Outcome Of Polymicrobial Sepsis Research Articles

Formyl Peptide Receptor Type 2 Deficiency in Myeloid Cells Amplifies Sepsis-Induced Cardiac Dysfunction

Using a global formyl peptide receptor (Fpr) 2 knockout mouse colony, we have reported the modulatory properties of this pro-resolving receptor in polymicrobial sepsis. Herein, we have used a humanized FPR2 (hFPR2) mouse colony, bearing an intact or a selective receptor deficiency in myeloid cells to dwell on the cellular mechanisms. hFPR2 mice and myeloid cell-specific hFPR2 KO (KO) mice were subjected to cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Compared with hFPR2 mice, CLP caused exacerbated cardiac dysfunction (assessed by echocardiography), worsened clinical outcome, and impaired bacterial clearance in KO mice. This pathological scenario was paralleled by increased recruitment of pro-inflammatory monocytes and reduced M2-like macrophages within the KO hearts. In peritoneal exudates of KO mice, we quantified increased neutrophil and MHC II+ macrophage numbers but decreased monocyte/macrophage and MHC II- macrophage recruitment. hFPR2 upregulation was absent in myeloid cells, and local production of lipoxin A4 was reduced in septic KO mice. Administration of the FPR2 agonist annexin A1 (AnxA1) improved cardiac function in hFPR2 septic mice but had limited beneficial effects in KO mice, in which the FPR2 ligand failed to polarize macrophages toward an MHC II- phenotype. In conclusion, FPR2 deficiency in myeloid cells exacerbates cardiac dysfunction and worsens clinical outcome in polymicrobial sepsis. The improvement of cardiac function and the host immune response by AnxA1 is more effective in hFPR2-competent septic mice.

Lactate induces vascular permeability via disruption of VE-cadherin in endothelial cells during sepsis.

Circulating lactate levels are a critical biomarker for sepsis and are positively correlated with sepsis-associated mortality. We investigated whether lactate plays a biological role in causing endothelial barrier dysfunction in sepsis. We showed that lactate causes vascular permeability and worsens organ dysfunction in CLP sepsis. Mechanistically, lactate induces ERK-dependent activation of calpain1/2 for VE-cadherin proteolytic cleavage, leading to the enhanced endocytosis of VE-cadherin in endothelial cells. In addition, we found that ERK2 interacts with VE-cadherin and stabilizes VE-cadherin complex in resting endothelial cells. Lactate-induced ERK2 phosphorylation promotes ERK2 disassociation from VE-cadherin. In vivo suppression of lactate production or genetic depletion of lactate receptor GPR81 mitigates vascular permeability and multiple organ injury and improves survival outcome in polymicrobial sepsis. Our study reveals that metabolic cross-talk between glycolysis-derived lactate and the endothelium plays a critical role in the pathophysiology of sepsis.

Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis

High circulating levels of lactate and high mobility group box-1 (HMGB1) are associated with the severity and mortality of sepsis. However, it is unclear whether lactate could promote HMGB1 release during sepsis. The present study demonstrated a novel role of lactate in HMGB1 lactylation and acetylation in macrophages during polymicrobial sepsis. We found that macrophages can uptake extracellular lactate via monocarboxylate transporters (MCTs) to promote HMGB1 lactylation via a p300/CBP-dependent mechanism. We also observed that lactate stimulates HMGB1 acetylation by Hippo/YAP-mediated suppression of deacetylase SIRT1 and β-arrestin2-mediated recruitment of acetylases p300/CBP to the nucleus via G protein-coupled receptor 81 (GPR81). The lactylated/acetylated HMGB1 is released from macrophages via exosome secretion which increases endothelium permeability. In vivo reduction of lactate production and/or inhibition of GPR81-mediated signaling decreases circulating exosomal HMGB1 levels and improves survival outcome in polymicrobial sepsis. Our results provide the basis for targeting lactate/lactate-associated signaling to combat sepsis.

Targeting CD39 Toward Activated Platelets Reduces Systemic Inflammation and Improves Survival in Sepsis: A Preclinical Pilot Study.

Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39). Experimental animal study and cell culture study. University-based experimental laboratory. Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice. Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo. Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase. Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application.

Dimethylarginine dimethylaminohydrolase 2 regulates nitric oxide synthesis and hemodynamics and determines outcome in polymicrobial sepsis.

Nitric oxide is a key to numerous physiological and pathophysiological processes. Nitric oxide production is regulated endogenously by 2 methylarginines, asymmetric dimethylarginine (ADMA) and monomethyl-L-arginine. The enzyme that specifically metabolizes asymmetric dimethylarginine and monomethyl-L-arginine is dimethylarginine dimethylaminohydrolase (DDAH). The first isoform dimethylarginine dimethylaminohydrolase 1 has previously been shown to be an important regulator of methylarginines in both health and disease. This study explores for the first time the role of endogenous dimethylarginine dimethylaminohydrolase 2 in regulating cardiovascular physiology and also determines the functional impact of dimethylarginine dimethylaminohydrolase 2 deletion on outcome and immune function in sepsis. Mice, globally deficient in Ddah2, were compared with their wild-type littermates to determine the physiological role of Ddah2 using in vivo and ex vivo assessments of vascular function. We show that global knockout of Ddah2 results in elevated blood pressure during periods of activity (mean [SEM], 118.5 [1.3] versus 112.7 [1.1] mm Hg; P=0.025) and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue asymmetric dimethylarginine (SEM) was 0.89 (0.06) versus 0.67 (0.05) μmol/L (P=0.02) and systemic nitric oxide concentrations. In a model of severe polymicrobial sepsis, Ddah2 knockout affects outcome (120-hour survival was 12% in Ddah2 knockouts versus 53% in wild-type animals; P<0.001). Monocyte-specific deletion of Ddah2 results in a similar pattern of increased severity to that seen in globally deficient animals. Ddah2 has a regulatory role both in normal physiology and in determining outcome of severe polymicrobial sepsis. Elucidation of this role identifies a mechanism for the observed relationship between Ddah2 polymorphisms, cardiovascular disease, and outcome in sepsis.

P150 - Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates nitric oxide production, haemodynamics and vascular responsiveness under basal conditions and outcome in polymicrobial sepsis

P150 - Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates nitric oxide production, haemodynamics and vascular responsiveness under basal conditions and outcome in polymicrobial sepsis

IRAK1-Dependent Signaling Mediates Mortality in Polymicrobial Sepsis

Interleukin-1 receptor-associated kinase (IRAK1) is a key regulatory protein in TLR/IL1R-mediated cell activation during inflammatory response. Studies indicated that pending on the nature of the used inflammatory model, downregulation of IRAK1 may be beneficial or detrimental. However, the role of IRAK1 in affecting outcome in polymicrobial sepsis is unknown. We tested this question using an IRAK1-deficient mouse strain and cecal ligation and puncture (CLP) procedure, which is a clinically relevant rodent septic model. Sepsis-induced mortality was markedly lower in IRAK1-deficient mice (35 %) compared to WT (85 %). Sepsis-induced increases in blood IL-6 and IL-10 levels were blunted at 6 h post-CLP in IRAK1 deficiency compared to WT, but cytokine levels were similar at 20 h post-CLP. Sepsis-induced blood granulocytosis and depletion of splenic B cells were also blunted in IRAK1-deficient mice as compared to WT. Analysis of TLR-mediated cytokine responses by IRAK1-deficient and WT macrophages ex vivo indicated a TLR4-dependent downregulation of IL-6 and IL1β in IRAK1 deficiency, whereas TLR2-dependent responses were unaffected. TLR7/8-mediated IL-6, IL1β, and IL-10 production was also blunted in IRAK1 macrophages as compared to WT. The study shows that IRAK1 deficiency impacts multiple TLR-dependent pathways and decreases early cytokine responses following polymicrobial sepsis. The delayed inflammatory response caused by the lack of IRAK1 expression is beneficial, as it manifests a marked increased chance of survival after polymicrobial sepsis.

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.

The Outcome of Polymicrobial Sepsis Is Independent of T and B CellS

The contribution of the adaptive and innate immune systems to the pathogenesis and outcome of sepsis remains a fundamental yet controversial question. Here, we use mice lacking the recombination activating gene 1 (Rag-1) to study the role of T and B cells in sepsis after cecal ligation and puncture (CLP). Spleens of Rag-1 mice were atrophic and completely devoid of CD3 T cells and CD19 B cells. Wild-type mice and Rag-1 mice (both on a C57BL/6J background) underwent CLP or sham surgery. Both wild-type and Rag-1 mice developed clinical signs of sepsis within the first day after CLP. This included severe hypothermia as measured by a decrease in body surface temperature and organ dysfunction as detected by plasma increases in blood urea nitrogen and lactate dehydrogenase levels. Survival curves of wild-type and Rag-1 mice after CLP were superimposable, with 35% survival in the wild-type group and 27% survival in the Rag-1 group, respectively (not significant, P = 0.875). Using multiplex bead-based assays, the mediator concentrations for 23 cytokines and chemokines were measured in plasma of wild-type and Rag-1 mice 8 h after CLP or sham surgery. Compared with sham surgery mice, the highest mediator levels were observed for granulocyte colony-stimulating factor, keratinocyte chemoattractant, IL-6, monocyte chemotactic protein 1, and IL-10. Levels for most mediators were unaffected by the absence of T and B lymphocytes. Only the concentrations of IL-6 and IL-17 were found to be significantly lower in Rag-1 mice compared with wild-type mice. In conclusion, the absence of T and B cells in the CLP model used does not appear to affect the acute outcome of severe sepsis.

Female X-Chromosome Mosaicism for NOX2 Deficiency Presents Unique Inflammatory Phenotype and Improves Outcome in Polymicrobial Sepsis

Cellular X-chromosome mosaicism, which is unique to females, may be advantageous during pathophysiological challenges compared with the single X-chromosome machinery of males, and it may contribute to gender dimorphism in the inflammatory response. We tested the hypothesis of whether cellular mosaicism for the X-linked gp91phox (NOX2) deficiency, the catalytic component of the superoxide anion-generating NADPH oxidase complex, is advantageous during polymicrobial sepsis. Deficient, wild-type (WT), and heterozygous/mosaic mice were compared following polymicrobial sepsis initiated by cecal ligation and puncture. Compared with WT littermates, sepsis-induced mortality was improved in deficient mice, as well as in mosaic animals carrying both deficient and WT phagocyte subpopulations. In contrast, blood bacterial counts were greatest in deficient mice. Consistent with poor survival, WT mice also showed the most severe organ damage following sepsis. In mosaic animals, the deficient neutrophil subpopulations displayed increased organ recruitment and elevated CD11b membrane expression compared with WT neutrophil subpopulations within the same animal. The dynamics of sepsis-induced blood and organ cytokine content and WBC composition changes, including lymphocyte subsets in blood and bone marrow, showed differences among WT, deficient, and mosaic subjects, indicating that mosaic mice are not simply the average of the deficient and WT responses. Upon oxidative burst, interchange of oxidants between WT and deficient neutrophil subpopulations occurred in mosaic mice. This study suggests that mice mosaic for gp91phox expression have multiple advantages compared with WT and deficient mice during the septic course.

Properdin Plays a Protective Role in Polymicrobial Septic Peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5alpha. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5alpha. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

Inhibiting early activation of tissue nuclear factor-κB and nuclear factor interleukin 6 with (1→3)-β-D -glucan increases long-term survival in polymicrobial sepsis

Background: Recent data implicate the activation of nuclear factor-κB (NF-κB) and nuclear factor interleukin 6 (NF-IL6) as important steps in the pathophysiologic mechanisms of adult respiratory distress syndrome and systemic inflammatory response syndrome. Methods: This study evaluated the effect of immunomodulating polysaccharides on transcription factor activation, cytokine expression, and mortality in a murine cecal ligation and puncture (CLP) model. ICR/HSD mice were treated with glucan (50 mg/kg) 1 hour before or 15 minutes after CLP. Liver and lung tissue were harvested at 3 hours and mortality trends were observed for 20 days. Results: CLP increased liver and lung NF-κB and NF-IL6 nuclear binding activity as well as tumor necrosis factor-α and interleukin 6 messenger RNA levels at 3 hours. Pretreatment or posttreatment with glucans inhibited tissue NF-κB and NF-IL6 nuclear binding activity and tissue cytokine messenger RNA levels. Prophylaxis with glucan phosphate or scleroglucan increased (P <.001) long-term survival (20% CLP vs 65% glucan phosphate, 75% scleroglucan). Posttreatment with glucan phosphate also increased (P <.05) long-term survival (20% vs 65%). Conclusions: Pretreatment or posttreatment with biologic response modifiers decreased tissue transcription factor nuclear binding activity and cytokine message in liver and lung of septic mice. Inhibiting early transcription factor activation and cytokine message expression correlates with improved outcome in polymicrobial sepsis as denoted by increased long-term survival. (Surgery 1999;126:54-65.)