divided into groups. 1. Control 2. Third degree burn over 40% of the total body surface (TBSA) 3. 40% TBSA+sepsis (intratracheal administration of Streptococcus pneumonia) 4. Sepsis. Cardiac function was measured. Serum, heart, and lung samples were taken at various times. Heart and lung protein was prepared for immunoblot for NLR factors, ASC and inactive/active caspase-1. Serum was used to correlate inflammation via ELISA monitoring TNF, IL-1B, IL-6, and IL-10. Controls showed no difference in cardiac ASC or NLRP1. 30m post burn, cardiac ASC doubled, and NLRP1 decreased 2.7%. After burn injury both NLRP1 and NLRP3 were present in cardiac tissue with active caspase-1. Predominant NLR at 4 and 8h post burn was NLRP3, a switch from NLRP1. Cardiac inflammasome changes observed in burned and burn/sepsis animals were similar, with a switch to NLRP3 at 2h post inoculation. Similar, but different changes occurred in sepsis animals, with NLRP3 predominant at 8h post inoculation. Serum was tested by ELISA. TNF peaked at 2h post injury (75±2.7pg/ml vs 342±5.8pg/ml). Increases in IL-6 and IL-1B were observed, with Il-1B being the latest peak at 8h (67±4.2 pg/ml vs 350 ±8.2 pg/ml). Burn or sepsis alone produced myocardial contractile defects and increases in pro-inflammatory cytokine secretion. Burn/Sepsis exacerbated the cardiac contractile defects seen with either injury alone.
In response to injury, there was a shift in cardiac expression from NLRP1 to NLRP3. This correlated with an increase in active caspase-1 and an increase in markers of inflammation. Burn/Sepsis that exacerbated the cardiac contractile defects seen with either burn or sepsis alone. We conclude that upon stimulation by initial injury there is a shift in the sensor protein in the inflammasome, triggering activity and the production of active cytokines. We further conclude that the initial injury may prime the patient such that a second insult produces significantly greater cardiac abnormalities than those seen with either burn alone or sepsis alone.
ERYTHRO-MYELOID COMMITMENT IN BURN PATIENTS IS UNDER BETA-ADRENERGIC CONTROL THROUGH MAFB REGULATION
S. Hasan, M. Mosier, R.L. Gamelli, P. Conrad, M. Halarz, and K. Muthumalaiappan. Loyola University Chicago, Maywood, IL
Introduction: Transfusion is the only viable option to treat anemia in burn patients. Despite the adverse consequences, lack of a reliable test platform to study the molecular mechanisms of impaired erythropoiesis in burn patients has been a limiting factor to consider alternate treatment strategies. We recently documented that MafB orchestrates the lineage bias restricting megakaryocyte-erythrocyte progenitor (MEP) production in burn patients within 1-2 weeks post burn (abstract accepted for 47th ABA meeting). Here we followed up the burn patients for 4-7 weeks post burn and found MEPs continued to decrease significantly with standard burn care. Conversely, in two patients who happened to receive adrenergic blockers during the course of burn care; erythroid commitment was restored as seen by an increase in MEPs with a concomitant drop in MafB expressing multi potent progenitors.
Method: Six burn patients (M=5, F=1) of 43±5.8 yrs age and 35±7.8%, TBSA burn were studied with informed consent and IRB approval. Blood samples collected within 4-7 weeks after burn (samples =12) and six healthy volunteers (C; 41±6.4 yrs, M=3, F=2) were analyzed. Ficoll separated PBMCs were placed in conducive growth factor cocktail to preserve and proliferate residing hematopoietic stem cells into erythro-myeloid progenitors. On day 5, lineage negative (lin-) cells were enriched using magnetic beads, incubated with a combination of surface Abs and intracellular MafB, and analyzed by flow cytometry. MEPs were identified by the differential expression of IL-3Rα and CD45RA and expressed as percentage of linnegCD34+CD38+ cells (multipotent progenitor cells, MPPs). Percentages of MafB+ cells in the MPPs were determined.
Results: In four burn patients who received standard care (B), PBMC derived MEPs continued to remain significantly low for up to 7 weeks. Whereas, in two other burn patients who received either propranolol or propranolol and labetolol along with standard burn care (B+P), the MEPs were significantly restored from 1-2 week levels. (B=*22.4±1.3%, B+P=*#65±5.8%, C=50.8±3.9%, * p<0.01 vs C; # p<0.001 vs B). With respect to myeloid transcription factor MafB, the percentage of MafB+ MPPs remained significantly higher in B compared to C at 4- 7 weeks post burn. Conversely, in patients on adrenergic blocker (B+P) there was a significant reduction in MafB+MPPs. (B=*74.9±1.5%, B+P=*#31.2±6.5%, C=48.8±4.2%, * p<0.01 vs C; # p<0.001 vs B). Results are in line with our previous reports in burn mice treated with propranolol.
Conclusion: Following burn injury, MafB plays a key role in controlling the hematopoietic commitment pattern away from erythroid lineage. β-adrenergic blockers can redirect the hematopoietic commitment toward erythroid lineage by lowering MafB expression in multipotent progenitors. β- blockade can be of potential therapeutic value targeted to treat anemia in burn patients.
LYOPHILIZED EXTRACT OF THE LARVA LUCILIA SERICATA FOR THE MANAGEMENT OF CHRONIC WOUNDS: AN UPDATE
E. Boelke1, B. Homey1, F. Gestmann1, H. Schrumpf1, N. Hoff1, H. Mehlhorn1, B.A. Buhren1, T. Jansen1, C. Matuschek1, M. van Griensven*2, and A. Gerber1. 1University of Duesseldorf, Duesseldorf, Germany, 2Technische Universität München, Munich, Germany
Background: Chronic wounds are a common problem in the 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. However, larval-therapy is associated with various problems, including the requirements for the transport of living larvae as well as low patient compliance due to disgust, putrid smell or pain/discomfort.
Objectives: Recently, we have generated a lyophilized extract of the larva of the greenbottle fly Lucilia sericata (Larveel®, Alpha-Biocare GmBH, Duesseldorf, Germany). Here, we present the results of the first nine 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®).
Methods and results: Nine patients with venous leg ulcers that did not respond sufficiently to standard wound management strategies were treated with Larveel®. Larveel® was resuspended with 2 ml of sodium chloride solution and directly applied on wound surface at dressing changes (3 x/ week) and covered with a non-adhesive dressing. Patients were treated with Larveel® for eight weeks. In seven 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 patients we did not observe any relevant adverse effects of Larveel®-therapy. In two patients 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 larva. 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 exclude colonizations with Gram-negative bacteria, since the potential selective antimicrobial effect of Larveel® on Gram-positive bacteria may promote the growths of Proteus spp. or Pseudomonas spp. strains.
DISRUPTION OF PULMONARY CAPILLARY ENDOTHELIUM BY STORED RED BLOOD CELLS
A.T. Makley, M.D. Goodman, R.A. Veile, L.W. Friend, E. Gulbins, and T.A. Pritts. University of Cincinnati, Cincinnati, OH
Objective: Acute lung injury is mediated by breakdown of pulmonary endothelial barrier function resulting in capillary leak and subsequent infiltration of fluid and inflammatory cells into the pulmonary interstitium. The transfusion of aged red blood cells (RBCs) is a proven risk factor for post-traumatic pulmonary infections and acute lung injury, but the effect of aged RBCs on pulmonary endothelium and normal barrier function is unknown. We hypothesized that exposure of endothelial cells to aged RBCs would disrupt the intact endothelial cell barrier and induce pulmonary vascular leak.
Methods: RBCs were harvested from C57BL/6 male mice, anti-coagulated, and stored in Nutricel (AS-3) storage media for 0 days (fresh) or 15 days (aged) at 4°C. Murine pulmonary microvascular endothelial cells were grown to confluence on Transwell membranes and treated with either fresh or aged RBCs for four hours. Endothelial barrier function was assessed by FITC-Dextran (40kDa) permeability across the Transwell membranes (n=4). Cellular supernatants were collected from a separate group of cells (n=5). Soluble thrombomodulin and sphingosine-1-phosphate levels were measured by ELISA to reflect markers of endothelial activation and destabilization. Endothelial cells exposed to media only served as controls.
Results: Endothelial cells exposed to aged RBCs exhibited higher levels of soluble thrombomodulin (323.70±35.64 pg/mL vs. 65.15±7.90 pg/mL, aged vs. fresh, p<0.05) and lower levels of extracellular sphingosine-1-phosphate (0.34±0.06 µM vs. 0.919±0.08 µM, aged vs. fresh, p<0.001) compared cells treated with fresh RBCs. FITC-Dextran transit across the Transwell membranes was increased after treatment with aged but not fresh RBCs (Figure 1).
Conclusion: The treatment of endothelial cells with aged RBCs resulted in global endothelial cell damage and disruption of intercellular interfaces, as demonstrated by increased soluble thrombomodulin levels and decreased sphingosine-1-phosphate levels. Exposure of pulmonary microvascular endothelial cells to aged RBCs induced breakdown of the normal endothelial protective barrier. These findings support that transfusion of aged RBCs may contribute to capillary leak and increase susceptibility to acute lung injury.
ENDOTHELIAL RESPONSE TO PHYSIOLOGIC CONDITIONS OF HEMORRHAGIC SHOCK - UNIQUE FIBRINOLYTIC RESPONSES TO DIFFERENT STIMULI IN-VITRO
E. Gonzalez1, E.E. Moore*1, H.B. Moore1, M.P. Chapman1, A.P. Morton1, S. Mitra1, M. Kehler1, 2, A. Banerjee*1, and C.C. Silliman*1, 2. 1University of Colorado, Aurora, CO, 2Bonfils Blood Center, Aurora, CO
Background: The role of endothelial cells as drivers of trauma-induced coagulopathy (TIC) has not been well characterized. We conducted a study to characterize the effect of plasma proteins elevated during hemorrhagic shock on cultured endothelial cells, with the hypothesis that they would yield distinct coagulopathic changes on whole blood thrombelastography(TEG).
Methods: Cryopreserved human liver sinusoidal endothelial cells(HLSEC) in culture were stimulated with tissue factor(TF), thrombin, epinephrine, nor-epinephrine, and vasopressin; the median concentration previously reported in the plasma of severely injured trauma patients was used. Medium supernatant from each well was flash-frozen at the following time-points: baseline, 15min., 30min., and 2hours after stimulant addition. Medium supernatant from HLSEC with no added stimuli was used as a control. This medium was later thawed and added to whole blood, and TEG was performed. To account for the direct effect of the stimulant on whole blood, TEG was performed in pairs with the medium supernatant containing the stimuli but not exposed to HLSEC. The %change in TEG variables between the paired assays was compared.
Results: R-time (time to beginning of clot formation) was reduced from baseline, and compared to control time-points with all stimuli (at 15min:TF -40.3%, thrombin -38.9%, epinephrine -15.9%, norepinephrine -17.0%, vasopressin -30.5%, control 2.0%). Angle(rate of clot formation) and MA(maximal clot strength achieved) were also increased from baseline, and compared to control time-points with all stimuli. LY30(percent fibrinolysis of clot 30min. after reaching MA) demonstrated distinct changes among stimuli: epinephrine and norepinephrine caused an increase in LY30 at 15min. of exposure, which later decreased at 30min. and 2h, when compared to baseline and control (Fig.1).
Conclusion: Catecholamines drive fibrinolysis in liver endothelial cells, while vasopressin, TF, and thrombin decrease fibrinolysis. Endothelial cells can be either pro- or anti-fibrinolytic depending on the balance of plasma proteins they are exposed to. With catecholamine surge being the direct result of hypotension, early control of blood pressure in trauma patients could prevent systemic hyperfibrinolysis associated with TIC.
TRAUMA-INDUCED COAGULOPATHY IN A NON-HUMAN PRIMATE (RHESUS MACAQUE) MODEL OF UNCONTROLLED HEMORRHAGE
D.M. Fryer1, A.R. Macko1, R. Crossland1, C.G. Fedyk2, M.R. Scherer2, A.P. Cap*2, and F.R. Sheppard*1. 1Naval Medical Research Unit - San Antonio, San Antonio, TX, 2US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: Uncontrolled hemorrhage is the leading cause (91%) of potentially survivable combat casualty trauma mortality, and is associated with trauma-induced coagulopathy (TIC). The objective of this study was to characterize the coagulation system response in a non-human primate (NHP) model of uncontrolled hemorrhage.
Methods: Uncontrolled hemorrhage was induced in anesthetized Rhesus macaques (7-12 kg; n=8) by a 60% left-lobe hepatectomy (T=0 minutes) under an IACUC approved protocol. From T=5-120 minutes, animals were resuscitated with 20cc/kg normal saline at a constant rate. At T=120 minutes, bleeding was surgically controlled and blood loss (BL) quantified. Blood samples were collected until T=480 minutes for analyses of clot strength (ROTEM®), coagulation function (STAGO®) and platelet aggregation (Multiplate®). Results are reported as mean±SEM. Statistical analyses utilized Student’s t-test, p<0.05 considered significant.
Results: BL averaged 47±3%. Compared to baseline (T=0 minutes), at T=20 minutes decreased clot strength (793±90 vs. 571±48 mm; p=0.04) platelet count (2.6x105 vs. 2.1x105 /µl; p=0.001), fibrinogen (215±16 vs. 155±9 mg/dL; p=0.003) and anti-thrombin III (96±3 vs. 72±2%; p<0.001) were observed, and prothrombin time (PT) was prolonged (13±0.2 vs. 14.5±0.3 sec; p<0.001). D-dimer increased after 120 minutes. Compared to baseline, reductions in platelet aggregation in response to collagen (5.3±0.5 vs. 4.5±0.5; p=0.03) and arachidonic acid (4.2±0.7 vs. 1.9±0.5; p=0.02) were observed post-injury.
Conclusion: These results indicate a consumptive coagulopathy with platelet dysfunction and fibrinolysis in response to uncontrolled hemorrhage consistent with previous descriptions of TIC. Further studies are warranted to establish pathophysiologic mechanisms and the effects of resuscitation.
GENETIC POLYMORPHISMS AND SEVERE SEPSIS - A MULTI-CENTER STUDY IN THE JAPANESE ICU POPULATION
E. Watanabe*1, S. Oda*1, T. Sakamoto2, O. Takasu*2, T. Ikeda4, J. Kotani*3, N. Kitamura5, and E. Schneider6. 1Chiba University, Chiba, Japan, 2Kurume University, Kurume City, Japan, 3Hyogo College of Medicine, Nishinomiya City, Japan, 4Tokyo Medical University Hachioji Medical Center, Hachioji City, Japan, 5Kimitsu Chuo Hospital, Kisarazu City, Japan, 6University of Ulm, Ulm City, Germany
Background: Some inflammation related genetic polymorphisms are suggested to be useful biomarkers as prognostic factors of severe sepsis. On the other hand, it is recognized that the genotypic distributions in Japanese population are quite different from those in the other racial population. Here, we conducted multi-center study to evaluate the influence of heredity on predisposition to poor outcome of severe sepsis in Japanese cohorts.
Patients and Methods: The study included 253 patients (of those 125 severe sepsis) who were admitted to the intensive care unit (ICU) of Chiba University Hospital between October 2001 and September 2008 (discovery cohort) and 767 (of those 271 severe sepsis) patients who were admitted to ICUs of 5 Japanese ICUs between October 2008 and September 2012 (multi-center validation cohort). We selected 254 patients from validation cohort by propensity score matching to equalize morbidity of severe sepsis and severity of both cohorts. Genotyping was performed using a microarray chip (Asper Biotech™) for 56 single nucleotide polymorphisms (SNPs) which are thought to be associated with sepsis pathophysiology. We examined whether these SNPs were associated with mortality of and/or susceptibility to severe sepsis.
Main Results and Discussion: Among the 56 SNPs and a variable number of tandem repeat polymorphism, a THBD (thrombomodulin gene) promoter SNP (-1748*G/C; rs2239562) was significantly associated with mortality of severe sepsis in discovery and combined cohorts (Fig. P=0.033, 0.028 in a dominant model with the correlation/trend test). A P2X7 exonic, His155Tyr, SNP (rs208294) was also significantly associated with mortality in validation and combined cohorts (P=0.049, 0.042 in a dominant model). Thrombomodulin has a pivotal role in the protein C system and the recombinant agent is thought to be a promising drug for sepsis-induced disseminated intravascular coagulation (DIC). P2X7 receptors have been implicated in ATP-mediated cell death, regulation of receptor trafficking, inflammation, and tissue repair. These pathways might be main streams in deterioration of severe sepsis from the results of this examination.
Conclusions: The data suggest that the THBD and P2X7 polymorphisms influence outcome in severely septic patients in the Japanese ICU patients.
SONOGRAPHIC MEASUREMENTS OF RIGHT INTERNAL JUGULAR VEIN AND INFERIOR VENA CAVA AND ITS CORRELATION WITH CVP MEASUREMENT
H.M. Jassim, M. Alkhatib, V.A. Naushad, M. Milad, P. Chandra, S. Koya, M. Allaithy, and H. Jaouni. Hamad Medical Corporation, Wakra, Qatar
Introduction: Hemodynamic monitoring is part and parcel of any critical care management whether in ICU, ED, operating rooms or pre hospital settings. Even though measuring Central Venous Pressure(CVP) is one of the key parameters for the monitoring it has got its own drawbacks. Bedside sonography of the inferior vena cava (IVC) has been shown to be useful in estimating intravascular volume status, however, it may be technically difficult and limited by patient factors such as obesity, bowel gas, or postoperative surgical dressings.
Aim: To compare the noninvasive bedside ultrasound estimation of the Antero-Posterior( AP) diameter, cross sectional area (CSA) and the derived collapsibility index of right internal jugular vein(IJV) and IVC with direct estimation of CVP.
Study design: Prospective observational study.
Method: Convenience sample of 58 spontaneously breathing patients who had an intrathoracic central venous catheter to allow measurement of CVP were enrolled from two medical ICU. Bedside ultrasound measurement of the right IJV maximum and minimum AP diameter and CSA in two positions (0 and 30 degree), the inferior vena cava maximum and minimum AP diameter in supine position. The derived collapsibility index was compared with invasive CVP measurement.
Results: Of 58 enrolled patients, 47(81%) were men and 11 (19%) were female. CVP mean of 9.88 cm H2O (range 1-25). Of the study group, 36 (62%) have CVP equal and less than 10 and 22 (38%) have CVP more than 10. IVC could not be examined in 13 (22%) out of 58 patients for different reasons. Correlation coefficient between CVP and collapsibility index of IJV CSA, AP diameter in supine position was -0.484(p 0.0001), -0.418(p 0.001) and -0.583(p 0.0001), -0.559(p 0.0001) with IJV CSA, AP diameter respectively in 30 degree position. The correlation coefficient between CVP and IVC collapsibility index was -0.540(p 0.0001).
The correlation coefficient between IVC and IJV collapsibility indices were [0.353(P 0.017)] in Supine IJV CSA, [0.335(p 0.024)] in supine IJV AP diameter,[0.489(p 0.001)] in 30 degree IJV CSA and [0.458(p 0.002)] in 30 degree IJV AP diameter.
Conclusion: Both IVC and IJV collapsibility indices have good negative correlation with CVP values and IJV CSA and AP diameter collapsibility indices at different body positions have good positive correlation with IVC collapsibility index. Hence, right IJV collapsibility index, especially CSA at 30 degree body position, can be used as an adjunct, or alternative to IVC collapsibility index in case of technical difficulties or in the hands of less experienced operator as a guide for a non-invasive CVP estimation and possible fluid responsiveness in patients with hypotension or shock state.
THE EVALUATION OF A PLATELET DERIVED HEMOSTATIC AGENT TO REDUCE BLOOD LOSS IN A NON-HUMAN PRIMATE (RHESUS MACAQUE) MODEL OF UNCONTROLLED HEMORRHAGE
R. Crossland1, A.R. Macko1, R.M. Paredes1, P.J. Vernon1, D.M. Fryer1, A.P. Cap*2, and F.R. Sheppard*1. 1Naval Medical Research Unit - San Antonio, San Antonio, TX, 2US Army Institute of Surgical Research, Fort Sam Houston, TX
Background: Hemorrhage remains the leading cause of potentially survivable mortality in trauma. Infusible platelet derived hemostatic agents (PDHAs) are being developed as therapeutics to reduce blood loss from traumatic hemorrhage. The objective of this study was to evaluate the efficacy of a PDHA to reduce blood loss in a non-human primate (Rhesus macaque) model of uncontrolled hemorrhage.
Methods: Rhesus macaques (7-12 kg) were assigned to receive either intravenous PDHA (2x1010 platelet equivalents; n=8) or vehicle control (5% albumin; n=8). Uncontrolled hemorrhage was initiated in anesthetized animals by a laparoscopic 60% left-lobe hepatectomy (T=0 minutes). At T=5 minutes, PDHA or control solution was administered. At T=120 minutes, laparotomy was performed, surgical hemostasis achieved, and blood loss (BL) quantified. Animals were observed to T=480 minutes (time of euthanasia), necropsies were performed, and tissue samples were collected for histopathological examination. Results reported as mean±SEM. Statistical analyses utilized Student’s t-test, p<0.05 considered significant.
Results: PDHA animals had 37±4% BL, while controls had 40±5% BL (p=0.53). Survival rates were 4/8 (50%) and 7/8 (87%) for PDHA animals and controls, respectively. Overall, BL was greater in non-surviving animals (n=5) compared to surviving animals (n=11), (55±2% vs. 42±3%; p<0.02). However, there was no evidence of micro-/macro-vascular thrombi or overt pathology in either PDHA or control treated animals.
Conclusion: Although more PDHA treated animals died, mortality was not attributable to treatment, as there were no overt pathological indications in either treatment. Further investigation of PDHA efficacy to reduce blood loss is warranted prior to transition to clinical trials.
NONINVASIVE DETECTION OF ACUTE VOLUME LOSS AND FLUID RESUSCITATION IN TRAUMA PATIENTS: RESULTS OF A PROSPECTIVE CLINICAL TRIAL
C. Stewart1, J. Mulligan2, G. Grudic2, M. Talley1, G. Jurkovich3, 1, V. Convertino4, and S.L. Moulton1, 2. 1University of Colorado School of Medicine, Aurora, CO, 2Flashback Technologies, Inc., Boulder, CO, 3Denver Health, Denver, CO, 4US Army Institute of Surgical Research, Fort Sam Houston, CO
Introduction: Humans are able to compensate for significant blood loss with little change in traditional vital signs. We hypothesized that a novel computational algorithm, which recognizes subtle compensatory changes in pulse oximetry (PPG) waveforms that are associated with changes in intravascular volume, would detect acute blood loss and trend volume resuscitation in acutely injured patients.
Methods: Eligible patients suffered blunt or penetrating trauma, were > 15 years old and managed at a level 1 trauma center. PPG data, collected on arrival and continuing for up to 24 hours using a custom designed pulse-oximeter, were post-processed by the algorithm to obtain a time history of compensatory reserve (CR, measured on a scale of 1 to 0, with 1 indicating fully compensated and 0 indicating no reserve, or decompensation). CR was compared to clinical findings and outcomes. Active bleeding was categorized by surgeon judgment as EBL > 500 ml (e.g. femur fracture, spleen/liver injury > grade 3 on CT, and/or intraoperative EBL of > 500 ml).
Results: 50 patients were enrolled in the study; 5 were excluded due to incomplete data/device malfunction. Of 45 patients, 12 were categorized as actively bleeding, 30 were not actively bleeding and 3 were indeterminate. CR values sampled before fluid resuscitation led to a classification accuracy between bleeding and non-bleeding patients of 93% with a sensitivity of 0.9333 and specificity of 0.9167. The AUC of the ROC was 0.975. The mean initial CR of the 12 bleeding patients was 0.17 (95% CI=0.1277 - 0.2219), std. dev. 0.0741 (95% CI 0.0525 - 0.1259). The mean initial CR for non-bleeding patients was 0.5602 (95% CI=0.4951 - 0.6252), std. dev. 0.1742 (95% CI=0.1387 - 0.2342). For bleeding patients, CR increased after receiving IVFs and subsequently decreased, suggesting continued blood loss and/or waning of compensatory mechanisms. Non-bleeding patients showed no change or an increase in CR with IVF fluids, which was sustained over the first 24 hours of resuscitation. The mean ISS for bleeding patients was 27 (95% CI=18.8995 - 35.1005), std. dev. 12.7493 (95% CI=9.0316 - 21.6468), whereas the mean ISS for non-bleeding patients was 7.5 (95% CI=4.2029 - 10.7304), std. dev. 8.7404 (95% CI=6.9610 - 11.7499).
Conclusion: A novel computational algorithm, which recognizes subtle compensatory changes in continuous PPG waveforms, detects acute blood loss and trends volume resuscitation in acutely injured patients. A multi-center phase II clinical trial to examine real-time results from the algorithm and its impact on clinical management and survival is warranted.
FREE HEME AS AN IMPORTANT CAUSE OF INJURY IN TRAUMATIC SHOCK
J.A. Luciano1, 2, P. Waltz1, 2, B. Kautza1, 2, H. Gomez1, and B.S. Zuckerbraun*1, 2. 1University of Pittsburgh Medical Center, Pittsburgh, PA, 2VA Pittsburgh Healthcare System, Pittsburgh, PA
Introduction: Tissue injury in a component of many disease processes and especially pronounced in trauma. This tissue injury leads to release of free heme as well as other damage associated molecular pattern molecules. Free heme leads to direct oxidative cytotoxicity as well as leading to an increased inflammatory state. Free circulating heme has been shown to increase leukocyte recruitment and activation, increase NADPH oxidase dependent generation of reactive oxygen species, and increased production of inflammatory cytokines including TNFa and IL-1. Heme scavengers including haptoglobin and hemopexin bind hemoglobin and free heme respectively removing them from circulation for heme oxygenase dependent heme metabolism thereby limiting their direct cytotoxic and inflammatory effects as well as resulting in cytoprotection via carbon monoxide production. These investigations tested the hypothesis that while free heme results an exacerbation of injury during traumatic shock, hemopexin is protective results in a reduced inflammatory state, reduced tissue injury, and improved survival.
Methods: C57BL/6 male mice were subjected to bilateral thigh crush injury, injection with a pseudofracture bone matric, and hemorrhaged to a MAP of 25 mmHg for 60 minutes via bilateral femoral cannulation and then resuscitated with 2X the maximum shed blood volume with lactated ringers and sacrificed 4 hours after resuscitation. Some mice were treated with hemin immediately following groin cannulation. Serum levels of ALT and hemopexin were measured. Pigs were subjected to bilateral groin cannulation and hemorrhaged to a MAP of 30mmHg for 90 minutes. Serum levels of free heme and hemopexin were measured. Bovine aortic endothelial cells were cultured and exposed to varying hemin concentrations and oxygen consumption was assessed by seahorse and reactive oxygen specie generation was assessed by DCF fluorescence. ANOVA and t-test was used for statistical analysis and significance was assumes as a P<0.05.
Results: Injection of hemin prior to induction of traumatic shock was associated with an exacerbation of hepatic injury at 10 and 70mmol/kg (serum ALT 163, 1314, 2667 IU/L at 0, 10, and 70mmol/kg respectively, p<0.05). Serum hemopexin levels were measured in mice not receiving hemin injection and we found an inverse relationship to the levels of circulating hemopexin and the severity of liver injury. Analysis of porcine serum undergoing traumatic shock indicates that those animals that experienced cardiovascular collapse had lower hemopexin levels at baseline as well as increased free heme levels 60 minutes after initial induction of hypotension.
Conclusion: Free circulating heme is an important cause of tissue injury in trauma. Heme scavengers such as hemopexin lead are protective and may serve as a potential for future therapeutics to limit the toxic affects of free heme.
PERIPHERAL VENOUS PRESSURE INDICATES PRELOAD EXCESS DURING VOLUME RESUSCITATION FROM HEMORRHAGE
M. Kinsky*1, W. Johnston2, M. Salter1, G. Kramer*1, N. Ruberio1, and M. Khan1. 1The University of Texas Medical Branch at Galveston, Galveston, TX, 2Scott and White, Temple, TX
There is poor consensus with regard to the amount of volume necessary to restore perfusion after hemorrhage. Substantial effort has focused on defining the relationship on how fluid increases hemodynamic function e.g., systolic function - “position on Starling curve”. However, under resuscitation and over resuscitation continue to plague resuscitation efforts. We believe that the physiologic cost associated with improper fluid resuscitation is due to diastolic, not systolic, function. We hypothesize that venous filling would translate into increased peripheral venous pressure [PVP] with excess volume due to reduced ventricular compliance. Anesthetized, ventilated, swine [n=8] were prepared with a: splenectomy, transducer tip catheters [Millar Inc., Houston TX] for measuring - central venous pressure [CVP] and PVP, pulmonary artery catheter - cardiac output (CO), stroke volume (SV) and pulmonary artery occlusion pressure (PAOP), inferior vena cava (IVC) Doppler [Transonic Ithaca NY], arterial catheters - blood pressure (MAP) and hemorrhage [Hem] line (whole blood stored) and ultrasound -preload by End-diastolic dimension (EDD). Hem was induced by removing blood to a MAP 40 mmHg ≈1000 mL/per 70kg. Then, albumin or blood in 500 mL, in alternated increments, infused to increase PAOP [5, 10, 15 and 20 mmHg] in graded steps. Measurements were recorded at Hem and each PAOP level. Table: mean ± SEM *, indicates statistical difference [p<0.05] for each variable sequentially compared at specific PAOP level. Volume significantly and continually increased PVP and CVP and reduced the gradient [PVP-CVP]. IVC flow, CO, MAP and EDD only increased until a PAOP 10 mmHg. Receiver operating curves (data not shown) showed PVP with highest AUC. Volume responsiveness occurred when EDD increased and PVP < 10 mmHg. When EDD did not increase and PVP >10 mmHg, lack of volume responsiveness was evident. SV vs EDD increased linearly (r2 =0.72: y = 24x-36), suggesting systolic function was not restrained. EDD vs PAOP was curve linear (r2 = 0.69: y =0.399x2.3), suggesting limitations to ventricular compliance with volume loading. Further assessment of diastolic function using echocardiography is needed.
PHYSIOLOGIC RESPONSE TO REBOA MAY BE BETTER CAPTURED BY CONTINUOUS VITAL SIGN AND VIDEO MONITORING
M. Brenner, P. Hu, S. Shackelford, M. Hoehn, and T. Scalea. Shock Trauma Center, Baltimore, MD
Introduction: Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) has been shown in both translational research and several case series to be a potentially life-saving procedure. The optimal methods of investigating this procedure and its initial physiologic consequences are unknown.
Methods: 2 methods of data collection were compared in 2 patients who underwent REBOA in Zone 3 (aortic bifurcation). We recorded invasive systolic blood pressure (iSBP) values every 2 seconds, for 10 minutes before after aortic occlusion (AO) in Case 1. We recorded cuff SBP (cSBP) 10 mins before and after AO in Case 2. For both patients, mean SBP, Shock Index (SI=HR/SBP), and Dose (pressure X dose, integrates duration and extent of insult for a certain parameter) SBP were calculated pre- and post- AO. Time of AO as determined by the physician performing the procedure was compared to time of AO as determined by review of the video captured in real-time.
Results: For Case 1, mean iSBP increased 86% from 57 to 106mmHg. Dose of iSBP<90 mmHg was reduced from 33 to 2. Mean MAP increased from 44 to 81 mmHg. It took only 1 min to reach iSBP of 100mmHg and reached a peak of 140mmHg in 3.3 min. The SI pre-and post-AO were 1.9 and 1.6, respectively.
For Case 2, mean cSBP increased from 82 to 92mmHg. Dose of cSBP<90 was reduced from 8 to 1. Mean MAP increased from 59 to 66mmHg. The SI pre-AO was 1.1 and 1.0 post-AO. It took 10 mins to reach cSBP of 100mmHg, and 21 mins to reach a peak of 121mmHg. Time of occlusion documented by the physician did not correlate with the time of occlusion as documented in the real-time video in either case.
Conclusion: Improvements in physiologic parameters occurs with REBOA, and may be captured by both invasive and non-invasive means. The data captured by non-invasive means may not accurately portray the initial physiologic outcome of AO. Continuous vital sign monitoring, along with video recording of the procedure, may provide the most reliable data in order to study the technique and physiologic parameters associated with REBOA. Protocols for REBOA should include invasive BP monitoring, and may be obtained by use of an upper extremity arterial line. An additional benefit of video monitoring of the procedure may include performance review for credentialing, and critique for quality assurance metrics.
OPTIMIZING COMBAT CAUSUALTY CARE: MODULATION OF MICROVASCULAR ENDOTHELIUM BY RESUSCITATION FLUIDS AFTER SEVERE BLOOD LOSS IN RATS
L.N. Torres, C. Salgado, C. Valdez, J.L. Sondeen, M.A. Dubick, and I. Torres Filho. US Army Institute of Surgical Research, San Antonio, TX
Background: Death from potentially survivable injuries on the battlefield is largely associated with severe hemorrhage. A transition in combat medicine practiced on the battlefield has occurred in recent years to reduce preventable death but questions regarding efficacy of resuscitation treatments in pre-hospital continue to arise. Successful resuscitation requires restoration of microcirculatory function, i.e. endothelial glycocalyx (EG) repair for less endothelial injury, edema and inflammation. This study goal was to demonstrate that small volume resuscitation can modulate the vascular endothelium in vivo (EG, leukocyte activation and permeability) after hemorrhagic shock in rats.
Methods: Over 100 venules of the cremaster muscle were studied in anesthetized rats (n=25) subjected to 1h of shock (40% estimated blood volume) followed by 1h resuscitation with fresh frozen plasma (FFP), normal saline (NS), or 3% hypertonic saline (3%HTS). SHAM rats were equally instrumented but not bled/resuscitated. Using intravital microscopy, microhemodynamics, EG thickness, permeability, leukocyte (WBC) rolling and adhesion were quantified. Dextrans bound to fluorescein isothiocyanate or Texas Red were injected to estimate EG thickness in vivo. Mean arterial pressure (MAP), heart rate and respiratory rate were measured continuously. Blood samples were collected before and after hemorrhage/resuscitation for blood gases and biochemistry analysis.
Results: Post-resuscitation MAP was significantly lower than baseline levels, but not statistically different among treatments. FFP repaired EG thickness (1.19 ± 0.09 μm), restored vascular permeability and decreased inflammation (rolling 8.5 ± 2.6 cells/min and adhesion 1.3 ± 0.3 cells/100 μm length) compared to SHAM (8.3 ± 2.0 and 2.2 ± 0.7, respectively). NS and 3%HTS significantly decreased blood hematocrit (26 ± 0.4% and 25 ± 0.6%, respectively) and total plasma protein (3.3 ± 0.1 and 3.4 ± 0.1 g/dl, respectively) compared to SHAM (40 ± 0.7, 4.1 ± 0.04, respectively). NS led to a significant EG shedding (0.44 ± 0.05 μm), higher WBC rolling (26.9 ± 5.2 cells/min) and adhesion (3.6 ± 0.4 cells/100um length) as well as > 50% increase in permeability compared to SHAM. Treatment with 3% HTS did not fully repair EG thickness compared to SHAM (0.88 ± 0.11 vs. 1.33 ± 0.08 μm, respectively), but significantly decreased WBC rolling (9.3 ± 5.0 cells/min) and adhesion (0.7 ± 0.3 cells/100 μm length) in vivo.
Conclusion: Modulation of vascular endothelium by resuscitation fluids is associated with microvascular functions, such as permeability/fluid exchange and inflammation. Restoration of EG layer might be an important therapeutic goal to select the most effective resuscitation strategy to severe blood loss with the best outcome.
Supported by US Army Medical Research & Materiel Command.
PLASMA RESUSCITATION PROMOTES COAGULATION HOMEOSTASIS FOLLOWING SHOCK-INDUCED HYPERCOAGULABILITY
J.C. Cardenas*1, A.P. Cap2, M. Huby1, L.A. Baer1, N. Matijevic1, B. Cotton1, J. Holcomb1, and C. Wade*1. 1The University of Texas Health Science Center at Houston, Houston, TX, 2US Army Institute of Surgical Research, San Antonio, TX
Background: Increased thrombin generation (TG) has been observed in injured patients possibly contributing to early consumption of coagulation factors and subsequently exacerbating hemorrhage. Identifying optimal resuscitation products for restoring plasma homeostasis following injury is important for improving management of these patients.
Objective: To determine the effects of crystalloid versus plasma resuscitation on TG in a rat model of gut distention and hemorrhagic shock (HS).
Patients/Methods: Rats were subjected to traumatic injury and HS followed by resuscitation with Lactated Ringer’s solution (LR) or fresh frozen plasma (FFP). Blood was collected at baseline, decompensation, and 3 hours post-resuscitation. TG was measured by calibrated automated thrombogram and antithrombin (ATIII) by ELISA. As part of a prospective observational study, admission blood samples were collected on highest-level activation trauma patients and treated with LR or FFP for TG analysis.
Results: HS significantly upregulated TG. Resuscitation with LR resulted in persistent hypercoagulability, however, FFP resuscitation reversed this hypercoagulable state to baseline TG or below. Plasma ATIII levels decreased following HS and remained low in rats that received LR, but were corrected in rats receiving FFP. Trauma patient plasma showed similar results, as LR increased TG while FFP reduced TG. The effects of FFP were abolished in the absence of ATIII.
Conclusions: HS is associated with faster and greater TG. Resuscitation with LR results in prolonged hypercoagulability whereas FFP resuscitation results in reversal of hypercoagulabilty, possibly through inhibition of TG by ATIII. Thus, ATIII is a key element in FFP restoring hemostatic balance following trauma and HS.
SHOCK CONDITIONS MODULATE LOW GRADE INFLAMMATORY STATE IN ADIPOSE TISSUE: A FACTOR IN POOR OUTCOME IN THE OBESE TRAUMA PATIENT.
M. Diebel, D.M. Liberati, and L. Diebel. Wayne State University/DMC, Detroit, MI
Objective: Obesity is an independent predictor of increased morbidity and mortality in the trauma patient. This is most apparent in the severely injured patient. Acute Respiratory Distress Syndrome (ARDS) and other pulmonary complications following injury are frequent morbidities and may significantly contribute to mortality. The obese patient may be at greater risk due to pre existing conditions. Chronic low grade inflammation is associated with obesity and may be potentiated by shock related tissue hypoxia and catecholamine surge. Adipocytes and macrophages are two important cell fractions of adipose tissue that may contribute to inflammation in obesity. We hypothesized that tissue hypoxia and/or catecholamine administration would impact adipokine and cytokine production by adipocytes and/or macrophages in a cell culture system. We therefore studied the effect of adipocyte/macrophage cell culture supernatants on human pulmonary microvascular endothelial cell (HMVEC) function in vitro.
Methods: Mature adipocyte (ADSC) and tissue macrophage (Raw 264.7) cell cultures were established and exposed to hypoxic conditions (1% O2) and/or epinephrine at 10-3 µM (epi). Cell culture supernatants were collected and leptin, TNFα, and IL-6 were quantitated by ELISA. In other experiments HMVEC monolayers were co cultured with these supernatants and HMVEC cellular apoptosis (%Apo), ICAM-1 expression, and monolayer permeability (FITC-dextran) were determined.
Conclusion: Low grade inflammatory response by adipocytes and macrophages were potentiated by either hypoxia or stress hormone conditions. The greatest response was noted by combined hypoxia and epi exposure. A similar effect was noted with HMVEC functional disturbances after co culture with the various adipocyte-macrophage treatment groups. The results of these experiments suggest that the impact of obesity on "early" onset ARDS in the trauma patient may be related to body mass index (a surrogate marker of adipose tissue mass) and shock severity.
Results: Mean ± S.D. (N = 5 for each group)
POST-HEMORRHAGIC SHOCK MESENTERIC LYMPH DRAINAGE REDUCES THE NLRP3 INFLAMMASOME EXPRESSION OF LUNG IN MICE
G. Liu, L. Jiang, L. Zhang, Z. Zhao, and C. Niu. Hebei North University, Zhangjiakou, China
Previous studies have indicated the post-hemorrhagic shock mesenteric lymph (PHSML) return is an important contributor to acute lung injury (ALI). More importantly, the activation of NLRP3 inflammasome is involved in the occurrence of ALI following hemorrhagic shock. Therefore, the aim of the present study was to investigate the relationship between PHSML return and NLRP3 inflammasome expression during hemorrhagic shock induced-ALI. To achieve this, we used a hemorrhagic shock model (40±2 mmHg for 90 min) and fluid resuscitation (the shed blood and equal Ringer’ solution), and drained the PHSML after resuscitation in the shock+drainage group. Then, we obtained the pulmonary tissue of mice for the examination of NLRP3, TLR2, TLR4, IL-1β, and IL-18 after 3 h of resuscitation. Result from Western blotting experiment showed that hemorrhagic shock induced increases in NLRP3, TLR2, and TLR4 protein expressions of pulmonary tissue, PHSML drainage decreased the expression of NLRP3 and TLR2, but no obvious affect on the TLR4 expression. Meanwhile, detection of qRT-PCR revealed that there were significant increases in NLRP3, TLR2, and TLR4 mRNA expressions of pulmonary tissue following hemorrhagic shock, which was reversed by PHSML drainage. In addition, using the ELISA analysis, we found that PHSML drainage abolished the role of hemorrhagic shock induced-increases in IL-1β, and IL-18 in pulmonary tissue. In summary, the current results demonstrated that the decreased expression of NLRP3 was involved in the PHSML drainage alleviating the ALI subjected to hemorrhagic shock. However, the further mechanism by which NLRP3 on the process of PHSML mediated-ALI needs investigation. This work was supported by the Key Research Program of Hebei North University (ZD201311).
ARTESUNATE PROTECTS AGAINST THE ORGAN INJURY AND DYSFUNCTION INDUCED BY SEVERE HEMORRHAGE AND RESUSCITATION
R. Sordi1, K. Nandra1, F. Chiazza2, N.S. Patel1, M. Collino2, K. Brohi1, and C. Thiemermann*1. 1Queen Mary University of London, London, United Kingdom, 2University of Turin, Turin, Italy
Introduction: Hemorrhagic shock (HS) is a common cause of death in severely injured patients and is associated with impairment of organ perfusion, systemic inflammatory response and multiple organ failure. There is no specific therapy that reduces organ dysfunction in HS. Artesunate is recommended by the WHO as the drug of choice for the treatment of falciparum malaria. Artesunate is superior to quinidine in improving survival in patients with complicated malaria, and also exhibits anti-inflammatory effects. The aim of the present study was to evaluate the effects of artesunate on organ injury and dysfunction associated with HS in the rat.
Methods: Male Wistar rats were subjected to HS under sodium thiopentone anesthesia (120 mg/kg; i.p.). The mean arterial pressure (MAP) was reduced to 30 ± 2 mmHg for 90 min, followed by resuscitation with the shed blood over 5 min. Rats were treated with artesunate (2.4 or 4.8 mg/kg; i.v.) or vehicle upon resuscitation. Four hours later, organ injury and dysfunction and the signaling events involved in the observed protective effects of artesunate were investigated.
Results: When compared to sham-operated rats, HS resulted in a significant decrease in creatinine clearance (from 1.36 ± 0.08 to 0.09 ± 0.03 mL/min) as well as rises in serum creatinine (from 32.5 ± 1.6 to 108.2 ± 5.5 µmol/L), aspartate aminotransferase (from 132.8 ± 22.9 to 1076 ± 109 U/L), alanine aminotransferase (from 47.9 ± 4.1 to 325.9 ± 34.2 U/L) and lactate (from 0.7 ± 0.3 to 2.9 ± 0.3 mmol/L), indicating the development of renal dysfunction, liver and muscular injury and organ ischemia (p<0.05 for all parameters). HS also caused a significant increase in lung myeloperoxidase activity (lung inflammation). Western blotting of kidney and liver tissue from HS rats revealed decreases in the phosphorylation of eNOS and glycogen synthase kinase-3β (GSK-3β), and increases in phosphorylation of IκB and nuclear translocation of NF-κB p65 subunit. Treatment of HS-rats with artesunate protected animals against the organ injury and dysfunction induced by HS. Artesunate increased the activation of Akt and eNOS, inhibited the activation of GSK-3β and NF-κB activation, and attenuated the increase in serum TNF-α associated with HS.
Conclusions: Administration of artesunate on resuscitation attenuated the organ injury and dysfunction associated with HS by a mechanism that may involve activation of the Akt-eNOS survival pathway, and/or inhibition of GSK-3β and NF- κB (reducing inflammation). Artesunate, a well-known, safe and low cost antimalarial drug, may represent a novel approach to reduce the organ injury and dysfunction associated with HS. A single-centre placebo-controlled randomized phase II clinical trial aimed at evaluating the effects of artesunate in patients with severe hemorrhage following trauma is planned at Barts Health NHS Trust.
EFFECT OF DIGESTIVE ENZYME INHIBITION IN THE INTESTINE ON PROTEASE ACTIVITY LEVELS IN THE HEART IN HEMORRHAGIC SHOCK
F. Aletti1, 2, M.H. Santamaria1, F. DeLano1, and G. Schmid-Schonbein1. 1University of California San Diego, La Jolla, CA, 2Politecnico di Milano, Milan, Italy
The mechanisms of heart injury during multiple organ failure after circulatory shock are still unresolved. We hypothesize that organ damage could be caused by uncontrolled proteolytic activity. In order to test it, protease activity levels were measured in heart homogenates from shock animals without and with an enteral protease inhibitor, tranexamic acid (TXA), and in sham controls.
Hemorrhagic shock was induced in Wistar rats (n=5/group) by progressive blood withdrawal from the left femoral vein, so that arterial blood pressure reached 35 mmHg for 2 hours. Then, the shed blood was returned, and the animals were euthanized at 2 hours after the initiation of reperfusion. The treatment group received enteral tranexamic acid by injections in the intestinal lumen during the ischemic phase. The injections were performed in short succession to achieve complete filling of the small intestine (total volume ∼ 15 cc). Hearts were excised following euthanasia, homogenized and frozen at -80C and the protease activity determined by gelatin zymography.
Analysis of the gels revealed characteristic bands of serine proteases, MMP-2, and MMP-9 with a significantly higher activity in shock compared to control. In the TXA group, no significant proteolytic activity for all three proteases was detected, similar to the controls.
These results suggest that the increase of protease activity levels may be a factor in the development of injury to the heart in hemorrhagic shock. Further analysis is necessary to understand the impact of proteolytic activity on the receptors which regulate heart function. Also, TXA was able to abolish protease activity, suggesting a potential role as a therapeutic agent in the management of shock and multiple organ failure.
This work was supported by the ShockOmics project of the European Union (Grant#602706), by the CelSys Shock Marie Curie International Outgoing Fellowship (PIOF-GA-2012-328796) of the European Union, and by GM 85072.
THE EFFECTS OF 17α-ETHINYLESTRADIOL-3-SULFATE AND 17β-ESTRADIOL-SO4 ON CARDIAC AND HEPATIC SIRT1 LEVELS FOLLOWING TRAUMA-HEMORRHAGE
B. Cam, S. Yang, S. Hu, R. Sirmali, W.J. Hubbard, K.I. Bland, and I.H. Chaudry. University of Alabama at Birmingham, Birmingham, AL
The NAD-dependent deacetylase sirtuin 1 (SIRT1), is one of deacylase proteins that have been shown to regulate variety cellular processes including inflammation, stress resistance, and aging. Our previous studies have shown that trauma-hemorrhage (T-H) produced cardiac depression which was correlated with T-H-induced decrease in cardiac SIRTs. We have also shown that 17β-estradiol-SO4 (E2) administration following T-H improved cardiac contractility and survival of animals after T-H. 17α-ethinylestradiol-3-sulfate (EE-3-SO4) is a highly water soluble synthetic estrogen and has a longer half-life compared to E2. The aim of our study therefore was to compare the effects of E2 and EE-3-SO4 on left ventricular (LV) performance, cardiac and hepatic SIRT1 levels in male adult S-D rat T-H model, who were treated with the above two hormones or other agents. A total of seven groups were used (n=4-6/group): sham, T-H+vehicle, T-H+E2 (1 mg/kg), T-H+ICI 182,780 (3 mg/kg)+E2, T-H+EE-3-SO4 (1 mg/kg), T-H+ICI 182,780+EE-3-SO4, and T-H+Sirtinol+EE-3-SO4. E2 or EE-3-SO4 were delivered iv at the onset of resuscitation. The estrogen receptor antagonist ICI 182,780 (ICI) was given intraperitoneally 30 min before estrogens administration and the sirtuins antagonist, sirtinol (1 mg/kg BW), was administered iv 5 min prior to resuscitation. Two hrs following T-H and resuscitation, LV performance was measured, and then heart and liver tissue were harvested. Cardiac and hepatic SIRT1 levels were determined by Western blot. Our results indicate that EE-3-SO4 and E2 restored LV performance and cardiac SIRT1 level following T-H. Similar results were observed for hepatic SIRT1 levels after T-H. However, ICI 182,780 and sirtinol blocked the salutary effects of both estrogens on left ventricular performance and SIRT1 levels. These results thus indicate that at 2 hrs after T-H and resuscitation, both forms of estrogens are equally effective in improving cardiac performance and SIRT1 levels after T-H. These studies also support the concept that further development of SIRT1 activation may be a potential approach for more selective targeting of trauma patients (NIH RO1GM39519).
COMPARISON OF GENE TRANSCRIPTION PROFILES INDUCED BY LIPOPOLYSACCHARIDE AND MONOPHOSPHORYL LIPID A IN HUMAN BLOOD
L. Luan*1, P. Lu1, X. Chen1, P. Enkhbaatar*2, and E. Sherwood*1. 1Vanderbilt University Medical Center, Nashville, TN, 2the University of Texas Medical Branch, Galveston, TX
Background: Monophosphoryl lipid A (MPLA) is a derivative of lipopolysaccharide (LPS) with useful immunomodulatory properties but less toxicity. As such, MPLA is currently employed as an FDA-approved vaccine adjuvant and under investigation as a non-specific immunomodulator. The goal of this study was to compare the differential genomic response of human blood to LPS and MPLA.
Methods: 2.5 mL of venous blood was drawn from six healthy volunteers (mean age 28 yrs.) and mixed with 30 µL of PBS, LPS (1 μg/ml) or MPLA (10 μg/ml). After incubation at 25 °C for 90 min, total RNA were extracted, quality of the total RNA samples was assessed and gene expression was determined using an Agilent Bioanalyzer with human 8x60K microarray chips (design ID 039494) at GenUs BioSystems. Probe-wise data was analyzed using R statistical language. Differentially expressed genes were identified with FDR cut-off 0.1.
Results: Among 21,103 qualified probes profiled, 130 and 136 genes were differentially expressed (fold change > 2) in human blood when stimulated by MPLA and LPS, respectively. Expression of 83 genes was equally regulated by both TLR4 ligands, while 47 genes were preferentially induced by MPLA and 53 genes were preferentially induced by LPS. A further comparison of the effects of MPLA and LPS showed a number of inflammatory mediators (IL18, IL23A, IL1A, IL12B, and IFNB1) were strongly induced by LPS, and 11 genes were intensely induced by MPLA. For example, CCL7, a small chemokine known to attract monocytes and regulate macrophage function, was not changed upon LPS challenge but was strongly upregulated by MPLA (>14-fold). In addition, a number of members of the CXCL family, CCL family and NF-kappaB signaling pathway were similarly induced by MPLA and LPS. Of note, IL-6 showed the greatest upregulation in both groups. Hierarchical clustering of eighteen samples resulted in three distinct clusters distinguishing the 6 MPLA-treated and 6 LPS-treated blood samples from the remaining 6 control samples.
Conclusion: Despite the vast similar genomic response of human peripheral blood to LPS and MPLA, multiple genes are strongly induced by MPLA but not LPS. Hence, more thorough preclinical studies are needed to characterize the molecular response to MPLA in humans.
RESOLVIN D1 PROTECTS THE MURINE LIVERS FROM ISCHEMIA/REPERFUSION INJURY VIA ACTIVATION OF SPHINGOSINE-1-PHOSPHATE SIGNALING PATHWAY
J. Kang*1, J. Shin*1, and S. Lee*. Sungkyunkwan University, Suwon, Republic of Korea
Introduction: Resolvins (Rvs) are a novel paradigm of endogenous lipid mediators that promote resolution of inflammation and stimulate the tissue return to homeostasis. We have recently revealed that RvD1 facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis via ALX/FPR2 in liver ischemia/reperfusion (IR) injury (unpublished data). However, the specific cellular or molecular targets for RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P1-S1P5), regulates lymphocyte circulation and various immune responses. In the present study, we investigated the molecular mechanisms of RvD1 in IR-induced hepatocellular damage, particularly focusing on the S1P signaling pathway.
Methods: Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (5, 10 and 15 μg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. For the depletion of KCs, liposome clodronate was administered (100 μL/mice, i.v.) 24 h prior to ischemia. VPC23019, an antagonist for S1P1/S1P3, was pretreated (100 μg/kg, i.p.) 10 min prior to initial RvD1 treatment.
Results: Serum and liver RvD1 concentrations peaked at 6 h and gradually declined by 24 h of reperfusion. 15-Lipoxygenase-1 (15-LOX1) and 5-LOX which involve in the conversion of ω-3 polyunsaturated fatty acids to RvD1 showed similar patterns with RvD1. Exogenous treatment of RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum ALT and AST activities and histopathological score. In addition, RvD1 reduced intracellular reactive oxygen species generation, lipid peroxidation and pro-inflammatory mediators. RvD1 attenuated the decrease in serum S1P concentration induced by IR. Moreover, in purified hepatocytes of mice exposed to IR, sphingosine kinase 1 (SK1), SK2, S1P1 and S1P3 mRNA expressions significantly decreased, which were attenuated by RvD1. KCs depletion by liposome clodronate did not influence the effect of RvD1 on SKs and S1P receptors, suggesting independency on KCs. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1.
Conclusion: Our findings suggest that RvD1 protects the liver against IR injury by activation of S1P signaling.
A CRITICAL ROLE OF TLR2 IN THE INFLAMMATORY RESPONSE AND ADVERSE REMODELING IN AGING HEARTS FOLLOWING ISCHEMIA/REPERFUSION
Y. Zhai, L. Ao, D. Fullerton, and X. Meng. University of Colorado Denver, Aurora, CO
The myocardial inflammatory response exacerbates heart failure following myocardial ischemia/reperfusion (I/R) injury. Aging is a risk factor for a worse post-ischemic cardiac outcome. Myocardial Toll-like receptor 2 (TLR2) can be activated by danger-associated molecular patterns released from injured or stressed cardiac cells and promotes myocardial inflammatory response and injury in adult animal models of myocardial I/R. Currently, the impact of aging on the signaling mechanism that mediates myocardial inflammatory response to I/R injury is unclear. We hypothesized that TLR2 play an important role in mediating the inflammatory response and matrix protein remodeling in the remote non-ischemic myocardium in aging hearts following regional I/R. Methods and results: Adult (4-6 months old) and aging (20-24 months old) C57BL/6 wild type (WT), TLR2 knockout (KO) mice were subjected to coronary artery ligation (30 min) and reperfusion (3 or 14 days). In aging WT hearts, levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the remote non-ischemic myocardium were significantly higher at day 3 of reperfusion compared to adult WT hearts. The enhanced inflammatory response in aging WT hearts was associated with greater production of matrix metalloproteinase 9 (MMP 9), larger infarct sizes and worse left ventricular performance at day 14 of reperfusion. TLR2 KO markedly downgraded the levels of ICAM-1, VCAM-1 and MMP 9 in the remote non-ischemic myocardium of aging hearts, reduced infarct sizes and improved left ventricular performance. In conclusion, TLR2 plays a role in mediating myocardial inflammatory response and matrix protein remodeling induced by I/R in aging hearts. TLR2 KO attenuates adverse cardiac remodeling in aging hearts following regional I/R injury, resulting in improved cardiac functional outcome.
COLD-INDUCIBLE RNA-BINDING PROTEIN ACTIVATES THE INFLAMMASOME FOR IL-1β RELEASE IN MICROGLIA
M. Zhou*1, W. Yang*1, X. Qiang1, J. Fan*2, and P. Wang*1. 1Hafstra North Shore-LIJ School of Medicine and The Feinstein Institute for Medical Research, Manhasset, NY, 2University of Pittsburgh School of Medicine, Pittsburgh, PA
Introduction: Neuroinflammation exacerbates brain injury in disease conditions such as trauma and stroke. Cold-inducible RNA-binding protein (CIRP) is a newly discovered inflammatory mediator. Our previous animal studies have shown that CIRP is upregulated and released during cerebral ischemic stress. The released CIRP stimulates inflammatory responses and causes neuronal damage. In the brain, microglia act as a major player for inflammatory activity. Here we investigated the role of CIRP on inflammasome activation in microglia.
Methods: Murine microglial BV2 cells were cultured in Dulbecco’s Modified Eagle medium and treated with recombinant murine (rm) CIRP (1 µg/ml) in the presence or absence of various inhibitors. After 4 or 8 hours of incubation, the treated and untreated BV2 cells and cultured media were harvested for analysis. The mRNA levels and protein expression of IL-1β in BV2 cells were determined by qPCR and Western blotting, respectively. IL-1β protein levels in cultured media were measured by ELISA. Cathepsin B activity was determined by the Magic Red cathepsin B detection kit.
Results: IL-1β mRNA levels in BV2 cells were increased by 3.8- and 3.3-fold after 4 and 8 hours of rmCIRP incubation, respectively (p < 0.05 vs. untreated). The protein levels of IL-1β precursor were also increased by 2.3- and 3.1-fold at 4 and 8 hours, respectively (p < 0.05 vs. untreated). Moreover, IL-1β levels in the cultured medium with 8 hours of rmCIRP incubation were 5.9-fold higher than those without rmCIRP (p < 0.05). Levels of the released IL-1β were reduced by 34% and 89% in the presence of the neutralizing anti-CIRP antibody (10 µg/ml) and P2x7 receptor inhibitor oxidized ATP (500 µM), respectively. In addition, rmCIRP incubation significantly increased cathepsin B activity in BV2 cells by 1.2-fold (p < 0.05). With the treatment of cathepsin B inhibitor Ca-074me (12.5 µM), the released IL-1β induced by rmCIRP were decreased by 63% (p < 0.05).
Conclusion: Extracellular CIRP stimulates the expression of IL-1β mRNA and protein and promotes the cleavage of IL-1β precursor for IL-1β release in microglia. Thus, activation of the inflammasome is one of the mechanisms for CIRP- induced inflammation. Development of an anti-CIRP therapy may reduce brain injury augmented by neuroinflammation.
T CELL ACTIVATION INVOLVES AUTOCRINE FEEDBACK THROUGH THE ADP RECEPTORS P2Y1, P2Y12, AND P2Y13
T. Woehrle1, 2, C. Ledderose2, Y. Sumi*2, L. Yip3, and W. Junger*2. 1Ludwig Maximilian University Munich, Munich, Germany, 2Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 3Stanford University, Stanford, CA
T cell receptor (TCR) stimulation leads to the release of adenosine-5’-triphosphate (ATP), which regulates T cell activation by autocrine and paracrine stimulation of P2X receptors. While these roles of ATP in T cell activation are well understood, the involvement of other nucleotides, specifically adenosine-5’-diphosphate (ADP) is less well characterized. Here, we show that ADP is released along with ATP from stimulated T cells. Jurkat T cells and primary human CD4+ T cells express the ADP-sensitive purinergic receptors P2Y1, P2Y12, and P2Y13. Stimulation of T cells with ADP receptor agonists or overexpression of P2Y1, P2Y12, or P2Y13 receptors promoted intracellular MAPK phosphorylation, IL-2 gene transcription, and T cell activation. Conversely, gene silencing of these receptors or pharmacological inhibition of P2Y1, P2Y12, or P2Y13 receptors with MRS2279, the platelet aggregation inhibitor clopidogrel, or MRS2211, respectively, blocked activation of p38 and ERK MAPKs and IL-2 production in response to TCR/CD28 stimulation. TCR stimulation increased the expression of P2Y1 and P2Y12 and blocked the expression of P2Y13 receptors on the surface of T cells. These data indicate that ADP and stimulation of P2Y1, P2Y12, and P2Y13 receptors contribute to the purinergic mechanisms that regulate T cell activation. Pharmacological targeting of these receptors may help modulate T cell responses in critical care patients and in patients with other acute or chronic inflammatory conditions.
MONOCYTES REQUIRE MITOCHONDRIA TO FUEL AUTOCRINE PURINERGIC SIGNALING AND INFLAMMASOME ACTIVATION
A.H. Lee1, L. Staudenmaier1, X. Li1, J. Zhang1, and W. Junger*1, 2. 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 2Ludwig Boltzmann Institute for Traumatology, Vienna, Austria
Previously, we have shown that autocrine purinergic signaling and mitochondria modulate immune cell functions that are critical for host defense. ATP generated by mitochondria is released from immune cells upon pathogen recognition. The released ATP triggers autocrine feedback signaling via purinergic receptors that regulate cell functions such as neutrophil chemotaxis and T cell proliferation. Here, we studied whether inflammasome activation in monocytes involves similar autocrine purinergic signaling mechanisms.
We found that toll-like receptor (TLR) stimulation of human monocytes causes an increase in extracellular ATP, which could be blocked with carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler that disrupts mitochondrial membrane ATP production (Fig. 1A). Inhibition of mitochondrial function with CCCP dose-dependently blocked TLR-induced IL-1β expression by monocytes stimulated with lipopolysaccharide (LPS; Fig. 1B).
Our data suggest that inflammasome activation in response to TLR stimulation involves mitochondria that regulate autocrine purinergic signaling pathways in monocytes.
This study was supported by NIH grants R01 GM51477, R01 GM60475, R01 AI080582, and T32 GM103702.
AUGMENTED HOST RESPONSE TO BACTERIA IS SPECIFIC TO MILD TRAUMATIC BRAIN INJURY
T. Hsieh, M. Vaickus, E.L. Chiswick, B. Lussier, E.R. Duffy, and D.G. Remick. Boston University School of Medicine, Boston, MA
Introduction: Our previous clinical study has shown reduced rates of pneumonia in traumatic brain injury (TBI) patients compared to blunt trauma patients. Work in our mild murine TBI model shows improved survival characterized by increased pulmonary cell recruitment and bacterial clearance in a Pseudomonas aeruginosa (PSD) pneumonia model compared to sham injury. We developed a blunt tail trauma model to mimic patient inflammatory responses to determine whether the immune priming is specific to TBI. This study investigates differences in host response to a pneumonia challenge after tail versus brain trauma.
Methods: Mild traumatic brain injury (mTBI) or tail trauma (TT) was induced using a weight drop model. 48 hours after mTBI or TT, Psd was intratracheally instilled into adult female ICR mice. Facial vein bleeds were collected prior to injury then 4, 24, 48, 54, and 72h post. Blood differential was obtained by Hemavet. Bronchoalveolar lavage (BAL) was performed 4h post-PNA (52h total) for cell counts and colony forming units. Cytokines were measured by ELISA.
Results: Both TT and MTBI induce similar hematological changes characterized by increased peripheral blood neutrophils and decreased lymphocytes 4h following injury, which resolved within the next 48 hours. Interestingly, IL-6 was elevated only in TBI mice 4 hours post-trauma (0.9251±0.2145ng/mL vs BLD). IL-1RA and TNF-α levels were undetectable prior to Psd challenge in both models. All 3 cytokines were undetectable by 48h post-injury. 4h after Psd pneumonia challenge, BAL data indicate mTBI-specific priming of the immune system. Plasma IL-6 and IL-1RA was higher in mTBI while TNFα was lower compared to TT. 4h post-Psd BALs reflect a lower inflammatory state in mTBI mice (Table 1). Both groups of mice exhibited similar levels of total blood WBCs and neutrophils 6h and 24h after pneumonia; however, the TBI mice had decreased lymphocyte numbers at 6h, but are similar at 24h compared to TT. mTBI mice showed increased cell recruitment to the alveolar space compared to TT. mTBI mice also had decreased bacterial burdens in their airspaces (Table 1).
Conclusions: Early increases in plasma IL-6 in mTBI mice may indicate immunological priming specific to brain injury. After pneumonia, mTBI mice reflect a lower inflammatory state compared to TT. TT mice also recruit fewer cells and have increased bacterial burden in the airspace compared to TBI. These data show mTBI provides specific augmentation of the host response to bacteria compared to blunt tail trauma.
POLYTRAUMA INDUCES EXPANSION OF HEMATOPOIETIC PROGENITORS
I.R. Turnbull, R.D.Winfield, A. Fuchs, S. Ghosh, C. Davis, S. Hughes, and G.V. Bochicchio. R.D. Winfield**1,2A. Fuchs*1,2, S. Ghosh*1,2, C. Davis*1,2, S. Hughes*1,2, G.V. Bochicchio*1,2. Washington University School of Medicine, Saint Louis, MO
Trauma remains a leading cause of death in adults in the US. More than 250,000 trauma deaths are reported each year. Improvements in trauma care have decreased early deaths; however, deaths from injury induced critical illness remain a significant problem. Previous work has shown that trauma predisposes to sepsis and that sepsis causes immunosuppression in-part by skewing hematopoiesis. We hypothesize that trauma skews hematopoiesis a priori and that the injury-induced changes in hematopoietic progenitors cause immunosuppression predisposing to sepsis. To test this we measured hematopoiesis in mice subjected to a polytrauma (PT) injury previously demonstrated to cause immunosuppression.
C57BL/6 mice were subjected to PT(n=13) consisting of 15% blood-volume hemorrhage, bilateral lower extremity pseudofracture and liver laceration; controls underwent sham laparotomy (S, n=10). 24 hours after injury, bone marrow was incubated with antibodies against Sca1, Flk2, CD34, cKit, and CD16/32; lineage negative (Lin-) cells were defined by antibodies to CD3, CD4, CD8, CD19, Gr-1, CD11b, TER119. Cells were analyzed by flow cytometry. Median % of total cells was compared by Kruskal-Wallis.
PT resulted in <5% mortality; there was no deaths in the shams. Our protocol allowed discrimination of hematopoietic stem cells (HSC, including short-term (ST-HSC) and long-term (LT-HSC) populations); multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), common myeloid progenitors (CMP), granulocyte/macrophage progenitors (GMP) and megakaryocyte/erythrocyte progenitors (MEP). Population definitions and results are shown in Table 1. We found that polytrauma induced expansion of the HSC pool, with significant increases in both total HSC and in the LT-HSC subset. There was a strong trend toward an increase in ST-HSC. Polytrauma caused a decrease in CMP numbers.
Non-lethal PT induces expansion of the hematopoietic progenitors and a decrement in myeloid progenitors. These results recapitulate those in murine models of sepsis. In contrast to those data, report skewed hematopoeisis with nonlethal sterile injury alone, without microbial challenge. These data provide a potential mechanism for the increased susceptibility to infection seen in patients with polytrauma injuries.
THE SPATIOTEMPORAL ACCUMULATION OF MYELOID LINEAGE CELLS IS MAPPING OUT FIBROSIS IN THE LIVER POST-THERMAL INJURY: A TARGETABLE PATHWAY USING A SEROTONIN ANTAGONIST
S. Amini-Nik*1, 2, A. Sadri2, C. Belo2, and M.G. Jeschke*1, 2. 1Department of Surgery, University of Toronto, Toronto, ON, Canada, 2Sunnybrook Research Institute, Toronto, ON, Canada
Severe burn injury results in local and systemic responses including profound hepatic alterations. Macrophages have an imperative role in promoting liver fibrosis by contributing to the transformation of hepatic stellate cells into myofibroblasts. However, the mechanism of action and the spatiotemporal role is not clear. As such, it is difficult to target this cell population in order to attenuate the pathology.
Through the use of Cre-transgenic mice that specifically mark myeloid lineage cells and burn as a model of systemic injury, we seek to ascertain the spatiotemporal role of myeloid lineage cells in liver fibrosis. Our Cre-transgenic mice permanently express enhanced yellow fluorescent protein (EYFP) under the control of the lysozyme (Lysz) promoter (Lysz-Cre; ROSA.EYFP) and label resident (Kupffer cells) and recruited myeloid cells in liver post thermal injury.
Here we report that thermal injury in mice (30% TBSA) induces fibrosis predominantly around portal venules whereas EYFP+ cells are enriched throughout the liver. This spatial pattern of fibrosis peaks around 7-14 day’s post-thermal injury. A robust increase of EYFP+ cells is observed and 87% of these cells are F4/80+. Gene expression microarray analysis of isolated myeloid cells, sorted using flow cytometry, from the liver of burned mice in comparison to the sham group suggests a dual signature of gene expression: pro-fibrotic as well as anti-fibrotic. The observed discrepancy between the spatial pattern of fibrosis and accumulation of myeloid cells suggests that they have pro-fibrotic effects around portal venules. This is due to recruitment of distinct myeloid cell subpopulations or due to enhanced phagocytic activity of myeloid cells at the portal venule sites, which shape their pro-fibrotic secretome. Aiming to target macrophage polarity in the liver of animal’s post thermal injury, we treated animals with serotonin antagonists. In animals treated with this drug, lower fibrosis was observed (p<0.02). While the drug did not change the number of F4/80+ cells in the liver of burned animals in comparison with PBS treated burned group, there is a significant increase (p<0.011) in the number of M2 macrophages, indicating that the anti-fibrotic effect is through promoting an M2 macrophage phenotype rather than affecting the recruitment or proliferation of macrophages. The later was addressed in vitro too.
Our data unravel a spatiotemporal signature for fibrosis with a mechanism mainly related to the phenotype of accumulated myeloid cells in liver post-thermal injury. Serotonin antagonist agents can modulate this phenotype.
ETAS, AN ENZYME-TREATED ASPARAGUS, ATTENUATES OXIDANT INJURY IN RAT EPITHELIAL CELLS
N. Sato*1, A. Kitaguchi1, T. Irahara1, 2, K. Ohtake1, 2, K. Inoue1, S. Murata1, S. Kawashima1, and K. Koike*1. 1Kyoto University, Kyoto, Japan, 2Nippon Medical school, Tokyo, Japan
Background: Gut ischemia/reperfusion injury causes mucosal damage and leads to systematic inflammation and multiple organ dysfunction syndrome in critically illness. It is important to reduce oxidant stress somehow to prevent further mucosal damage. We have previously shown that oxidant injury using H2O2 in rat intestinal epithelial cell (IEC-6). Asparagus has been known to reduce oxidative stress and recently, Enzyme-Treated Asparagus extract (ETAS) has been produced as functional food. The purpose of this study was to investigate the effect of ETAS in rat intestinal epithelial cell (IEC-6) under oxidative stress.
Methods: IEC-6 cells were cultured to confluence then added different concentrations of ETAS (0.2 or 0.8 mg/mL) with or without oxidant stress induced by H2O2 (0.5mM). Cytotoxity was measured in medium by LDH after 24 hours. The production of HO-1 and COX-2 mRNA were measured by real time RT-PCR and wound restitution was measured using microscopy.
Results: LDH without oxidant stress was 0.15±0.02. LDH addition of ETAS 0, 0.2mg/mL, 0.8 mg/mL with oxidant stress were 0.25±0.02, 0.25±0.02, 0.21±0.02*, p<0.05 respectively. HO-1 mRNA addition of ETAS 0.8 mg/mL without oxidant stress was significantly increased compared to addition of ETAS 0, 0.2mg/mL without oxidant stress. On the contrary HO-1 mRNA addition of ETAS 0.8 mg/mL was significantly decreased compared to addition of ETAS 0, 0.2 mg/mL with oxidant stress. COX-2 mRNA with oxidant stress was significantly higher than without oxidant stress, but was not affected by addition of ETAS. Wound restitution with oxidant stress was 19.3±1.5 μm, which was significantly lower than without oxidant stress 36.6±2.9 μm. Addition of ETAS 0.2 mg/mL and 0.8 mg/mLwith oxidant stress were 26.1±2.1 μm and 27.5±3.3 μm.
Conclusions: We demonstrated that ETAS decreased cytotoxicity and enhanced wound restitution. ETAS affected HO-1 mRNA production, but not COX-2 mRNA. ETAS might have protective role under oxidant stress in intestinal epithelial cell.
M2 MACROPHAGE DIFFERENTIATION AFTER ELECTROPORATION-MEDIATED GENE DELIVERY OF THE NA+/K+-ATPASE PUMP DOES NOT INCREASE VULNERABILITY TO GRAM NEGATIVE PNEUMONIA AFTER LUNG CONTUSION IN MICE
D.A. Machado-Aranda, N. Talarico, V. Dolgachev, B. Thomas, S. Madathilparambil, M. Hemmila, and K. Raghavendran. University of Michigan, Ann Arbor, MI
Objective: Lung contusion (LC) is a leading cause of admission into the ICU and is a significant risk factor for the development of pneumonia. Their combination can lead into severe respiratory failure (ARDS). Our laboratory has recently shown that electroporation-mediated delivery of genes for the Na+/K+-ATPase Pump (EP-Pump) can reduce lung injury and induce a significant increase in M2 Macrophage cells recovered in bronchial alveolar lavage (BAL) fluid from lungs treated with EP-pump accelerating recovery after LC. However this raises concerns as we have shown that an unbalanced increase of M2 Macrophages (impaired intra-cellular killing phenotype) could make the host more susceptible to a gram-negative pneumonia. We set out to investigate if EP-pump would render the lung vulnerable to gram-negative Pneumonia after LC.
Methods: After inducing right-sided LC injury, plasmid DNA encoding for the a/b subunits of the Na+/K+-ATPase pump were delivered using tongue pull hypopharynx aspiration followed by immediate transthoracic electroporation (EP-pump). A lung contusion only group was used as a control (LC-only). At 24 hr after LC, all animals were inoculated intra-tracheally with 500 CFU of Klebsiella pneumoniae and survival curves were performed. Parallel experiments were performed in order to assess for duration of expression using luciferase and quantitative cellular population(s) analysis was performed using Flow Cytometry.
Results: A survival benefit was seen in the EP-pump group up to 72 hr after LC (48 hr after pneumonia) (100% vs. 60%). This protective effect correlates to the duration of the transgene (luciferase) expression after lung contusion (48 hr). Additionally, EP-pump does induces M2 phenotypic alteration in BAL, with an absolute rise in the numbers of Neutrophils (PMNs) with diminished apoptosis as compared to control.
Conclusion: EP-mediated delivery of genes for the Na+/K+-ATPase pump induced polarization of BAL macrophages towards an M2 phenotype, but does not make the host more susceptible to gram-negative pneumonia.
P2Y12 RECEPTOR AS A THERAPEUTIC TARGET IN SEPSIS
E. Liverani, M.C. Rico, S.P. Kunapuli, and L. Kilpatrick. Temple University, Philadelphia, PA
Introduction: Sepsis and sepsis-induced lung injury is a multifactorial pathological condition in response to bacterial infections. An important component of host tissue damage is excessive neutrophil migration across vascular endothelium. The mechanisms which regulate neutrophil activation and migration in sepsis are incompletely understood but there is growing evidence that platelets are actively involved in neutrophil activation and play a key role in microvascular permeability and neutrophil-mediated organ damage. ADP receptors regulate platelet activation and P2Y12 receptors have been implicated in pulmonary inflammation. In addition, P2Y12 receptor antagonists (i.e. thienopyridines) can reduce sepsis severity. However, thienopyridines also exert P2Y12-independent effects on immune cells. Thus the role of these receptors in sepsis is not well understood. In this study we investigated the effect of P2Y12 receptor deficiency and antagonism during sepsis.
Methods: Sepsis was induced by cecal ligation and puncture (CLP) and the role of the P2Y12 receptor in neutrophil migration and lung injury was investigated using wild type (WT) and P2Y12 null mice. P2Y1 null mice, the other ADP receptor expressed on platelets, were also used to discriminate between P2Y12 deficiency and a reduced response to ADP. WT and P2Y12 null mice were also treated with the thienopyridine, clopidogrel (30mg/kg loading dose; 10 mg/kg maintenance dose), to compare receptor antagonism with receptor deficiency.
Results: Twenty-four hours post CLP or sham surgery, circulating levels of white blood cells, activated platelets (increased P-selectin expression), neutrophil-platelet aggregates, and the cytokines TNFα, IL-6, IL-10 and MIP-1β, were significantly elevated in WT septic mice as compared to sham surgery mice. These systemic alterations were attenuated in CLP-P2Y12 null mice as compared to CLP-WT mice. In the lung, CLP-P2Y12 null mice had decreased platelet and neutrophil infiltration as compared to CLP-WT mice. Furthermore, there was decreased lung injury as assessed by cell infiltration, tissue architecture and edema in CLP-P2Y12 null mice as compared to CLP-WT mice. Similar lung protective effects were observed when WT mice were treated with clopidogrel. No significant differences were observed in clopidogrel treated P2Y12 null mice as compared to clopidogrel treated WT mice indicating the protective effects were P2Y12 receptor-mediated and P2Y12-independent effect of clopidogrel. In contrast, there were no significant differences observed in P2Y1 null mice during sepsis as compared to WT mice, indicating that the observed protective effects were not mediated by an altered response to ADP.
Conclusions: Taken together these data indicate that during sepsis activated platelets and the P2Y12 receptor play an important role in sepsis-mediated pulmonary inflammation and inflammatory cell recruitment.
THE EFFECT OF LL-37, A PORE-FORMING PEPTIDE ON CHEMOTAXIS OF NEUTROPHILIC GRANULOCYTES IN PATIENTS WITH POLYTRAUMA, SEPSIS, AND AUTOINFLAMMATORY DISEASES
E. Schneider1, A. Weins2, S. Denk3, M. Weiss4, and M. Huber-Lang*3. 1Sektion Experimentelle Anaesthesiologie, Ulm, Germany, 2Dept. Dermatology, University Hospital Ulm, Germany, 3Department of Traumatology, Hand, Plastic, and Reconstructive Surgery, University Hospital Ulm, Germany, 4Department of Anesthesiology, University Hospital Ulm, Germany
Inflammation either induced by trauma or infections may lead to increased concentrations of the pore forming peptide, LL-37. This peptide influences chemotaxis and neutrophil extracellular trap (NET) formation. Using ELISA techniques, we determined inflammatory cytokines, lipopolysaccharide binding protein (LBP), ferritin, HMGB1 and LL-37 concentrations in patients with trauma, sepsis/septic shock, and in patients with autoinflammatory diseases and macrophage activation syndromes (MAS). Highest concentrations of LL-37 were detected in polytrauma patients (5-30 ng/ml). Autoinflammatory diseases and MAS presented with high LL-37 as well (<20 ng/ml) and septic patients ranged in between. We then tested isolated neutrophils of patients with sepsis and septic shock for chemotaxis against synthetic LL-37 (0.2-2 ng/ml), recombinant C5a (1-10ng/ml) and recombinant IL-8 (10-50 ng/ml). Despite of a high variability in different patients, all patients’ derived PMN were more active in migration than neutrophils prepared from healthy donors. When 0.2-2 ng/ml of LL-37 were added into the migration assay, the chemotactic response by the majority of sepsis isolates was impaired by exogenous LL-37, whereas healthy donors had an increased chemotactic response when LL-37 was present. Results suggest that LL-37 is a biomarker and effector of sterile as well as infection-associated inflammation. Significant concentrations of LL-37 may impair the chemotactic response of LL-37 against other stimuli. LL-37 synergistically activates chemotaxis in healthy donors but attenuates migration in an infectious microenvironment. Moreover, increased plasma concentration of LL-37 in inflammasome-driven autoinflammatory diseases may explain neutrophilia, vasculitis and skin manifestation.
SKELETAL MUSCLE INSULIN RESISTANCE AND INCREASED UBIQUITIN LIGASE GENE EXPRESSION FOLLOWING POLYTRAUMA
R.M. Akscyn1, J.L. Franklin1, T.A. Gavrikova1, and J.L. Messina*1, 2. 1University of Alabama at Birmingham, Birmingham, AL, 2Veterans Affairs Medical Center, Birmingham, AL
Polytrauma is 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. Resistance to anabolic hormones, loss of muscle mass, and metabolic dysfunction occurs following severe injury. To investigate the effects of combined injuries, we have developed a highly reproducible rodent model of polytrauma. This model combines burn injury and soft tissue trauma with penetrating injury to the gastrointestinal (GI) tract. 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). The single injuries are minor and highly survivable when occurring separately. Furthermore, 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 (≥ 90%). However, when these injuries were combined survival was greatly decreased (∼30% within 48h). Our previous work demonstrated that polytrauma induced hepatic insulin resistance and metabolic dysfunction. In the current studies, insulin signaling was examined in skeletal muscle (triceps). Changes in the abundance of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) were observed at the protein and mRNA levels. Furthermore, insulin resistance was observed following polytrauma as evidenced by decreased IR and AKT phosphorylation upon exogenous insulin treatment. In addition to insulin resistance, dramatic increases in expression of the E3 ubiquitin ligases Atrogin-1/FBX032 and TRIM63/MuRF1 were measured in skeletal muscle. These changes in gene expression were injury and timepoint dependent. Lastly, changes in the skeletal muscle mRNA levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 were observed following both single injuries and polytrauma.
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. We believe our model is ideal for studying the complex sequelae of polytrauma, which differs from single injuries. Insights gained from using this model may suggest better therapies with improved patient prognosis.
REPAIR OF FAULTY FATTY LIVER REGENERATION AND IMPROVED SURVIVAL AFTER HEPATECTOMY THROUGH EPIDERMAL GROWTH FACTOR RECEPTOR RESTORATION BY GENE THERAPY IN OBESE MICE
L.G. Koniaris*1, X. Jin2, Z. Zhang2, Y. Jiang1, and T. Zimmers*1. 1Indiana University, Indianapolis, IN, 2Thomas Jefferson University, Philadelphia, PA
Steatosis is the most common histological finding in liver and the incidence is increasing with obesity. Even mild steatosis is associated with delayed hepatic regeneration and poor outcomes following liver resection. We sought to identify and target molecular pathways that mediate this dysfunction. Lean mice and mice made obese through feeding of a high fat, hyper-caloric diet were subjected to 70% or 80% hepatectomy. While no mortality was observed in either group after 70% resection, obese mice experienced increased liver injury, reduced energy stores, reduced mitoses, increased necroapoptosis and delayed recovery of liver mass versus lean mice. Increasing resection to 80% of liver mass was associated with mortality of nearly 40% in lean mice, which doubled to 80% in obese mice. We screened for defects in mitogenic pathways by expression profiling. Epidermal Growth Factor Receptor (EGFR), known to be essential for normal regeneration, was decreased in fatty liver in all conditions. Data mining and meta-analysis of expression studies inrodents and patients consistently demonstrated reduction of EGFR in fatty liver. Furthermore, reduced EGFR protein was observed in steatotic human liver. Thus there is a cross-species association of high fat and low EGFR. In mice both EGFR and pEGFR decreased with increasing percentage body fat. RNA and protein levels were coordinately decreased, suggesting transcriptional suppression of EGFR expression. Transgenic replenishment of EGFR to the liver by hydrodynamic transfection of plasmid DNA encoding full length EGFR corrected fatty liver regeneration, reduced liver injury and increased hepatocyte proliferation. More importantly, EGFR gene therapy increased survival after 80% resection in obese mice from 25% to 70% (P < 0.05; n = 10 per group). These data indicate that chronically low EGFR expression in hepatosteatosis is rate limiting for liver regeneration in obesity. Thus therapies directed at increasing EGFR could promote regeneration and survival in obesity and steatosis.
COMPARISON OF TWO DIFFERENT INSTRUMENT SYSTEMS FOR MEASURING INTRACRANIAL PRESSURE FOLLOWING TRAUMATIC BRAIN INJURY
B. Cam, T. Yu, W.J. Hubbard, K.I. Bland, and I.H. Chaudry. University of Alabama at Birmingham, Birmingham, AL
Measurement of intracranial pressure (ICP) is an extremely important parameter for assessing the severity or treatment efficacy of traumatic brain injury (TBI) in both clinical and experimental settings. With our experimental TBI research program, we had successfully adapted a clinical instrument and probe (Integra Camino patient monitor MPM-1-6P in combination with a 110-4L monitoring catheter) to a rat moderate TBI model using lateral fluid percussion-induced injury. The data obtained fit expectations for sham; vehicle and 17α-ethinyl estradiol-3-sulfate (EE-3-SO4) rats treated with 1 mg/kg BW delivered IV 1 hr post-TBI (see Fig. 1, solid black bars). However, the clinical system proved prohibitively expensive due to having a costly single-use probe, which was not amenable to cleaning for reuse. As an alternative, we evaluated a Harvard Apparatus instrument (FISO FP1-LS monitor) coupled with a 75-0706 probe. The advantages for this instrument and probe, besides being designed for small animal use, are that it employs robust fiber optical signal transmission with a protected sensor tip, which is appreciably smaller than the clinical probe, and thus should reduce mechanical damage artifacts (see photograph on left for comparison). The probe is multiple-use, and can be cleaned/sterilized and calibrated between uses. Data obtained 24 hr after TBI and treatment with the Harvard instrument are comparable to the clinical instrument, and are shown below (Fig. 1, solid grey bars). In our hands, there was a further advantage to the Harvard system in that after placement it required a maximum of 10 min for ICP readings to stabilize, while the Integra instrument typically took 30 min to attain stabilization. In conclusion, we have confirmed that the Harvard Instrument small animal monitor is an excellent, cost-effective choice for measuring ICP in rat TBI models (DOD CDMRP Grant # PT075653).
ULTRASTRUCTURAL ANALYSIS OF ENDOTHELIAL DISORDER IN ACUTE KIDNEY INJURY BY SEPSIS
K. Suzuki*1, H. Okada*1, G. Takemura2, Y. Hotta3, C. Takada1, S. Yoshida1, H. Ushikoshi1, I. Toyoda1, and S. Ogura*1. 1Department of Emergency and Disaster Medicine, Gifu University Graduate School of Medicine, Gifu, Japan, 2Department of Internal Medicine, Asahi University School of Dentistry, Mizuho, Japan, 3Central Research Institute of Oral Science, Asahi University School of Dentistry, Mizuho, Japan
Introduction: Acute kidney injury (AKI) lead to a number of complications, including metabolic acidosis, high potassium levels, uremia, changes in body fluid balance, and effects to other organ systems. Sepsis is whole-body inflammation caused by an infection. Sepsis often caused AKI. Sepsis caused vascular endothelial disorders, subsequently the damaged endothelial surface inhibits anticoagulant properties as well as increases antifibrinolysis, which can lead to intravascular clotting, the formation of blood clots in small blood vessels, and multiple organ failure.
We herein analyzed the ultrastructure of renal vascular endothelial disorders under septic condition, chronologically.
Methods: Lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 20 μg/kg/body weight corresponding. The mice were sacrificed at 0h, 3h, 6h, 12h and 24h after LPS injection. As control mice, we injected PBS instead of LPS. Lung, liver and kidney were obtained from these mice and the samples for scanning electron microscopy (SEM) were made by frozen fracture method. Likewise, vascular corrosion casting was performed by resin injection to the artery.
Results: Survival rate was 62.5 % 24h after LPS injection. In optical microscope analysis including HE, Masson’s Trichrome, PAS staining, glomerular basement membrane thicking and mesangial cell proliferation were not observed in both groups. In the SEM analysis, renal vascular endothelial disorder was not observed 6h after LPS injection in glomerule. On the other hands, in the liver and lung, vascular endothelial disorder have already detected SEM. Vascular corrosion casting analysis was performed by SEM and computed tomography. The number of vessel in kidney was decreased in the LPS injected group compare with control group. This result suggested that LPS caused microangiopathy in septic kidney. In SEM analysis using frozen fractured kidney sample, the microstructure of the glomerular vessel was disrupted that edematous endothelial cell and vascular occlusion by extracted fibrin caught red blood cells in the vessel 12h after LPS injection. (FIGURE)
Conclusion: The present study showed that organ disorders were appeared that in the order of lung, liver, and kidney. Edematous endothelial cells and occluded vessels by fibrin clots were found in septic kidney. These result demonstrated ultrastructural that AKI was caused microangiopathy in sepsis.
LACTOBACILLUS RHAMNOSUS GG TREATMENT INCREASES COLONIC RETINALDEHYDE DEHYDROGENASE 2 LEVELS IN MURINE PSEUDOMONAS AERUGINOSA INDUCED PNEUMONIA
C.H. Baird, L. Khailova, and P. Wischmeyer. University of Colorado, Aurora, CO
Background: More than 50,000 deaths each year are attributed to pneumonia in the USA. Clinical data suggests probiotic administration may prevent nosocomial infections, ventilator associated pneumonia and improve clinical outcome in critical illness. More recent data show enhanced regulatory T cell (T-reg) responses correlate with resistance to invasive bacterial pneumonia. We have previously shown an increase in lung T-reg cells in mice treated with Lactobacillus Rhamnosus GG (LGG) in an experimental model of pseudomonas-induced pneumonia, which correlates with a significant increase in survival. The mechanism of LGG induced protection in the lung and its relationship to T-reg cell response remains to be elucidated. LGG has been shown to increase IL-10 levels in murine colon. Retinaldehyde Dehydrogenase 2 (ALDH1A2) is secreted by macrophages in the presence of IL-10 and TGF-b and catalyzes formation of retinoic acid, which increases T-Reg cell differentiation.
Purpose: To investigate the mechanism of enhanced T-reg cell presence in mice treated with LGG.
Methods: 6-week old FVB/N mice were treated (o.g.) with or without LGG (109CFU/ml) and intratracheally injected with 4x108CFU P. aeruginosa or saline. At 12 and/or 24 h, colon samples were evaluated for IL-10, FOXP3 mRNA, and protein expression of ALDH1A2, and FOXP3. Blood was evaluated via flow cytometry for T-reg cells.
Results: Mice treated with LGG showed a trend of increasing IL-10 mRNA and a significant increase in ALDH1A2 protein (p<0.001 vs. pneumonia) in the colon at 24 h. FOXP3 mRNA was significantly increased at 12 h in colons (p<0.05) in LGG-treated animals. Colonic FOXP3 protein showed a strong trend of increasing at 12 h but did not reach significance. Flow cytometry showed blood T-reg cells increased in LGG-treated animals vs. pneumonia alone (p<0.05) at 24 h.
Conclusion: LGG treatment may decrease mortality in this model by modulating immune signaling in the gut that leads to an enhanced T-reg cell induction, thus protecting the lung and other vital organs. Since IL-10 and ALDH1A2 levels increase with LGG, it is possible macrophages are involved in regulating T-reg cell numbers. Understanding mechanisms of LGG-induced protection may help guide use of LGG/probiotics as a therapeutic agent to reduce mortality and improve outcome in pneumonia patients.
HEXOKINASE INHIBITION ATTENUATES CARDIAC DYSFUNCTION IN POLYMICROBIAL SEPSIS
H. Ma, X. Wang, T. Ha, J. Kalbfleisch, R. Kao, D.L. Williams, and C. Li. East Tennessee State University, Johnson City, TN
There is compelling evidence that cardiovascular dysfunction is a major complication associated with sepsis induced morbidity and mortality. Cardiac dysfunction in sepsis is referred to as “septic cardiomyopathy”. Cardiomyopathy is present in >40% of sepsis patients. Septic cardiomyopathy is associated with mortality rates of up to 70%. Despite the severity of this condition, the mechanisms by which septic cardiomyopathy occur remain unclear. Recent evidence suggests that metabolic reprogramming plays a critical role in host defense and inflammation. Activation of TLRs on immune cells can induce metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis. Hexokinase inhibition with 2-deoxy-D-glucose (2-DG) modulates glycolytic activity. This study investigated what, if any, role hexokinase inhibition plays in septic cardiomyopathy. C57B/J male mice were treated with a hexokinase inhibitor (2-deoxy-D-glucose, 2-DG). Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Untreated septic mice served as control. Sham surgical operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. We found that polymicrobial sepsis induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction (EF%) and fractional shortening (%FS) in 2-DG treated septic mice were significantly (p<0.05) better than in untreated CLP mice. Hexokinase inhibition also significantly attenuated CLP-induced increases in serum TNFα, IL-1β and IL-6. In addition, sepsis induced increases in the production of lactate were markedly attenuated in 2-DG treated CLP mice. The data suggests that one or more of the glycolytic pathways may play an important role in sepsis induced septic cardiomyopathy as well as inflammatory cytokine production.
MITOCHONDRIAL DNA REGULATES TNF-α MRNA STABILITY
B. Sarkar, S. Bond, S. Mitra, E.L. Chiswick, D.G. Remick, and K. Desai. Boston University School of Medicine, Boston, MA
Background: Sepsis is a disease state defined by systemic inflammation in the setting of an infectious source. It is a major healthcare issue associated with significant mortality with an annual cost in the billions. Survival largely depends on early intervention in the form of empiric antibiotic therapy and aggressive fluid resuscitation, as treatments targeting specific inflammatory regulators have failed to show any survival benefit. Elevated levels of circulating mitochondrial DNA (mtDNA) have been correlated with mortality in septic ICU patients, but the role of mtDNA in sepsis is largely unknown. The focus of this study was to recreate abdominal sepsis in mice using the cecal ligation and puncture (CLP) model and measure plasma mtDNA levels, as well as to investigate the effect of mtDNA stimulation on macrophages, specifically looking at up regulation of inflammatory cytokines.
Methods: Mouse liver was used to purify mtDNA for comparison to plasma samples and also to stimulate primary peritoneal macrophages extracted from naïve mice. CLP was performed on mice, and plasma was extracted 24 hours later. Levels of inflammatory cytokine TNF-α protein and mRNA were measured with ELISA and quantitative PCR. The RNA binding protein, AUF1, was immunoprecipitated from macrophage cell extracts and assessed for binding to TNF-α mRNA following stimulation with mtDNA.
Results: Mitochondrial DNA resulted in induction of TNF-α protein and increased steady-state levels of TNF-α mRNA similar to LPS treatment when compared with control macrophages. Half-life analysis using actinomycin D, showed that both mtDNA and mitochondrial debris caused an increase in the stability of TNF-α transcript. Stimulation with these mitochondrial DAMPs resulted in direct binding of AUF1 to TNF-α mRNA. In vivo, plasma was purified from mice at 24 hours post-CLP, and elevated amounts of circulating mtDNA were detectable using quantitative PCR.
Conclusions: These results demonstrate that mitochondrial DAMPs regulate TNF-α mRNA expression through a post-transcriptional mechanism involving the AUF1 protein. This is a novel mechanism that contributes to sepsis pathophysiology. The CLP model produces circulating plasma mtDNA similar to septic human ICU patients. Further studies will define the exact role of mtDNA in sepsis.
LATE INTERVENTION WITH REPEATED ANTIBIOTIC AND FLUID RESUSCITATION RESCUES MICE FROM LETHAL CECAL SLURRY-INDUCED SEPSIS
A.M. Steele*1, M. Starr*2, and H. Saito*1, 2. 1University of Kentucky, Department of Physiology, Lexington, KY, 2University of Kentucky, Department of Surgery, Lexington, KY
Background: Antibiotic treatment is often included in animal studies on sepsis to mimic clinical situations, but intervention is commonly initiated very early, blunting the progression of systemic inflammation and downstream pathology. As interest grows regarding the pathophysiological mechanisms during late phases of sepsis, the development of a new model which allows for a longer disease course would prove beneficial.
Objective: The objective of this study is to establish a clinically relevant sepsis model that includes multiple therapies initiated at late time-points to rescue animals from otherwise completely lethal sepsis while allowing a longer disease course.
Methods: Cecal slurry (CS) was prepared and stored by our newly developed highly reproducible protocol in which cecal contents from donor mice were mixed with glycerol-PBS and stored at -80°C PMID: 25531402). Different amounts of CS were injected intraperitoneally to 4-month-old male C57BL/6 mice to induce intra-abdominal polymicrobial sepsis and survival was monitored for 10 days. In vitro analyses including liquid and agar bacterial culture were performed to test the effectiveness of the broad-spectrum antibiotic imipenem (IPM) in preventing growth of CS bacterial populations. During in vivo experiments, IPM was injected (1.5mg/mouse i.p. every 12 h) with or without fluid resuscitation (1mL saline, s.c.) beginning at 1, 6, 12, and 24 h after injection of CS. To assess bacterial load, blood samples were obtained by tail vessel microsampling immediately before antibiotic treatment and again 36 h later.
Results: The minimum lethal dose of CS (without any re suscitation) used in the subsequent experiments was determined to be 500µL as it caused 100% mortality whereas 400µL caused 60% mortality. Treatment with IPM (which killed all CS bacteria in vitro), starting at 1 and 6 h after CS injection lead to 100 and 60% survival, respectively. IPM treatment started at 12 h was ineffective at preventing mortality, but delayed time of death. When fluid resuscitation was initiated in addition to IPM beginning at 12 h, hypothermia was significantly improved after only one treatment (fluids and IPM vs. no treatment, p< 0.001), and bacteria load was reduced from 3.6X103 before treatment to 0.0 CFU/mL after 3 treatments. Ultimately, survival rates of mice with the combined treatment initiated at 12 and 24 h was 80 and 40%, respectively (p< 0.05, compared to antibiotics alone and non-treated).
Conclusions: A significant number of mice were rescued by repeated treatment with antibiotics and fluid resuscitation starting as late as 12 and 24 h after injection of 100% lethal dose of CS. This new late-resuscitation model of sepsis more closely mimics clinical conditions, and thus may be particularly useful for investigating pathophysiological mechanisms in later stages of severe sepsis.
RED BLOOD CELL TRANSFUSION IN PREMATURE INFANTS MAY BE ASSOCIATED WITH INCREASED NECROTIZING ENTEROCOLITIS SEVERITY
F.C. Okolo1, K. Mollen2, and M. Good*2. 1University of Pittsburgh Medical Center, Pittsburgh, PA, 2Children’s Hospital of Pittsburgh, Pittsburgh, PA
Objective: To determine the outcomes of premature infants who received blood transfusions prior to the onset of necrotizing enterocolitis (NEC).
Study Design: Premature neonates who developed NEC (Bell’s Stage 2 or greater) over a 16-month period were included in the study. Key variables studied included blood transfusions, severity of NEC, need for surgical intervention and mortality.
Results: One Hundred and seven infants (107) were reviewed over a period of 16 months (7/2010 - 11/2014). 43 (40%) of these patients received blood transfusions, 28 (26%) of whom received transfusion within 72hrs prior to the diagnosis of NEC. Of patients who received blood transfusion, 31 (72%) required surgical intervention. Of the patients who did not receive blood transfusion (n=64), 13 (20%) underwent surgical intervention. There were a total of 11 deaths, 10 (91%) of whom were in the blood transfusion group.
Conclusion: The findings suggest that in our cohort of infants, blood transfusion was associated with development of NEC as well as the need for surgical intervention. Furthermore, the high mortality rate noted in infants who received a blood transfusion prior to NEC development may support the correlation between blood transfusion and NEC severity. Since there are multiple variables associated with the development of NEC including prematurity, low birth weight, gestational age, administration of breast milk, as well as maternal comorbidities, a larger scale prospective trial would be beneficial to determine the true relationship between NEC and blood transfusions.
CHRONIC ALCOHOL INGESTION DELAYS MEMORY CD8+ T CELL ACTIVATION AND EFFECTOR FUNCTION IN SEPSIS
L. Margoles, Z. Liang, J. Lyons,* R. Mittal, C.M. Coopersmith, and M.L. Ford. Emory University, Atlanta, GA
Introduction: Chronic alcohol ingestion causes baseline immune deficits and increased mortality from sepsis in humans and murine models. We examined alterations in T cells associated with this increased mortality.
Methods: Mice received 20% ethanol or water for 12 weeks followed by cecal ligation and puncture or sham laparotomy. Splenocytes were harvested for surface staining of activation markers (n=4-9/group) at 24 and 72 hours and intracellular cytokine staining following ex vivo stimulation at 24, 72, and 120 hours. CD8 expression of CD44 (memory), CD69 (activation), CD43 (effector function), and intracellular IL2 were assessed by flow cytometry followed by Kruskal-Wallis test with multiple comparisons (groups: H2O sham, EtOH sham, H2O CLP, EtOH CLP).
Results: There were no significant differences in CD44 expression due to sepsis in the water or alcohol-fed groups at either 24h or 72h. In memory CD8s at 24h, the water septic group increased CD69 expression (H2O sham 6.9±1.3% vs H2O CLP 21±2.3%, p=0.01) while the alcohol septic group did not. By 72h, both the water and alcohol fed groups upregulated CD69 on memory CD8s in sepsis (H2O sham 8.9±0.6% vs H2O CLP 19.7±1%, p=0.005; EtOH sham 11.5±0.5% vs EtOH CLP 26.2±3.1%, p=0.02). ) In memory CD8s at 24h, the CD69+CD43+ population increased in water sepsis (H2O sham 0.2±0.04% vs H2O CLP 13.6±2.8% p=0.04) but not alcohol sepsis. By 72h, both water and alcohol septic groups show significant increase in this population above sham controls (H2O sham 0.2±0.05% vs H2O CLP 11.1±0.6%, p=0.01; EtOH sham 0.3±0.05% vs EtOH CLP16.8±2.5%, p=0.01). Concurrently at 72h, CD8 intracellular IL2 production was decreased in alcohol septic mice compared with the water septic group (2.81±0.79% vs 16.87±2.0%, p=0.0004).
Conclusions: Chronic alcohol fed animals exhibited increased expression of the activation markers CD44, CD69, and CD43 within the CD8+ T cell compartment during sepsis, but this increased expression was delayed relative to water-fed controls. Importantly, this delay corresponded to decreased IL2 production in alcohol septic animals at 72h. In sum, the observed altered kinetics of immune activation in alcohol-fed septic animals suggests a potentially important qualitative difference in the immune response in this highly morbid condition.
ENDOTHELIAL RELEVANT MIRNAS ARE ALTERED IN PLASMA FROM MICE WITH POLYMICROBIAL SEPSIS AND PATIENTS WITH SEPSIS
H. Fan*1, A.J. Goodwin1, C. Guo1, 2, 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 an etiologic factor in the transition from sepsis to multiple organ dysfunction syndrome. Our previous studies demonstrated that the CXCL12 analogue CTCE-0214 (CTCE) has beneficial effects in cecal ligation and puncture (CLP)-induced polymicrobial sepsis. We examined the hypothesis that endothelial relevant miRNAs are altered in plasma from septic mice treated CTCE and patients with sepsis. CD-1 mice were subjected to CLP and treated in vivo with CTCE (10mg/kg) at 2 and 6 hours post-CLP. MiRNA levels in mouse serum were determined. In subsequent studies, fifty four patients with sepsis and thirty two healthy volunteers were recruited. MiRNAs were isolated from plasma samples and quantitated by real-time PCR. CTCE suppressed miR-34a (23±6%) and 15a (9±2%) in serum of septic mice. In human plasma, our findings demonstrated that endothelial relevant miRNAs 126-3p, 126-5p, 34a, 146a, 155, 125b, 27a, 15a, 16, and 181b were significantly increased in patients with sepsis compared to healthy controls. Among the patients with sepsis miR-34a and 15a were significantly associated with shock. Since miR-34a and 15a target Sirt1 and Bcl-2 and contribute to endothelial cell apoptosis, further examination of these miRNAs in the etiology of septic shock is merited. (Supported by NIH GM27673, GM67202, KL2 TR000060 and UL1 TR 000062)
EARLY ANGIOGENIC BALANCE AND ADVERSE LONG-TERM OUTCOMES IN SEPSIS
A. Bihorac1, P.A. Efron*3, A. Agudelo1, R. Ungaro3, K.M. Marquez1, T. Ozrazgat-Baslanti1, L. Moldawer*3, F. Moore*3, and M.S. Segal4, 2. 1University of Florida, Gainesville, FL, 2Malcolm Randall VA Medical Center, Gainesville, FL, 3Department of Surgery, University of Florida, Gainesville, FL, 4Department of Medicine, University of Florida, Gainesville, FL
Objective: Maladaptive repair in sepsis may lead to hospital death, chronic critical illness and long-term morbidity and mortality. The appropriate balance of angiogenic growth factors in early sepsis may determine whether the resulting angiogenesis will lead to adequate repair.
Methods: In a prospective study of 50 septic patients we have classified the early changes in soluble angiogenic growth factors (vascular endothelial growth factor [VEGF], angiopoietins and erythropoietin) into four angiogenic profiles (quiescent endothelium [QEP], angiogenic [AP], anti-angiogenic [AAP], and intermediate [IP] profiles) and determined their association with adverse outcomes.
Results: Only 20% of the cohort had low levels of all factors consistent with a quiescent endothelium profile while 30% had an anti-angiogenic profile, categorized by suppressed VEGF-A and elevated angiopoietin II levels. A significantly higher percentage of septic patients with this profile had persistently elevated lactic acid levels and increased hospital mortality. In addition, these patients were two times more likely to develop chronic critical illness and had a decreased one-year survival compared to patients with a quiescent endothelium (Figure 1).
Conclusion: We have determined a new phenotype of early angiogenic imbalance in sepsis that may be associated with adverse short and long-term outcomes likely through the process of maladaptive repair and persistent inflammation.
DIVERGENT EFFECT OF AGONISTIC BTLA ENGAGEMENT ON INNATE IMMUNE RESPONSE TO THE COMBINED INSULTS OF HEMORRHAGIC SHOCK/SEPSIS
T. Cheng1, J. Bai1, C. Chung*2, Y. Chen2, and A. Ayala*2. 1Department of Emergency Internal Medicine, Shanghai East Hospital, Tongji University, Shanghai, China, 2Division of Surgical Research, Department of Surgery, Rhode Island Hospital, Brown University School of Medicine, Providence, RI
Sepsis following hemorrhagic shock has high morbidity and mortality, which is to some extent related to severe innate immunosuppression. B and T lymphocyte attenuator (BTLA) is an immune-regulatory receptor expressed not only on adaptive immune cells but also on innate cells. Our previous data showed that BTLA gene deficient mice were protected from septic mortality when compared with WT control C57BL/6 mice. Here we extended our study by treating C57BL/6 mice with an agonistic anti-BTLA antibody (6A6) in a mouse model of hemorrhagic shock (Hem) followed by cecal ligation and puncture (CLP), positing that agonist BTLA engagement, unlike gene deficiency, would have the inverse affect on the inflammatory response/morbidity in these mice after such insults. Here we report that BTLA expression was elevated in innate immune cells after Hem/CLP. Anti-BTLA antibody treatment increased the cytokine (TNF-a, IL-12, IL-10)/ chemokine (KC, MIP-2, MCP-1) levels and inflammatory cells (neutrophils, macrophages, dendritic cells) recruitment in peritoneal cavity, but aggravated organ injury and elevated its mortality in Hem/CLP mice. These results indicate that agonistic BTLA engagement during Hem/CLP has divergent effects, as it appears capable of potentiating the innate inflammatory response while concomitantly aggravating organ injury, culminating in a high morbidity and mortality.
HAPTOGLOBIN BINDS AND SEQUESTERS HMGB1 TOXICITY VIA CD163: IMPLICATIONS FOR HMGB1 SERUM CLEARANCE AND A NOVEL ANTI-INFLAMMATORY PATHWAY
H. Yang*, Y. Wang, H. Wang*, S.S. Chavan*, and K.J. Tracey*. The Feinstein Institute of Medical Research, Manhasset, NY
Severe sepsis accounts for over 200,000 deaths in the United States annually. The mechanism of this lethal syndrome involves endogenous molecules that impair organ functions. Extracellular hemoglobin is one such factor that enhances tissue damage during sepsis (Larsen R et al, Sci Translational Med, 2010). Previous studies showed that haptoglobin, the primary scavenger of hemoglobin in circulation, plays an important anti-inflammatory role in endotoxemia and sepsis (Lim SK et al, Blood, 1998). Here we demonstrate that haptoglobin also binds and sequesters HMGB1, an endogenous mediator in sepsis (Yang H et al, J Leuk Biol, 2013, J Exp Med, 2015). The addition of haptoglobin suppressed HMGB1-stimulated TNF and IL-8 release in cultured macrophages. Haptoglobin knockout mice subjected to cecal ligation and puncture (CLP)-induced sepsis had significantly higher serum HMGB1 levels and higher mortality rate compared to wild type animals (75% survival in wild type vs. 34% in haptoglobin knockout mice; n=15/group, P<0.05). Treatment with anti-HMGB1 antibodies significantly reduced the CLP-induced sepsis mortality in haptoglobin knockout mice (12% survival in control vs. 56% in animals receiving HMGB1 antibodies; n=16/group, P<0.05). Furthermore, the complex of HMGB1 and haptoglobin signals via a novel pathway through binding to a cell surface receptor CD163, thus triggers an anti-inflammatory response via the expression of heme oxygenase 1 and IL-10 release from macrophages. These findings indicate that haptoglobin is an endogenous modulator of HMGB1 that is capable of reducing the toxicity of HMGB1 in sepsis (Supported by grants from NIH, NIGMS RO1GM62508 to KJT and RO1GM098446 to HY).
THE KINETICS AND FUNCTIONAL ROLE OF 2B4 EXPRESSION ON CD8+ T CELLS DURING SEPSIS
C. Chen*, J. Lyons*, Z. Liang*, C.M. Coopersmith*, and M.L. Ford*. Emory University, Atlanta, GA
Background: Lymphocyte apoptosis plays a major role in sepsis pathogenesis, in that experimental evidence from animal models reveals significant apoptosis in the T cell compartment following sepsis induction and that blocking this apoptosis results in improved survival. In addition, T cell exhaustion markers are up-regulated and may contribute to septic immune dysfunction. We found that expression of 2B4, an immunomodulatory receptor known to be expressed on NK cells, increases on CD8+ T cells during sepsis. Importantly, 2B4-deficiency dramatically improved sepsis survival. However, the functional role of 2B4 on T cells during sepsis is incompletely understood.
Methods: C57BL/6 mice were subjected to either 2x25 cecal ligation and puncture (CLP) or sham operation. Splenocytes were collected at different days post-CLP. CD8 T cells were stained for 2B4, CD62L and CD44 and assayed by flow cytometry(N=3∼11). For the apoptosis assay, splenocytes were stained for Annexin V and 7-AAD at 24hr post CLP(N=9). Human PBMC were obtained from septic patients and healthy donors under an IRB-approved protocol and stained extracellularly for 2B4, CCR7 and CD45RA.
Results: Studies in our murine CLP model revealed that 2B4 expression on total CD8+ T cells was upregulated and peaked at 24hr after CLP, and was subsequently downregulated after day 3. At all time points analyzed, central memory T cells (CD62L+CD44+) and effector memory T cells (CD62L-CD44+) exhibited a higher frequency of 2B4+ cells relative to naïve T cells, and the kinetics of expression of 2B4 on memory CD8+ T cells was similar to total CD8+ T cells. The finding that 2B4 is upregulated on CD8+ T cells during sepsis in mice was mirrored in human septic patients, in that we observed that human septic patients contained higher frequencies of 2B4+ CD8+ T cells than healthy donors. As observed in septic animals, 2B4+ T cells isolated from septic patients were enriched within the effector memory and TEMRA T cell compartments (CCR7-CD45RA- and CCR7-CD45RA+). In order to dissect the mechanism underlying the increased survival in 2B4-/- mice following CLP, we investigated the frequencies of apoptotic cells within the 2B4+ vs. 2B4- CD8+ T cell subsets in septic animals. Importantly, we observed that 2B4+ CD8 T cells exhibited significantly higher frequencies of AnnexinV+7-AAD+ cells 24hr post-CLP.
Conclusions: After CLP, 2B4 expression on CD8+ T cells is increased, peaks at 24hr and is downregulated after day 3. The frequency of 2B4+ among CD8+ T cells in septic animals is significantly greater within the memory T cell subset relative to naïve T cells in both septic animals and septic patients. Moreover, our data reveal that 2B4+ CD8+ cells have an increased propensity to undergo apoptosis during sepsis relative to 2B4- cells, which suggests a possible mechanism underlying the survival benefit observed in 2B4 deficient mice.
IL-18 INCREASES MURINE NEONATAL SEPSIS MORTALITY VIA IL-17A
J.L. Wynn*1, C. Wilson1, J. Hawiger1, P.O. Scumpia2, J. Liu1, H. Wong*3, P. Lahni3, J.T. Benjamin1, E. Plosa1, J. Weitkamp1, E. Sherwood*1, L. Moldawer*4, R. Ungaro4, H. Baker4, M. Lopez4, S. McElroy5, and D. Moore1. 1Vanderbilt University, Nashville, TN, 2University of California, Los Angeles, Los Angeles, CA, 3Cincinnati Children’s Hospital, Cincinnati, OH, 4University of Florida, Gainesville, FL, 5University of Iowa, Iowa City, IA
Sepsis is the predominant cause of mortality and lifelong morbidity in neonates. These adverse outcomes are mediated by an aberrant innate immune system but the molecular mechanism of neonatal injury to sepsis remains unexplained. Interleukin (IL)-18 is an important effector of innate immunity and canonically produced by the inflammasome. The role of IL-18 and its downstream mediators in the development and progression of neonatal sepsis remain unanswered. Here, we find that IL-18 null neonatal mice are protected from sepsis, whereas IL-18 delivery increases lethality with endotoxemia or polymicrobial sepsis in wild-type neonatal mice. IL-18 and IL-18 binding protein (BP) concentrations are both increased in plasma from septic human and murine neonates, while the IL-17 receptor emerged as a critical regulatory node in genome-wide analysis of human neonatal blood mRNA. Indeed, IL-18 increased IL-17A production by murine Ly6G+ myeloid cells, and targeting IL-17 improved survival to both endotoxemia and neonatal sepsis, in part through preventing sepsis-induced gut damage. We conclude that IL-17A is a previously unrecognized effector of IL-18-mediated inflammation, and that disruption of the IL-18/IL-17 signaling axis represents a novel therapeutic approach to improve outcomes for human neonates with sepsis.
INFLAMMATORY CYTOKINES AND REACTIVE OXYGEN AND NITROGEN SPECIES ARE DIFFERENTIALLY MODULATED IN MONOCYTES DURING CLINICAL SEPSIS
S. Santos1, M. Brunialti1, S. Trevelin2, A. do Carmo1, M. Assuncao3, F. Machado1, L. Azevedo*4, F. Cunha2, and R. Salomao*1. 1Escola Paulista de Medicina, Sao Paulo, Brazil, 2Faculdade de Medicina USP -RP, Ribeirao Preto, Brazil, 3Albert Einstein Hospital, Sao Paulo, Brazil, 4Sírio Libanês Hospital, Sao Paulo, Brazil
Objectives: To evaluate the modulation of monocytes functions in patients with severe sepsis and shock.
Methods: Blood samples were obtained from septic patients (SP) at admission (N=30), and after 7 days of follow-up (N=12); 10 healthy volunteers (HV) were enrolled as controls. Monocytes cell functions were evaluated in whole blood, using flow cytometry. Cytokines, ROS and NO were measured at baseline and following lipopolysaccharide (LPS), Pseudomonas aeruginosa and Staphylococcus aureus stimulation. TNF-alpha and IL-6 were detected intracellular using conjugated anti-TNF-alpha PE-Cy7 and anti-IL-6-APC antibodies. ROS and NO were detected using the substrates 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FMDA) respectively. Co-localization of gp91phox and p47phox, subunits of NADPH-oxidase, was determined in a subgroup of SP (N=10) and HV (N=6) by confocal microscopy.
Results: Detection of intracellular TNF-alpha and IL-6 was lower in SP than in HV after LPS, P. aeruginosa and S.aureus stimulation, in contrast with NO and ROS, which were higher in SP (table). Co-localization of gp91phox and p47phox was found in 8 out of 10 SP and in none of 6 HV. Detection of TNF-alpha and IL-6 were higher at D7 than at D0.
Conclusion: Inflammatory cytokines and ROS / NO generation are differentially modulated in monocytes from septic patients. Inhibition of cytokines and preserved capacity to generate ROS / NO further support a reprogramming of cell functions aiming to control the inflammatory response, while preserving the ability to fight the infecting microorganisms.
ANALYSIS OF INFLAMMATORY AND PRO-COAGULANT FACTOR GENE EXPRESSION IN VISCERAL ADIPOSE TISSUE OF PATIENTS WITH INTRA-ABDOMINAL SEPSIS: ADIPOSE TISSUE IS A MAJOR SOURCE OF SYSTEMIC PAI-1 WHICH CORRELATES WITH SEPSIS SEVERITY
M. Starr*, B. Zwischenberger, A.J. Stromberg, P.K. Chang, and H. Saito*. University of Kentucky, Lexington, KY
Background: Adipose tissue has become increasingly recognized as an important contributor to chronic inflammatory diseases. Using a murine endotoxemia model, we previously reported that visceral adipose tissue, more so than subcutaneous adipose tissue, is also highly active during the initial stages of acute inflammation. We further identified visceral adipose tissue as a major source of inflammatory cytokine interleukin-6 (IL-6), and pro-coagulant factors plasminogen activator inhibitor (PAI)-1 and -2, and thrombospondin-1 (Thbs-1) during endotoxemia [Aging Cell 2013, PMC3633415].
Objective: The purpose of this study was to evaluate the clinical significance of inflammatory cytokines and pro-coagulant factors that are produced by visceral adipose tissues during sepsis.
Methods: Surgical patients (N=33, Age range: 24-88, BMI: 19-34, 52% female) undergoing an abdominal procedure were enrolled in this study and categorized into four groups: Control (elective non-inflammatory conditions, N=10), Local Inflammation (appendicitis, diverticulitis, N=9), Sepsis (abdominal source of infection, N=6), and Severe Sepsis (abdominal source of infection with organ failure, N=8). Visceral adipose tissues (mesenteric fat, epiploic appendages, and omentum) and blood samples were collected. Histological analyses were performed on formalin fixed adipose tissues. Quantitative real-time RT-PCR analysis was utilized to determine the expression level of inflammatory and thrombotic factors in adipose tissue samples. Concentration of plasma proteins were measured by ELISA.
Results: Histologically, adipose tissue samples from patients with sepsis or severe sepsis were absent of marked inflammatory cell infiltration confirming our previous data obtained from mouse models. All sources of visceral adipose tissue from sepsis and severe sepsis groups showed trends of increased mRNA levels of IL-6, IL-1β, PAI-1, PAI-2, and Thbs-1. Among these, PAI-1 was consistently and significantly (p<0.05) upregulated more than 20-fold in the severe sepsis group compared to controls. Patients with local inflammation did not show a significant increase for any gene measured. Plasma concentration of PAI-1 strongly correlated with PAI-1 mRNA levels in each of the adipose depots sampled (r=0.9: mesenteric, r=0.7: epiploic, r=0.8: omentum). Circulating procalcitonin concentration, used as a marker of sepsis severity, was significantly elevated in the severe sepsis group only (p<0.001), and correlated strongly with plasma PAI-1 concentration (r=0.8).
Conclusion: The severity of intra-abdominal sepsis is strongly associated with high plasma concentrations of PAI-1, likely derived from upregulated PAI-1 production by resident cells within visceral adipose tissues.
TETRAHYDROBIOPTERIN TREATMENT IN PERITONITIS-INDUCED EXPERIMENTAL SEPSIS IN RATS
A. Maxan, T. Dumbarton, N. Farah, N. Sharawy, J. Zhou, and C. Lehmann. Dalhousie University, Halifax, NS, Canada
Background: Sepsis is a disease of the microcirculation (1). By modifying pathways involved in inflammatory response and endothelial dysfunction, improvement of the tissue perfusion may be achieved. Tetrahydrobiopterin (BH4), an endogenous nucleic acid derivative, acts as a cofactor for numerous endothelial enzymes was reported to be dysregulated in sepsis, such as nitric oxide synthase (2).
Objective: To demonstrate the effect of tetrahydrobiopterin on global hemodynamics and the microcirculation in peritonitis-induced sepsis in rats.
Design: Randomized, controlled animal study.
Intervention: Male Lewis rats were anesthetized and subsequently underwent colon ascendens stent peritonitis (CASP) surgery to induce sepsis (3). Experiments were conducted in three groups (n=5 in each group). One experimental group underwent sham surgery, one underwent CASP surgery without treatment and the remaining group underwent CASP surgery with BH4 treatment (60 mg/kg, intraperitoneal).
Outcome measures: Eighteen hours after surgery, intravital microscopy of the microcirculation was performed in the terminal ileum. Outcomes of microvascular function were quantified by measuring leukocyte activation (rolling, adhesion to the endothelium) and functional capillary density in intestinal muscle layers and mucosa. Mean arterial pressure and heart rate were monitored continuously.
Results: CASP surgery significantly increased leukocyte adherence in both intestinal submucosal V1 and V3 venules. In V1 venules leukocyte adhesion increased from 62.06 to 350.70 cells/mm2. The functional capillary density significantly decreased in the longitudinal and circular muscle layers after CASP surgery. In animals receiving BH4, we observed a significant decrease in leukocyte adhesion in V1 vessels, to 159.30 cells/mm2. We also observed an improved functional capillary density in the longitudinal and circular muscle layers.
Conclusion: BH4 treatment in peritonitis-induced experimental sepsis decreased intestinal leukocyte activation and increased the functional capillary density, suggesting a potential therapeutic role in treating the microcirculatory dysfunction of sepsis. Further studies are needed to provide evidence on BH4 as a possible therapeutic approach for treating sepsis.
1. Spronk, PE., Zandstra, DF., & Ince, C. (2004). Bench-to-bedside review: sepsis is a disease of the microcirculation. Critical care (London, England), 8(6), 462-8.
2. Bendall, JK., Douglas, G., McNeill, E., Channon, KM., & Crabtree, MJ. (2014). Tetrahydrobiopterin in cardiovascular health and disease. Antioxidants & redox signaling, 20(18), 3040-77.
3. Lustig MK, Bac VH, Pavlovic D, Maier S, Grundling M, Grisk O, Wendt M, Heidecke CD, Lehmann C. (2007) Colon ascendens stent peritonitis–a model of sepsis adopted to the rat: physiological, microcirculatory and laboratory changes. Shock 28:59-64.
NRF2-MEDIATED PENTOSE PHOSPHATE PATHWAY ACTIVITY PROMOTES MACROPHAGE INFLAMMASOME ACTIVATION AND BACTERIAL KILLING IN MICROBIAL SEPSIS
X. Li, D. Gillette, B. Cairns*, and H. Wen*. University of North Carolina at Chapel Hill, Chapel Hill, NC
Classical activation of the innate immune cells such as macrophages and dendritic cells causes dramatic metabolic changes towards increased glucose uptake, glycolysis and pentose phosphate pathway (PPP) activity. However, the role and mechanism of this metabolic reprogramming in the regulation of macrophage function and its disease relevance is poorly understood. Here we report a critical role of PPP activity in inflammasome activation. Pharmacological inhibition of PPP resulted in a defective inflammasome activation. Macrophages with a mutation in G6pd, the gene encoding a rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase, showed decreased inflammasome activation. Mechanistically, PPP activity was required for the prevention of excessive reactive oxygen species generation and safeguarding an intact extracellular Ca2+ influx, a critical step for inflammasome activation. Nuclear factor E2-related factor-2 (Nrf2) regulates the expression of several key enzymes involved in PPP. Furthermore, Nrf2-/- macrophages showed defective caspase-1 activation after E. coli infection, which caused decreased phagosome acidification and bacterial killing. Nrf2-/- mice and mice with PPP inhibition exhibited an increased bacterial burden in E. coli-induced and cecal ligation and puncture-induced microbial sepsis. In sum, these findings identify a novel function of PPP in inflammasome activation and suggest PPP as a therapeutic target in the treatment of microbial sepsis.
SYSTEMIC ATP IN SEPSIS PREVENTS BACTERIAL CLEARANCE BY IMPAIRING NEUTROPHIL HOST DEFENSE
X. Li1,2, A.H. Lee1, L. Staudenmaier1, J. Zhang1, Y. Bao1, C. Ledderose1, and W. Junger*1. 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 2West China Hospital, West China School of Medicine, Sichuan University, Chengdu, China
We have previously shown that endogenous release of ATP though pannexin-1 (panx1) channels are an essential requirement for neutrophil activation and that autocrine purinergic feedback triggered by the released ATP regulates neutrophil chemotaxis. However, extracellular ATP can also serve as a danger signal. We previously found that sepsis increases plasma ATP levels, which may interfere with neutrophil function by disrupting autocrine purinergic signaling. Here we studied how removal of endogenous or exogenous ATP affects neutrophil function and outcome in a mouse sepsis model.
Male wild-type (WT) and panx1 knockout (KO) mice were subjected to polymicrobial sepsis using cecal ligation and puncture (CLP). Mice were treated with the purinergic receptor antagonist suramin or with apyrase to block purinergic signaling or to remove extracellular ATP. Blood was collected at different times to assess neutrophil activation, organ damage markers, and bacterial counts. We also assessed survival rates in the different treatment groups.
CLP increased plasma ATP levels and promoted neutrophil activation as indicated by a 4-fold increase in CD11b levels on the cell surface. Neutrophil activation and plasma levels of the liver damage marker AST were significantly lower in panx1 KO mice or WT mice treated with suramin when compared to WT mice (P<0.05). However, systemic bacterial counts and mortality rates in panx1 KO and suramin-treated mice were significantly higher than in control mice (P<0.05). By contrast, apyrase lowered bacterial burden and significantly improved survival, while neutrophil activation and plasma AST levels remained unchanged compared to untreated control animals.(Figure 1)
We conclude that inhibition of autocrine purinergic signaling impairs neutrophil activation and worsens outcome after sepsis, while removal of systemic ATP improves neutrophil immune defenses. Targeting excessive systemic ATP levels in plasma may thus be a potential novel therapeutic strategy for the treatment of sepsis.
This work was funded in part by grants from the NIGMS, NIAID, National Natural Science Foundation of China, and Deutsche Forschungsgemeinschaft.
EXTRACELLULAR ATP PROTECTS AGAINST SEPSIS THROUGH MACROPHAGE P2X7 RECEPTORS BY ENHANCING INTRACELLULAR BACTERIAL KILLING
G. Hasko*1, B. Csóka1, Z. Németh2, G. Törö1, M. Idzko3, A. Zech3, and Z. Spolarics1. 1Rutgers New Jersey Medical School, Newark, NJ, 2Morristown Medical Center, Morristown, NJ, 3Freiburg University Medical Center, Freiburg, Germany
Extracellular ATP binds to and signals through P2X7 receptors (P2X7R)s to modulate immune function in both inflammasome-dependent and independent manners. We show here using P2X7-/- mice as well as pharmacological receptor and channel ligands that ATP release through connexin/pannexin channels and subsequent P2X7R activation are crucial for the control of mortality, bacterial dissemination and inflammation following sepsis induced by cecal ligation and puncture. Our results with P2X7-/- bone-marrow chimeras, adoptive transfer of macrophages, and myeloid-specific P2X7-/- mice indicate that P2X7R signaling on macrophages is required for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, but independently of the inflammasome. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the therapy of sepsis.
THE CARDIAC DYSFUNCTION CAUSED BY SEPSIS IN ANIMALS WITH CHRONIC KIDNEY DISEASE IS ATTENUATED BY INHIBITING IκB KINASE
J. Chen1, J. Kieswich1, F. Chiazza2, T. Gobbetti1, N.S. Patel1, M. Perretti1, M. Collino2, M.M. Yaqoob1, and C. Thiemermann*1. 1Queen Mary University of London, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 2University of Turin, Department of Drug Science and Technology, Turin, Italy
Patients with chronic kidney disease (CKD) requiring dialysis have a higher risk of sepsis and a 100-fold higher mortality than the general population with sepsis. The severity of cardiac dysfunction is an important predictor of mortality among patients with sepsis. Having discovered that pre-existing CKD worsens the cardiac outcome in mice with sepsis (1), we have investigated whether inhibition of IκB kinase (IKK) reduces the cardiac dysfunction in these animals.
Male C57BL/6 mice were subjected to a two-stage, 5/6th nephrectomy (SNX). After 8 weeks, mice with SNX/CKD were subjected to either low dose LPS (2mg/kg) or cecum ligation and puncture (CLP) (fluid and antibiotics given at 6h and 18h after CLP). At 18h after LPS injection or 24h after CLP, cardiac function was evaluated by echocardiography.
When compared to sham mice, SNX for 8 weeks resulted in a significant rise in urea and creatinine and a small (P<0.05) reduction in ejection fraction (EF). In sham mice without CKD, low dose LPS or CLP had no effect on EF, lung myeloperoxidase (MPO) activity or systemic cytokine levels. In CKD mice, LPS or CLP caused profound cardiac dysfunction (Fig. 1A), increased lung MPO activity and increased plasma cytokine levels (TNF-α, IL-1β, IL-6, IL-10). Compared with sham mice, cardiac biopsies obtained from CKD mice showed increases in the phosphorylation of IkBa, nuclear translocation of the NF-kB subunit p65, increased iNOS expression, and significant increases in phosphorylation of Akt and ERK1/2. Moreover in CKD mice, LPS or CLP further increased the phosphorylation of IkBa, the nuclear translocation of p65 and the iNOS expression. Treatment of CKD mice with the IKK inhibitor IKK 16 (1 mg/kg, 1 h after CLP) attenuated cardiac dysfunction (Fig. 1B), increase in lung MPO activity and cytokine formation caused by CLP. IKK 16 also reduced the i) increased phosphorylation of IκBα; ii) increased nuclear translocation of p65; and iii) significant increase in iNOS expression in CKD hearts subject to CLP.
In conclusion, the presence of CKD aggravates the cardiac dysfunction caused by LPS or CLP in the mouse; and this may (at least in part) be due to increased cardiac activation of NF-κB and increased iNOS expression.
(1) Chen et al, Shock 2014;41(Supplement 2):40
MYOCARDIAL RECOVERY MECHANISMS UNDERLYING THE RESOLUTION OF ENDOTOXEMIC CARDIOMYOPATHY IN MICE
I.A. Hobai*1, 2, J.C. Morse2, D.R. Siwik2, and W.S. Colucci2. 1Massachusetts General Hospital, Boston, MA, 2Boston University Medical Center, Boston, MA
Background: Sepsis induced cardiomyopathy (SIC) complicates the management and worsens prognosis of septic patients. SIC is associated with dysregulated myocardial calcium (Ca2+) handling, including redox-mediated dysfunction of sarcoplasmic reticulum (SR) Ca2+ ATP-ase (SERCA) and downregulation of L-type Ca2+ channels (LTCC).
In surviving patients, SIC resolves spontaneously within 7-10 days from sepsis onset. In the absence of any experimental data, it is generally believed that SIC resolution reflects the remission of septic shock. Here we aimed to challenge this belief, and hypothesized that SIC resolution is due to the activation of specific recovery mechanisms, that up-regulate cardiac Ca2+ handling and contractility.
Results: Male C57Bl6 mice were administered lipopolysaccharide (LPS, 7 μg/g, ip), which induced an inflammatory shock syndrome associated with cardiomyopathy (endotoxemic cardiomyopathy, EC) and 59% mortality (n=34 mice). Left ventricle ejection fraction (EF) was measured by echocardiography. Cardiomyocytes were isolated at various times after LPS challenge, and we measured sarcomere shortening (SS) and Ca2+ transients (ΔCai) elicited by external pacing at 37 °C.
12h after LPS administration, EF was depressed to 63 ± 7% of baseline (B, n = 5 mice each group; p<0.05 for this and the following). SS and ΔCai were decreased to 53 ± 10% and 78 ± 5% of B, respectively (n >30 cells from 3 mice, 30/3, for each group;). Consistent with previous studies, SERCA and LTCC activities were depressed to 88 ± 5% and 66 ± 10% of B, respectively (n > 18/3 for each group).
In mice that survived 3 days after LPS administration, EF was increased to 125 ± 5% of B (n=5 mice each group). SS, ΔCai and LTCC function had all recovered to B values (n>19/3). Importantly, SERCA showed a supranormal activation to 138 ± 4% of B (n > 28/3), which was associated with the under-expression of phospholamban (PLB, SERCA main inhibitory subunit) to 35 ± 9% of control (n = 6 hearts each group).
Six days after LPS challenge, ΔCai was increased to 124 ± 7 % of B (n > 18/3), in conjunction with an increase in SR Ca2+ load (as measured with caffeine) to 126 ± 6% of B (n>11/2) and inhibition of the membrane Na+/Ca2+ exchange (NCX) to 51 ± 4 % of baseline (n > 12/3). Twelve days after LPS, SS and ΔCai were again, back to B levels (n>16/2).
Conclusion: Myocardial recovery during the resolution phase of murine endotoxemic cardiomyopathy is associated with (and likely due to) up-regulated cellular Ca2+ handling. Underlying mechanisms include SERCA activation, PLB down regulation, and NCX inhibition, which are distinct from the initiating mechanisms (such as SERCA oxidative inhibition). Therefore, SIC spontaneous resolution may not be simply due to the remission of the inciting factors, but, instead, reflect the activation of a specific myocardial recovery process.
IMPACT OF THE LECTIN-LIKE DOMAIN OF THROMBOMODULIN IN MURINE SEPSIS AND HEART DYSFUNCTION
N. Hechaichi1, 2, B. Ndongson Dongmo1, 3, E. Conway4, R. Claus1, 2, M. Bauer1, 2, G. Theilmeier5, R. Bauer1, 3, R. Heller1, 3, and S. Stehr1, 6. 1Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany, 2Clinic for Anesthesiology and Intensive Care, Jena University Hospital, Jena, Germany, 3Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany, 4UBC Centre for Blood Research, Vancouver, BC, Canada, 5Faculty of Health Sciences, University of Oldenburg, Oldenburg, Germany, 6Clinic for Anesthesiology and Intensive Care, University Hospital Schleswig-Holstein, Lübeck, Germany
Background: Sepsis-induced cardiomyopathy is commonly observed as a feature of severe sepsis and septic shock. Its occurrence is highly correlated with a poor outcome. Although Thrombomodulin (TM) has been identified as a potential therapeutic option during sepsis, no attention has been focused on the impact of the lectin-like domain of TM (TM-LLD) in sepsis-induced cardiomyopathy by modulating HMGB1 bioavailability.
Aim: The aim of the current study was to assess 1) the in vitro influence of the TM-LLD in cardiomyocyte cell culture after stimulation and 2) whether TM-LLD affects in vivo myocardial function and survival after polymicrobial sepsis.
Methods: All animal experiments were approved by the local regulatory board. Mice, lacking the TM-LLD (TM-LeD mice) and their wildtype (WT) counterparts were used for in vivo experiments as well as a source of cardiac myocytes for in vitro experiments. We examined the expression of inflammatory mediators in neonatal cardiomyocytes after stimulation with cytokinemix. Furthermore, we used the animals in a sepsis model with polymicrobial contamination and infection (PCI), to study the effect on survival and cardiac immune response at several time points during the septic insult. Additional assessment of in vivo myocardial function after 12 h PCI with a Millar Tip transducer catheter was performed.
Results: The absence of TM-LLD in cardiomyocytes results in a significantly increased expression of the pro-inflammatory factor il-1β after stimulation in vitro, reflecting the cardio-protective and immuno-modulating effect of TM-LLD. TM-LeD mice had a significantly increased mortality within 24 h after PCI (67 % in WT and 85 % in TM-LeD mice) as well as an earlier increase and overall higher score of clinical disease severity, revealed significantly different starting at 12 h after PCI. The myocardial mRNA expression and plasma protein analysis of inflammatory markers showed a substantial increase in IL-1β, IL-6 and MCP-1 levels in TM-LeD mice in the early inflammatory response. Accordingly, plasma troponin and HMGB1 concentrations were significantly increased in TM-LeD mice after PCI. These genotype-specific observations are time-dependent and disappear within 24 h, indicating a more substantial pro-inflammatory reaction among the TM-LeD mice during the early acute phase after PCI. Additionally, sepsis induction decreased myocardial function as assessed 12 h after challenge in both genotypes, but consistently deeper in TM-LeD mice.
Conclusion: The findings of the current study support the notion that TM-LLD 1) affects the development of cardiac dysfunction in vitro and 2) confers beneficial effects on survival, disease severity and cardiac function in polymicrobial sepsis in vivo. Based on these results, we suggest that administration of TM-LLD should be further investigated as a potential therapy in septic cardiomyopathy.
EPIDERMAL GROWTH FACTOR PROTECTS MICE WITH CHRONIC ALCOHOL USE IN SEPSIS
Z. Liang. Emory University, Atlanta, GA
Background: Patients admitted to the intensive care unit (ICU) with alcohol use disorders have increased morbidity and mortality. Our previous work demonstrated that chronic alcohol consumption increases mortality in a murine model of septic peritonitis and EGF, a cytoprotective polypeptide, restores gut integrity and improves survival by 50% in septic mice.
Objectives: The purpose of this study was to determine if EGF has the same beneficial effects in septic mice with chronic alcohol ingestion as in septic mice without alcohol ingestion.
Methods: FVB/N mice received alcohol (20% w/v) for 12 weeks and were then subjected to CLP. Half of the mice received post-CLP EGF (total dose 150ug/kg/day, divided in 2 doses) via systemic administration, half received the same amount of water. Mice were followed seven days for survival (n=24-27/group) or sacrificed after 24 hours for functional studies (n=6-7/group). Villus length was determined by measuring the distance in micrometers from the crypt neck to the villus tip using Image J software. Apoptotic cells were quantified using both H&E staining and active caspase 3 staining in paraffin-embedded sections. To characterize intestinal proliferation, BrdU was given to label S-phase cells 90 minutes before mice were sacrificed. To test permeability, fluorescein isothiocyanate-conjugated dextran (FD-4, 22mg/ml, molecular mass 4.4 kDa) was orally gavaged. Blood was collected 5 hours later and the concentration of FD-4 was determined by fluorospectrometry.
Results: EGF improved 7-day survival in septic mice to 51.9% while septic mice that received water had a 20.8% survival (p<0.05). EGF increased villus length significantly. The villus length of mice with and without EGF treatment was 344.1±45.1 µm vs. 268.7±45.5 µm, respectively (p<0.05). Intestinal apoptosis was significantly decreased in septic mice with EGF treatment compared to septic mice that received water (26.3±5.6 vs. 36.0±5.6 cells/100 crypts, p<0.05 for active caspase 3, 26.6±7.1 vs. 35.6±7.3 cells/100 crypts, p<0.05 for H&E). Proliferation in intestine was higher in septic mice with EGF treatment vs. water injected mice (889.6±59.1 vs. 746.7±40.7 cells/100 crypts, p<0.05). Intestinal permaibility was lower in septic mice with EGF than in septic mice with water (473.7±267.8 µg/ml vs. 820.9±273.4 µg/ml, p<0.05).
Conclusions: EGF has the same beneficial effects in septic mice with chronic alcohol ingestion as in septic mice without alcohol ingestion. EGF treatment decreases mortality and is associated with improved gut integrity in sepsis. EGF thus may be a potential therapeutic agent for the treatment of patients in the ICU with chronic alcohol use.
BURN TRAUMA ACUTELY INCREASES RESPIRATORY CAPACITY AND FUNCTION IN LIVER MITOCHONDRIA
F.J. Bohanon, R.S. Radhakrishnan, D. Herndon*, X. Wang, N. Bhattarai, T. Toliver-Kinsky*, L. Sidossis, and C. Porter*. The University of Texas Medical Branch, Galveston, TX
Introduction: The liver likely plays a pivotal role in the hypermetabolic stress response to burns due to its metabolic, inflammatory and immune functions. However, comprehensive data on the impact of burns on hepatic bioenergetics are currently lacking. Here, we set out to characterize the acute impact of severe burn trauma on liver mitochondrial function. We hypothesize that increased ATP demand following burn trauma will augment the respiratory capacity and function of hepatic mitochondria.
Methods: Male BALB/c mice (8-10 wk old) were used in the current study. Fresh liver samples were collected from six sham and five burn treated mice (30% of the total body surface area scald burn) 24-hr post-treatment. Mitochondrial respiratory capacity and function were determined in fresh tissue by high-resolution respirometry following the titration of substrates, ADP and the uncoupler Carbonyl cyanide m-chlorophenyl hydrazone (CCCP).
Results: Respiration coupled to ATP production was 2-fold higher in burned vs. sham mice (133±14 vs. 65±7 pmols/mg/sec, P<0.01). Respiratory capacity, maximum electron flux through the electron transport chain when the system is uncoupled following CCCP titration, was greater in burn vs. sham mice (165±28 vs. 92±10 pmols/mg/sec P<0.05). The respiratory control ratio (RCR) for ADP, an indicator of respiratory function, was 43% greater in burn vs. sham mice (P<0.05). The flux control ratio (coupled respiration normalized to respiratory capacity), an indicator of mitochondrial efficiency, was 25% greater in burned vs. sham mice (88±4 vs. 71±3%, P<0.01).
Conclusions: Burn trauma results in an acute increase in mitochondrial capacity and coupling control in the liver. Specifically a doubling of coupled respiration per mg of tissue, a 43% increase in mitochondrial function (ADP sensitivity), and a 25% increase in phosphorylation efficiency combine to augment hepatic ATP production post-burn. These data support the notion that the liver contributes to post-burn hypermetabolism through increased ATP turnover.
PLANTARIS MICRORNA AND TARGET GENE PROFILE AFTER EXERCISE TRAINING IN AN ANIMAL MODEL OF BED REST AND SEVERE BURN
M.R. Saeman1, J. Song*1, L.A. Baer2, K. Despain1, C. Wade*2, and S.E. Wolf*1. 1University of Texas Southwestern Medical Center, Dallas, TX, 2University of Texas Health Science Center at Houston, Houston, TX
Introduction: Muscle atrophy in severe burn is exacerbated with bed rest. Exercise is known to ameliorate disuse atrophy. MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional gene expression. The study aim was to investigate if exercise alters the muscle epigenetic profile in burn with bed rest using a clinically relevant animal model.
Methods: Male Sprague-Dawley rats received 40% total body surface area (TBSA) scald burn and were placed in a hindlimb unloading traction system. Half (n=6) exercised twice daily by climbing 1 meter 5 times (EX) the others did not exercise (NX). On day 14, plantaris was harvested and stored in RNA later at -80°C. Samples from each group were pooled. Total RNA was extracted with Qiagen miRNeasy Mini kit. MicroRNA profiles were measured using Affymetrix miRNA 4.0 Array chips at our institutional microarray core facility. One chip, including 363,353 small non-coding RNA probes with varied species and controls, was used per sample. Chip reproducibility (intra and inter-lot) was greater than 0.95. Genomic profile was examined from RNA samples running with Affymetric Rat gene 2.0 ST arrays. Raw data was normalized with Robust Multi-array Average (RMA). Data was analyzed with Transcriptome Analysis Console (TAC 2.0).
Results: In each group 36,222 miRNAs were detected; 1,218 miRNAs were rattus norvegicus. 623 miRNAs were upregulated and 587 were down regulated in exercise. 31 probes increased over 2 fold and 21 probes decreased over 2 fold with exercise. Of these, 6 upregulated and 7 down regulated miRNAs corresponded with target gene profile changes (Table). All gene expression except Rrm2 was down regulated. Type I collagen gene was associated with several miRNAs. Down regulation of mir-182, mir-92b-3p and up-regulation of mir 409a-3p together inhibits Col1a2 expression; mir-138-5p inhibits Col1a1 expression.
Conclusion: Exercise in disuse atrophy after burn altered the miRNA profile with down regulation of target genes. Mechanisms of miRNA regulation are complicated. Redundant epigenetic pathways provide flexibility to adapt to injury. Targeting miRNA could be useful as a therapeutic approach.
SEVERE BURN INJURY INDUCES THERMOGENICALLY FUNCTIONAL MITOCHONDRIA WITHIN THE INGUINAL WHITE ADIPOSE TISSUE OF MICE
C. Porter*1, 2, N. Bhattarai1, 2, M.K. Saraf1, 2, B. Szczesny1, 2, C. Szabo*1, 2, D. Herndon*1, 2, T. Toliver-Kinsky*1, 2, and L. Sidossis1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals For Children, Galveston, TX
Chronic cold exposure results in adrenergic stress, which leads to the induction of functionally thermogenic mitochondria in the inguinal white adipose tissue (iWAT) of mice. Whether this response occurs in pathophysiological states associated with an adrenergic stress response remains unclear. Here, we determined the impact of severe burns on iWAT mitochondrial function in mice.
Male balb c mice (6-8 weeks) were burned on the dorsum by immersion in 99°C water for 10 seconds, creating a full-thickness burn on ∼30% of the body surface area. Sham treated mice underwent the same experimental procedure without the immersion in water. iWAT was harvested from mice at 1, 4, 10, 20, and 40 days post burn/sham. Mitochondrial respiration in the uncoupled state was determined before and after titration of the uncoupling 1 protein (UCP1) inhibitor GDP. Citrate synthase (CS) activity was analyzed in iWAT as a proxy of mitochondria abundance. Immunohistochemistry was performed to access iWAT morphology and UCP1 expression.
UCP1 dependent respiration was significantly greater at 4 and 10 days post burn vs. sham, peaking at 20 days post burn (1.52±0.34 vs. 8.47±1.36 pmols/mg/sec; P<0.001). CS activity was 3-fold greater at 4, 10, 20 and 40 days post-burn vs. sham (P<0.05). iWAT from burned mice exhibited numerous multi-locular lipid droplets and stained positive for UCP1 from 4 days post-burn onwards.
The current findings demonstrate the induction of thermogenically competent mitochondria within iWAT in a clinically relevant model of burn trauma. These data identify a specific pathology which induces the browning of WAT in vivo, and may offer a mechanistic explanation for the chronic hypermetabolism observed in burn victims.
SIMILAR PHYSIOLOGIC RESPONSES IN TAIL TRAUMA AND MILD TRAUMATIC BRAIN INJURY, FUTURE TRAUMA CONTROL MODEL?
M. Vaickus, T. Hsieh, B. Lussier*, E.L. Chiswick, and D.G. Remick*. Boston University Medical School, Boston, MA
Introduction: Our murine model of mild traumatic brain injury (mTBI) has shown improved survival and bacterial killing after Pseudomonas aeruginosa pneumonia (Psd) compared to naïve mice. To determine if this phenomenon is specific to mTBI, we designed a control model of blunt tail trauma (TT).
Methods: Mild traumatic brain injury or blunt trauma to the tail was induced using a weight-drop model under anesthesia. TT was performed at a drop height of 2.5 cm from the base of the tail. After trauma, mTBI and TT mice were handled by the scruff to avoid repeated tail trauma. Physiologic parameters (heart rate, 02 saturation, weight, breath rate, and pulse distension) were measured with non-invasive cervical collar telemetry (MouseOx). Respiratory parameters (respiratory rate, tidal volume, time inspiration, and time expiration) were measured with whole body plethysmography (Buxco).
Results: Within 4h post-trauma, TT showed increased pulse distension, breath rate, and a decreased O2 saturation compared to baseline. Respiratory parameters at 4h post-trauma exhibit increased respiratory rate and tidal volume, and a decreased Ti and Te for TT compared to mTBI. All physiologic parameters returned to baseline by 48h. mTBI mice average a 10-20% reduction in body weight over 48h while TT has a 5% reduction in body weight over 48h.
Conclusion: Our TT model elicits a similar physiological response compared to mTBI although TT did have slightly faster breathing rates in the first 4h as compared to mTBI. Based on early physiological characterization, the TT model is comparable to the mTBI model and can be utilized for further studies as a control to examine whether the novel findings of survival and bacterial killing after Psd are exclusive to mTBI.
Physiologic measures of mTBI (n=5-8) and TT (n=5-8) mice post-trauma.
PLATELET DYSFUNCTION IS POORLY RECOGNIZED BY CLINICIANS DURING BURN SURGERY
H.F. Pidcoke, M.C. Herzig, B.S. Schaffer, C.G. Fedyk, K.K. Chung*, and A.P. Cap*. US Army Institute of Surgical Research, San Antonio, TX
Hemostatic resuscitation of acutely hemorrhaging trauma patients with balanced blood products to achieve whole blood functionality is associated with better outcomes. Although such a strategy is not used during burn surgery, patients experience considerable blood loss and may develop coagulopathy. We compared surgeon and anesthesiologist assessments of patient coagulation status to: 1. measured coagulation factors, thromboelastometry and platelet function; and 2. blood product utilization to determine awareness of clot forming status.
Consent was obtained and patients who were expected to need blood transfusions during surgery were enrolled into an IRB-approved prospective observational study. Before blood was drawn for coagulation assays (baseline, pre-transfusion, and post-transfusion), surgeons and anesthesiologists were queried regarding the patient’s perceived coagulation status. PT, aPTT, thromboelastometry (ROTEM: EXTEM reagent), and aggregations studies (Multiplate, collagen) were compared to clinician assessment. Blood products were recorded, and samples amalgamated for analysis, maintaining the volume ratio of transfused products. Data were analyzed with Fisher’s exact test, Student’s t-test, one-way ANOVA, or parametric equivalents, as appropriate, and significance set at p<0.05.
Of the 32 burn surgeries studied, 407 responses were obtained from surgeons and anesthesiologists over the course of each case. Sixteen (4%) were excluded due to insufficient data for analysis, and of the remainder, 197 were obtained from anesthesiologists and 194 from surgeons. Differences between surgeon and anesthesiologist impressions of overall clot forming ability did not differ significantly (clotting ability considered abnormal in 24% of instances versus 30%, respectively, p=0.385); however, anesthesiologists were more familiar with aPTT reference ranges compared to surgeons (p<0.001). When asked to predict the subject’s PT and aPTT, clinician results were not statistically different than actual values regardless of specialty ( p≥0.05), but neither group were aware of platelet dysfunction, particularly in patients who received blood products (EXTEM MCF 48.9±7.8 versus 55.9±7.3 mm, p=0.006; Multiplate collagen response 29.6±3.8versus 58.5±6.6 U, p=0.011), as evidenced by the infrequent use of platelet products. A majority of patients received blood products (91%, range: 0-17 units), but only 18.8 percent received platelets. Despite little evidence of significant factor deficiencies (mean PT 16.4±0.3 s, PTT 32.9±0.6 s), a majority of 75% patients received plasma during surgery.
Burn surgeons and anesthesiologists, while able to predict coagulation status, are unaware of the prevalence of platelet dysfunction during surgical hemorrhage. Burn patients exhibit significant platelet dysfunction and might benefit from a balanced transfusion strategy that includes platelets.
EVIDENCE THAT THE INITIAL HMGB1 SURGE CONTRIBUTES TO TRAUMA-INDUCED IMMUNE RESPONSES IN HUMANS
A. Wei Chen, R.A. Namas*, O.M. Abdul-Malak*, Y. Vodovotz*, and T. Billiar*. University of Pittsburgh, Pittsburgh, PA
Background: Dysregulated or excessive inflammation post-injury is associated with a complicated clinical course in a subset of patients, i.e. patients who develop in-hospital organ dysfunction and nosocomial infection (NI). High-mobility Group Protein B1 (HMGB1) has emerged as a prototypical damage-associated molecular pattern molecule that activates inflammatory signaling when released from damaged tissues. We hypothesized that higher levels of circulating HMGB1 early post-injury would correlate with circulating mediator profiles and stratify trauma patients that went on to develop a complicated clinical course.
Methods: In a cohort of 472 blunt trauma survivors (age: 48.8 ± 0.8, males [M]/females [F] 330/142, injury severity score [ISS] 19.6 ± 0.5), we performed a retrospective case-control study, in which 44 NI trauma patients (age: 48 ± 3; M/F 27/17; ISS: 26.3 ± 1.7) were identified and compared to 44 no-NI (age: 47.3 ± 2; M/F 27/17; ISS: 26.1 ± 0.9), matched for demographics, ISS, and mechanism of injury. Plasma was sampled 3 times within the first 24 h and then daily up to day 7 post-injury and assayed for 23 inflammation biomarkers using Luminex™. HMGB1 measurement was performed using ELISA. Two-way analysis of variance (ANOVA) and area-under-curve (AUC) analysis were used to determine statistical significance (p<0.05) between the NI and no-NI sub-groups. Spearman’s correlation coefficient (cc) was calculated to determine associations among HMGB1, inflammatory mediators, and ISS.
Results: ICU length of stay (LOS), total LOS, and days on ventilation were statistically significantly greater in the NI patients. HMGB1 levels were significantly elevated at 4 h post-injury in patients who went on to develop NI which was also associated with higher organ dysfunction suggested by the Marshall MODScore. AUC analysis showed 2 fold higher levels in circulating HMGB1 in the NI patients at 4 h post-trauma when compared to the no-NI patients. In addition, HMGB1 levels correlated positively with ISS (cc:0.5, p= 0.002), IL-6 (cc:0.4, p=0.01), MCP-1 (cc:0.4, p=0.01), IL-10 (cc:0.4, p=0.01), and IL-25 (cc:0.5, p=0.03) in patients who went on to develop a complicated clinical course.
Conclusion: Our results point to the potential role of an early surge in HMGB1 release in driving worse clinical outcomes post-trauma. In addition, HMGB1 may be integral to the upregulation of specific components of the immune response following injury and therefore could be a potential therapeutic target.
THE IMMUNOSUPPRESSED PHENOTYPE IN SEVERE BURN INJURY
C.P. Henson*, L. Luan*, and E. Sherwood*. Vanderbilt University, Nashville, TN
Background: There is an increased incidence in late infectious complications in patients following severe burn injury1. PD-1 and PD-L1 are cell surface receptors that down-regulate T lymphocyte activity, and altered expression of PD-1 has been observed in other patient populations with an increased incidence of late infectious complications2,3. Also, a relationship has been demonstrated between peripheral blood T lymphocyte number and immune dysfunction in critically ill patients4. Our aim was to demonstrate whether patients with large total surface area burns exhibit signs of immune dysfunction characterized by decreased peripheral blood T lymphocyte numbers, increased expression of PD-1 and PD-L1 and altered cytokine production.
Methods: Cell surface expression of PD-1 and PD-L1 on T lymphocytes and inflammatory monocytes, respectively, was measured in 18 patients with severe burn injury (>30% TBSA) at four time points (post-burn Day 0, 3, 7, and 14) with flow cytometry. Age and gender-matched control volunteers were used for comparison. Total CD4+ and CD8+ T lymphocyte and inflammatory monocyte numbers were also measured. Ex vivo expression and plasma concentration of the cytokines IFN-gamma, IL-6 and IL-10 was observed.
Results: Time-dependent changes in number and proportion of CD4+ and CD8+ T lymphocytes and inflammatory CD14+/CD16+ monocytes were demonstrated in patients following severe burn injury, as well as altered expression of PD-1 and PD-L1 on these cells. These changes were evident early in the course of injury and persisted through day 14 post-burn.
Conclusions: Given the high rate of late infection patients with severe burn injury, the presence of a functionally immunosuppressed state is possible. Injury-induced alterations in expression of the T lymphocyte co-inhibitory receptor PD-1 and its ligand, PD-L1, may contribute to this process in patients following severe burn injury. Furthermore, alterations in CD4+ and CD8+ T lymphocyte number and function remain evident out to 14 days post-injury. Our study outlines a possible time course for altered lymphocyte function in a population of severely burned patients. We posit that functional immunosuppression is present as early as 3 days following severe burn injury.
1. Rex S. Burn injuries. Curr Opin Crit Care. 2012;18(6):671-676.
2. Boomer JS et al. A prospective analysis of lymphocyte phenotype and function over the course of acute sepsis. Crit Care. 2012;16(3):R112-R112.
3. Monaghan SF et al. Programmed death 1 expression as a marker for immune and physiological dysfunction in the critically ill surgical patient. Shock. 2012;38(2):117-122.
4. Boomer JS et al. Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA. 2011;306(23):2594-2605.
REDUCED ACID SPHINGOMYELINASE ACTIVITY PROMOTES DECREASED NEUTROPHIL-DERIVED MICROPARTICLE NUMBERS IN BURN-INFECTED MICE
C.C. Caldwell*1, 2, T. Rice1, E.F. Midura1, and T.A. Pritts*1. 1University of Cincinnati, Cincinnati, OH, 2Shriners Hospitals for Children — Cincinnati, Cincinnati, OH
Background: Burn injury can lead to an immunosuppressive state resulting in increased susceptibility to nosocomial infections such as pneumonia. Control of the inflammatory response is critical to promote an antimicrobial response and minimize lung injury during pneumonia. Neutrophils have a key role in the antimicrobial response. Previously, neutrophils have been shown to produce microparticles (MPs) and these MPs have been demonstrated to be elevated in critically ill patients. The MPs from critically ill patients were demonstrated to influence leukocyte function. Here, we sought to elucidate the presence of MPs in pneumonia following scald injury. We hypothesized that reduced acid sphingomyelinase (Asm) activity observed following scald would result in a decrease in the generation of MPs at the site of injury.
Methods: Outbred mice were subjected to a 28% total body surface area, full thickness scald injury. On post burn day (PBD) 1, mice were intratracheally inoculated with 105 CFU Pseudomonas aeruginosa. Bronchoalveolar lavage (BAL) fluid was collected 24 hours following inoculation. Microparticles were isolated and characterized using Nanoparticle Tracking Analysis.
Results: On PBD1, we observed that infected burn-injured mice had increased mortality as compared to infected sham mice (50.0% vs 18.2%, p=0.048). Neutrophil accumulation in BAL fluid was not significantly different between injured and sham infected mice. However, decreased neutrophil derived MPs were enumerated in the BAL fluid of the burn-injured mice.
Conclusion: We have previously demonstrated that scald injury reduces Asm activity in burned mice. Further, we have observed that reduced Asm activity can result in decreased MP production. These data suggest a working model where neutrophils from scald-injured mice produce fewer MPs due to decreased Asm activity with altered immune dysfunction leading to increased mortality.
DYNAMIC INFLAMMATORY NETWORKS SUGGEST AN EARLY DIVERGENCE OF CLINICAL OUTCOMES IN BLUNT TRAUMA SURVIVORS VS. NON-SURVIVORS
A.N. Abboud*, R.A. Namas*, Q. Mi*, K.W. Almahmoud*, O.M. Abdul-Malak*, D. Barclay, J. Yin, R. Zamora*, T. Billiar*, and Y. Vodovotz*. University of Pittsburgh, Pittsburgh, OH
Introduction: Human blunt trauma patients presenting with similar demographics and injury severity often diverge in clinical outcomes despite receiving equivalent medical care. We hypothesized that early trauma-induced inflammation can foreordain similar patients for survival versus mortality, and carried out combined clinical and in silico studies to gain insights into this process.
Methods: From a cohort of 493 blunt trauma patients studied following IRB approval, 19 non-survivors ([NS]; 3.9%; 16 males and 3 females; age: 58.8±4.5; Injury Severity Score [ISS]: 23.3±2.1) were stringently matched to 19 trauma survivors ([S]; 16 males and 3 females; age: 59.0±3.0; ISS: 23.4±2.0; p=NS). Plasma (3 samples within the first 24 h, then from days 1 to 7 post-injury) was assayed for 27 inflammatory mediators by Luminex™ and ELISA. These data were analyzed by 2-way ANOVA as well as by Dynamic Network Analysis (DyNA).
Results: No statistically significant differences were observed in ISS, ICU length of stay (LOS), or total LOS between S and NS; yet, the Marshall MODScore rose continuously over 7 days in NS but not S, and was significantly different (p<0.05) between groups by 2-way ANOVA. NS also exhibited higher HMGB1 levels than S in the first 24 h, statistically significant (p<0.05) between groups by 2-way ANOVA. DyNA network complexity mimicked the MODScore, rising over 7 days in NS in a manner suggesting self-sustaining inflammation. In contrast, S exhibited a relatively static network. Inferred dynamic networks in S involved lymphoid-polarizing cytokines (IL-4, IL-5, IL-13, IL-17A, IL-22, IL-23) discernible as early as 24 h and consistently connected over 7 days. In contrast, NS were characterized by both innate mediators (Eotaxin, IL-8, MIP-1α, MIP-1β, IL-1β, TNF-α, IFN-α, IFN-γ) and lymphoid mediators (IL-2, IL-4, IL-5, IL-7, IL-13, IL-17A, IL-22, IL-23) interconnected early, and which evolved into much larger, innate-dominant networks by Day 3. IL-17A in NS was positively correlated with GM-CSF and negatively correlated with IL-10, suggesting the presence of pathogenic Th17 cell populations in NS.
Conclusion: Increased network complexity and paradoxically decreased lymphoid activation combined with the emergence of pathogenic Th17 cells were associated with elevated organ dysfunction and fatal clinical trajectories in moderately-injured blunt trauma patients. These studies suggest a novel, early role for lymphoid cells in mediating a key inflection point in the response to blunt trauma, and highlight the power of combining stringently-matched sub-cohorts with in silico modeling.
MILD TRAUMATIC BRAIN INJURY (MTBI) IN THE MURINE MODEL IS ASSOCIATED WITH ALVEOLAR MACROPHAGE EVIDENCE OF FAT EMBOLI
B. Lussier*, T. Hsieh, and D.G. Remick*. Boston University Medical Center, Boston, MA
Background: Fat emboli and fat emboli syndrome are feared complications of burn and trauma. Though seen frequently in systemic trauma, pulmonary fat emboli have never been studied in traumatic brain injury. The mechanism by which fat emboli alter immune function is poorly understood. Based on prior research, pulmonary host immunologic response is altered significantly after traumatic brain injury. This study investigated whether fat embolization occurs after traumatic brain injury.
Methods: Outbred ICR mice were subjected to a model of MTBI or blunt tail injury by weight drop model, or sham injury. After 24 hours, mice were sacrificed and bronchoalveolar lavage was analyzed for macrophage inclusion of lipids by Oil Red O staining. Lung histology was stained for Oil Red O to evaluate anatomy and intravascular disease.
Results: In sham trauma there was no evidence of fat staining within any of the alveolar macrophages. The lipid index, derived by assessment of 100 consecutive alveolar macrophages from each subject and scored from 0 to 4 based on degree of intracellular lipid content, was increased by MTBI. Within 24 hours of MTBI, bronchoalveolar lavage lipid index was significantly higher than in sham (45.7±10.8 vs. 0.5± 0.5; p< 0.05 compared to sham). Blunt trauma resulted in a lower lipid index compared to MTBI.
Conclusions: Mild traumatic brain injury leads to evidence of fat emboli in cells recovered from the bronchoalveolar lavage in the murine model. This novel finding suggests that fat emboli should considered as a potential factor contributing to altered pulmonary pathophysiology after traumatic brain injury.
TRAUMATIC BRAIN INJURY INDUCED NEUTROPHIL RECRUITMENT CONFERS INCREASED SURVIVAL TO PNEUMONIA
E.L. Chiswick, T. Hsieh, M. Vaickus, G. Baldeon Vaca, and D.G. Remick*. Boston University, Boston, MA
Background: Mild Traumatic Brain Injury (mTBI) results in decreased incidence of hospital acquired pneumonia, as compared to patients matched for blunt trauma. Similarly, a murine model of TBI results in increased survival from subsequent pneumonic challenge which is associated with increased neutrophil (PMN) recruitment to the airways, and decreased bacterial CFUs. This study examined how mTBI primes the immune system to increase survival from pneumonia.
Methods: Mice received mTBI, Tail Trauma (TT), or sham injury under anesthesia by a controlled weight impact. At 6, 24, and 48hr post-injury, mice were exsanguinated and their lungs perfused via the right ventricle. Flow cytometry was used to identify up to 11 leukocyte populations in the spleen, blood, and total lung digests. Mild PMN depletion was achieved via 10ug anti-Ly6G i.p., followed by TBI 24hr later. Pneumonia was given 48hr post TBI/sham with 5*107 CFU Pseudomonas aeruginosa (Psd). 500ng KC was given i.t. 4hr prior to Psd to mimic TBI induced PMN recruitment. Airway PMNs were enumerated via BAL and cytospins.
Results: mTBI resulted in increased PMN as compared to sham in the lung (51.4 vs 11.3%**), blood (59.0 vs 13.0%**), and spleen (16.6 vs 3.5%*) within 6hr post injury. These increases were sustained through 48hrs post injury.
PMN recruitment was also greater in TBI vs TT mice at 6hr post injury: lung (51.4 vs. 18.0%**); blood (59.0 vs 24.3 %***); spleen (16.6 vs 6.4 %**). Similar differences were found at 24hr, thus suggesting that injury induced PMN recruitment to the lung and spleen is specific to mTBI, but not peripheral trauma. No significant differences were found for other phagocytes at 6hr post-injury comparing mTBI vs TT: Lung- Alveolar Macs (7.3 vs 7.0%), inflammatory monocytes (iM)(5.3 vs 7.8%), Ly6C low monocytes/macrophages (rM) (3.5 vs 5.9); Spleen- iM (2.0 vs 1.7%), and rM (1.7 vs 1.1%).
4hr post-Psd challenge mTBI mice recruit significantly more PMNs into the airways, compared to sham (5.9 vs 3.2 *106/mL***). KC (i.t.) therapy also enhanced recruitment in mTBI vs. sham (5.7 vs 2.0 *106/mL***). However, there was not a significant difference in the percentage or numbers of PMNs in the lung digests 6hr post-Psd (mTBI-23.2*106, sham 22.4*106). This suggests that mTBI induced pulmonary recruitment of PMNs facilitates PMN recruitment into the airways post-Psd. In support of this, mild PMN depletion pre-mTBI resulted in decreased survival to pneumonia (0% survival), while KC recruitment increased survival (57% survival).
Conclusion: TBI primes the host immune system to recruit more PMNs into the airways following pneumonic challenge, resulting in decreased bacterial burden and increased survival relative to sham. This is due in part to the PMN influx observed from mTBI itself, which if blocked via PMN depletion, abrogates the survival benefit of mTBI.
BURN WOUND INFECTION CAUSES SIGNIFICANT ADAPTIVE IMMUNE SYSTEM DYSFUNCTION
N.K. Patil*, J. Bohannon*, L. Luan*, Y. Guo*, and E. Sherwood*. Vanderbilt University Medical Center, Nashville, TN
Background: Burn patients are at increased risk of developing wound infection and sepsis leading to multi-organ failure and death. Previous studies show that T cell dysfunction and low absolute lymphocyte counts are major contributing factors to increased mortality in non-burned septic patients. Although impaired antimicrobial immunity has been described in burn patients, the changes in adaptive immunity after burn injury and infection have not been investigated. We used a clinically relevant mouse model of burn wound infection to assess the adaptive immune system dysfunction.
Methods: A 35% TBSA full thickness burn was induced and the wound was infected with Pseudomonas aeruginosa on day 4 post-burn. Lymphocyte, macrophage and dendritic cell counts and phenotype in spleen and wound draining lymph nodes were characterized by flow cytometry.
Results: We observed a significant decline (>80%) in blood absolute lymphocyte count and CD4+ and CD8+ T cell counts in the spleen and burn-draining lymph nodes of wound infected mice (WI) as compared to non-infected burn mice (see figure). WI also lead to a significant decrease in the expression of the co-stimulatory receptor CD28 on both CD4+ and CD8+ cells in spleen and lymph nodes and an increase in the expression of the co-inhibitory receptor CTLA4 on CD4+ cells in the spleen (2.9 vs 1.7%) and lymph nodes (3.7 vs 1.3%) compared to control. PD-1 expression was not affected. Burn injury alone led to a significant decline in dendritic cell counts in spleen on day 4 (27x105 vs 39x105 in sham) which recovered on day 6; and WI decreased dendritic cell numbers in lymph nodes (2x105 vs 6x105) as compared to control. WI also lead to a significant decrease in F4/80+ macrophage cell counts (48x105 vs 81x105); decrease in expression of MHCII on dendritic cells (29 vs 42 %); and an increase in the expression of PDL1 on dendritic cells (31 vs 23%), macrophages (25 vs 10%) and Ly6C+ inflammatory monocytes (19 vs 11%) in the spleen, as compared to control. These changes were associated with significant kidney and liver injury and approximately 75 % mortality at day 3 after WI.
Conclusion: We show that adaptive immune system functions are significantly impaired following burn injury and wound infection and associated with a decrease in host resistance to infection.
NEBULIZED EPINEPHRINE REDUCES BURN AND SMOKE INHALATION INJURY INDUCED VASCULAR HYPER-PERMEABILITY VIA ITS ALPHA-1 EFFECT
S. Fukuda1, K. Ihara1, E. Lopez*1, D. Herndon*1, 2, D. Prough*1, and P. Enkhbaatar*1. 1University of Texas Medical Branch Galveston, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX
Rationale: Combined burn and smoke inhalation injury is associated with bronchoconstriction, airway mucosal hyperemia and pulmonary edema. Previously, we have reported superior effects of nebulized epinephrine (Epi) (nonspecific adrenergic agonist) compared to nebulized albuterol (β-2 adrenergic agonist) in reducing pulmonary microvascular hyper-permeability in an ovine model of burn and smoke inhalation. In the present study, we tested the hypothesis that salutary effects of nebulized Epi on vascular hyper-permeability are attributable to its α-1 agonist property.
Methods: To test the hypothesis, we utilized our well-characterized ovine model of skin flame burn (40% TBSA, 3rd degree) and smoke inhalation (48 breaths of cooled cotton smoke) and compared the effects of nebulized Epi and α-1 adrenergic agonist phenylephrine (PE). Efferent thoracic lymph node was cannulated to assess pulmonary transvascular fluid flux (lung lymph). Urinary output was measured to calculate fluid net balance. After the injury, all sheep were randomly allocated to 3 groups; 1) Epi: nebulized with 4 mg epinephrine, n=6; 2) PE: nebulized with 10 mg of phenylephrine, n=6; and control: nebulized with saline, n=8. The treatment was started 1 h post injury and repeated every 4 hrs. All sheep were placed on mechanical ventilation and monitored in a conscious state for 48 hrs. Pre/post injury analgesia was maintained with buprenorphine. Statistical analysis: Two-way ANOVA.
Results: 63 % of control sheep survived 48 hrs, while all animals survived in both treated groups. Nebulized Epi significantly reduced lung lymph flow and pulmonary permeability index (= lung lymph × plasma protein / lung lymph protein) (see Table) and significantly improved pulmonary mechanics and gas exchange that prevented the progression of moderate ARDS (PaO2/FiO2 >200 at 48 hrs) as compared to control group. Nebulized PE tended to attenuate PaO2/FiO2 (>100 at 48 hrs), but not pulmonary mechanics. Both treatments significantly increased urinary output vs. control group (at 36 hrs).
Conclusions: Vasoconstriction by α-1 adrenergic activation is not the sole mechanism explaining salutary effects of nebulized Epi on pulmonary vascular hyper-permeability. However, increasing urinary output in this model is mediated by α-1 adrenergic effect. Nebulized Epi more effectively ameliorates the degree of acute lung injury than PE. Nebulized Epi should be considered as an effective and safe therapy for burn patients with smoke inhalation. (Support: W81XWH-12-2-0086 and SHC84050)
DIVERGENT MECHANISMS UNDERLY INCREASED COAGULATION IN A MURINE MODEL OF BURN INJURY
E.F. Midura1, M.D. Goodman*1, 2, and C.C. Caldwell*1, 2. 1University of Cincinnati, Cincinnati, OH, 2Shriners Hospitals for Children — Cincinnati, Cincinnati, OH
Background: After scald injury, a subacute, hypercoagulable state develops which increases microvascular thrombosis, hypo-perfusion and multi-system failure leading to worsened outcomes. The mechanisms leading to these alterations remain poorly understood. Recently, the role of microparticles (MPs), specifically platelet derived MPs (PMPs), has been suggested based on their contribution to hypercoagulable states in other disease processes. In the current study, we hypothesized that scald injury would result in changes in circulating PMP populations and that these changes would contribute to the post-burn, hypercoagulable state.
Methods: A murine scald model with 28% TBSA full thickness burn injury was induced in CF-1 mice. Blood samples were collected one and six days after scald. Circulating MP populations and platelet counts were determined. Whole blood coagulation was analyzed by thromboelastometry. Platelet function was evaluated by impedance aggregometry.
Results: Burn injury led to hypercoagulability on post-burn day one (PBD1), which persisted six days after injury (PBD6). On PBD1, there was a significant decrease in platelet counts as well as a reduction in platelet contribution to clot formation. By contrast, there was a significant increase in pro-coagulant PMPs at this time-point. On PBD6, there was a significant increase in platelet count with no change in PMP numbers compared to sham. Furthermore, ADP-induced platelet aggregation was decreased on PBD1 but increased on PBD6 compared to sham.
Conclusions: After scald injury there is a hypercoagulable state that is maintained almost one week after injury. On PBD1, we postulate that increased PMP numbers compensate for the decreased platelet functionality and ultimately contribute to increased coagulation. Conversely, hypercoagulability on PBD6 may be due to increased platelet activation. We therefore conclude, 1) the mechanisms underlying the coagulopathy seen after scald injury are altered temporally, 2) changes on PBD1 are likely PMP induced and 3) platelet aggregation via ADP plays a significant role in these changes.
CENTRAL ROLE OF CHEMOKINES IN MODULATING THE INFLAMMATORY RESPONSE IN TRAUMA-INDUCED COAGULOPATHY
X. Zhu1, R.A. Namas*1, Q. Mi1, O.M. Abdul-Malak*1, Y. Vodovotz*1, J. Sperry*1, Q. Feng2, and T. Billiar*1. 1University of Pittsburgh, Pittsburgh, PA, 2Peking University People’s Hospital, Beijing, China
Background: Coagulopathy in the setting of blunt trauma can induce physiological alterations accompanied by a broad systemic inflammatory response that can predispose patients to worse clinical outcomes. Despite significant recent advancements associating post-traumatic inflammation and trauma-induced coagulopathy (TIC), a better understanding of this complex interaction is needed. We sought to gain insights on the systemic inflammatory response accompanying TIC. Accordingly, we carried out an extensive time course analysis of circulating inflammatory mediators coupled with data-driven modeling.
Methods: In a cohort of 472 blunt trauma survivors (age: 48.8±0.8, males [M]/females [F] 330/142, injury severity score [ISS] 19.6±0.5), we performed a retrospective case-control study, where 27 patients with TIC (admission INR>1.3, age: 43.0±4.3, M/F 23/4, ISS: 20.6±1.5) were matched to 27 patients without TIC (age: 43.8±3.3, M/F 23/4, ISS: 21.2±1.5) for demographics, ISS, and mechanism of injury. Plasma was sampled 3 times within the first 24 h and then daily up to day 7 post-injury and assayed for multiple inflammation biomarkers using Luminex™. NO2-/NO3- was measured using the nitrate reductase/Griess assay. Two-way analysis of variance (ANOVA) and area-under-curve (AUC) analysis were used to determine statistical significance (p<0.05) between the TIC and non-TIC sub-groups. Dynamic network analysis (DyNA) was used to suggest dynamic connectivity and complexity of the inflammatory mediators.
Results: ICU length of stay (LOS), total LOS, and days on ventilation were statistically significantly prolonged in the TIC patients when compared to non-TIC patients. In addition, the TIC sub-group had a greater requirement of blood products transfusion within the first 24 h post-admission. The TIC sub-group had a higher degree of organ dysfunction from days 1 to 5 when compared to the non-TIC sub-group. Importantly, circulating levels of MIG, IP-10, IL-10, NO2-/NO3-, MCP-1, IL-8 and GM-CSF were significantly elevated in the TIC sub-group. Moreover, DyNA suggested that the inflammatory response in non-TIC had a high degree of interconnection/connectivity while the response in TIC consisted of different multiple isolated nodes and exhibited substantial reduction of interconnectivity from time of admission and up to day 7 post-injury.
Conclusion: These results suggest that post-traumatic coagulopathy, identified by elevated admission INR, is associated with a markedly different inflammatory response when compared to patients that present without TIC despite similar injury patterns. The early mediator profiles point to differences and a key role of specific chemokines in patients with TIC.
HEMORRHAGIC SHOCK STIUMLATES ORGAN TISSUE RELEASE OF TPA INDEPENDENT OF DISSEMINATED INTRAVASCULAR COAGULATION
H.B. Moore1, 2, E.E. Moore*1, 2, A.P. Morton1, 2, E. Gonzalez1, 2, M.P. Chapman1, 2, M. Fragoso1, 2, F. Gamboni1, S. Mitra1, C. Anderson1, A. Sauaia1, A. Banerjee*1, and C.C. Silliman1, 3. 1University of Colorado, Denver, CO, 2Denver Health, Denver, CO, 3Bonfils Blood Center, Denver, CO
Introduction: Trauma-induced coagulopathy (TIC) has been hypothesized to be a consumptive process related to disseminated intravascular coagulation (DIC). However, evidence for the acute onset of DIC after trauma is lacking. The hyperfibrinolytic phenotype of TIC is associated with elevated tissue plasminogen activator (tPA) with concurrent depletion of its inhibitor plasminogen activator inhibitor -1(PAI-1). We hypothesize that tPA is released in response to hemorrhagic shock and is not a process driven by microvascular thrombi.
Methods: Experimental groups (n=6) of rats included; 1) tissue injury (laparotomy evisceration/bowel crush), 2) shock (hemorrhage to mean arterial pressure <25 mmHG), 3) sham (arterial cannulation and tracheostomy). Blood sampling was done after cannulation and 30 minutes post intervention. Plasma was assayed for total tPA and PAI-1 concentration with simultaneous heart, lung, liver, and kidney tissue assays. Lung, liver, and kidney were evaluated histologically for evidence of microthrombi.
Results: Plasma tPA was elevated in the shock group vs trauma (p=0.009) and sham (p=0.012). Plasma PAI-1 concentrations were not detected. Overall tPA differed by organ (figure 1). PAI-1 tissue levels were also variable. Shock was associated with a reduction in tissue tPA levels in lung vs sham (p=0.004); and reduced in liver (p=0.004) and kidney (p=0.007) compared to tissue injury. PAI-1 tissue levels did not differ significantly among groups. When adjusting for specific organ median tPA levels, overall tPA levels were reduced in the shock group compared to sham (p=0.024) and tissue injury (p<0.001). There was no evidence of thrombi in the kidney or liver, while the lung of both the shock and tissue injury groups had evidence of thrombus.
Conclusion: Increases in plasma tPA is associated with shock and reduction in tissue tPA concentrations without evidence of diffuse microvascular thrombosis in multiple organs. Tissue injury does not increase plasma tPA. Dynamic changes in organ tissue tPA and PAI-1 levels are reflective of a complex system that cannot be explained by DIC.
THE ENDOCANNABINOID N-ARACHIDONOYL DOPAMINE (NADA) REGULATES ENDOTHELIAL PROSTANOID METABOLISM
S. Khakpour*, K. Wilhelmsen*, A. Tran, and J. Hellman*. University of California San Francisco, San Francisco, CA
Background: Endocannabinoids are endogenous, arachidonic acid-derived lipid mediators that display a diverse array of immunomodulatory activities. We have previously shown that the endocannabinoid N-arachidonoyl dopamine (NADA) reduces endothelial cell (EC) activation induced by inflammatory agonists (Wilhelmsen, JBC, 2014), however the precise mechanism of this activity remains unclear. 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 NADA’s immunomodulatory activity in the endothelium is dependent upon its regulation of other arachidonic acid-derived lipids, including the prostanoids.
Methods: To determine the effects of endocannabinoids on the expression of genes involved in arachidonic acid metabolism, a qPCR gene array was performed using cDNA derived from primary human lung microvascular endothelial cells (HMVEC-Lung) treated with vehicle or LPS in the presence or absence of the endocannabinoids anandamide, 2-arachidonoylglycerol, and NADA (3h). Genes showing significant changes in expression (> 3-fold) were verified by immunoblot. The levels of prostanoids were quantified in supernatants and cell culture lysates by ELISA after similar treatments (20h). To determine if prostanoids contribute to the immunomodulatory activity of NADA, HMVEC-Lung were treated with NADA and LPS in the presence of the COX-1/2 inhibitor indomethacin, and cytokine levels were quantified in culture supernatants by ELISA.
Results: In contrast to other endocannabinoids, NADA reduces LPS-induced prostacyclin (PGI2), thromboxane A2 (TXA2), and prostaglandin E2 (PGE2) production by HMVEC-Lung. In addition, NADA is sufficient to dramatically upregulate prostaglandin D2 (PGD2), a prostanoid with characterized roles in inflammatory resolution. Furthermore, NADA induces a robust upregulation of COX-2 expression, while non-selective inhibition of the COX enzymes dose-dependently reversed the NADA-mediated decrease in LPS-induced cytokine secretion.
Conclusion: Our results suggest that the immunomodulatory activity of NADA depends upon its regulation of COX-2-derived lipid mediators. By elucidating the mechanism by which endocannabinoids modulate prostanoid metabolism in endothelial cells, we hope to better understand the therapeutic potential of manipulating the endocannabinoid system in acute inflammatory disorders that are characterized by endothelial activation.
EXTRACELLULAR HISTONES INDUCE ENDOTHELIAL BARRIER DYSFUNCTION AT HIGH DOSES, BUT TIGHTEN BARRIER JUNCTIONS AT LOWER DOSES, AND IS ATTENUATED BY ACTIVATED PROTEIN C
B. Miyazawa, and M.J. Cohen*. University of California San Francisco, San Francisco, CA
Background: Extracellular histones released in response to inflammatory challenge have been shown to contribute to endothelial dysfunction and organ failure. Activated Protein C (aPC) has been reported to cleave these histones. We have previously shown an endothelial barrier breakdown after treatment with trauma patient plasma, which can be attenuated by aPC. Therefore, we hypothesized stimulation of the endothelial barrier with histones would induce a similar dysfunction, and in turn, be attenuated by aPC.
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. Primary histones and activated Protein C were purchased from Sigma.
Results: Treatment of HUVEC monolayers with 100ug/ml of histones resulted in a maximal increase in resistance of 49.6%. One hour preincubation of the 100ug/ml dose of histones with 100nM of aPC resulted in a maximal increase of 42.7%. In contrast, 1000ug/ml of histones induced a maximal decrease in resistance of 29.3%. One hour preincubation of the 1000ug/ml dose of 100nM of aPC resulted in a maximal decrease in resistance of 12.1%.
Conclusions: Extracellular histones do indeed appear to induce endothelial barrier breakdown, but only at a relatively high dose. At the dose reported in other studies on extracellular histones, the histones surprisingly appear to protect the endothelium. Furthermore, as expected, preincubation with aPC inhibits the effect of the histones at both doses. Further studies are warranted and underway to examine the disparity between the effects of the histone doses and its relationship with a PC.
PATTERNS OF COAGULATION GENE EXPRESSION IN LEUKOCYTES FROM TRAUMA PATIENTS
R.R. Meyer, M. Bhattacharjee, and B.H. Brownstein*. Washington University School of Medicine, St Louis, MO
Introduction: One of the major objectives of this research is to learn why, as a consequence of traumatic injury, individual types of White Blood Cells (WBCs) up- or down-regulate their expression of genes specifying many coagulation proteins involved in coagulopathy. For this study, we looked at 117 genes known to be part of the entire coagulation process.
Methods: Supervised and unsupervised hierarchical clustering of mRNAs specifying proteins, which mediate the entire coagulation process, was performed with 244 Glue Grant patient samples. Blood was sampled up to 24 hours’ time from injury (TFI); monocytes, neutrophils and T-cells were purified by microfluidics, and total RNA purified. The RNA was labeled and hybridized against a custom GG chip, and mRNAs were examined for abundance starting with TFI up to 24 hours.
Results: Hierarchical clustering was performed with patient samples grouped according to TFI. Supervised clustering showed no correlation between mRNA profiles and TFI irrespective of cell type. Unsupervised, clustering revealed distinct sets of differentially regulated genes, which stratified patient mRNAs into distinct groups specific for each cell type.
The abundance of individual mRNAs from each cell type was surveyed by principal component analysis (PCA). For example, by PCA, F3 mRNA is primarily down-regulated in monocytes, about 50% up-regulated in neutrophils and about 25% up-regulated in T-cells over TFI out to 12 hours. For another case, SerpineB10 mRNA is about 35% up-regulated in monocytes, 20% up-regulated in neutrophils and mostly down-regulated in T-cells.
An issue of concern was whether blood transfusions confound the abundance of coagulation mRNAs since all of the patients had some form of blood transfusion. Consequently, we compared mRNA abundance for all WBC cell types with the nature of transfusion type. Very simply, no differences were seen correlated to type of transfusion from TFI to 12 hours.
Discussion: We do not understand the role of specific coagulation genes in the three distinct WBC types. The mRNA profiles of genes involved in coagulation from TFI up to 24 hours post trauma show cell type specificity and gene specificity. All of the data derived so far that focuses on groups of genes from the distinct cell types represent part of an initial process to develop a panel of mRNA biomarkers that could be diagnostic for coagulopathy.
A NETWORK BASED RNA-SEQ ANALYSIS OF EFFECTS OF HTS ON INFLAMMATORY TRANSCRIPTOME
S. Mitra, K.L. Jones, A. D’Alessandro, F. Gamboni, C. Anderson, and A. Banerjee*. University of Colorado Denver, Aurora, CO
Introduction:Hypertonic Saline (HTS) has been used as an alternative resuscitation fluid in trauma and hemorrhagic shock. In addition to restoring circulating volume several studies have reported beneficial immunomodulatory effects of HTS administration. The mechanisms by which HTS modulates the immune response are poorly characterized. We have shown that HTS affects inflammatory signaling pathways and transcriptional activation of several proinflammatory cytokines, thus we hypothesize that HTS elicits a genome-wide transcriptional response. To explore these possibilities, we used RNAseq to explore early changes in global transcriptional response in lung epithelial cells that were pretreated with HTS in the presence or absence of cytomix (TNFα, IL-1β, IFNγ).
Methods: SAECs (Small Airway Epithelial Cells) were pretreated with 400 mOSM HTS in the presence and absence of cytomix for 4 hours. Total RNA was isolated and used to prepare the Illumina HiSeq libraries. mRNA template libraries were sequenced on an Illumina HiSeq2000 platform. A custom computational pipeline consisting of the open-source gSNAP, Cufflinks, and R was used for alignment and discovery of differential gene expression, Pathway analyses was done by Ingenuity’s Pathway Analysis software (IPA).
Results: HTS treatment in the presence and absence of cytomix show very distinct differences in gene expression. HTS treatment alone showed changes in the expression of 3500 genes (q < 0.05) compared to control cells. Using IPA, 25 different molecular interactions were identified, these included but were not limited to proteins regulating cell cycle progression and survival, carbohydrate, amino acid and lipid metabolism, oxidative stress response genes and protein ubiquitination pathways.HTS pretreatment prior to cytomix stimulation show a decreased level of change (750 genes, q < 0.05) when compared to cytomix treated cells. The top molecular interactions identified cytokines/chemokines involved in cell- cell communication, ligand activated nuclear receptors, kinases and transcriptional regulators amongst several others. IPA also identified about 25 independent transcription factor modules (eg. STAT, IRF, Rel) which could explain several fold changes in the expression of as a many as 29 messages. (eg.22-fold change in RANTES P< 0.01)
Conclusion: High throughput RNA sequencing has allowed us to identify novel interactions, upstream transcriptional elements and molecular networks that are perturbed during inflammation and subsequently the effects of HTS on these molecular perturbations. HTS pretreatment alone seems to act as a stress which biases the cell towards a survival mode. HTS pretreatment reduces the effect of a second inflammatory exposure by changing the trancriptome profile that abrogates inflammation, as well as allows communication between cells to further propagate the anti-inflammatory response.
ACID SPHINGOMYELINASE INHIBITION STABILIZES ERYTHROCYTE MEMBRANES DURING STORAGE
R.S. Hoehn1, P.L. Jernigan1, A.L. Chang1, M.J. Edwards1, C. Caldwell*1, E. Gulbins1, 2, and T.A. Pritts*1. 1University of Cincinnati, Cincinnati, OH, 2University of Duisburg-Essen, Essen, Germany
Objective: As human red blood cell units (pRBCs) age during storage they develop biochemical and physical changes known as the red blood cell storage lesion. A major component of this lesion is erythrocyte membrane destabilization leading to hemolysis and subsequent formation of reactive oxygen species, scavenging of nitric oxide, and impairment of vasodilation and tissue perfusion in transfusion recipients. We hypothesized that by inhibiting acid sphingomyelinase, an enzyme known to contribute to programmed cell death, we could mitigate aspects of the red blood cell storage lesion.
Methods: Human and murine pRBCs were obtained and stored under standard blood banking conditions with and without the addition of amitriptyline, a known acid sphingomyelinase inhibitor. Standard erythrocyte parameters were measured with an electronic hematology analyzer. Flow cytometry was used to measure erythrocyte size, complexity, externalization of phosphatidylserine, and presence of band 3 protein. Blood smears were fixed and stained for light microscopy analysis.
Results: Cell-free hemoglobin, a marker of hemolysis, increased during storage of pRBCs. Mouse and human units treated with amitriptyline showed decreased hemolysis in a dose-dependent manner. Standard pRBC storage led to loss of biconcave disc appearance and increased echinocytosis as determined by light microscopy and increased phosphatidylserine expression, as well as decreased size and band 3 protein integrity as determined by flow cytometry. Each of these changes was dose-dependently reduced in the presence of amitriptyline. Other erythrocyte parameters (hemoglobin, hematocrit, erythrocyte number, mean corpuscular hemoglobin) were not affected by amitriptyline.
Conclusions: As shown previously, the red blood cell storage lesion is associated with increased hemolysis as well as changes in cell size, complexity, and membrane molecular composition. Treatment with an acid sphingomyelinase inhibitor reduced these age-related changes in a dose-dependent manner. Our data suggest a novel mechanism by which stored pRBCs may be treated in order to prevent the deleterious consequences following transfusion of aged blood products.
PREVIOUS CRYOPRESERVATION ACCELERATES SUBSEQUENT LIQUID-PHASE DEVELOPMENT OF THE RED BLOOD CELL STORAGE LESION
A.L. Chang1, R.S. Hoehn1, P.L. Jernigan1, M. Schreiber2, B.R. Robinson1, and T.A. Pritts*1. 1University of Cincinnati, Cincinnati, OH, 2Oregon Health & Science University, Portland, OR
Objective: Cryopreservation is an important technique to extend the shelf life of packed red blood cell units (pRBCs). Recent clinical studies in trauma patients indicate that treatment of anemia by transfusion with previously cryopreserved pRBCs is superior to that of aged units stored under standard conditions, but the FDA currently restricts their use to 14 days after thawing. Use of these units beyond this limit would increase the supply of pRBCs for transfusion, but the effect of longer-term liquid storage on development of the biochemical, metabolic, and morphologic changes collectively known as the “red blood cell storage lesion” is unknown. We investigated the effect of previous cryopreservation on the long-term development of the red blood cell storage lesion.
Methods: Human pRBC units (n=6) cryopreserved in 40% glycerol at -80°C were thawed, deglycerolized, and prepared for transfusion. Age-matched pRBC units stored under standard conditions (n=6) served as controls. The red blood cell storage lesion was evaluated after 14, 21, 28, and 42 days of storage at 4°C in AS-3 storage medium. Red blood cell counts (RBC), hemoglobin (Hgb), cell free hemoglobin, hematocrit (HCT), lactic acid concentration (LA) and cell free potassium (K+) were measured using clinical blood analyzers. Phosphatidylserine (PS) expression and production of red blood cell derived microparticles were measured by flow cytometry. Osmotic fragility of red blood cells in hypotonic saline was determined by spectrophotometric analysis.
Results: Previously frozen pRBCs (FpRBC) showed significantly lower initial RBC counts (3.7 vs 5.3 x10^6 cells/uL, p<0.01), Hgb (12.0 vs 16.5 g/dL, p<0.001), and HCT (33.0 vs 46.5%, p<0.01) than standard-storage pRBC units. Cryopreservation was associated with a lower initial pH (6.27 vs 6.72, p<0.01) and potassium (8.8 vs 9.5 meq/L, p=0.02). Free hemoglobin was increased in FpRBCs as compared to controls by day 28 of storage (0.7 vs 0.3 g/dL, p<0.01). PS exposure on the extracellular membrane was significantly greater in FpRBCs after 28 days of storage (10.1 vs 3.3%, p<0.01), however this difference was not significant by 42 days of storage (21.4 vs 18.6%, p=0.38). Microparticle concentrations increased by day 28 in FpRBCs as compared to control (30,836 vs 1,802 MP/uL, p<0.01) and persisted throughout storage. Osmotic fragility, as measured by the EC50 of hypotonic osmotic stress in saline solutions, was higher in FpRBCs than controls at 28 days of storage (0.54 vs 0.49%, p<0.01).
Conclusion: The development of the red blood cell storage lesion is accelerated during long-term liquid storage of previously frozen pRBCs. Our data suggests that a liquid storage time of less than 28 days at 4° Celsius is needed to achieve similar characteristics as standard storage.
EFFECTS OF PLATELET APHERESIS ON COAGULATION IN PIGS
W.Z. Martini*, C. Rodriguez, J. Richardson, A.P. Cap*, and M.A. Dubick*. US Army Institute of Surgical Research, Ft Sam Houston, TX
Introduction: Platelet apheresis is a routine clinical practice, but the ex vivo effects of apheresis on coagulation are unclear. This study investigated the effects of platelet apheresis on coagulation in pigs during 4 hour platelet apheresis as a prelude to initiating studies of hemorrhage and resuscitation in swine with reduced platelets.
Methods: Forty pigs (39.8±0.6 kg) were anesthetized and catheterized with an apheresis catheter in the femoral vein for blood-withdrawal from pigs to separate platelets and returning remaining RBCs and plasma back to the pigs, using Haemonetics 9000. Hemodynamics was recorded during the apheresis process. Blood samples were collected before and after apheresis to assess changes in coagulation by PT, aPTT (Start®), and using Rotem® thrombelastogram, and platelet aggregation using a Chrono-Log 700 aggregommeter.
Results: A total of 12 cycles of apheresis in 4 hours were performed in pigs. Platelet count decreased from baseline 383±20 109/L to 141±14 109/L and plasma fibrinogen concentration reduced from 225±5 mg/dL to 195±5 mg/dL (both p<0.05). During each cycle of apheresis, MAP was decreased by blood-withdrawal from baseline 74±2 to 45±1 mmHg (p<0.05), but recovered after blood-returning. Rotem alpha (clotting speed) decreased from 79±0 to 69±1 degrees and MA (clot strength) decreased from 71±1 to 57±1 mm (both p<0.05), after the 4 hour apheresis period. No changes were observed in PT or aPTT. Platelet aggregation induced by arachidonic acid was decreased to 28%±6% of baseline values (p<0.05) by apheresis.
Conclusion: In addition to reducing platelet counts, platelet apheresis caused fluctuations in MAP and compromised platelet aggregation and clotting function. The observations warrant consideration in humans undergoing apheresis over extended periods.
HEXTEND IS SUPERIOR TO HYPERTONIC SALINE FOR RESUSCITATION OF HEMORRHAGIC SHOCK
P.L. Jernigan1, 2, R.S. Hoehn1, 2, J. Heyl1, 2, W. Dorlac1, 2, and T.A. Pritts*1, 2. 1University of Cincinnati Department of Surgery, Cincinnati, OH, 2University of Cincinnati Institute for Military Medicine, Cincinnati, OH
Objectives: Military medical teams routinely care for injured patients in austere far forward environments. Teams currently carry Hextend (6% hetastarch in lactated Ringers’ solution) for treatment of hypovolemia and 3% saline for treatment of intracranial hypertension. We hypothesized that equivalent volumes of 3% saline could be used to resuscitate from hemorrhagic shock, thus allowing team members to carry a single resuscitation fluid under these conditions.
Methods: Female Yorkshire swine (35 ± 5 kg) were sedated and intubated and invasive catheters were placed for hemodynamic monitoring and hemorrhage. Animals underwent laparotomy with splenectomy followed by a pressure-controlled hemorrhage to a mean arterial pressure (MAP) of 30 ± 5 mmHg for 30 minutes. They were then resuscitated with either 3% saline or Hextend (n=4 per group) to a MAP of 50 ± 5 mmHg. They were maintained at this MAP for 4 hours, stopping fluids if they were within the target MAP range and infusing fluid if they fell below it. Point of care lab values and cardiac output were measured every hour and routine vital signs were recorded every 15 minutes. Total volumes of shed blood and resuscitation fluid were recorded. All values are given as mean ± SEM.
Results: There were no significant differences between groups in initial hemoglobin (Hgb), shed blood volume, or spleen weight, and all animals survived for the full four-hour resuscitation period. The mean volume of 3% saline required to maintain target MAP was significantly higher than that of Hextend (906±171 vs 215±29 mL, p=0.007). The MAP was significantly lower in the 3% group after the first hour of resuscitation (48±1 vs. 55±2 mmHg, p=0.007) although the groups converged at subsequent time points. After 4 hours of resuscitation the 3% group had lower values than the Hextend group for base excess (4±1 vs. 9±1 mEq/L, p=0.02), serum bicarbonate (28.8±1.3 vs. 33.5±1.4 mEq/L, p=0.04), and Hgb (5.6±0.6 vs. 8.0±0.4 g/dL, p=0.02). The 3% group had significantly higher values than the Hextend group for lactate (2.0±0.2 vs. 1.4±0.2 mmol/L, p=0.02), serum sodium (151±3 vs. 135±1 mEq/L, p=0.004) and serum chloride (120±3 vs. 102±2 mEq/L, p=0.003).
Conclusions: Hextend is superior to 3% saline as a volume expanding resuscitative fluid for initial treatment of hemorrhagic shock. Resuscitation with 3% saline led to exacerbation of anemia and derangements in serum electrolytes. Based on our data, we would not recommend the use of 3% saline as the sole resuscitation fluid after hemorrhage.
USE OF EARLY TRANEXAMIC ACID TREATMENT IN SWINE SUBJECTED TO UNCONTROLLED HEMORRHAGE
M.A. Dubick*, M. Prince, I.A. Polykratis, R. De Guzman, A.P. Cap, and J.L. Sondeen. US Army Institute of Surgical Research, San Antonio, TX
Background: Tranexamic acid (TXA) has been reported to increase survival in both military and civilian trauma patients, but the mechanisms remain unclear. The purpose of this double blinded, randomized placebo controlled pre-clinical study was to determine if TXA reduced blood loss and improved survival times in a swine model of uncontrolled hemorrhage when used as a component of hypotensive resuscitation with Hextend (HEX) or fresh frozen plasma (FFP).
Methods: Instrumented, anesthetized pigs (n=11/gp) were subjected to 24 ml/kg controlled hemorrhage, followed by transection of the spleen. After 15 minutes of uncontrolled bleeding, TXA (1.43 mg/kg/min) or normal saline (NS) was given over 10 minutes, and then 15 ml/kg of HEX or FFP was administered at 1 ml/min. At 90 minutes, a second 10 minute infusion of TXA or NS was given. Post resuscitation blood loss, hourly samples for coagulation status, and 5-hour survival were determined. Tissue plasminogen activator (tPA) was added to blood samples collected before and after TXA to confirm that the TXA inhibited fibrinolysis. In addition, a comparison of a dose response to tPA-induced fibrinolysis was made between swine and human plasma in vitro.
Results: TXA prevented the rise in D-dimers that occurred 180 min after spleen injury. However, there was no significant effect of TXA on survival or blood loss compared to NS with HEX (NS- HEX:17±2vs TXA-HEX:17±2 ml/kg) or FFP (NS-FFP: 7±2vsTXA-FFP:12±3 ml/kg) while FFP significantly reduced blood loss and increased survival compared to HEX in the NS treated animals. The tPA-induced fibrinolysis was completely inhibited in blood from TXA- treated animals, yet in fibrinolysis sensitivity studies, human plasma was greater than 30 times more sensitive to tPA-induced fibrinolysis than swine plasma, in vitro.
Conclusions: TXA did not reduce post resuscitation blood loss in our splenic uncontrolled hemorrhage swine model even though TXA was anti-fibrinolytic in the pigs. The possibility remains that the pig is highly resistant to fibrinolysis and not a good model to study the effects of antifibrinolytics or that fibrinolysis is not a major factor in bleeding from splenic injury.
EPITHELIAL CYTOKINES AND GROUP 2 INNATE LYMPHOID CELLS (ILC2S) ARE INCREASED IN LUNGS IN A MODEL HEMORRHAGIC SHOCK WITH TRAUMA
S. Chen1, 2, M.H. Ramadan1, R. Hoffman1, L. Kohut1, P. Loughran1, A. Frank1, and T. Billiar*1. 1University of Pittsburgh Medical Center, Pittsburgh, PA, 2Department of Biochemistry, School of Life Science, Central South University, Changsha, China
Background and Objective: Accumulating evidence suggests that hemorrhagic shock and trauma produce a bias toward a T helper 2 cell (Th2) response. Group 2 innate lymphoid cells (ILC2s) represent a recently discovered cell population which are critical for the initiation of type 2 immunity. The epithelial cell-derived cytokines IL-33, IL-25 and TSLP regulate the activation and effector functions of ILC2s. However, it is unknown if ILC2 are upregulated in the setting of systemic injury. The aim of this study was to determine if epithelial cytokines and ILC2s are upregulated following hemorrhagic shock and trauma.
Methods: C57BL/6 mice (n=6/group) were subjected to resuscitated hemorrhagic shock (2h,25mmHg) with bilateral lower extremity injury (HS+T) and sacrificed at 6h and 24h. ILC2s were identified as CD45+Lin-CD90.2+CD25+CD127+ cells by flow cytometry, which also expressed T1/ST2 and c-Kit. Absolute lung leukocyte counts as well as the proportion of leukocytes that were ILC2 were determined by cell counting and flow cytometry. Lung levels of IL-33, IL-25 and TSLP were measured by ELISA at 6h and 24h after HS+T. Immunofluorescence to localize IL-33, IL-25 and TSLP in the lungs and lung histology were assessed.
Results: Lung ILC2 frequency at 24h (1.233±0.42%) was markedly increased compared to that at 6h (0.59±0.17%) and baseline (0.516±0.177%), while the absolute number of leukocytes((10.54±0.3847) x106) was not changed by HS+T. Levels of IL-33 at 6h (814.42±79.701 pg/ml/100ug protein vs. 543.535±86.636pg/ml/100ug protein, P<0.05) and IL-25 at 24h(201.059±30.612 pg/ml/100ug protein vs. 94.889±4.657pg/ml/100ug protein, P<0.05) in the lung tissue were significantly elevated compared to that at baseline. The levels of TSLP at 6h and 24h in the lung tissue were also higher than that at baseline. Immunofluorescence confirmed that the expression of all three cytokines was enhanced in the airway epithelium following HS+T.
Conclusions: These data establish that epithelial cytokines and one of their target cells, ILC2, are upregulated in the lungs of mice subjected to HS+T. Thus, ILC2 could contribute to the immuno-inflammatory response in the lung following severe injury.
ROLE OF POST-HEMORRHAGIC SHOCK MESENTERIC LYMPH DRAINAGE ON SPLENIC DENDRITIC CELLS FUNCTION IN MICE
H. Liu, J. Li, L. Zhang, H. Wang, G. Liu, Z. Zhao*, and C. Niu*. Hebei North University, Zhangjiakou, China
Previous studies demonstrated that immune cell dysfunction contributed to the development of immunosuppression, which is associated with organ injury and mortality induced by hemorrhagic shock. Dendritic cells (DCs) are the key antigen presenting cells for the initiation of T-cell dependent immune response, and previous investigations have shown that hemorrhagic shock suppresses splenic DCs maturation and antigen presentation capacity. Meanwhile, our studies indicated the post hemorrhagic shock mesenteric lymph (PHSML) return promotes the development of hemorrhagic shock mediated systemic inflammatory response syndrome and organ injury. However, it remains unclear whether PHSML modulates splenic DCs functions under such condition. The aim of this study therefore was to investigate the role of PHSML drainage on splenic DCs ability to produce inflammatory cytokines in mice. Firstly, we established a hemorrhagic shock model (40±2 mmHg for 60 min), followed by fluid resuscitation with the shed blood and equal Ringer’ solution, and drained the PHSML after resuscitation in the shock+drainage group. At 3 h after resuscitation, we harvested the splenic tissue, and isolated splenic DCs using anti-CD11c immunomagnetic beads. After cultivation with DMEM for 24 h, we detected the TNFα, IL-10, and IL-12 levels in the culture supernatants of CD11c-positive cells obtained from mice of the sham, shock, and shock+drainage groups, respectively. However, we have not found the obvious differences in these indices among there groups. After incubation with LPS (10 μg/mL, 10 μL) for 24 h, the TNFα and IL-12 in the sham group, the TNFα and IL-10 in the sham group, and the TNFα, IL-10, and IL-12 in the shock+drainage group were obviously increased compared to the same group without incubation with LPS, respectively. Meanwhile, after incubation with LPS, the TNFα and IL-12 in shock group were significantly decreased, and IL-10 was increased than that of the sham group, which was reversed in shock+drainage group. In summary, our findings in the current investigation demonstrated that the decrease in PHSML return depressed the decreased susceptibility of DCs producing inflammatory cytokines subjected to LPS stimulation induced by hemorrhagic shock. However, the role of DCs on PHSML-induced immunosuppression should be further research. This work was supported by the Key Research Program of Education Department in Hebei Province (ZH2012004).
PRE-INJURY METABOLIC STATE PREDICTS MORTALITY IN PORCINE HEMORRHAGIC SHOCK AND POLYTRAUMA
A. Wolf, N. Witowski, E.R. Lusczek*, C.E. Determan, B.G. Ostrowski, and G. Beilman*. University of Minnesota, Minneapolis, MN
Introduction: Significant, severity-independent variation in outcome in traumatic injury is related to genetic, environmental and other factors. One component of this variability is the effect of metabolism. We have previously described the effect of carbohydrate pre-feeding in our porcine model of hemorrhagic shock and injury. We hypothesize that individual variability in metabolic markers in this model predicts outcome.
Methods: Pigs (n=32) received a standardized dose of carbohydrate (28 cal/kg bodyweight) 60 minutes before anesthesia. Pigs underwent laparotomy and splenectomy, then received pulmonary contusion, standardized hemorrhage to a systolic blood pressure<60 mmHg and liver crush injury. After 45min of shock, we initiated resuscitation with shed blood and Ringer’s Lactate. Tissue, serum and urine samples were collected at baseline, end of shock and during early resuscitation and analyzed via nuclear magnetic resonance spectroscopy and Chenomx software. Metabolite levels and differences between time points were compared using Partial Least Squares Discriminant Analysis and metabolites were ranked by their variable importance of projection (VIP) scores. Binary logistic regression of baseline metabolites was performed to predict mortality. Physiologic data were compared using Mann Whitney U test.
Results: Nearly half (15/32) of the animals died within 24h of resuscitation. Before injury the metabolic profile in non-survivors suggests increased glycogen production, lower pentose-phosphate pathway activity and higher muscle ATP production. Pigs that died experienced decreased muscle ATP levels during shock despite evidence of increased substrate level phosphorylation, anaerobic glycolysis, lipolysis and proteolysis. Logistic regression identified five markers at baseline (Table 1) predictive of mortality after shock and injury (p<0.001, R2= 0.75).
Conclusions: Metabolite variability at baseline predicted mortality. We propose that these variations are a result of differences in glucose processing, with a potential mechanism being a decrease in the threshold for opening of the mitochondrial permeability transition pore during shock and resuscitation.
TISSUE INJURY AND HEMORRHAGIC SHOCK EVOKE DIFFERENTIAL METABOLIC PATHOLOGY IN AN ANIMAL MODEL
A.L. Slaughter1, A. D’Alessandro3, 1, A. Banerjee*1, E.E. Moore*2, 1, K. Hansen3, A. Sauaia*5, C.C. Silliman*4, 6, H.B. Moore1, K. Leasia1, M. Fragoso2, 1, and E.D. Peltz*1. 1University of Colorado Denver, School of Medicine; Department of Surgery/Trauma Research Center, Aurora, CO, 2Denver Health Medical Center, Denver, CO, 3University of Colorado Denver, School of Medicine; Department of Biochemistry and Molecular Genetics, Aurora, CO, 4University of Colorado Denver, School of Medicine; Department of Pediatrics, Hematology/Oncology, Aurora, CO, 5University of Colorado Denver, School of Medicine; Department of Health Care Policy and Research, Aurora, CO, 6Belle Bonfils Blood Center, Denver, CO
Introduction: Severe metabolic aberrancy contributes to patient morbidity and mortality following major traumatic injury. While the metabolic response to trauma has been a long-standing focus of investigation, the independent contributions of tissue injury and hemorrhagic shock to metabolic deregulation remain undefined. We hypothesize that tissue injury and shock will generate distinct metabolic profiles in an animal model.
Methods: Sprague-Dawley rats (n=8) were subjected to tissue injury/hemorrhagic shock (T/HS) by laparotomy and hemorrhage followed by resuscitation (R) with normal saline and shed blood. Sham shock (SS) animals (n=6) were subjected only to tissue injury. Plasma samples were collected for metabolomic analysis by Ultra High Pressure Liquid Chromatography (UPLC). Repeated measures ANOVA with Tukey multiple column comparison test (significance threshold for p-values < 0.05) and partial least squares discriminant analysis (PLS-DA) compared change in metabolites from baseline to post-injury, shock and resuscitation time points.
Results: Tissue injury and hemorrhagic shock resulted in clustered metabolic phenotypes. PC1 + PC2 accounted for 25.8% total covariance (PC1 = 20.3%; PC2 = 5.5%) (Figure A). Metabolites showing highest covariance across the principle components were determined. Pathway-wise analysis highlighted specific metabolite changes uniquely following hemorrhagic shock including increases in lactate, citric acid cycle intermediates, markers of oxidative stress, osmoregulators and purine catabolites (p<0.05).
Conclusion: Tissue injury and hemorrhagic shock instigate distinct metabolic aberrancies. Hemorrhagic shock disturbs acid-base milieu and immediately provokes altered energy pathways, catabolism, osmoregulation and nitrogen imbalance. Metabolomic analysis can be a valuable tool in identifying early, global metabolic derangement following shock, and can provide the foundation for studies that define resuscitation strategies to restore baseline metabolic profiles and prevent systems pathology.
IS THE CARDIOPROTECTION BY THE ESTROGEN RELATED RECEPTOR (ERR) β AND γ AGONIST DY131 FOLLOWING TRAUMA-HEMORRHAGIC SHOCK (T-H) ESTROGEN RECEPTOR (ER)-DEPENDENT?
S. Yang, S. Hu, B. Cam, W.J. Hubbard, K.I. Bland, and I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
Alterations in the myocardium occur during trauma-hemorrhagic shock (T-H), resulting in diminished cardiac performance. Cardiac autophagy is a maladaptive response to T-H-induced hemodynamic stress. Our previous studies have shown that administration of the estrogen related receptor (ERR) β and g agonist, DY131, after T-H restored cardiac function and down-regulated plasma and heart proinflammatory cytokines. However, it remains unclear whether T-H induces autophagy and if so, whether administration of DY131 has any effects on cardiac autophagy under those conditions. To study this, left ventricular (LV) performance and cardiac autophagy related signaling were determined in male adult Sprague-Dawley rats after T-H (60% blood loss, BP maintained at 35 mmHg for 90 min) in rats that were treated with DY131 or other agents. Seven groups were used (n=6/group): sham, sham + DY131, T-H + vehicle, T-H + β-estradiol-water soluble (E2; 1 mg/kg) (Sigma), T-H + ICI 182,780 (ICI; 3mg/kg) + E2, T-H + DY131 (40µg/kg), and T-H + ICI + DY131. DY131 or E2 was delivered IV at the onset of resuscitation and the estrogen receptor (ER) antagonist ICI was given IP 30 min before DY131 or E2 administration. Two hrs following T-H and resuscitation, LV performance was measured; blood and heart tissue were then harvested. Plasma and heart tissue cytokines (TNF-α, IL-6 and IL-10) were measured by ELISA. Cardiac AKT/PI3K, mTOR, and P53 were determined by Western blot (WB). Our results indicate that T-H induced a significant decrease in LV performance, cardiac PI3K and P-AKT. In contrast, T-H induced a significant increase in cardiac P-mTOR and P-P53. Similar to E2, DY131 administration normalized LV performance, cardiac PI3K and P-AKT. DY131 also down-regulated T-H-induced increase in cardiac P-mTOR and P-P53. Although the ER antagonist ICI abolished the salutary effect of E2 on cardiac function and the above signaling; it did not affect the salutary effects of DY131 on cardiac function or cardiac signaling. Thus, it can be concluded that the salutary effect of DY131 on cardiac function and cardiac autophagy is ER-independent (NIH R01 GM39519).
PHYSIOLOGICAL RESPONSE TO MITOQ AFTER HSR IS ORGAN SPECIFIC AND HIGHLY VARIED
R.D. Powell*, D.A. Goodenow, I.H. McKillop, and S.L. Evans*. Carolinas Medical Center, Charlotte, NC
Background: Hemorrhagic shock and reperfusion (HSR) injury leads to a cascade of events including reactive oxygen species (ROS) production, mitochondrial dysfunction, cell death, systemic inflammation and multiple organ dysfunction (MOD). MitoQ is a mitochondrial targeted antioxidant; a ubiquinone moiety attached to triphenylphosphonium (TPP). We sought to determine if MitoQ would alter initial ROS production, endogenous antioxidant activity or the inflammatory response in a rat model of HSR in a tissue specific manner.
Methods: Male Sprague-Dawley rats were hemorrhaged to a MAP of 25±2.0mmHg for 1-hr prior to resuscitation. MitoQ (5mg/kg), TPP (5mg/kg) or saline (0.9%) was administered (iv) 30-mins prior to resuscitation, followed by IP administration (MitoQ, 20mg/kg; TPP, 20mg/kg; saline, 0.9%) immediately after resuscitation (n=5/group). Rats were sacrificed by cardiac puncture 2-hrs post-surgery. Tissue was collected to determine necrosis (H&E), mitochondrial membrane integrity (cytochrome c staining) or inflammatory cell presence (CD68 stain) and assayed for lipid peroxidation (TBARS), endogenous antioxidant activity (glutathione peroxidase (GPx)), and cytokine expression (TNF-α and IL-6).
Results: HSR-MitoQ significantly increased pulmonary lipid peroxidation (TBARS) vs sham and HSR (7.3±0.53 vs 4.5±0.42 vs 4.6±0.38; p<0.05; n=5) and hepatic TBARS vs sham (15±1.3 vs 10±1.1; p<0.05; n=5). HSR-MitoQ resulted in increased GPx activity in the liver vs sham and HSR (38±3.0 vs 25±6.7 vs 20±3.5nmol/min/ml; p<0.05; n=5). CD68 staining demonstrated a significant increase in liver macrophages in HSR-MitoQ vs sham, HSR and HSR-TPP (1.3±0.37 vs 0.0±0.0 vs 0.5±0.06 vs 0.25±0.13; p<0.05; n=5). In liver, both HSR-TPP and HSR-MitoQ animals exhibited lower TNF-alpha expression vs HSR (580±5.0 vs 660±16 vs 920±19pg/mg; p<0.05; n=5). Conversely, in lung, HSR-TPP and HSR-MitoQ demonstrated significantly increased TNF-alpha levels vs sham and HSR (2637±141 vs 2410±370 vs 1470±116 vs 1932±196pg/mg; p<0.05; n=5). IL-6 levels were decreased in the liver of HSR-TPP and HSR-MitoQ vs HSR (6700±1300 vs 7300±1700 vs 11000±860pg/mg; p<0.05; n=5), while HSR-MitoQ increased IL-6 expression in the lung vs sham, HSR and HSR-TPP (2116±473 vs 591±253 vs 829±283 vs 611±154pg/mg; p<0.05; n=5).
Conclusions: MitoQ is associated with a decrease in pro-inflammatory cytokines and an increase in GPx in the liver. This response is potentially organ specific, as is indicated by the differences in the lung. These data suggest distinct physiology among organs and that systemic administration of therapeutics may need to be modified with future development focusing on organ specific delivery mechanisms.
ORAL TRANEXAMIC ACID: A GUT PROTEASE INHIBITOR THAT MITIGATES LOCAL AND DISTANT ORGAN INJURY AFTER HEMORRHAGIC SHOCK
R. Kozar*1, Z. Peng1, A. LeBlanc3, and K. Ban2. 1Shock Trauma, Baltimore, MD, 2Baylor College of Medicine, Houston, TX, 3University of Texas Houston, Houston, TX
Introduction: Intravenous tranexamic acid (TXA) is an effective adjunct to hemorrhage control as an antifibrinolytic. In injured patients TXA has been shown to decrease mortality. Recent laboratory data demonstrated that luminal TXA inhibited digestive proteases and protected the gut. TNFα converting enzyme (TACE) is an effective sheddase of intestinal syndecan-1 (sdc-1). Ectodomain shedding of sdc-1 exposes the underlying intestinal epithelium to digestive proteases and subsequent systemic insult. We therefore hypothesized that luminal TXA mitigates hemorrhagic shock (HS)-induced intestinal scd-1 shedding by inhibiting TACE and TNFα, thereby protecting the gut and lessening distant organ (lung) injury.
Methods: Mice underwent HS (MAP of 35 ± 5 mmHg) and trauma (laparotomy). After 90 min of HS, TXA was delivered intraluminally into the jejunum and compared to animals that underwent shock alone or shams±TXA. After 3 hrs, animals were sacrificed and jejunum and lungs harvested and blood obtained for analysis. TNFα, TACE protein and activity were quantitated by ELISA. Sdc-1 was measured in gut tissue by immunostaining and by ELISA in gut and serum. In the gut and lung, histologic injury was quantitated and inflammation assessed by myeloperoxidase activity (mU/mg).
Results: Gut injury and inflammation were significantly increased by HS (% injured villi 38.8±8±3.17 and inflammation 1.37±19) compared to shams (injury 1.75±0.59 sham and 1.38±0.46 sham TXA; inflammation 0.25±0.03 sham and 0.17±0.03 sham TXA) but significantly reduced by TXA (injury 11.75±1.96 and inflammation 0.66±0.09) with similar findings in the lung. Gut sdc-1 protein was preserved by TXA (240±22 sham and 231±29 sham-TXA vs 109±10 HS and 180±18 HS-TXA; p<0.05) with similar findings for immunostaining. TXA lessened shed sdc-1 and correlated with a reduction in gut, lung, and serum TNFα and TACE activity (Table) and protein.
Conclusion: In a mouse model of HS, luminal TXA lessened gut and lung injury and inflammation. These protective effects were associated with preservation of intestinal sdc-1 and a reduction in gut, lung, and systemic TACE and TNFα. As oral TXA is FDA approved, additional studies are warranted to determine if it may not only play a role in injured patients with bleeding, but also be of benefit in reducing subsequent multiple organ failure. This may be of particular relevance to patients or soldiers who may not have access to timely medical care.
EXTRACELLULAR RNA INDUCES INFLAMMATORY RESPONSE VIA TLR7 AND CONTRIBUTES TO MYOCARDIAL ISCHEMIA-REPERFUSION INJURY
Y. Feng, H. Chen, L. Zou, G. Xu, and W. Chao*. Massachusetts General Hospital, Charlestown, MA
Introduction: We have recently reported that necrotic cell-associated extracellular RNA (exRNA) induces a cytokine response in cardiomyocytes and that eliminating circulating exRNA with exogenous RNase confers cardiac protection against ischemia/reperfusion (I/R)-induced myocardial inflammation and injury. The molecular mechanism by which exRNA exhibits the proinflammatory effect is unknown.
Methods:I/R model: Mice were subjected to sham procedure or coronary occlusion for 45 min followed by reperfusion. exRNA extraction and quantification: RNA in media and sera was extracted using Trizol LS and quantified using Quant-iTTM RNA assay kit. Circulating microRNA (miRNA) array: 68 miRNAs in plasma were quantified using miRNA assay kit without RNA extraction (Firefly, Cambridge, MA).
Results: Twenty-four hours after hypoxia/serum-deprivation (H/SD), RNA in cardiomyocyte culture medium rose from 12 ng/ml to approx. 600 ng/ml. In vivo, 24 h after I/R injury, serum RNA level was increased from 247±46 to 464±92 ng/ml. Unbiased plasma miRNA array data indicate that 31 out of 68 miRNAs tested were significantly increased by at least two-fold at 4 h following myocardial I/R compared to the sham mice. To test the ability of exRNA to induce inflammatory response, cardiomyocytes and immune cells (neutrophils and macrophages) were treated with RNA (2.5 - 10 μg/ml) extracted from mouse heart or miRNA mimics (10 - 1500 nM) selected from the miRNA array data. Purified RNA and six miRNA mimics (miR-34a, -122, -133a, 142a, -146a, -208a) were found to induce MIP-2 production in a dose-dependent manner. The effects were abolished by pre-treatment of RNase, but not DNase. The RNase effect was specific for RNA/miRNAs as it had no effect on Pam3cys (a TLR2 ligand). Moreover, RNA-induced cytokine production, both in cardiomyocytes and immune cells, was significantly inhibited by a specific TLR7 antagonist or in TLR7-/- or MyD88-/- macrophages, but not TLR3-/- or Trif-/- cells. Similarly, miRNA-induced cytokine production was completely diminished in TLR7-/- or MyD88-/- macrophages. Finally, i.p. injection of RNA or miRNAs induced acute peritoneal inflammation as characterized by IL-6 production and neutrophil activation.
Conclusions: Our data demonstrate that 1) RNA, including miRNAs, are released from hypoxic cardiomyocytes in vitro and ischemic myocardium in vivo; 2) exRNA, both purified RNA from the heart and miRNA mimics, induce cytokine production through TLR7-MyD88 signaling; 3) exRNA induces acute peritonitis after i.p. injection. These data suggest that exRNA is a potent proinflammatory ligand that signals through TLR7-MyD88.
INVOLVEMENT OF PROTEASOME/IMMUNOPROTEASOME PROTEASE SUBUNITS IN DIFFERENTIATION OF CELL TYPES
N. Silswal, J. Reis, X. Guan, and N. Qureshi*. University of Missouri Kansas City, Kansas City, MO
We have previously shown that cellular proteasomes/immunoproteasomes are pivotal regulators of LPS-induced inflammation and degradation of mediators involved in multiple signaling pathways that ultimately lead to shock and mortality. However, there is not much known about the expression and function of different proteasome/immunoproteasome subunits (proteasomes X, Y, Z and immunoproteasomes LMP7, LMP2, LMP10) in various cell types and why murine and human cells behave differentially. We first examined the protein expression of all six proteasome subunits in different murine and human cell lines by using Western blot analyses. We then compared the expression of proteasome/immunoproteasome subunits in resident and thioglycolate (TG)-elicited peritoneal macrophages using C57Bl/6 mice and later examined the chymotrypsin-like (CT-like), trypsin-like (T-like), and post-acidic (PA) activities of proteasome subunits by luminometry. Lastly, we measured induction of TNF-α and nitric oxide (NO) in supernatants of resident and TG-elicited peritoneal macrophages obtained from wild type, LMP7-/-, LMP2-/- and LMP7-/- / LMP2-/- double knockout mice of same age by ELISA and Griess reagent after LPS stimulation. Our data established that various types of human and murine cells possess different proteasome/immunoproteasome subunits and therefore displayed different protease activities. The CT-like and T-like activities were robust in TG-elicited C57Bl/6 peritoneal macrophages, whereas, PA activity was robust in resident peritoneal macrophages. TG-elicited macrophages also exhibited robust induction of TNF-α, as compared to resident macrophages after LPS treatment, however, no difference was observed in macrophages obtained from LMP knockout mice as compared to wild type mice. Moreover, LPS stimulation of TG-elicited macrophages from WT mice displayed robust NO release in supernatants. In contrast, there was none or very little increase in NO levels in supernatants obtained from TG-elicited and resident peritoneal macrophages obtained from LMP knockout mice in response to LPS. Most of the monocytes predominantly displayed presence of immunoproteasomes instead of proteasomes, while the murine macrophages showed both proteasomes and immunoproteasomes. In conclusion, we are the first to demonstrate proteasome/immunoproteasome subunit expression varies with cell-types, as well as with macrophage differentiation (TG-elicited), which leads to changes in the specificities of proteasome/immunoproteasome protease activities and inflammatory response to LPS in various cell-types. These studies will therefore lead to novel strategies for preventing septic shock.
RAPID DETECTION OF LEUKOCYTE OXIDATIVE BURST CAPACITY CORRELATES WITH TH1-POLARIZING CYTOKINE SECRETION AFTER EX-VIVO LPS STIMULATION OF NON-HUMAN PRIMATE (RHESUS MACAQUE)
P.J. Vernon1, R.M. Paredes1, R. Crossland1, L.J. Schaub1, J.J. Dalle Lucca*2, and F.R. Sheppard*1. 1Naval Medical Research Unit - San Antonio, San Antonio, TX, 259th Medical Wing, US Air Force, JBSA Fort Sam Houston, TX
Background: Maladaptive immune responses, particularly Th1-driven, are a significant contributor to the deleterious inflammation present in immune-mediated pathologies, which may be blunted by immuno-modulators if detected early. Commercial cytokine arrays are routinely used to assess inflammation, requiring 18-24 hours. Applications to rapidly detect inflammation would allow earlier identification of patients at risk for exacerbated Th1-responses and guide application of immuno-modulators. Here we evaluate leukocyte oxidative burst (LOX) capacity as a strategy to rapidly detect inflammation.
Methods: Fresh whole blood was collected from healthy anesthetized Rhesus Macaques (n=25) and simultaneously analyzed for plasma cytokine secretion (23-plex Luminex assay) and LOX. For cytokine secretion, paired samples were either unstimulated or ex-vivo LPS-stimulated (100µg/mL/24h). LOX capacity was measured in dihydrorhodamine-123 loaded samples following PMA/ionomycin treatment. Pearson’s test was utilized to correlate LOX capacity with cytokine secretion, p<0.05 considered significant.
Results: LPS-stimulation increased secretion of several inflammatory cytokine and chemokines, including G-CSF, IL-1β, IL-1Ra, IL-6, IL-10, IL-12, IL-18, MIP-1α, MIP-1β, and TNFa. Although values were highly variable, several cytokines highly correlated with LOX capacity with p-values of ≤0.0001 (IL-1β [r=0.66], IL-6 [r=0.74], IL-12/23 (p40) [r=0.78], and TNFα [r=0.76]), one of which, IL-12/23 (p40), is a Th1-polarizing cytokine.
Conclusion: LOX capacity highly correlated with inflammatory and Th1-polarizing cytokine secretion, indicating its ability to rapidly detect inflammatory responses. Moreover, these data suggests that LOX may rapidly identify patients at risk for maladaptive immune responses, which may benefit from immuno-modulation treatment. Future studies will assess the in-vivo predictive value of LOX in animal models of immune-mediated pathologies.
RESPONSE OF THE IL-6:IL-10 RATIO BY ETHNICITY IN OVERWEIGHT/OBESE POSTMENOPAUSAL WOMEN FOLLOWING A 20-WEEK DIET AND EXERCISE WEIGHT LOSS INTERVENTION
C.D. Markert*1, M. McKenzie1, and B. Nicklas2. 1Winston-Salem State University, Jamestown, NC, 2Wake Forest University Health Sciences, Winston-Salem, NC
Specific Objectives: The IL-6:IL-10 ratio relates a measure of a circulating pro-inflammatory cytokine (IL-6) to that of an anti-inflammatory cytokine (IL-10), providing insight into whole-body inflammation. Our specific objectives were to assess IL-6:IL-10 in Caucasian and African American overweight and obese postmenopausal women following an intervention of diet and exercise intended to cause weight loss. We hypothesized that the intervention would lead to a decrease in the ratio, indicating salutary effects of the intervention on this index of whole-body inflammation, and that ethnicity would not play a role.
Methods: Demographics and physical characteristics were assessed at baseline and post-intervention. Baseline and post-intervention plasma samples were obtained from 9 Caucasian (C) and 9 African American (AA) subjects. A panel of cytokines, including IL-6 and IL-10, in each of the samples was queried in duplicate using a multiplex immunoassay.
Summary of Results: Mean bodyweight at baseline was 90.3 ± 11.2 kg. All subjects lost weight as a result of the intervention (mean 12.1 ± 4.5 kg). Of the 18 samples analyzed, 12 samples (6 C, 6 AA) provided pre-post data within the detection limits of the assay. These limited data indicated a tendency for the IL-6:IL-10 ratio to decrease in C (increase in 2 subjects, decrease in 4) and to increase in AA (increase in 4, decrease in 1, no change in 1), but this tendency was not significant at the p ≤ 0.05 level.
Statement of Conclusions: Future studies will follow up using an assay with higher sensitivity. Current data do not suggest that ethnicity differentially affects the IL-6:IL-10 ratio following weight loss in overweight/obese postmenopausal women.
DEPLETION OF PERIPHERAL MONOCYTES RESULTS IN A PARADOXICAL INCREASE IN INFLAMMATORY MACROPHAGES AFTER TRAUMATIC BRAIN INJURY
D.M. Trahanas, K.S. Kim, H. Perlman, and S.J. Schwulst*. Northwestern University, Chicago, IL
Background: The central nervous system (CNS) is an immune privileged site restricting peripheral monocyte-derived cells (MDC) from interaction with the CNS. After traumatic brain injury (TBI), the blood brain barrier is compromised allowing peripheral MDCs to infiltrate into the injured brain and directly interact with the CNS. The current study is focused on the role these MDCs play once infiltrated into the injured brain.
Hypothesis: We hypothesized that depletion of peripheral MDCs will prevent the generation of an inflammatory milieu within the injured brain after TBI.
Methods: C57/B6 male mice (n=18) underwent depletion of peripheral MDCs via intravenous injection of clodronate-encapsulated liposomes (200μl) vs. PBS (200μl) at day -1 and day 0. The mice then underwent TBI via an open controlled cortical impact vs. sham injury. Blood was collected immediately prior to TBI to confirm depletion of monocytes. Brains were harvested 48 hours post-injury and MDCs were identified via flow cytometry and counted via an automated cell counter. Data was analyzed with the software program PRISM and are reported as the mean ± SEM.
Results: Treatment with clodronate resulted in near complete depletion of monocytes from the peripheral blood as compared to vehicle treated mice (0.3 ± 0.1% vs. 9.8 ± 0.6% monocytes; p< 0.0001). However, brain- injured mice depleted of peripheral monocytes demonstrated substantially more inflammatory (CD64+MHCII+) macrophages within the injured brain as compared to vehicle treated mice (3.4e6 ± 1.3e6 vs. 1.5e4 ± 2.2e3 cells; p<0.02).
Conclusions: Depletion of monocytes from peripheral blood prior to the induction of TBI resulted in a significant increase of CD64+MHCII+ macrophages within the injured brain. This finding is counterintuitive as the traditional route for the generation of CD64+MHCII+ macrophages is from peripheral monocytes trafficking to the site of injury. These results suggest that infiltrating monocytes may initially play an anti-inflammatory role within the acutely injured brain.
MITOCHONDRIAL DAMAGE ASSOCIATED MOLECULAR PATTERNS (DAMPS) AND GAMMA DELTA T-CELLS: POTENTIAL EFFECTORS IN THE WOUND HEALING RESPONSE
M. Rani*1, Q. Zhang1, T.L. Holloway1, S. Sordo1, and M.G. Schwacha*1, 2. 1University of Texas Health Science Center, San Antonio, TX, 2US Army Institute of Surgical Research, Fort Sam, Houston, TX
Introduction: Gamma delta T-cells have been shown to be important to the early immune inflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with DAMPs generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). Mitochondrial DAMPs (MTDs) have been shown to activate splenic γδ T-cells. Nonetheless, it is unknown whether MTDs can activate γδ T-cells that reside in skin.
Methods: Gamma delta T-cells were isolated from the skin of naïve male C57BL/6 mice by enzymatic digestion. Mitochondria were isolated from mouse livers and MTDs were generated by sonication and centrifugation. Purified γδ T-cells were incubated with various concentrations of MTDs (0-500 μg/ml) for 24 h and cells and supernatants were collected. T-cells were phenotyped for TLR expression by flow cytometry and the supernatants were assayed for cytokine and growth factor content.
Results: MTDs activated dermal γδ T-cells, as evidenced by a dose-dependent increase in TLR-2 and TLR-4 expression. MTDs also induced the production of inflammatory cytokines (IL-1β, IL-6 and IL-10), chemokines (RANTES) and growth factors (PDGF and VEGF) by γδ T-cells.
Conclusions: These findings support the concept that MTDs released after tissue/cellular injury, such as burn, are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes.
PATTERNS OF GENE EXPRESSION AMONG MURINE MODELS OF HEMORRHAGIC SHOCK TRAUMA AND SEPSIS
B.E. Szpila, D.C. Nacionales, C. Lopez, L.F. Gentile, B. Mathias, R. Ungaro, H. Baker, L. Moldawer, and P.A. Efron*. University of Florida, Gainesville, FL
Murine models have received scrutiny for their ability to recapitulate human trauma and sepsis. In humans, it has been shown that the initial genomic response by blood leukocytes is remarkably similar between trauma and burns. This is believed to be due to an overlap in the early signals, including damage associated molecular patterns (DAMPs), that initiate the inflammatory response. However, studies in mice have shown little correlation between models of trauma, burns and endotoxemia in terms of leukocyte genomic responses (Seok J, PNAS, 2013). By increasing the severity of the murine trauma model, we have previously shown improved genomic correlations between murine and human trauma (PT) (Gentile LF, Crit Care Med 2013). Here, we attempt to clarify whether the early genomic response to severe trauma, presumably initiated by DAMPs, is quantitatively similar to two models of sepsis, cecal ligation and puncture (CLP), and Pseudomonas pneumonia (Pp) 1 day following PT.
Methods: 6-10 week old B6 mice either underwent PT or CLP. These mice were then sacrificed at 2 hrs, 1 and 3 days. For the PT+Pp, mice were intranasally instilled with 107 cfu