25 SYNDECAN-1 MEDIATES GUT PROTECTION BY ENTERAL GLUTAMINE IN A RODENT MODEL OF GUT ISCHEMIA/REPERFUSION
K. Ban1, A. Sen1, Z. Peng1, R.J. Grill2, P. Park3, and R.A. Kozar*1. 1University of Texas Health Science Center at Houston, Houston, TX, 2University of Texas Health Science Center at Houston - Integrative Biology & Pharmacology, Houston, TX, 3Harvard Medical School Children’s Hospital-Medicine, Boston, MA
Objective: Syndecan-1 is the predominant heparan sulfate proteoglycan on epithelial cells, and like glutamine, is essential in maintaining the intestinal epithelial barrier. We therefore hypothesized that syndecan-1 contributed to enteral glutamine’s protection of the postischemic gut.
Methods: Wild type (WT) and syndecan-1 knockout (KO) mice were administered ± enteral glutamine followed by 60 min of gut ischemia/reperfusion (IR). Intestinal injury was assessed by fluorescent dye clearance from an intestinal sac using In vivo Imaging System (IVIS); permeability as mucosal to serosal clearance by ex vivo everted sacs, and inflammation by myeloperoxidase (MPO) activity. Results were analyzed by ANOVA then Tukey, n=6/group, mean ±SEM. Means with different letters are significantly different.
Results: Gut IR resulted in a significant increase in intestinal permeability, inflammation, and injury which were further increased in syndecan- KO mice. Glutamine, however, decreased intestinal permeability, inflammation, and injury in WT mice but these protective effects were abolished in syndecan-1 KO mice (table and figure).
Conclusions: Syndecan-1 plays a novel and critical role in the protective effects of enteral glutamine in the postischemic gut and supports the use of glutamine as a gut specific pharmaconutrient. Further investigation into the precise mechanism by which glutamine modulates syndecan-1 warrants further investigation.
26 HYPERTONIC SALINE RESUSCITATION WITHOUT DEXTRAN INHIBITS NEUTROPHIL ACTIVATION
W. Junger*1, S.G. Rhind*2, R.B. Sandro*3, J. Cuschieri*4, S.Y. Maria3, A.J. Baker*5, L. Li1, P.N. Shek*2, D.B. Hoyt*6, and E.M. Bulger*4. 1Dept. of Surgery, Beth Israel Deaconess Medical Center & Harvard Medical School, Boston, MA, 2Defence Research & Development Canada, Toronto, ON, Canada, 3Dept. of Surgery & Critical Care Medicine, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 4Dept. of Surgery, University of Washington, Harborview Medical Center, Seattle, WA, 5Brain Injury Laboratory, Cara Phelan Centre for Trauma Research Keenan Research Centre, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada, 6American College of Surgeons, Dept. of Surgery, University of California, Irvine, CA
Background: Hypertonic saline (HS) resuscitation improves outcome in animal models at least in part by reducing neutrophil activation. However, in a recent clinical trial with prehospital bolus infusion of 250 ml HS (7.5% NaCl) or HS+6% dextran-70 (HSD) we have not been able to reproduce the beneficial effects observed in animal studies.
Objectives: To determine possible reasons for such disparate results, we studied how HS and HSD influence neutrophil activation in trauma patients using blood samples from a subset of patients enrolled in Toronto and Seattle as part of the HS Resuscitation Outcomes Consortium (ROC) trial.
Design: Shock patients (SBP<70 mmHg) were randomly assigned to receive HS (n=9), HSD (n=8), or normal saline (NS; n=17). Neutrophil activation (oxidative burst, myeloperoxidase, metallomatrix protease 9) was assessed at admission to the emergency department (ED≤3h) and 12 and 24 h after admission and compared to values of age-matched healthy controls (n=20).
Results: Trauma caused neutrophil priming and activation that was significantly higher in the NS group compared to healthy controls. This upregulation was inhibited in the HS group, but not in the HSD group.
Conclusions: These findings suggest that hypertonic resuscitation with HS but not HSD can reduce post-traumatic inflammation in shock patients. However, these results cannot explain why hypertonic resuscitation failed to improve clinical outcome in the parent ROC trial.
Funding: Defence R&D Canada and NIH R01GM076101.
27 ALTERED GUT FLORA IN PATIENTS WITH SEPSIS: SURVIVORS VS. NON-SURVIVORS
T. Yamada1, K. Shimizu1,2, H. Ogura1, T. Asahara3, K. Nomoto3, M. Morotomi3, K. Yamakawa1, Y. Ogawa1, T. Hirose1, M. Ohnishi1, Y. Kuwagata1, and T. Shimazu1. 1Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School, Suita City, Osaka, Japan, 2Department of Clinical Quality Management Osaka University Hospital, Suita City, Osaka, Japan, 3Yakult Central Institute for Microbiological Research, Kunitachi-shi, Tokyo, Japan
Background: The gut is considered an important target organ of injury after severe insult such as sepsis, trauma, and shock. The role of gut flora has been focused on in perspective of immunity such as regulatory T cells. Gut flora in patients with sepsis has not been thoroughly clarified. In the present study, we quantitatively evaluated changes in the gut microflora including Clostridium species in patients with sepsis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique.
Methods: Sixty-two patients with sepsis were included in our study (mean age 62.1±20.6 years, APACHE II score 16.7±9.1). A fecal sample was used for quantitative evaluation of 13 kinds of microflora by reverse transcription quantitative polymerase chain reaction (RT-qPCR) technique. Data obtained from patients were compared between survivors (n=46) and non-survivors (n=16).
Results: Analysis of fecal flora confirmed that septic non-survivors had significantly lower obligate anaerobes (especially Clostridium coccoides group (survivors 7.8±1.8 vs. non-survivors 6.1±1.7)), C. leptum subgroup (7.7±1.8 vs.6.1±1.8), Bacteroides fragilis group (8.3±1.6 vs. 6.7±2.2), Bifidobacterium (7.3±1.8 vs. 6.2±1.8), Atopobium cluster (7.3±1.8 vs.5.9±1.3), and facultative anaerobes (Lactobacillus(6.6±1.8 vs. 5.5±1.6), Enterobateriaceae (6.8±1.5 vs. 5.8±1.4) and Enterococcus (7.5±1.8 vs. 6.0±2.0)) than those in survivors. The number of pathogenic bacteria (Staphylococcus (5.1±1.7 vs. 5.2±1.5), Pseudomonas (3.7±1.7 vs. 3.7±1.3) had no significant difference between the two groups. (Log10 counts/g feces).
Conclusions: The gut flora is significantly altered in patients with sepsis especially in non-survivors. Altered gut flora may disrupt immunity and affect systemic inflammatory response in sepsis.
28 IMMUNE-ENHANCING PROPERTIES OF STATIN AND COENZYME Q10 TREATMENT IN SEPSIS
A. Osuka*1,3, M. Yamada2, V.M. Stoecklein*1, M. Kaneki2, and J. Lederer*1. 1Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, 2Massachusetts General Hospital/Harvard Medical School, Boston, MA, 3Social Insurance Chukyo Hospital, Nagoya, Japan
We recently reported that treating septic mice with the farnesylation inhibitor drug, FTI-277, could improve mouse survival. Statins and FTI-277 modulate the mevalonate pathway and accordingly, we found that treating mice with Atorvastatin was nearly as effective as using FTI-277. One complication of statin treatment is the marked reduction of coenzyme Q10 (CoQ10) levels in cells leading to loss of mitochondrial function and integrity. In this study, we tested whether combined CoQ10 and Atorvastatin treatment might enhance the effectiveness of Atorvastatin treatment alone. Groups of mice underwent cecal ligation and puncture (CLP) sepsis or sham CLP and were treated with 20 mg/kg Atorvastatin and 40 mg/kg CoQ10 at 1 day after CLP. At 48 hours, we measured the effects of treatment on systemic cytokine levels and immune cell subset changes. To test for potential immune-enhancing activity, sham or CLP mice were immunized with the T cell dependent antigen, TNP-haptenated ovalbumin, and treated with Atorvastatin, CoQ10, or a combination. Antigen-specific antibody isotype formation was measured at 7 days after CLP to judge helper T cell responses in vivo. Combined Atorvastatin and CoQ10 treatment prevented sepsis-induced immune cell subsets losses. Plasma cytokine profiling showed that Atorvastatin/CoQ10 treatment significantly boosted systemic IFNγ, IL-10, IL12, and IL13 levels in septic mice. Immunized CLP mice showed marked suppression of antigen-specific IgG formation, which was partially restored by combined treatment, but not single drug therapies. Collectively, these findings demonstrate that combined Atorvastatin/CoQ10 treatment has immune system enhancing effects in a mouse sepsis model and suggests a potential for using statin with CoQ10 as treatment to improve immune function in patients that develop sepsis.
29 THE EFFECT ON GLYCEMIC CONTROL OF A LOW-CARBOHYDRATE, HIGH-FAT ENTERAL FORMULA IN CRITICALLY ILL PATIENTS ADMITTED TO A TRAUMA AND CRITICAL CARE CENTER
N. Nakamoto, T. Mouri, D. Wada, T. Kiguchi, H. Matsuda, N. Kubo, N. Inadome, Y. Nakamori, and S. Fujimi*. Osaka General Medical Center, Osaka, Japan
Hyperglycemia is considered to be associated with the mortality and morbidity of critically ill individuals. We employed a high-fat low-carbohydrate enteral formula, reported to be useful for controlling blood glucose in diabetes, for patients admitted to our trauma and critical care center from April 2010 instead of the standard enteral formula. The aim of this study was to clarify the effect on glycemic control of this high-fat low-carbohydrate enteral formula in comparison with the standard enteral formula. A total of 147 mechanically ventilated patients hospitalized between April 2009 and March 2011 were enrolled in this study. Among them, 74 patients hospitalized between April 2009 and March 2010 received the standard enteral formula (Group S), while 73 patients hospitalized between April 2010 and March 2011 received the high-fat low-carbohydrate enteral formula (Group G). Enteral feeding was started within 7 days after admission. Each formula was given continuously starting at a rate of 20 mL per hour, which was increased to provide an appropriate calorie intake within a few days of starting nutritional support. We measured the maximum blood glucose level and need for insulin therapy after starting enteral nutrition in both groups. There were no significant differences between the two groups with regard to age, sex, underlying diseases, and blood glucose at the start of feeding. The maximum blood glucose level of group S (163±32.0 mg/dL) was significantly higher than that of group G (151±28.4mg/dL) (p=0.022). The rate of starting insulin therapy was significantly higher in group S (12.2%) than in group G (1.37%) (p=0.018). In conclusion, a high-fat low-carbohydrate enteral formula is more effective for glycemic control in critically ill patients compared with a standard enteral formula.
30 PROTECTIVE ROLE OF THE GUT MICROBIOME IN THE HOST DEFENSE AGAINST STREPTOCOCCUS PNEUMONIAE INDUCED PNEUMONIA
T. Schuijt, T. van der Poll*, and J. Wiersinga. AMC Amsterdam, Amsterdam, Netherlands
For many years it has been hypothesized that in the critically ill the gut has an important detrimental role in promoting systemic inflammation and infection. More recently however, the gut microbiome has emerged as a key defense system by local support of mucosal defense systems and systemic priming of antibacterial effector mechanism. In this study, we aimed to investigate the role of the gut microbiome in the host defense against severe pneumonia. Therefore, we inoculated mice intranasally with Streptococcus pneumonia and depleted the gut microbiome prior to infectious challenge using antibiotic treatment with vancomycin, ampicillin, neomycin and metronidazole. In the gut microbiome depleted mice, we found significantly increased pulmonary outgrowth of S. pneumonia when compared to controls. This corresponded with increased production of interleukin (IL)-10, IL-6, IL-1β, chemokines (e.g. KC) and neutrophil effector proteins (e.g. myeloperoxidase). Interestingly however, tumor necrosis factor (TNF)-α levels were downregulated in gut microbiome depleted mice. We conclude that the intestinal microbiome plays a protective role in the host defense against S. pneumonia induced pneumonia. Manipulating the intestine microbiome by gut flora replacement or the use of specific bacterial components and/or metabolites might be a new treatment modality for critically ill patients with pneumonia in order to restore gut homeostasis and possibly prevent septic complications and multi-organ failure.
31 BENEFICIAL EFFECT OF A STROMAL CELL-DERIVED FACTOR (SDF)-1α ANALOGUE IN CLP-INDUCED SEPSIS
H. Fan*1, K. Borg1, B. Zingarelli*2, P. Halushka1, and J. Cook*1. 1Medical University of SC, Charleston, SC, 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Previous studies demonstrated that the SDF-1α analogue CTCE-0214 (CTCE) suppresses plasma inflammatory mediators in acute endotoxemia, zymosan-induced multiple organ dysfunction syndrome, and severe sepsis induced by cecal ligation and puncture (CLP). In the present study we examined the hypothesis that the beneficial effect of CTCE is a result of increase recruitment of neutrophils and endothelial progenitor cell (EPC)s. Severe sepsis was induced by CLP with two punctures from a 22-gauge needle. CTCE (10 mg/kg) or vehicle PBS was given by s.q. injection at 2, 6, 24 and 48 h after CLP. The antibiotic Imipenem (25mg/kg) was administrated by s.q. injection at 6, 24 and 48 h after CLP. Mortality was monitored every 12 h until 168 h. In another group of experiments CTCE-0214 (10 mg/kg) or vehicle PBS was given by s.q. injection at 2 and 6 h after CLP. Peripheral blood mature neutrophils (CD11b+/GR-1+ cells) and EPCs (CD34+/VEGFR2+ cells) were determined by flow cytometry. Bacterial load in peritoneal fluid, blood and lung tissue was determined by colony-forming units (CFU) count. CTCE improved survival to 73% compared to 33% in the PBS-CLP group (p<0.05). CTCE significantly increased the percentage of neutrophils and EPCs in peripheral blood compared to the PBS group (p<0.05). CTCE significantly increased neutrophil recruitment (2 fold) in the peritoneal cavity compared to the PBS-CLP group (p<0.05). The increased recruitment of neutrophil during CLP with CTCE greatly accelerated bacterial clearance in peritoneal fluid, peripheral blood and lung with a greater than 77% reduction in CFUs compared to untreated septic mice (p<0.05). These data demonstrate that CTCE increases neutrophil and EPC recruitment, accelerates bacterial clearance during CLP, and improves sepsis outcome. (Supported by NIH GM27673, GM67202).
32 THE PROTECTIVE EFFECT OF EPIDERMAL GROWTH FACTOR DURING SEPSIS DEPENDS ON THE ADAPTIVE IMMUNE SYSTEM
L.S. Jaffree, Z. Liang, E.R. Breed, and C.M. Coopersmith*. Emory University SOM, Atlanta, GA
Background: Lymphocytes aid in mediating gut epithelial apoptosis during sepsis. Epidermal growth factor (EGF) is a cytoprotective polypeptide that restores gut integrity and improves survival by 50% in septic mouse models.
Objective: To determine if EGF interacts with lymphocytes to improve survival and protect gut integrity in sepsis.
Methods: Rag-1-/- mice were subjected to 2×25 cecal ligation and puncture and randomized to receive 150 μg kg1d1 i.p. EGF or an equivalent volume of saline. Groups were either compared at 24 hrs for villus length, permeability, apoptosis, and serum and peritoneal fluid inflammatory cytokines (n = 10-12) or followed for survival (n=18). Mann Whitney analysis was used for all comparisons except survival, which was analyzed using the Log rank test.
Results: EGF worsened 7-day survival in septic Rag-1-/- mice to 16% while mice receiving saline had 61% survival (p= 0.005). This is contrary to what has previously been shown in septic mice with intact immune systems where EGF improved survival by 50%. EGF did, however, decrease gut apoptosis in both H&E and active caspase 3 analyses in Rag-1-/- mice. This was accompanied by a decrease in intestinal Bax expression to undetectable levels. EGF treatment groups had no difference in villus length or intestinal permeability when compared to saline controls. Serum IL-1 and TNFα and peritoneal fluid TNFα were significantly decreased in mice receiving EGF.
Conclusion: EGF drastically diminishes survival in sepsis when lymphocytes are not present, however, retains its ability to improve gut apoptosis. These findings suggest that EGF’s ability to decrease apoptosis may not be responsible for its role in survival in mice with intact immune systems and demonstrate the critical role the adaptive immune system plays in the protective action of EGF in sepsis.
33 DISCOVERY OF A NOVEL SMALL MOLECULE INHIBITOR OF TOLL-LIKE RECEPTOR 4 (TLR4) WITH ANTI-INFLAMMATORY EFFECTS IN MURINE AND HUMAN TISSUE
M.D. Neal1, S. Kim1, C.P.1, P. Wipf2, J. Brodsky3, T.R. Billiar*1, and D.J. Hackam*1. 1University of Pittsburgh School of Medicine, Department of Surgery, Pittsburgh, PA, 2University of Pittsburgh Department of Chemistry, Pittsburgh, PA, 3University of Pittsburgh Department of Biological Sciences, Pittsburgh, PA
Introduction: TLR4 signaling plays a critical role in the pathogenesis of many inflammatory and infectious disorders. We sought to identify novel TLR4 inhibitors with potential anti-inflammatory properties and to test them directly in models of TLR4-mediated inflammation using an in silico to in vivo approach.
Methods: An in silico screen of 26 million compounds was performed to identify potential TLR4 antagonists based on homology to the TLR4-LPS binding domain. Selected compounds were screened for efficacy in vivo. Secondary screens for TLR4 inhibition were performed using in vivo imaging (IVIS, Xenogen) in an NFκB luciferase reporter mouse. Tertiary screens included murine models of TLR4 mediated inflammation: hemorrhagic shock and necrotizing enterocolitis (NEC).
Results: Sixty-five (65) compounds were identified as potential TLR4 antagonists and 21 compounds reduced the severity of endotoxemia in vivo. Reduction of NFκB expression as a marker of TLR4 signaling occurred in 8 compounds, but one compound (C34) demonstrated robust TLR4 inhibition. C34 dramatically reduced disease severity and the expression of mucosal iNOS (p<0.01) in murine NEC. Additionally, pretreatment with C34 resulted in a significant reduction of liver injury as measured by AST/ALT expression (p<0.05) in mice subjected to hemorrhagic shock. To test the physiologic efficacy of C34, freshly isolated human intestinal mucosa from an infant with NEC was exposed to C34 ± LPS ex vivo. Strikingly, the production of mucosal iNOS was significantly inhibited by pretreatment with C34 (p<0.01).
Conclusions: We have discovered a unique inhibitor of TLR4 with potent anti-inflammatory effects using a combination of in silico and in vivo screening. These data suggest that C34 may provide a novel therapy in diseases of TLR4 mediated inflammation.
34 INHIBITION OF LIPOGENESIS REDUCES INFLAMMATION AND ORGAN INJURY INDUCED BY SEPSIS
J.P. Idrovo1,2, W. Yang1, 2, J. Nicastro1, G.F. Coppa1, and P. Wang*1,2. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Sepsis is a life-threatening acute inflammatory disease and associated with metabolic complications. Accumulation of free fatty acids (FAA) induces inflammation and causes lipotoxic effects in the liver. Fatty acid synthase is a rate-limiting enzyme of generating FAA or lipogenesis and its activity can be inhibited by C75, a synthetic compound. We hypothesized that administration of C75 could alleviate the injury caused by sepsis.
Methods: Male mice were subjected to sepsis by cecal ligation andpuncture (CLP). At 4 h after CLP, different doses of C75 (1 or 5 mg/kg BW) or vehicle (20% DMSO in saline) was injected intraperitoneally. Blood and liver tissues were collected at 24 h after CLP for various measurements. Murine macrophage RAW 246.7 cells were used to analyze the effect of C75 on TNF-α release induced by LPS (10 ng/ml).
Results: The levels of FAA in the liver of C75-5 mg group were lower than those in the vehicle group (29.1 ± 3.5 vs. 53.5 ± 3.8 pmol/g tissue, p < .05). Administration of C75 reduced the levels of serum proinflammatory cytokines and organ injury indexes in a dose-dependent manner. The measurements among the sham, vehicle, and C75 treated groups 24 h after CLP are summarized in the table below. Moreover, the levels of TNF-α release in RAW 246.7 cells after 4 h-exposure to LPS were reduced by 32% and 74% in the presence of 10 and 50 μM C75, respectively (p < .05). These doses of C75 did not affect the viability of RAW 246.7 cells.
Conclusions: C75 effectively lowered FAA accumulation in the liver, which was associated with inhibition of inflammation and reduction of organ injury after CLP. In addition, C75 had a direct activity on inhibiting inflammatory responses to LPS in macrophages. Thus, C75 has a potential to be developed as a novel therapeutic agent for treating sepsis.
35 RESVERATROL IMPROVES COGNITIVE PERFORMANCE IN CONCUSSED ATHLETES
J. Gatson*1, V. Warren1, J. Barillas1, K. Abdelfattah*1, S. Wolf*1, J. Simpkins2, M. Cullum1, J. Wigginton*1, and J. Minei*1. 1UT Southwestern Medical Center, Dallas, TX, 2University of North Texas Health Science Center, Fort Worth, TX
Background: Each year, approximately three million sports-related concussions occur in athletes. Only about 5% of these concussions are treated in the hospital setting. To date, there are no effective interventions used at decreasing the levels of oxidant injury and inflammation within these athletes. In previous studies, following stroke and TBI, resveratrol decreased the levels of oxidative stress and lesion volume in the brain. These studies suggest that resveratrol may afford protection from secondary injury in individuals suffering from brain injuries such as sports concussions.
Methods: Boxers (n=12) were consented to participate in the Resveratrol and Sports Concussion Study (REPAIR) and prior to competition (within 2 weeks), a baseline Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT) test was administered to the athletes. After injury (within 2 hours), the boxers that experienced a concussion were enrolled in this study. The boxers were randomized to either placebo or resveratrol treatment. At day 2, 7 and 30 after injury, the ImPACT test was administered.
Results: In the resveratrol group, the boxers had improved reaction time, speed, and accuracy. With respect to reaction time, in the placebo group, there was an approximate 4-fold increase in reaction time. The boxers that received resveratrol treatment had post-injury reaction times similar to their baseline levels. In addition, the boxers that received resveratrol performed better (∼2-fold increase) on the symbol match test (speed and accuracy).
Conclusion: In this safety and feasibility pilot clinical trial, we found that there was a modest improvement with reaction time, speed, and accuracy. These results suggest that resveratrol treatment after mild TBI (concussion) may afford athletes protection from secondary brain injury.
36 CANNABINOID 2 RECEPTOR ACTIVATION IS BENEFICIAL DURING A MURINE MODEL OF SEPSIS
C. Caldwell*, K.R. Kasten, and P.S. Prakash University of Cincinnati College of Medicine, Cincinnati, OH
Our previous studies have demonstrated that endogenous cannabinoids, signaling through the cannabinoid receptor 2 (CB2R), are critical regulators of the immune response during experimental sepsis. We hypothesized that during sepsis, CB2R gain-of-function would improve the murine response to sepsis. To induce sepsis, mice were subjected to a cecal ligation and puncture (CLP). Consistent with our previous data, CB2R-agonist treatment in wild-type mice increased the mean survival time in response to CLP, while decreasing serum IL-6 levels, bacteremia, and damage to the lungs (Figure 1) compared to vehicle-treated mice. We observed that macrophages isolated from GP1a-treated septic mice demonstrated decreased MHC II expression and increased phagocytic ability. Previous biochemical studies showed that CB2R is a Gi-coupled receptor known to negatively regulate adenylyl cyclase. The adenylyl cyclase/cAMP circuit is a critical regulator of NF-kB and p38, molecules that play essential roles in inflammation and immune responses. Indeed, we found that CB2R agonist treatment decreased neutrophil recruitment into the peritoneal cavity, increased p38 activity, and increased phagocytic function. Altogether, we show that the CB2R plays a critical role in myeloid cell recruitment and function, thereby acting upon a major regulatory pathway of mortality in sepsis.
37 A CARDIOVASCULAR HERBAL MEDICINE (TANSHINONE IIA) FACILITATES CELLULAR HMGB1 UPTAKE
Y. Zhang1,2, W. Li1,2, S. Zhu*1,2, J. Li1, A. Jundoria1,2, H. Yang*1, K. Tracey*1, P. Wang*1, A. Sama1,2, and H. Wang*1,2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Department of Emergency Medicine, North Shore University, Manhasset, NY
The discovery of HMGB1 as a late mediator of lethal systemic inflammation has initiated a new field of investigation for the development of experimental therapeutics. We previously reported that a major ingredient of Danshen (Tanshinone IIA sodium sulfonate, TSN-SS) rescued mice from lethal sepsis by specifically inhibiting endotoxin-induced HMGB1 release.
Objectives: To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release.
Methods: We determined whether TSN-SS stimulated HMGB1 uptake in macrophage cultures; and whether genetic depletion of HMGB1 receptors (e.g., TLR2, TLR4, or RAGE) or pharmacological inhibition of clathrin-mediated endocytosis impaired TSN-SS-facilitated HMGB1 uptake.
Results: TSN-SS facilitated the uptake of recombinant HMGB1 protein into macrophage LC3-positive cytoplasmic vesicles (likely autophagosomes) in a time- and dose-dependent fashion. Simultaneously, it dramatically enhanced HMGB1-induced production of an autophagy marker, LC3-II, even in the presence of an autophagy inhibitor, bafilomycin A1, possibly facilitating autophagic degradation of cytoplasmic HMGB1 in macrophages. Genetic depletion of TLR2, TLR4, and/or RAGE did not affect TSN-SS-mediated enhancement of HMGB1 uptake, eliminating the potential involvement of these HMGB1 receptors in its cellular uptake. In contrast, a specific clathrin inhibitor, chlorpromazine, effectively abolished TSN IIS-SS-mediated HMGB1 uptake.
Conclusions: It is possible to employ herbal medicine to pharmacologically “recycle” potentially injurious proinflammatory mediators.
(Supported by the National Institutes of Health Grants R01GM063075 and R01AT05076).
38 MUNG BEAN (VIGNA RADIATA) EXTRACT IS PROTECTIVE AGAINST LETHAL SEPSIS IN MICE
S. Zhu*1,2, J. Li1, W. Li1,2, M. Ashok2, A. Jundoria1,2, Y. Zhang1,2, A. Sama2, and H. Wang*1,2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2North Shore University Hospital, Manhasset, NY
The mung bean is commonly used as a food source worldwide, but has also been prescribed as a traditional Chinese herbal medicine in the treatment of a number of inflammatory ailments since 1050’s. Recent evidence has suggested a pathogenic role for HMGB1 as a “late” mediator of lethal systemic inflammation with a relative wider therapeutic window for pharmacological interventions.
Objectives: To explore the HMGB1-inhibiting capacity and therapeutic potential of Mung bean hull extract (MBH) in vitro and in vivo.
Methods: Human U937 or murine RAW 264.7 macrophage-like cell cultures were stimulated with bacterial endotoxin (LPS) in the absence or presence of MBH, and HMGB1 release was assessed 16 hours later. Male Balb/C mice (20-25 g, 6-7 weeks old) were subjected to experimental sepsis by cecal ligation and puncture (CLP), and MBH extract was orally administered at 24, 48, and 72 h post CLP.
Results: MBH extract dose-dependently abrogated LPS-induced HMGB1 release in both murine and human macrophage cultures. Meanwhile, it stimulated HMGB1 protein aggregation, and elevated LPS-induced production of an autophagy marker, LC3-II, thereby facilitating autophagic HMGB1 degradation in human and murine macrophage cultures. Oral administration of MBH extract significantly increased animal survival rates from 29.4% (in control saline group, N=17 mice) to 70% (in experimental Mung bean extract group, P < 0.05).
Conclusion: These data suggest that Mung bean hull extract is protective against lethal sepsis partly by attenuating endotoxin-induced HMGB1 release via stimulating its autophagic degradation.
(Supported by the National Institutes of Health Grants R01GM063075 and R01AT05076).
39 RESVERATROL REDUCES ENDOPLASMIC RETICULUM STRESS FOLLOWING TRAUMA-HEMORRHAGE
B. Jian S. Yang, I.H. Chaudry*, and R. Raju*. University of Alabama at Birmingham, Birmingham, AL
Resveratrol (RSV), a plant polyphenol, is an antioxidant and specific activator of Sirt1. Our functional mitogenomics experiments demonstrated a down regulation of Sirt1 following trauma-hemorrhage (T-H). Our subsequent experiments showed that RSV prevents T-H-induced Sirt1 downregulation, and has salutary effect on left ventricular contractility and mitochondrial (mt) function following T-H. However, the specific influence of RSV on metabolic perturbation following T-H remains unknown. Accordingly, we sought to determine the effect of RSV on endoplasmic reticulum (ER) stress following T-H. Previous studies from our laboratories have demonstrated the initiation of ER stress following T-H. In this study, we specifically tested the level of expression of ER stress proteins in the left ventricular tissue of Sprague Dawley rats subjected to T-H, followed by RSV (8 mg/Kg body weight;), sirtinol (a specific inhibitor of Sirt1) or vehicle administration. RSV or vehicle was administered I.V. at 10 minutes after the onset of resuscitation. mt complex I activity and total ATP content were determined. Total proteins were tested for Bip (immunoglobulin heavy chain binding protein), IRE-1a (inositol-requiring kinase 1) and PDI (protein disulphide isomerase); and the pro-apoptotic protein, CHOP (C/EBP homologous protein, also known as GADD153) (n=3-4/group) by Western blot. RSV treatment improved total ATP content (p<0.05) and complex I activity (p<0.05). RSV treatment also abrogated the elevated expression of Bip, IRE-1a, PDI and CHOP following T-H. Sirtinol abolished the salutary effect of RSV. The results demonstrate that RSV reduces ER stress, when administered as an adjunct to resuscitation fluid. We conclude that RSV may be a useful adjunct to resuscitation fluid.
40 WATER SOLUBLE ESTROGENS PROLONG PERMISSIVE HYPOTENSION FOLLOWING MAJOR BLOOD LOSS EVEN WITHOUT FLUID RESUSCITATION
W.J. Hubbard, J. Robertson, A. Ayub, A. McBrayer, and I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
A DARPA research program termed SBL (surviving blood loss) targeted a practical means to enable warfighters to survive on the battlefield. Success was defined initially as 3-hour and subsequently as 6-hr survival from 60% blood loss in rats without resuscitation. We examined use of small volume estrogen (0.4 ml/kg) to facilitate life support, adding an additional “real world” injury of soft tissue trauma (midline laparotomy). Preliminary testing with this model revealed that efficacy hinged on delivering the hormone rapidly in a supraphysiologic dose in a water-soluble form. We explored 6 routes of delivery: intravenous (IV), intraosseous (IO), sublingual, intranasal, subcutaneous and intramuscular; we found IV and IO routes to be superior in attaining a rapid, high blood level. We selected IV administration and tested 3 soluble forms of estrogen (E2-cyclodextrin, E2-sulfate and ethynyl estradiol sulfate [custom synthesis, EE-SO4]). EE-SO4 was selected as the ultimate test article owing to its greater half-life and biological activity. Tests from a panel of 4 doses (0.1, 0.3, 1.0 and 3.0 mg/kg) ascertained that the 1 mg/kg dose was superior, based on 6-hr survival percentages of 16, 50, 87 and 50, respectively. The greater biological activity of EE-SO4 was confirmed through the use of isolated rat aortic rings in a vasorelaxation test system. Titrating the EE-SO4 in a logarithmic series from 1x10-9 to 1x10-4 M, we found that EE-SO4 was most effective at the lowest doses as compared to E2-SO4; it additionally demonstrated a steeper response curve. Our examination of the mechanism for the relaxation response revealed that it was endothelium-dependent and mediated by NO. Thus, this synthetic estrogen holds high potential for SBL therapy for both military and civilian use (DARPA W911NF-06-1-0219, W911NF-10-1-0130).
41 TLR4 ANTAGONISM RESTORED CARDIAC FUNCTION AFTER TRAUMA-HEMORRHAGE
X. Zhang, C. Lu, M. Gao, T. Ha, J. Kalbfleisch, W. Browder, D.L. Williams**, C. Li* and R.L. Kao. East Tennessee State University, Johnson City, TN
Trauma-hemorrhage (TH) induced cell and tissue injuries can release endogenous damage-associated molecular patterns (DAMPs) that are recognized by Toll-like receptors (TLRs) to initiate immune and inflammatory responses. Uncontrolled immune and inflammatory responses can lead to tissue damage, multi organ failure or death and cardiovascular collapse is one of the major factors contributing to the demise of TH patients. Our recent studies indicate that TLR4 deficient mice show attenuation of cardiac dysfunction due to TH. Eritoran tetrasodium (E5664), a TLR4 antagonist, has been evaluated in phase III trial as a treatment for severe sepsis. The primary objective of this study was to evaluate therapeutic effect of E5664 on cardiac dysfunction in our mouse model of TH. Trauma by soft tissue injuries and hemorrhage by blood withdrawal with mean arterial pressure maintained at 35±5 mmHg were established. Arterial and left ventricular (LV) pressures, as well as LV volumes were determined. Our results show that E5564 treatment significantly improved hemodynamic functions compared to placebo group after 60 min of TH in a dose dependent fashion (n=10/group). Using the optimal dose of E5564 (3 mg/kg), a single intravenous administration of the drug without fluid resuscitation prevented hemodynamic collapse when given at 1, 2, 3, or 4 hours after TH (n=10/group). E5564 also significantly attenuated TH-decreased phospho-Akt/Akt ratio and ameliorated TH-increased NF-κB binding activity in the myocardium. In summary, TLR4 antagonist E5564 significantly attenuated TH-induced cardiac dysfunction. TLR4 antagonism may be a novel treatment strategy for managing cardiac dysfunction and enhancing recovery in TH patients.
42 ADIPONECTIN (ADPN) IS A KEY COMPONENT IN FRESH FROZEN PLASMA (FFP) RESPONSIBLE FOR ITS ENDOTHELIAL PROTECTIVE EFFECTS AFTER HEMORRHAGIC SHOCK (HS)
X. Deng1, M. Huby1, L. Baer1, G. He1, W. Sun2, Y. Cao1, J. Dong2, R.A. Kozar*1, C.E. Wade*1, J.B. Holcomb*1, and T.C. Ko1. 1The University of Texas Health Science Center at Houston, Houston, TX, 2Baylor College of Medicine, Houston, TX
Objective: Previously, we have shown that FFP resuscitation restores vascular barrier and vasomotor function in a mouse model of laparotomy/HS and AMPK is a key signaling molecule for FFP’s barrier-protective effects in vitro. We have also demonstrated that plasma levels of Adpn, the most abundant AMPK activator in plasma, are lower in HS patients prior to resuscitation compared with healthy donors. We hypothesize that Adpn is a key component in FFP responsible for its endothelial protective effects.
Methods: FFP was fractionated using fast performance liquid chromatography (FPLC) and tested for Adpn level and AMPK activation. Adpn was depleted from FFP by immunoprecipitation with an anti-Adpn antibody [FFP(Adpn-)] using an isotype IgG as control [FFP(IgG)]. Endothelial protective effects were evaluated using in vitro permeability assay and the mouse model of laparotomy/HS.
Results: The only FFP fraction (#6) that contained the highest level of Adpn induced AMPKalpha phosphorylation in human pulmonary microvascular endothelial cells (HPMECs). Immunodepletion resulted in >90% removal of Adpn from FFP. In vitro, phosphorylation of AMPKalpha and inhibition of hypoxia-induced permeability were reduced by 70.8% and 73.8%, respectively, in HPMECs treated with FFP(Adpn-) compared with FFP(IgG). Laparotomy/HS mice resuscitated with FFP(Adpn-) had increased pulmonary vascular permeability as determined by Evans blue dye extravasation (0.28±0.11 vs 0.59±0.06 OD620, P=0.068), and attenuated vasodilation as determined by acetylcholine injection (22.0±3.2 vs 9.0±3.9 mmHg, P=0.042) compared with FFP(IgG).
Conclusions: Our study suggests that Adpn is a key component in FFP responsible for its endothelial protective effects after HS. Restoring plasma Adpn level may be a novel strategy in the treatment of trauma/HS patients.
43 SERUM METABOLOMIC ANALYSIS OF THE EFFECTS OF GLUCOSE PRE-FEED ON METABOLISM IN A PORCINE MODEL OF POLYTRAUMA AND HEMORRHAGIC SHOCK
D.R. Lexcen, E.R. Lusczek, N. Witowski, J. Ashgar, P. Iyegha, K. Mulier, and G. Beilman. University of Minnesota, Minneapolis, MN
Hemorrhagic shock is a leading cause of trauma related deaths. Small animal studies demonstrated that pretreatment with glucose prior to hemorrhage results in a survival benefit. The mechanism of benefit is not completely understood. Thus, it was our goal to elucidate the metabolic response and effects on survival of a glucose pre-feed in a porcine polytrauma and hemorrhagic shock model.
Thirty-two male Yorkshire pigs underwent pulmonary contusion, liver crush injury, and controlled bleed. After polytrauma and shock, animals received 1 hour of limited resuscitation, followed by twenty hours of a standard resuscitation protocol. 1H-NMR spectroscopy was used to analyze serum samples from animals that were randomized to receive glucose prior to experiment (n=16) or fasted (n=16). Partial least squares discriminant analysis (PLS-DA) was used to identify differences in metabolic profiles.
Mortality was not significantly different between glucose pre-fed animals compared to fasted animals (50.0% vs. 31.3%, p=0.36). PLS-DA evaluation revealed significant separation between the fasted and pre-fed groups. The most discriminant metabolites contributing to the model were glucose and metabolites related to alternate energy production pathways. PSL-DA also revealed distinct metabolic changes associated with mortality. Specific biomarkers of mortality were succinate, choline, hypoxanthine and uridine.
Contrary to previous studies, we observed no survival benefit with pretreatment of glucose in this study. Glucose prefeed was associated with a significantly altered metabolic profile in response to hemorrhagic shock. Serum biomarkers predictive of mortality in this large animal model include succinate, choline, hypoxanthine, and uridine. Future work will include confirmation of these biomarkers in human studies.
44 HYPEROXEMIC REOXYGENTATION DOES NOT INCREASE ROS FORMATION DURING REPERFUSION AFTER HEMORRHAGIC TRAUMATIC SHOCK ASSOCIATED WITH HYPOXIA
C. Penzenstadler*, A. Zifko*, M. Jafamadar, A. Klotz, A. Kozlov*, and S. Bahrami*. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Trauma Research Center, Vienna, Austria
Aim: Reperfusion injury is in part ascribed to the formation of reactive oxygen species (ROS) during reoxygenation subsequent to a hypoxic state. We aimed to determine the effects of 3 reoxygenation strategies on ROS formation after hemorrhagic traumatic shock (HTS).
Methods: Ventilated rats were subjected to HTS. Hypoxia was induced by hypoventilation to achieve an arterial pO2 of 55mmHg. At end of shock animals were randomized to receive 3 different reoxygenation strategies (n=10 each) during reperfusion (75ml/kg/h, Ringer’s solution). Normoxemic group (NG): immediate return to baseline (pO2>80mmHg). Hyperoxemic group (HG): gradual decrease from 100% oxygen in respiration air over 50min until baseline pO2 values were reached. Gradual reoxygenation group (GG): gradual increase to baseline pO2 values over the first 50min. Organ specific ROS formation was determined by infusion of ROS scavenger CPH during resuscitation time (100min) followed by electron paramagnetic resonance spectroscopy.
Results: During reperfusion mean arterial pressure was higher in HG/NG compared to GG (p<.05). ROS were increased in GG versus both NG (plasma, 1.4fold) and HG (ileum, 1.5fold). Although thiobarbituric acid-reactive substances in ileum were higher in HG/NG compared to lab controls (p<.05), there was no difference in ileum, kidney and liver between groups. Lung myeloperoxidase activity was increased in all groups compared to lab controls (p<.05). Respiratory burst capacity increased at the end of experiment equally in all groups. Cellular/organ (liver, kidney) damage was higher in GG compared to other groups (p<.05).
Conclusions: Gradual reoxygenation after HTS and hypoxia is linked to increased ROS formation and cellular/organ damage. Hyperoxemic and normoxemic strategies may be preferred to a restricted reoxygenation.
45 DILUTING THE SURVIVAL BENEFIT OF HEMOSTATIC RESUSCITATION
C. Guidry, E. Gleeson, P. Meade, N. McSwain, and J. Duchesne Tulane University, New Orleans, LA
Background: High ratios of FFP:PRBC in Damage Control Resuscitation (DCR) are associated with increased survival. The impact of volume and type of resuscitative fluid used during this high ratio transfusion has not been analyzed. We hypothesize a direct mortality correlation between quantity and type of resuscitative fluids used in patients that received high ratios of FFP:PRBC.
Methods: This 4 year retrospective study included patients who received ≥ 10 units of PRBC with high ratio resuscitation, >1:2 of FFP:PRBC. Demographics and outcomes of the quantity and type of resuscitative fluids used were compared and analyzed. To further compare quantity of fluids given, Kaplan-Meier survival analysis was computed.
Results: There were 56 patients included (28 in crystalloid group, 28 in colloid group). Demographics were statistically similar. Mean units of PRBC: crystalloid vs colloid was 14.8 (SD 9.4) vs 17.3 (SD 8.0), p=0.246; mean units of FFP: 13.0 (SD 6.8) vs 14.0 (SD 5.9), p=0.33. Hospital and ICU LOS was similar. Odds Ratio for 10 day mortality in the crystalloid group was 8.41 [95% CI 1.65-42.76 (p=0.01)]. Kaplan-Meier survival curve compared four groups: colloids (mean quantity = 1.4L), <3L crystalloid, 3-6L crystalloid, and >6L crystalloid. Lowest mortality was in the colloid group and higher mortality when increasing the amount of crystalloid (p=0.029).
Conclusion: In high ratio DCR: higher volume of crystalloids decreased survival and low effective colloid use increased survival. With better understanding of limiting crystalloids, we postulate future guidelines will incorporate protocols targeting an effective low volume resuscitation with high ratio component resuscitation to improve outcomes without diluting the survival benefit of hemostatic resuscitation.
46 HMGB1 CYSTEINE 106 IS REQUIRED FOR BINDING TO MD-2 IN THE TLR4/MD2 COMPLEX TO ELICIT INFLAMMATORY RESPONSES
H. Yang*1, P. Lundback2, L. Ottosson2, Y. Al Abed*1, M. Ochani*1, J. Li1, B. Lu1, S. Chavan1, D.J. Antoine3, H. Harris2, U. Andersson2, and K.J. Tracey*1. 1Feinstein Institute, Manhasset, NY, 2Karolinska Institute, Stockholm, Sweden, 3University of Liverpool, Liverpool, United Kingdom
High mobility group box 1 (HMGB1) is a cytokine mediator in the pathogenesis of inflammatory diseases (Yang et al, BBA, 2009). HMGB1 contains three conserved cysteine residues at position 23, 45 and 106. Previously, we showed that HMGB1 binds to TLR4/MD2 and that mutation of cysteine 106 (C106) prevents the binding interaction and subsequent stimulation of cytokine release from macrophages (Yang et al, PNAS, 2010). Recently we further demonstrated that both reduced C106 and a disulfide bond between C23 and C45 are required for HMGB1 cytokine activity (Yang et al, Mol Med, 2011). It is known that TLR4 activity and interaction with its ligands depends on the extracellular adaptor MD-2 (Visintin et al, 2006). Using biosensor-based surface plasmon resonance (BIAcore), we found that HMGB1 binds human MD-2 with high affinity (apparent Kd = 8nM), and it does not bind to TLR4 alone (data not shown). Inactive HMGB1, created by either exposure to mercury which reacts to C106 thiol or by mutation of C106 or C45 to alanine in full length HMGB1, abolished the binding interaction with MD-2. This modification also prevents HMGB1-induced TNF release in cultured human macrophages (Yang et al, Mol Med, 2011). Thus, our data indicate that HMGB1 binds to MD-2 in the TLR4/MD2 complex to elicit inflammatory responses and C106 is important for this binding interaction. Supported in part by grants from NIH, NIGMS (RO1GM62508 to KJT and RO1GM098446 to HY).
47 HISTONES ACTIVATE NLRP3 INFLAMMASOME THROUGH TOLL-LIKE RECEPTOR 9-DEPENDENT PATHWAY IN STERILE INFLAMMATORY LIVER INJURY
H. Huang, H. Chen, W. Yan, G. Nace, L. Zhang, and A. Tsung. University of Pittsburgh Medical Center, Pittsburgh, PA
Purpose: NLRP3 inflammasome activation induces pro-inflammatory and innate immune responses in stressful conditions. We recently showed that extracellular histones act as a new class of DAMPs and mediate inflammation and organ damage through TLR9 after liver ischemia/reperfusion (I/R). We sought to determine the mechanisms by which NLRP3 inflammasome activation results in organ damage and inflammatory response following liver I/R.
Methods: NLRP3 KO, TLR9 KO, and WT mice were subjected to a segmental (70%) warm hepatic I/R. Anti-histone antibodies or exogenous histones were administered in selected I/R groups. Liver damage was assessed by ALT levels and histology. Protein, cytokines, mRNA and innate immune cell population was evaluated.
Results: NLRP3 and TLR9 KO mice w/o histone treatment protected liver from I/R injury. Histone-treatment upregulated protein levels of NLRP3 inflammasome (NLRP3, ASC and pro-caspase-1) and downstream activated caspase-1, IL-1β and IL-18 following liver I/R. Conversely, reduction of these proteins was observed in anti-histone-treated mice. Exogenous histones increased the production of reactive oxygen species (ROS) that promoted the association of thioredoxin-interacting protein (TXNIP) with NLRP3. This association was reduced by inhibiting TLR9, a receptor for histones. Furthermore, activation of NLRP3 inflammasome and its production of IL-1β and IL-18 in TLR9 KO mice were significantly less than in WT mice after I/R.
Conclusion: NLRP3 inflammasome is activated by endogenous histones during liver I/R, which contributes to organ damage through activation of caspase-1 and increased proinflammatory IL-1β and IL-18 production. Activation of NLRP3 inflammasome by histones is through a TLR-9-dependent pathway that mediates ROS production and subsequently promotes the association of TXNIP to NLRP3.
48 PKR IS REQUIRED FOR NLRP3 INFLAMMASOME ACTIVATION AND HMGB1 RELEASE
B. Lu1,4, T. Nakamura2, K. Inouye2, S. I. Valdes-Ferrer1,4, P.S. Olofsson1, J. Li1, H. Wang*1, U. Andersson3, H. Yang*1, S.S. Chavan1, G.S. Hotamisligil2, and K. Tracey*1. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Harvard School of Public Health, Boston, NY, 3Karolinska Institute, Stockholm, Sweden, 4The Elmezzi Graduate School of Molecular Medicine, Manhasset, NY
The NLR family pyrin domain-containing 3 (NLRP3) inflammasome, an intracellular oligomeric protein complex, regulates release of caspase-1 activation-dependent cytokines, including IL-1β, IL-18, and high-mobility group box 1 (HMGB1). During the course of studying HMGB1 release mechanisms from macrophages, we unexpectedly observed that double-stranded RNA dependent protein kinase (PKR) is required for NLRP3 inflammasome activation. PKR inactivation by genetic deletion or pharmacological inhibition blocked NLRP3 inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, and live E coli. Overexpression of PKR significantly enhanced the reconstituted NLRP3 inflammasome-induced caspase-1 activation and IL-1β maturation, whereas blocking PKR significantly inhibited the reconstituted NLRP3 inflammasome activation. PKR physically interacts with NLRP3, and that PKR autophosphorylation is required for efficient PKR and NLRP3 interaction. Importantly, PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. Together, these results indicate that PKR is required for inflammasome activation, and that it should be possible to target this molecule to inhibit inflammasome activity during inflammation. Supported by grant from NIH (RO1GM62508 to KJT).
49 LOSS OF MYD88 OR TRIF DOES NOT IMPACT SURVIVAL TO NEONATAL POLYMICROBIAL SEPSIS
D.N. Joiner, A. Cuenca, J.L. Wynn, K. Kelly-Scumpia, P.O. Scumpia, D.C. Nacionales, P.A. Efron* and L.L. Moldawer*. University of Florida, Gainesville, FL
Despite improvements in perinatal care, neonatal infection and sepsis are still global health concerns that claims 1 million lives a year. Neonates have distinct immunological responses relative to adults. In the adult, survival to polymicrobial sepsis has been demonstrated to be dependent on the downstream TLR signaling protein, MyD88; however, the role of MyD88 or the other TLR downstream signaling adaptor protein in the survival to neonatal polymicrobial sepsis is unknown. To address this, 5-7 day old wild-type (WT) B6, MyD88-/-, or TRIF-/- mice (on a B6 background) were inoculated intraperitoneally (ip) with a cecal slurry (CS), cecal contents from adult mice, and followed for survival. Surprisingly, WT, MyD88-/- and TRIF-/- neonates, had similar survival to a LD70 dose of CS (25% vs 22% vs 18% respectively, p= NS). Similar data were obtained using a LD30 dose. In addition, there were no differences in the percentage of macrophages and neutrophils recruited to the peritoneum 24 hours following CS challenge in WT vs MyD88-/- vs TRIF-/- neonates (70% vs 68% vs 68% respectively, p=NS). There were also no differences in the baseline respiratory burst and production of reactive oxygen species of neutrophils and macrophages in WT, TRIF-/- and MyD88-/- neonates in response to phorbol ester. These data suggest that neonates are able to compensate for the loss of one these critical downstream TLR signaling adaptor proteins in the face of polymicrobial sepsis. This is contrast to adult data that has suggested that MyD88 is critical for survival to polymicrobial sepsis. Further studies that delete both of these proteins will elucidate which compensatory mechanisms elaborated by either TRIF and/or MyD88 are critical for the survival of neonates to polymicrobial sepsis.
50 THE ALARMIN S100A8/A9 AMPLIFIES VENTILATOR-INDUCED LUNG INJURY
M.T. Kuipers1,2, T. Vogl4, H. Aslami1, G. Jongsma1, E. van den Berg1, A.P. Vlaar1, J.J. Roelofs3, N.P. Juffermans1, M.J. Schultz1, T. van der Poll*2, J. Roth4, and C.W. Wieland1. 1Laboratory of Experimental Intensive Care and Anesthesiology Academic Medical Center, Amsterdam, Netherlands, 2Center of Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands, 3Department of Pathology, Academic Medical Center, Amsterdam, Netherlands, 4Institute of Immunology, University of Münster, Münster, Germany
Objective: Mechanical ventilation (MV) can induce lung injury in which innate immunity plays an important role. However, the exact triggers of the response remain unclear. S100A8/A9 (Mrp8/14) proteins are endogenous danger signals that activate innate immunity via TLR4. We hypothesized that S100A8/A9 proteins are released in acute lung injury (ALI) and mediate lung inflammation in a 2-hit model of LPS-induced lung injury combined with MV.
Methods: S100A8/A9 levels were measured in bronchoalveolar lavage fluid (BALF) of patients with and without ALI. Next, wild-type (WT) and S100A9 knock-out mice, naïve and with induced ALI (intranasal LPS), were randomized to 5 h of spontaneously breathing or MV. S100A8/A9 levels were measured, other endpoints were: lung wet/dry ratio, total protein level, neutrophil influx, cytokine and chemokine levels, and histology. In addition, healthy spontaneously breathing and ventilated WT mice received S100A8/A9, S100A8 or zvehicle intratracheally. Neutrophils, total protein, cytokine and chemokine levels were measured in BALF.
Results: S100A8/A9 proteins were increased in BALF of ALI patients and in mice with LPS- or MV-induced lung injury. S100A8/A9 levels further increased upon LPS/MV double hit. Targeted deletion of S100A9 attenuated lung inflammation in the 2-hit model. Spontaneously breathing mice receiving S100A8/A9 or S100A8 had higher KC levels and more neutrophil influx. In mice undergoing MV, S100A8/A9 or S100A8 instillation amplified lung inflammation; neutrophil influx, IL-6, KC, IL-1β, and MIP-2 levels were increased compared to ventilated vehicle-treated mice.
Conclusion: S100A8/A9 proteins are released in ALI and their presence enhances inflammation in a LPS/MV model. S100A8/A9 administration during MV amplifies the pulmonary inflammatory response towards ventilation.
51 ROLE OF CELL-FREE DNA IN SEPSIS PATHOPHYSIOLOGY: NOVEL STUDIES IN MURINE MODELS OF SEPSIS
S. Mai1,3, A.L. Patrick1,2, A. Fox-Robichaud*1,2, and P. Liaw1,3. 1McMaster University, Hamilton, ON, Canada, 2Hamilton Health Science, Hamilton, ON, Canada, 3David Braley Cardiac, Vascular & Stroke Research Institute, Hamilton, ON, Canada
Introduction: Sepsis is characterized by an exaggerated host response to microbial infection resulting in the systemic activation of coagulation and inflammation. We have recently observed that high levels of cell-free DNA (cfDNA) released in the circulation is a powerful prognostic marker in severe sepsis patients. Our objective was to investigate the role of cfDNA in inflammation during sepsis.
Methods: Healthy C57BL/6 male mice were subjected to cecal ligation and puncture (CLP), involving a ligation distal to the ileocecal valve and two punctures of the ligated cecum (mortality rate of 100% at 24 hrs). Mice were exsanguinated and organs harvested at times between 0 to 10 hours post-surgery and plasma levels of cfDNA, IL-6, TNF, and protein C were quantified. This was repeated in a lipopolysaccharide (LPS, 0.04 ug/g) model of inflammation.
Results: Baseline levels of cfDNA in healthy mice were 1.1±0.2 ug/mL. At 6 hours post-CLP, cfDNA levels increased more than two-fold compared to sham mice (5.5±0.4 ug/mL vs. 2.4±0.2 μg/mL; p < 0.01) and remained elevated for 10 hours post-CLP. IL-6 and TNF levels increased and protein C decreased in mice subjected to CLP while these levels did not change significantly in sham-operated mice over 10 hours. Similarly, LPS-challenged mice had significant elevations in cfDNA versus healthy controls over 8 hours (6.24±0.3 μg/mL vs. 2.1±0.2 μg/mL respectively; p < 0.01).
Conclusion: These studies are the first to show rapid elevations of cfDNA associated with an early proinflammatory response in experimental sepsis, supporting the importance of early intervention (ie. administration of resuscitation fluids and antibiotics within 1 hour of diagnosis). These findings suggest that early therapeutic interventions which modulate cfDNA levels may improve clinical outcome of septic patients.
© 2012 by the Shock Society