NETs form in the pancreata of mice during the development of AP, and NET levels are increased in plasma from patients with AP, compared with controls. NETs regulate organ inflammation and injury in mice with AP, and might be targeted to reduce pancreatic tissue damage and inflammation in patients.

Abdominal sepsis is associated with dysfunctional hemostasis. Thrombin generation (TG) is a rate-limiting step in systemic coagulation. Neutrophils can expell neutrophil extracellular traps (NETs) and/or microparticles (MPs) although their role in pathological coagulation remains elusive. Cecal ligation and puncture (CLP)-induced TG in vivo was reflected by a reduced capacity of plasma from septic animals to generate thrombin. Depletion of neutrophils increased TG in plasma from CLP mice. Sepsis was associated with increased histone 3 citrullination in neutrophils and plasma levels of cell-free DNA and DNA-histone complexes and administration of DNAse not only eliminated NET formation but also elevated TG in sepsis. Isolated NETs increased TG and co-incubation with DNAse abolished NET-induced formation of thrombin. TG triggered by NETs was inhibited by blocking factor XII and abolished in factor XII-deficient plasma but intact in factor VII-deficient plasma. Activation of neutrophils simultaneously generated large amount of neutrophil-derived MPs, which were found to bind to NETs via histone-phosphatidylserine interactions. These findings show for the first time that NETs and MPs physically interact, and that NETs might constitute a functional assembly platform for MPs. We conclude that NET-MP complexes induce TG via the intrinsic pathway of coagulation and that neutrophil-derived MPs play a key role in NET-dependent coagulation.

Excessive neutrophil activation is a major component in septic lung injury. Neutrophil-derived DNA may form extracellular traps in response to bacterial invasions. The aim of the present study was to investigate the potential role of neutrophil extracellular traps (NETs) in septic lung injury. Male C57BL/6 mice were treated with recombinant human (rh)DNAse (5 mg/kg) after cecal ligation and puncture (CLP). Extracellular DNA was stained by Sytox green, and NET formation was quantified by confocal microscopy and cell-free DNA in plasma, peritoneal cavity, and lung. Blood, peritoneal fluid, and lung tissue were harvested for analysis of neutrophil infiltration, NET levels, tissue injury, as well as CXC chemokine and cytokine formation. We observed that CLP caused increased formation of NETs in plasma, peritoneal cavity, and lung. Administration of rhDNAse not only eliminated NET formation in plasma, peritoneal cavity, and bronchoalveolar space but also reduced lung edema and tissue damage 24 h after CLP induction. Moreover, treatment with rhDNAse decreased CLP-induced formation of CXC chemokines, IL-6, and high-mobility group box 1 (HMGB1) in plasma, as well as CXC chemokines and IL-6 in the lung. In vitro, we found that neutrophil-derived NETs had the capacity to stimulate secretion of CXCL2, TNF-α, and HMGB1 from alveolar macrophages. Taken together, our findings show that NETs regulate pulmonary infiltration of neutrophils and tissue injury via formation of proinflammatory compounds in abdominal sepsis. Thus we conclude that NETs exert a proinflammatory role in septic lung injury.

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.

Sepsis-triggered immune paralysis including T-cell dysfunction increases susceptibility to infections. Statins exert beneficial effects in patients with sepsis, although the mechanisms remain elusive. Herein, we hypothesized that simvastatin may attenuate T-cell dysfunction in abdominal sepsis. Male C57BL/6 mice were pretreated with simvastatin (10 mg/kg) before cecal ligation and puncture (CLP). Spleen CD4 T-cell apoptosis, proliferation, and regulatory T cells (CD4CD25Foxp3) were quantified by use of flow cytometry. Formation of interferon γ (IFN-γ) and interleukin 4 (IL-4) in the spleen and plasma levels of high-mobility box group 1 (HMBG1) and IL-6 were determined using enzyme-linked immunosorbent assay. Cecal ligation and puncture caused a clear-cut increase in apoptosis and decrease in proliferation in splenic CD4 T cells. It was found that simvastatin markedly reduced apoptosis and improved proliferation in CD4 T cells in septic mice. Moreover, CLP-induced formation of regulatory T cells in the spleen was abolished in simvastatin-treated animals. Cecal ligation and puncture greatly decreased the levels of IFN-γ and IL-4 in the spleen. Simvastatin completely reversed this sepsis-mediated inhibition of IFN-γ and IL-4 formation in the spleen. We observed that CLP increased plasma levels of HMBG1 by 25-fold and IL-6 by 99,595-fold. Notably, treatment with simvastatin abolished this CLP-evoked increase in HMBG1 and IL-6 levels in the plasma, suggesting that simvastatin is a potent inhibitor of systemic inflammation in sepsis. Lastly, it was found that simvastatin reduced CLP-induced bacteremia. In conclusion, these novel findings suggest that simvastatin is a powerful regulator of T-cell immune dysfunction in abdominal sepsis. Thus, these protective effects of simvastatin on T-cell functions help to explain the protective effect of statins in patients with sepsis.

Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro-inflammatory compounds, including the chemokines CCL5 and CXCL4, although their role in acute colitis remains elusive. The aim of this study is to examine the role of platelets and platelet-derived chemokines in acute colitis. Acute colitis is induced in female Balb/c mice by administration of 5% dextran sodium sulfate (DSS) for 5 days. Animals receive a platelet-depleting, anti-CCL5, anti-CXCL4, or a control antibody prior to DSS challenge. Colonic tissue is collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin-6 (IL-6), and CCL5 levels as well as morphological analyses. Platelet depletion reduce tissue damage and clinical disease activity index in DSS-exposed animals. Platelet depletion not only reduces levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduces tissue damage, CXC chemokine expression, and neutrophil recruitment in DSS-treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration, and tissue damage in the colon. Moreover, our results suggest that platelet-derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis.

Systemic inflammatory response syndrome and severe infections are associated with major derangements in the coagulation system. The purpose of this study was to examine the dynamic alterations in thrombin generation in abdominal sepsis. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57/Bl6 mice. Cecal ligation and puncture caused a systemic inflammatory response, with neutrophil recruitment and tissue damage in the lung as well as thrombocytopenia and leukocytopenia. Thrombin generation, coagulation factors, lung histology, and myeloperoxidase activity was determined 1, 3, 6, and 24 h after induction of CLP. It was found that thrombin generation was increased 1 h after CLP and that thrombin generation started to decrease at 3 h and was markedly reduced 6 and 24 h after CLP induction. Platelet-poor plasma from healthy mice could completely reverse the inhibitory effect of CLP on thrombin generation, suggesting that sepsis caused a decrease in the levels of plasma factors regulating thrombin generation in septic animals. Indeed, it was found that CLP markedly decreased plasma levels of prothrombin, factor V, and factor X at 6 and 24 h. Moreover, we observed that CLP increased plasma levels of activated protein C at 6 h, which returned to baseline levels 24 h after CLP induction. Finally, pretreatment with imipenem/cilastatin attenuated the CLP-evoked decrease in thrombin generation and consumption of prothrombin 24 h after CLP induction. Our novel findings suggest that thrombin generation is initially increased and later decreased in abdominal sepsis. Sepsis-induced reduction in thrombin generation is correlated to changes in the plasma levels of coagulation factors and activated protein C. These findings help explain the dynamic changes in global hemostasis in abdominal sepsis.

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