Polymicrobial Sepsis In Mice Research Articles

The Bacterial Component Flagellin Induces Anti-Sepsis Protection Through TLR-5, IL-1RN and VCAN During Polymicrobial Sepsis in Mice

Background: The present study was designed to observe the effects of the bacterial component flagellin on anti-sepsis protection through TLR-5, VCAN and IL-1RN. Methods: A clinically relevant model of sepsis was induced by cecal ligation and puncture (CLP). An in vitro culture of endothelial cells was analyzed. Results: Flagellin induced anti-sepsis protection through inhibition of inflammation and induction of endothelial proliferation by down-regulating the expression of TLR 3, TLR 4, and IL-1RN and promoting the expression of VCAN in mice 24 h post-CLP. In vitro, flagellin promoted the proliferation of endothelial cells. These effects could be inhibited by transfection of endothelial cells with VCAN siRNA or IL-1RN over-expression constructs. VCAN expression decreased after transfection of the cells with an IL-1RN over-expression construct and increased after transfection of the cells with an IL-1RN siRNA construct. IL-1RN expression remained unchanged after transfection of the cells with VCAN over-expression or siRNA constructs. Conclusions: These data suggest that flagellin pretreatment promoted anti-sepsis protection through the TLR-5, IL-1RN and VCAN pathway. This pathway is necessary to mediate endothelial repair and thereby promote survival following sepsis challenge.

Circulating microRNA-223 serum levels do not predict sepsis or survival in patients with critical illness.

Background and Aims. Dysregulation of miR-223 was recently linked to various diseases associated with systemic inflammatory responses such as type 2 diabetes, cancer, and bacterial infections. However, contradictory results are available on potential alterations of miR-223 serum levels during sepsis. We thus aimed to evaluate the diagnostic and prognostic value of miR-223 serum concentrations in patients with critical illness and sepsis. Methods. We used i.v. injection of lipopolysaccharide (LPS) as well as cecal pole ligation and puncture (CLP) for induction of polymicrobial sepsis in mice and measured alterations in serum levels of miR-223. These results from mice were translated into a large and well-characterized cohort of critically ill patients admitted to the medical intensive care unit (ICU). Finally, results from analysis in patients were correlated with clinical data and extensive sets of routine and experimental biomarkers. Results. Although LPS injection induced moderately elevated serum miR-223 levels in mice, no significant alterations in miR-223 serum levels were found in mice after CLP-induced sepsis. In accordance with these results from animal models, serum miR-223 levels did not differ between critically ill patients and healthy controls. However, ICU patients with more severe disease (APACHE-II score) showed moderately reduced circulating miR-223. Strikingly, no differences in miR-223 levels were found in critically ill patients with or without sepsis, and serum levels of miR-223 did not correlate with classical markers of inflammation or bacterial infection. Finally, low miR-223 serum levels were moderately associated with an unfavorable prognosis of patients during the ICU treatment but did not predict long-term mortality. Conclusion. Recent reports on alterations in miR-223 serum levels during sepsis revealed contradictory results, preventing a potential use of this miRNA in clinical routine. We clearly show that miR-223 serum levels do not reflect the presence of sepsis neither in mouse models nor in a large cohort of ICU patients and do not indicate clinical outcome of critically ill patients. Thus miR-223 serum levels should not be used as a biomarker in this setting.

Betulinic acid attenuates lung injury by modulation of inflammatory cytokine response in experimentally-induced polymicrobial sepsis in mice

Sepsis commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity and mortality. Septic ALI is characterized by excessive production of proinflammatory mediators. It remained refractory to present therapies and new therapies need to be developed to improve further clinical outcomes. Betulinic acid (BA), a pentacyclic lupane group triterpenoid has been shown to have anti-inflammatory activities in many studies. However, its therapeutic efficacy in polymicrobial septic ALI is yet unknown. Therefore, we investigated the effects of BA on septic ALI using cecal ligation and puncture (CLP) model in mice. Vehicle or BA (3, 10, and 30mg/kg) was administered intraperitoneally, 3 times (0, 24 and 48h) before CLP and CLP was done on 49thh of the study. Survival rate was observed till 120h post CLP. Lung tissues were collected for analysis by sacrificing mice 18h post CLP. BA at 10 and 30mg/kg dose significantly reduced sepsis-induced mortality and lung injury as implied by attenuated lung histopathological changes, decreased protein and neutrophils infiltration. BA also decreased lung NF-κB expression, cytokine, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and matrix metalloproteinase-9 levels. These evidences suggest that, the protective effects of BA on lungs are associated with defending action against inflammatory response and BA could be a potential modulatory agent of inflammation in sepsis-induced ALI.

Double blockade of CD14 and complement C5 abolishes the cytokine storm and improves morbidity and survival in polymicrobial sepsis in mice.

Sepsis and septic shock, caused by an excessive systemic host-inflammatory response, are associated with high morbidity and mortality. The complement system and TLRs provide important pattern recognition receptors initiating the cytokine storm by extensive cross-talk. We hypothesized that double blockade of complement C5 and the TLR coreceptor CD14 could improve survival of experimental polymicrobial sepsis. Mice undergoing cecal ligation and puncture (CLP)-induced sepsis were treated with neutralizing anti-CD14 Ab biG 53, complement C5 inhibitor coversin (Ornithodoros moubata C inhibitor), or a combination thereof. The inflammatory study (24-h observation) revealed statistically significant increases in 22 of 24 measured plasma biomarkers in the untreated CLP group, comprising 14 pro- and anti-inflammatory cytokines and 8 chemokines, growth factors, and granulocyte activation markers. Single CD14 or C5 blockade significantly inhibited 20 and 19 of the 22 biomarkers, respectively. Combined CD14 and C5 inhibition significantly reduced all 22 biomarkers (mean reduction 85%; range 54-95%) compared with the untreated CLP group. Double blockade was more potent than single treatment and was required to significantly inhibit IL-6 and CXCL1. Combined inhibition significantly reduced morbidity (motility and eyelid movement) and mortality measured over 10 d. In the positive control CLP group, median survival was 36 h (range 24-48 h). Combined treatment increased median survival to 96 h (range 24-240 h) (p = 0.001), whereas survival in the single-treatment groups was not significantly increased (median and range for anti-CD14 and anti-C5 treatment were 36 h [24-48 h] and 48 h [24-96 h]). Combined with standard intervention therapy, specific blockade of CD14 and C5 might represent a promising new therapeutic strategy for treatment of polymicrobial sepsis.

Levels of Circulating miR-133a Are Elevated in Sepsis and Predict Mortality in Critically Ill Patients

Serum levels of microRNA have been proposed as biomarkers in various inflammatory diseases. However, up to now, their clinical relevance in critical illness and sepsis is unclear. Single-center clinical study. Fourteen-bed medical ICU of the University Hospital Aachen, university laboratory research unit. Experimental sepsis model in C57Bl/6 mice; 223 critically ill patients in comparison with 76 healthy volunteers. We used the model of cecal pole ligation and puncture for induction of polymicrobial sepsis in mice and measured alterations in serum levels of six different microRNAs with a known function in inflammatory diseases upon induction of septic disease. These results from mice were translated into a large and well-characterized cohort of critically ill patients admitted to the medical ICU. Serum miR-133a was then measured in 223 critically ill patients (138 with sepsis and 85 without sepsis) and 76 controls and associated with disease severity, organ failure, and prognosis. Significant alterations of miR-133a, miR-150, miR-155, and miR-193b* were found in mice after cecal pole ligation and puncture-induced sepsis. Among all regulated microRNAs, miR-133a displayed the most prominent and concordant up-regulation in sepsis, and this microRNA was therefore chosen for further investigation in the human. Here, significantly elevated miR-133a levels were found in critically ill patients at ICU admission, when compared with healthy controls, especially in patients with sepsis. Correlation analyses revealed significant correlations of miR-133a with disease severity, classical markers of inflammation and bacterial infection, and organ failure. Strikingly, high miR-133a levels were predictive for an unfavorable prognosis and represented a strong independent predictor for both ICU and long-term mortality in critically ill patients. miR-133a serum levels were significantly elevated in critical illness and sepsis. High miR-133a levels were associated with the severity of disease and predicted an unfavorable outcome of critically ill patients.

Uncoupling of Peripheral and Master Clock Gene Rhythms by Reversed Feeding Leads to an Exacerbated Inflammatory Response After Polymicrobial Sepsis in Mice

Reversed feeding uncouples peripheral and master clock gene rhythms and leads to an increased risk of disease development. The aim of this study was to determine the effects of clock gene uncoupling on sepsis-induced inflammation using a mouse cecal ligation and puncture (CLP) model. C57BL/6N mice were entrained to a 12-h light-dark cycle (lights on at 7 AM). Mice were permitted ad libitum feeding either during the night (7 PM-7 AM) or the nonphysiological light phase (7 AM-7 PM) for a week before CLP. In daytime-fed mice, phase inversion of clock gene expression was observed in the liver, but not in the suprachiasmatic nucleus. Daytime-fed mice also had decreased body weight and food intake. Survival rate was significantly lower in daytime-fed mice (29%) compared with nighttime-fed mice (54%) 72 h after CLP (P = 0.03). Serum levels of interleukin 6 (IL-6), tumor necrosis factor α, high mobility group box 1, IL-1α, IL-9, eotaxin, and granulocyte colony-stimulating factor increased in daytime-fed mice compared with nighttime-fed mice after CLP. Baseline expression levels of sirtuin peroxisome 1 and proliferator-activated receptor γ coactivator 1α in the liver decreased in daytime-fed mice compared with nighttime-fed mice. Thus, daytime feeding induces clock gene uncoupling, which leads to decreased expression of longevity-related and energy metabolism proteins. Daytime feeding may also increase the levels of inflammatory cytokines, thereby increasing mortality in a mouse sepsis model. Our findings suggest that uncoupling of peripheral and master clock gene rhythms by reversed feeding exacerbates inflammatory responses.

Heparanase mediates renal dysfunction during early sepsis in mice

Heparanase, a heparan sulfate-specific glucuronidase, mediates the onset of pulmonary neutrophil adhesion and inflammatory lung injury during early sepsis. We hypothesized that glomerular heparanase is similarly activated during sepsis and contributes to septic acute kidney injury (AKI). We induced polymicrobial sepsis in mice using cecal ligation and puncture (CLP) in the presence or absence of competitive heparanase inhibitors (heparin or nonanticoagulant N-desulfated re-N-acetylated heparin [NAH]). Four hours after surgery, we collected serum and urine for measurement of renal function and systemic inflammation, invasively determined systemic hemodynamics, harvested kidneys for histology/protein/mRNA, and/or measured glomerular filtration by inulin clearance. CLP-treated mice demonstrated early activation of glomerular heparanase with coincident loss of glomerular filtration, as indicated by a >twofold increase in blood urea nitrogen (BUN) and a >50% decrease in inulin clearance (P < 0.05) in comparison to sham mice. Administration of heparanase inhibitors 2 h prior to CLP attenuated sepsis-induced loss of glomerular filtration rate, demonstrating that heparanase activation contributes to early septic renal dysfunction. Glomerular heparanase activation was not associated with renal neutrophil influx or altered vascular permeability, in marked contrast to previously described effects of pulmonary heparanase on neutrophilic lung injury during sepsis. CLP induction of renal inflammatory gene (IL-6, TNF-α, IL-1β) expression was attenuated by NAH pretreatment. While serum inflammatory indices (KC, IL-6, TNF-α, IL-1β) were not impacted by NAH pretreatment, heparanase inhibition attenuated the CLP-induced increase in serum IL-10. These findings demonstrate that glomerular heparanase is active during sepsis and contributes to septic renal dysfunction via mechanisms disparate from heparanase-mediated lung injury.

Physical Exercise Induces Specific Adaptations Resulting in Reduced Organ Injury and Mortality During Severe Polymicrobial Sepsis

High physical activity levels are associated with wide-ranging health benefits, disease prevention, and longevity. In the present study, we examined the impact of regular physical exercise on the severity of organ injury and survival probability, as well as characteristics of the systemic immune and metabolic response during severe polymicrobial sepsis. Animal study. University laboratory. Male C57BL/6N mice. Mice were trained for 6 weeks by treadmill and voluntary wheel running or housed normally. Polymicrobial sepsis in mice was induced by injection of fecal slurry. Subsequently, mice were randomized into the following groups: healthy controls, 6 hours postsepsis, and 24 hours postsepsis. Blood and organ samples were collected and investigated by measuring clinical chemistry variables, cytokines, plasma metabolites, and bacterial clearance. Organ morphology and damage were characterized by histological staining. Physical exercise improved survival and the ability of bacterial clearance in blood and organs. The release of pro- and anti-inflammatory cytokines, including interleukin-6 and interleukin-10, was diminished in trained compared to untrained mice during sepsis. The sepsis-associated acute kidney tubular damage was less pronounced in pretrained animals. By metabolic profiling and regression analysis, we detected lysophosphatidylcholine 14:0, tryptophan, as well as pimelylcarnitine linked with levels of neutrophil gelatinase-associated lipocalin representing acute tubular injury (corrected R=0.910; p<0.001). We identified plasma lysophosphatidylcholine 16:0, lysophosphatidylcholine 17:0, and lysophosphatidylcholine 18:0 as significant metabolites discriminating between trained and untrained mice during sepsis. Regular physical exercise reduces sepsis-associated acute kidney injury and death. As a specific mechanism of exercise-induced adaptation, we identified various lysophosphatidylcholines that might function as surrogate for improved outcome in sepsis.

Lipopeptides rather than lipopolysaccharide favor the development of dendritic cell dysfunction similar to polymicrobial sepsis in mice

We investigated whether the dysfunction of dendritic cells (DC) that develops during polymicrobial sepsis is mimicked by systemic administration of the Toll-like receptor (TLR) 4 agonist lipopolysaccharide (LPS) or of the TLR2 agonist Pam3-Cys-Ser-Lys4 (P3CSK4). BALB/c mice underwent cecal ligation and puncture (CLP) or sham operation or received a single i.p. injection of LPS (30 mg/kg body weight), P3CSK4 (10 mg/kg body weight), or saline as control. Purified splenic DC and in-vitro-generated DC from bone marrow were analyzed in terms of surface marker expression, cytokine secretion, and antigen-specific T-cell activation in vivo. Splenic DC were suppressed in IL-12 secretion 12 h after LPS and P3CSK4 administration but released increased levels of IL-12 4 days after TLR agonist application, unlike DC from CLP mice. Polymicrobial sepsis and TLR agonists caused a loss of DC in the spleen but led to the expansion of diverse DC subsets. DC that differentiated from bone marrow after P3CSK4 but not after LPS application resembled DC from CLP mice regarding cytokine secretion and impaired Th1-cell polarization. The development of DC dysfunction during sepsis is at least partly mimicked by TLR2 agonists rather than TLR4 agonists.

End Binding protein 3 regulates calcium signaling and permeability of the endothelial barrier

End binding (EBs) proteins bind growing microtubule (MT) plus ends and promote MT growth. We have recently shown that Vascular Endothelial (VE)‐cadherin outside‐in signaling regulates phosphorylation of EB3 to suppress MT persistent growth and thus allowing for the assembly of adherens junctions (AJs). Depletion of EB3 has no effect on either MT dynamics or formation of (AJs) in confluent endothelial monolayers. However, depletion of EB3 significantly attenuates cell shape change and permeability increase in cells subjected to pro‐inflammatory mediator thrombin. EB3 interacts with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) and facilitates agonist‐induced clustering of IP3R3 and IP3‐dependent release of calcium from intracellular stores. Cell permeant variant of IP3R3‐derivative peptide containing EB3 consensus motif reduces the interaction between EB3 and IP3R3 and mitigates increased endothelial permeability response. Furthermore, infusion of this peptide in ex vivo murine lung preparation attenuates increase in lung vascular liquid permeability (Kf,c) in response to activation of the Protease‐Activated Receptor‐1 (PAR‐1). Intravenous injection of this peptide prevents lung injury and reduces the lethality of polymicrobial sepsis in mice. These data indicate that EB3 regulates calcium signaling in endothelium and facilitates pulmonary vascular leak in disease states.This work was supported by NIH grants R01 HL103922 and Giles F. Filley Memorial Award to Y. K.; R01 HL 45638 to A.B. M.; T32 HL07829–17 support to M. G.

Effects of dexmedetomidine on early and late cytokines during polymicrobial sepsis in mice

We investigated whether dexmedetomidine provided protective effects on cecal ligation and puncture (CLP)-induced septic mice, through suppressing the expression of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6)] and high mobility group box 1 (HMGB1). The model of sepsis was set up by CLP in 136 male BALB/c mice (40 mice for survival studies and 96 for cytokine studies) which were divided into four groups, including a C, CLP, DEX+CLP and CLP+DEX group. The serum levels of TNF-α, IL-6 and HMGB1 were detected at 6, 12, 24 and 48h after operations, and lung HMGB1 mRNA were analyzed at 24 and 48h. The mortality rates were calculated 7days after the operations. The mortality rates 7days after operations were significantly lower in the CLP+DEX (50%) and DEX+CLP (30%) groups than in the CLP group (90%). Serum concentrations of IL-6 and TNF-α decreased significantly in dexmedetomidine administration groups compared with the CLP group. The levels of HMGB1 and lung HMGB1 mRNA were lower in the dexmedetomidine administration groups than in the CLP group. There was a significant correlation between lung HMGB1 mRNA and serum HMGB1(r=0.858). Dexmedetomidine could reduce the mortality rate and inhibit pro-inflammatory cytokine responses during polymicrobial sepsis in mice.

Transgenic Expression of FoxM1 Promotes Endothelial Repair following Lung Injury Induced by Polymicrobial Sepsis in Mice

Enhancing endothelial barrier integrity for the treatment of acute lung injury (ALI) is an emerging novel therapeutic strategy. Our previous studies have demonstrated the essential role of FoxM1 in mediating endothelial regeneration and barrier repair following lipopolysaccharide-induced lung injury. However, it remains unclear whether FoxM1 expression is sufficient to promote endothelial repair in experimental models of sepsis. Here, employing the FoxM1 transgenic (FoxM1 Tg) mice, we showed that transgenic expression of FoxM1 promoted rapid recovery of endothelial barrier function and survival in a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP). We observed lung vascular permeability was rapidly recovered and returned to levels similar to baseline at 48 h post-CLP challenge in FoxM1 Tg mice whereas it remained markedly elevated in WT mice. Lung edema and inflammation were resolved only in FoxM1 Tg mice at 24 h post-CLP. 5-bromo-2-deoxyuridine incorporation assay revealed a drastic induction of endothelial proliferation in FoxM1 Tg lungs at 24h post-CLP, correlating with early induction of expression of FoxM1 target genes essential for cell cycle progression. Additionally, deletion of FoxM1 in endothelial cells, employing the mouse model with endothelial cell-restricted disruption of FoxM1 (FoxM1 CKO) resulted in impaired endothelial repair following CLP challenge. Together, these data suggest FoxM1 expression in endothelial cells is necessary and sufficient to mediate endothelial repair and thereby promote survival following sepsis challenge.

Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis

Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased. Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS. Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis. Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.

A Historical Perspective on Sepsis

In North America, approximately 700,000 cases of sepsis occur each year, with mortality ranging between 30% and 50%. The American Journal of Pathology has featured numerous articles on the topic, revealing mechanistic insights gleaned from both experimental rodent models and human sepsis. Nonetheless, there remains urgent need to determine the basis for sepsis-related complications and how they can be avoided, as well as how they can be most effectively treated once recognized. This historical perspective reviews what we currently understand about the mechanisms of sepsis, as well as the barriers that remain in our treatment strategies.

Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice

IntroductionAlthough the formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. This study was performed in order to evaluate the influence of NETs depletion by administration of recombinant human (rh)DNase on bacterial spreading, PMN tissue infiltration and inflammatory response in a mouse model of polymicrobial sepsis.MethodsIn a prospective controlled double-armed animal trial, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, mice were treated with rhDNase or phosphate buffered saline, respectively. Survival, colony forming unit (CFU) counts in the peritoneal cavity, lung, liver and blood were determined. PMN and platelet counts, IL-6 and circulating free (cf)-DNA/NETs levels were monitored. PMN infiltration, as well as organ damage, was analyzed histologically in the lungs and liver. Capability and capacity of PMN to form NETs were determined over time.Resultscf-DNA/NETs were found to be significantly increased 6, 24, and 48 hours after CLP when compared to the levels determined in sham and naïve mice. Peak levels after 24 hours were correlated to enhanced capacity of bone marrow-derived PMN to form NETs after ex vivo stimulation with phorbol-12-myristate-13-acetate at the same time. rhDNase treatment of mice resulted in a significant reduction of cf-DNA/NETs levels 24 hours after CLP (P < 0.001). Although overall survival was not affected by rhDNase treatment, median survival after 24 hours was significantly lower when compared with the CLP group (P < 0.01). In mice receiving rhDNase treatment, CFU counts in the lung (P < 0.001) and peritoneal cavity (P < 0.05), as well as serum IL-6 levels (P < 0.001), were found to be already increased six hours after CLP. Additionally, enhanced PMN infiltration and tissue damage in the lungs and liver were found after 24 hours. In contrast, CFU counts in mice without rhDNase treatment increased later but more strongly 24 hours after CLP (P < 0.001). Similarly, serum IL-6 levels peaked after 24 hours (P < 0.01).ConclusionsThis study shows, for the first time, that depletion of NETs by rhDNase administration impedes the early immune response and aggravates the pathology that follows polymicrobial sepsis in vivo.

Protective effects of finasteride on the pulmonary immune response in a combined model of trauma-hemorrhage and polymicrobial sepsis in mice

Literature supports findings about a gender specific outcome following multiple trauma. Male sex hormones such as dihydrotestosterone (DHT) exert deleterious effects on the posttraumatic immune response whereas increased estradiol concentrations are correlated with improved outcome. Pretreatment with the 5α-reductase inhibitor finasteride resulted in an improved outcome following trauma-hemorrhage (TH) in mice. The present study tested the hypothesis that finasteride exerts beneficial effects on the posttraumatic immune response also in a combined setting of TH and sepsis when administered during the resuscitation process. Material and MethodsMale C57BL/6N-mice were subjected to TH (blood pressure, 35mm Hg, 60min) followed by finasteride application and fluid resuscitation. Thereafter, finasteride was administered every 12h. 24h after TH, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Plasma cytokines (MIP-1α, MIP-1β, TNF-α, MCP-1, IL-6), productive capacity by alveolar macrophages (AM) and systemic estradiol levels were determined 4h thereafter. The expression of pro-inflammatory mediators in lung tissue was evaluated by PCR. Pulmonary infiltration of PMN was determined by immunohistochemical staining. ResultsFinasteride treatment resulted in a reduced posttraumatic cytokine secretion of AM as well as in a decreased concentration of MCP-1 and MIP-1β in lung tissue. Systemic estradiol levels were increased following finasteride treatment. ConclusionFinasteride mediates salutary effects on the pulmonary immune response using a therapeutical approach following TH–CLP in mice. Thus, finasteride might represent a relevant therapeutic substance following major trauma also in the clinical setting.

Microtubule‐associated protein EB3 regulates calcium signaling and facilitates increase in endothelial permeability

End binding proteins (EBs) are highly conserved microtubule plus‐end tracking accessory factors that bind to growing microtubule ends and suppress microtubule catastrophe events. We investigated the roles of EB1 and EB3 in regulating endothelial cytoskeletal dynamics and cell shape change, the primary determinants of the permeability of endothelial barrier. Protein depletion experiments showed that EB3, but not EB1, facilitates thrombin‐induced release of calcium from endoplasmic reticulum (ER), and thereby activates calcium entry from extracellular stores. We observed that EB3 interacts with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) in lung microvasculature. Cell permeant variant of IP3R3 derivative peptide containing EB3 consensus motif reduced the interaction between EB3 and IP3R3 and mitigated increased endothelial permeability response associated with the cell shape changes. Furthermore, infusion of IP3R3 derivative peptide in ex vivo murine lung preparation attenuates the increase in lung vascular liquid permeability (Kf,c) in response to activation of the Protease‐Activated Receptor ‐1. Intravenous injection of this peptide prevents lung injury and reduces the lethality of polymicrobial sepsis in mice. These data indicate that EB3 regulates calcium signaling in endothelium and facilitates pulmonary vascular leak in disease states.

Peroxisome Proliferator–activated Receptor γ–induced T Cell Apoptosis Reduces Survival during Polymicrobial Sepsis

Despite intensive research, sepsis displays the most prevalent cause of death on intensive care units. The hallmark of sepsis is an overshooting T-cell death that reduces host defense mechanisms and that is associated with poor patient survival. Previous in vitro studies revealed that the expression of the transcription factor peroxisome proliferator-activated receptor (PPAR) γ was increased in isolated T cells of patients with sepsis. We determined the importance of targeting PPARγ for sepsis treatment and underlying molecular mechanisms for T-cell apoptosis in vivo. To mimic human systemic inflammation and septic conditions, we used a nonlethal endotoxemia and a lethal cecum ligation and puncture polymicrobial sepsis model. PPARγ inhibition in T cells with either the PPARγ antagonist GW9662 or a newly generated T cell-specific PPARγ knockout (Tc-PPARγ(-/-)) mice provided a survival advantage during polymicrobial sepsis in mice, which correlated with abrogated T-cell depletion in both in vivo models. Pathway analysis revealed increased antiapoptotic IL-2 and Bcl-2 expression, and activated prosurvival PI3K/Akt signaling under PPARγ-deficient conditions. In line, neutralizing IL-2 in Tc-PPARγ(-/-) mice resulted in T-cell apoptosis and increased mortality. Our results provide evidence for a pivotal involvement of PPARγ in T-cell depletion by activating two important apoptosis pathways, and subsequently provoking the breakdown of defense mechanisms during systemic inflammation and sepsis.

NOS3 PROTECTS AGAINST SYSTEMIC INFLAMMATION AND MYOCARDIAL DYSFUNCTION IN MURINE POLYMICROBIAL SEPSIS

NO has been implicated in the pathogenesis of septic shock. However, the role of NO synthase 3 (NOS3) during sepsis remains incompletely understood. Here, we examined the impact of NOS3 deficiency on systemic inflammation and myocardial dysfunction during peritonitis-induced polymicrobial sepsis. Severe polymicrobial sepsis was induced by colon ascendens stent peritonitis (CASP) in wild-type (WT) and NOS3-deficient (NOS3KO) mice. NOS3KO mice exhibited shorter survival time than did WT mice after CASP. NOS3 deficiency worsened systemic inflammation assessed by the expression of inflammatory cytokines in the lung, liver, and heart. Colon ascendens stent peritonitis markedly increased the number of leukocyte infiltrating the liver and heart in NOS3KO but not in WT mice. The exaggerated systemic inflammation in septic NOS3KO mice was associated with more marked myocardial dysfunction than in WT mice 22 h after CASP. The detrimental effects of NOS3 deficiency on myocardial function after CASP seem to be caused by impaired Ca handling of cardiomyocytes. The impaired Ca handling of cardiomyocytes isolated from NOS3KO mice subjected to CASP was associated with depressed mitochondrial ATP production, a determinant of the Ca cycling capacity of sarcoplasmic reticulum Ca-ATPase. The NOS3 deficiency-induced impairment of the ability of mitochondria to produce ATP after CASP was at least in part attributable to reduction in mitochondrial respiratory chain complex I activity. These observations suggest that NOS3 protects against systemic inflammation and myocardial dysfunction after peritonitis-induced polymicrobial sepsis in mice.

Saponin fraction from Astragalus membranaceus roots protects mice against polymicrobial sepsis induced by cecal ligation and puncture by inhibiting inflammation and upregulating protein C pathway

Sepsis remains the leading cause of death in intensive care units. Uncontrolled systemic inflammation and an impaired protein C pathway are two important contributors to sepsis pathophysiology. Based on the beneficial effects of the saponin fraction from Astragalus membranaceus roots (SAM) against inflammation, liver dysfunction, and endothelium injury, we investigated the potential protective roles and underlying mechanisms of SAM on polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. SAM, orally administered 1 h before and after CLP, significantly elevated the survival rate of mice. At 96 h after CLP operation, all mice in the model group died, whereas 33.3% of mice in the SAM (400 mg/kg)-treated group survived. SAM attenuated both inflammatory factors and their abilities to induce tissue dysfunction, which was mainly evidenced by decreased infiltration of polymorphonuclear leukocytes, tissue edema, and lung wet-to-dry weight ratio, lowered levels of myeloperoxidase (MPO), nitric oxide (NO), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum, as well as downregulated expressions of iNOS and IL-1beta mRNA in livers. Furthermore, we addressed the effects of SAM on the protein C (PC) pathway, closely linked with sepsis. In CLP-induced septic mice, SAM elevated the impaired expression of PC mRNA in livers. In vitro, SAM reversed the decreased expressions of thrombomodulin (TM) and endothelial PC receptor (EPCR) mRNA induced by lipopolysaccharide (LPS) in endothelial cells. These findings suggest that SAM is able to restore the impaired protein C pathway. Taken together, the current study demonstrates that SAM has protective effects on polymicrobial sepsis in mice. The mechanisms of action involve anti-inflammation and upregulation of the PC pathway.