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32.3. Citrullinated Histone H3 - A Novel Target for Treatmentof Septic Shock. Y. LI,1 Z. Liu,1 B. Liu,2 T. Zhao,1 Y. Wang,3

G. Velmahos,1 H. Alam2; 1Massachusetts General Hospital -Trauma/Surgery/Harvard Medical School, Boston, MA, USA;2University Of Michigan - General Surgery/University OfMichigan Medical School, Ann Arbor, MI, USA; 3Penn StateUniversity College Of Medicine - Biochemistry And MolecularBiology, Hershey, PA, USA

Introduction:Wehave recently demonstrated that in a rodent model oflipopolysaccharide (LPS)-induced shock, an increase in blood citrulli-nated histone H3 (Cit H3) is associated with lethality of sepsis, andtreatment with suberoylanilide hydroxamic acid (SAHA), a histonedeacetylase inhibitor (HDACI), significantly improves survival. How-ever, the role of Cit H3 in pathogenesis and therapeutics of sepsis arelargely unknown. The present study was designed to test whether theHDACI could inhibit cellular Cit H3 production and neutralization ofblood Cit H3 with anti-Cit H3 antibody could improve survival ina clinically relevant mouse model of cecal ligation and puncture(CLP) induced septic shock. Methods: HL-60 neutrophilic cells grownon a coverslip were treated with LPS (100 ng/ml) in the presence orabsence of SAHA (5 mmol) for 3 h, and subjected to immuno-stainingwith anti-Cit H3 antibody to assess effect of SAHA on Cit H3 produc-tion under a fluorescencemicroscope. The ratio of Cit H3 positive cellswas calculated as mean 6 SD (n¼3). In a separate experiment, maleC57B1/6J mice were divided into control and treatment groups, andsubjected to CLP-induced septic shock. Two hours later, vehicle andSAHA (50 mg/kg i.p.; n¼10/group) or immunoglobulin (IgG) and CitH3 antibody (20 mg/kg iv; n¼5/group) were injected into the controland treatment groups, respectively. Survival was monitored for 5-10days.Results:LPS induced Cit H3 production in theHL-60 cells, whileSAHA treatment inhibited H3 citrullination significantly (Fig. 1). Inan in vivo HDACI study, all vehicle injected mice died within 3 dayswith increased blood Cit H3, whereas treatment with the HDACI no-tably improved long term survival (p < 0.01). In addition, IgG did notprolong animal life, but treatment with Cit H3 specific antibody, evenonly once, significantly improved survival (p<0.014). Conclusions: In-hibition of histone deacetylase significantly suppresses Cit H3 pro-duction in vitro and improves survival in vivo. Neutralization ofblood Cit H3 remarkably prolongs life of septic mice. Collectively,our findings have indicated for the first time that Cit H3 could notonly be a potential biomarker but also a novel therapeutic target forsepsis. The further investigation is ongoing.

FIG. 1. SAHA Suppresses LPS-induced Cit H3 productionin HL-60 cells. (A) A representative experiment of Cit H3 staining.Nuclei stained with diamidino-2-phenylindole (DAPI) were in blue.Cit H3 positive cells were in red. (B) The ratio of Cit H3 positive cellswere calculated as mean 6 SD (n¼3). *p<0.01.

32.4. TLR4 on Dendritic Cells Regulates Organ Damage AfterTrauma. K. R. Zettel,1 S. Korff,1 W. Shufesky,1 M. Scott,1 A.Morelli,1 T. R. Billiar1; 1University Of Pittsburgh - Surgery,Pittsburgh, PA, USA

Introduction:Using chimeric mice, we have previously shown that theacute organ injury following soft tissue injury and hemorrhagic shock(HS+STI) is dependent on TLR4 on both bone marrow-derived cellsand parenchymal cells. More recently we determined that, surpris-ingly, TLR4 on dendritic cells (DC)may play a role in early organdam-age in our mouse model of trauma. DCs are CD11c+ cells that arewidely known as antigen presenting cells, which modify T-cell re-sponses to infectious pathogens, although less is understood abouttheir role in regulating early inflammatory responses. Here we testthe hypothesis that DCs are the specific CD11c+ cell type that medi-ates early organ injury in a TLR4-dependent manner by using a novelstrategy in chimeric mice to selectively delete TLR4 fromCD11c+ DC.Methods:C57BL/6 (WT)micewere irradiated followed by the adoptivetransfer bone marrow derived from WT, DC TLR4 cell-selectiveknockout mice (DCTLR4ko), or mice containing diphtheria toxin-re-sponsive CD11c-DCs (CD11c-EGFP-DTR). When treated with diph-theria toxin, the CD11c+ cells in these mice are specifically ablated.These mice were then subjected to soft tissue injury and 2 hours ofhemorrhage shock (HS+STI), with 3x-shed blood volume resuscita-tion with lactated ringer’s solution. AST/ALT levels were analyzedat 6 hours asmarkers of early organ injury.Next, we adoptively trans-ferred WT or TLR4ko bone marrow-derived DC into TLR4ko mice 24hours prior to HS+STI and sacrificed them at 6 hours after HS+STIfor the analysis of early organ injury. Results: Mice depleted of DCs(CD11c-EGFP-DTR) had significantly decreased liver injury follow-ing STI+HS compared to WT chimeric mice (AST 1868 vs. 5442 U/L,p<0.05; ALT 2860 vs. 17572 U/L, p<0.05; n¼5). A similar reductionin liver injury was seen in chimeric mice that have DCs, but areTLR4-deficient on these DCs (DCTLR4ko chimeras) compared toWT chimeras (AST 1440 vs. 5442 U/L, p<0.05; ALT 2671 vs. 17572U/L, p<0.05, n¼6). Furthermore, adoptively transferring WT DCsinto TLR4komice prior to HS+STI caused an increase in organ injurycompared to transferring TLR4ko DCs into TLR4ko mice (AST 2654vs. 2541 U/L, p<0.05; ALT 7662 vs. 2728, p <0.05; n¼8).Conclusions: Surprisingly, using chimeric mice, we demonstratethat CD11c-positive cells play a TLR-4 dependent role in early organinjury following trauma and hemorrhagic shock. We further showthat of these cells, DCs are a subset of CD11c-positive cells that medi-ate this TLR-4 dependent organ injury.

32.5. AMPK Activation Minimizes Organ Injury by Decreas-ing Endothelial Activation and Inflammation. D. A.Escobar,1 H. Gomez,2 A. M. Botero-Quintero,1 B. C. Kautza,1

S. Stratimirovic,1 B. S. Zuckerbraun1,3; 1University OfPittsburgh - Surgery, Pittsburgh, PA, USA; 2University OfPittsburg - Critical Care Medicine, Pittsburgh, PA, USA; 3VAPittsburgh Healthcare System - Surgery, Pittsburgh, PA, USA

Introduction:Sepsis is a leading cause of deathworldwide.Mortality insepsis is most often attributed to the development of multiple organdysfunction and consequent organ failure. Previous studies in hemor-rhagic shock have demonstrated that activation of AMPactivated pro-tein kinase (AMPK) decreases inflammation and protects againstorgan injury. In sepsis, inflammation-mediated endothelial activationhas been associatedwith severity of disease. Thus, the objective of thisstudy was to show that AMPK activation regulates inflammation, en-dothelium activation and protects against organ injury in sepsis.Methods: Thirty C57BL/6 male mice, 6-8 weeks of age, and weighing20-25 g were divided into six groups: A. Cecal Ligation and Puncture(CLP); B. CLP+AICAR (100 mg/kg; AMPK stimulant); C. CLP+Com-pound C (30mg/kg; CoC; AMPK inhibitor); D. Sham; E. Sham+AI-CAR; F. Sham+CoC. Tissue and blood were collected at 8 hourspost-CLP. Endothelium activation was measured by I-CAM expres-sion via immunofluorescence. Renal functionwasmeasuredwith Cys-tatin C (ng/mL) and BUN/creatinine. Cytokine expression wasmeasured by ELISA kits and luminex assay. Data is presented asmean 6SD. Statistical significance is defined as p value <0.05. A

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similar experiment was conducted on isolated peritoneal macro-phages, divided into six groups. 1. Control; 2. LPS (100ng/mL); 3. Con-trol+AICAR (1mM); 4. LPS+AICAR; 5. Control+CoC (10mM); 6.LPS+CoC. Cells were pretreated with AICAR or CoC for 1 hour beforeLPS, and then treated for 4 hours. Cytokine levels were measuredwith ELISA kits. Results: Creatinine and cystatin C were elevatedin CLP as compared to sham (0.4460.18 vs 0.1360.115; p¼0.04, and317661297 vs 733.8682.2; p¼0.03, respectively), but not in CLP+AI-CAR vs CLP (0.260.0; p¼0.04, and 14026741; p¼0.01). Immunofluo-rescence images of liver and kidney tissue show an upregulation ofendothelial cell activation marker I-CAM in CLP groups as comparedto sham, but not in CLP+AICAR. TNF-a and IL-6 levels were213.86172.3 and 11066210.7, respectively in the CLP groups, butwere not elevated with the addition of AMPK activator AICAR(CLP+AICAR vs CLP) (39.97616.6 vs 213.86172.3; p¼0.03, and817.66146.5 vs 11066210.7; p¼0.04, respectively). When measuringthe same cytokines in isolated macrophages, LPS group has higherlevels of TNF-a and IL-6, as compared to control (18826583.3 vs0.20560.007; p¼0.044 and 241.76122.1 vs 5.6165.41; p¼0.11, respec-tively), but these were not elevated with the addition of AICAR(LPS+AICAR vs LPS) (7.1956122.1; p¼0.11, and 82.68628.25;p¼0.04, respectively). Conclusions: In this model, we demonstratethat upregulation of AMPK by AICAR minimizes organ injury by de-creasing inflammatory cytokines and decreasing endotheliumactivation. These data suggest that energetic pathways asAMPKmodulation play an important role in the development of organinjury.

32.6. SIRS Following Trauma Induces Loss Of Both CD3+ Andgd-T-Lymphocytes, But Clinical Resolution Is Associ-atedWith Differential Recovery Of Lymphocyte Popula-tions. W. A. Young,1 J. S. Young,1 S. F. Monaghan,1

C. S. Chung,1 W. G. Cioffi,1 A. Ayala,1 D. S. Heffernan1;1Brown University School Of Medicine - Division Of SurgicalResearch, Department Of Surgery, Providence, RI, USA

Introduction: Trauma, a leading cause of death, induces a SystemicInflammatory Response Syndrome (SIRS) with immune-paralysisand T-lymphocyte anergy. We have previously demonstratedtrauma induces significant lymphocyte dysfunction. A dis-coordi-nated trauma immune response associates with organ failure,sepsis and mortality. GammaDelta-T-cells (gd T-cells), innate reg-ulatory lymphocyte subpopulations play essential roles coordinat-ing immune response to sepsis, burns and wound healing. Thereis, however, a paucity of data on early trauma-induced gd T-cellsalterations, specifically absolute numbers of gd T-cells. Further,there is no data addressing differences in lymphocyte and subpop-ulation responses in relation to resolution of clinical SIRS state.Methods: Prospectively enrolled Trauma Intensive Care Unit(TICU) patients displaying SIRS criteria [2 or more of: tachycardia,respiratory distress, hyper- or hypo-thermia, leukocytosis or leucope-nia] within 24 hours of admission. Patients were followed into SIRSresolution. Daily white cell count allowed calculation of absolute cellcounts. Monoclonal antibody whole blood staining with CD3, TCR-pan gd, and CD69 (marker of early activation) was analyzed usingFACS flow cytometry. 10 healthy controls were also enrolled. Statisti-cal analysis was Chi-squared, Mann-Whitney-U and one way AN-OVA. Results: 52 enrolled TICU patients (67% male; aveage¼46years), 29 followed through SIRS resolution. SIRS patients,compared with healthy controls, displayed leukocytosis (12.6 vs6.3x10^9; p¼0.006), lymphopenia (CD3+) as a percentage (8.4% vs26.4%; p<0.001), and absolute number (8.1 vs 16.4x10^8; p<0.001)with increased lymphocte activation (CD69+CD3+) (10.5% vs 3.5%;p¼0.02). Similarly, the gd-T-cell subpopulation of CD3+Lymphocytes,SIRS induced marked reduction in gd-T-cells both as % of CD3+cells(2.3% vs 5.2%; p<0.001) and absolute number (1.8 vs 8.1x10̂7;p<0.001) with increased activation of these gd-T-cells (18.6% vs

5.7%; p¼0.003). Within the 29 patients followed into resolution ofSIRS, compared to the SIRS state, there was normalization of the leu-kocytosis (7.2 vs 12.6; p¼0.03) but persistence of CD3+lymphocyteloss as a percent (5.1% vs 8.4%; p¼0.15) and absolute number (4.7vs 7.5x10̂8; p¼0.15). Unlike the persistent lymphocyte loss, patientswho resolved SIRSdisplayed near normalization of gd-T-cells as a per-centage (4.6% vs 2.3%; p¼0.73) and absolute number (7.3 vs 1.8x10̂7;p<0.001) with a persistence of the percentage of activated gd-T-cells(16.6% vs 18.6%; p¼0.84). Conclusions: SIRS in trauma patients is as-sociated with loss of CD3+T-cells and innate regulatory gd-T-cells.During resolution of trauma related SIRS, differential preservationand recovery mechanisms exist, potentially critical to the regulatoryrole of gd-T-cells. Understanding these mechanisms will be essentialfor future regulatory and therapeutic targets.

32.7. NF-kB and Heme Oxygenase-1, but not Activator Pro-tein-1, Mediate Interferon Gamma Induced Productionof Macrophage Derived Chemokine (MDC/CCL22) in Al-veolar Macrophages. J. M. Sutton,1 P. L. Jernigan,1 R. S.Hoehn,1 J. R. Richter,1 R. M. Schuster,1 A. B. Lentsch,1 T. A.Pritts1; 1University Of Cincinnati - Department Of Surgery,Division Of Trauma And Critical Care, Cincinnati, OH,USA

Introduction: Data from our and other laboratories suggest thatpulmonary macrophages play a critical role in the pathogenesisof acute respiratory distress syndrome (ARDS) after hemorrhagicshock and resuscitation. Recent data from our laboratory indicatea novel and critical role for Macrophage Derived Chemokine(MDC/CCL22) as a proximal regulator of acute lung inflammationfollowing resuscitation, with further experiments implicating alve-olar macrophages as a significant source of MDC. However, littleis currently known regarding the transcription factors regulatingMDC production in alveolar macrophages in this setting. As NF-kB and heme oxygenase-1 pathways have previously been impli-cated in regulation of MDC production in keratinocytes, we hy-pothesized that similar pathways are utilized in the productionof MDC within alveolar macrophages. Methods: An immortalizedmurine line of alveolar macrophages (AMJ2-C11) were cultured permanufacturer recommendations. Cultured alveolar macrophageswere incubated with increasing doses of pyrrolidine dithiocarbamate(PDTC), sulforaphane, or SR11302, which are inhibitors of NF-kB,heme oxygenase-1, and activator protein-1 transcription factors, re-spectively. Following a 2h incubation period, cells were then treatedwith IFNg (10 ng/mL), as we have demonstrated that hemorrhagicshock followed by resuscitationwith packed red blood cells leads to in-creased systemic levels of IFNg. Cellular media was collected at 24hand analyzed by ELISA to determine supernatant MDC levels. One-way ANOVA with Tukey post-hoc tests were used to compare MDClevels across treatment groups.Results: IFNg-induced activation of al-veolar macrophages resulted in significantly increased MDC produc-tion compared to control cells (Figure 1A-B). Pretreatment withPDTC, an NF-kB inhibitor, resulted in significantly attenuatedMDC production at the 50 mM and 100 mM doses (p < 0.001) but notat the 1 mM and 10 mM doses (Figure 1A). Pretreatment with sulfora-phane, an inhibitor of heme oxygenase-1, also resulted in significantlyreducedMDC levels in a dose-dependent fashion (Figure 1B; 10 mM, p< 0.05; 20 and 50 mM, p < 0.001). Alternatively, treatment with theactivator protein-1 inhibitor SR11302 had no effect on MDC produc-tion even at doses as high as 100 mM (data not shown). Conclusions:Alveolar macrophages significantly increase their production ofMDC after exposure to IFNg, contributing to subsequent pulmonaryinflammation. NF-kB and heme oxygenase-1 and are key transcrip-tion factors in MDC production in these cells and provide potentialtherapeutic targets to mitigate acute lung injury after hemorrhagicshock and resuscitation.