Antioxidant and food additive BHA prevents TNF cytotoxicity by acting as a direct RIPK1 inhibitor.

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.

Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

RIP kinase 1-dependent endothelial necroptosis underlies systemic inflammatory response syndrome.

Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context-dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory "cytokine storm" has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase-inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.

Predicting CYP3A-mediated midazolam metabolism in critically ill neonates, infants, children and adults with inflammation and organ failure.

AIMS: Inflammation and organ failure have been reported to have an impact on cytochrome P450 (CYP) 3A-mediated clearance of midazolam in critically ill children. Our aim was to evaluate a previously developed population pharmacokinetic model both in critically ill children and other populations, in order to allow the model to be used to guide dosing in clinical practice. METHODS: The model was evaluated externally in 136 individuals, including (pre)term neonates, infants, children and adults (body weight 0.77-90 kg, C-reactive protein level 0.1-341 mg l-1 and 0-4 failing organs) using graphical and numerical diagnostics. RESULTS: The pharmacokinetic model predicted midazolam clearance and plasma concentrations without bias in postoperative or critically ill paediatric patients and term neonates [median prediction error (MPE) <30%]. Using the model for extrapolation resulted in well-predicted clearance values in critically ill and healthy adults (MPE <30%), while clearance in preterm neonates was over predicted (MPE >180%). CONCLUSION: The recently published pharmacokinetic model for midazolam, quantifying the influence of maturation, inflammation and organ failure in children, yields unbiased clearance predictions and can therefore be used for dosing instructions in term neonates, children and adults with varying levels of critical illness, including healthy adults, but not for extrapolation to preterm neonates.

Reduced serum cholinesterase activity indicates splenic modulation of the sterile inflammation.

BACKGROUND: Sterile inflammation is an immediate and well-coordinated immune response to surgical injury. The cholinergic system plays a pivotal role in the inflammatory response. Induced inflammation stimulates the vagus nerve, which in turn activates anti-inflammatory nonneuronal processes. Serum cholinesterase (butyrylcholinesterase [BChE]) is an enzyme that hydrolyzes acetylcholine. Measuring the activity of the BChE in blood might indicate the level of the nonneuronal cholinergic activity. The spleen is a major organ of the immune system playing an important role during inflammation. A functional connection of the neuroimmune reflex has thus far been described only in experimental settings. MATERIALS AND METHODS: In 48 patients receiving major pancreatic surgery, BChE activity was measured by applying point-of-care-testing, in addition to standard laboratory tests. RESULTS: The BChE activity decreased in patients receiving surgery. This reduction emerged much earlier than changes in C-reactive protein concentration, an inflammatory biomarker broadly used in the clinical environment. A milder reduction in the BChE activity was observed in patients subjected to surgery with splenectomy than in those with a preserved spleen. CONCLUSIONS: The use of the point-of-care-testing system for quick bedside diagnostics and the rapid effects of inflammation on BChE levels provide a method and a marker to facilitate the early detection of systemic inflammation. Furthermore, this study provides evidence that the experimentally documented neuroimmune interaction is part of the physiological response to surgery-induced sterile inflammation. Splenic function plays an essential role in modulating the cholinergic anti-inflammatory response.

SPATA2 regulates the activation of RIPK1 by modulating linear ubiquitination.

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.

The mechanisms regulating cyclin-dependent kinase 5 in hippocampus during systemic inflammatory response: The effect on inflammatory gene expression.

Cyclin-dependent kinase 5 (Cdk5) is critical for nervous system's development and function, and its aberrant activation contributes to pathomechanism of Alzheimer's disease and other neurodegenerative disorders. It was recently suggested that Cdk5 may participate in regulation of inflammatory signalling. The aim of this study was to analyse the mechanisms involved in regulating Cdk5 activity in the brain during systemic inflammatory response (SIR) as well as the involvement of Cdk5 in controlling the expression of inflammatory genes. Genetic and biochemical alterations in hippocampus were analysed 3 and 12 h after intraperitoneal injection of lipopolysaccharide. We observed an increase in both Cdk5 gene expression and protein level. Moreover, phosphorylation of Cdk5 on Ser159 was significantly enhanced. Also transcription of Cdk5-regulatory protein (p35/Cdk5r1) was augmented, and the level of p25, calpain-dependent cleavage product of p35, was increased. All these results demonstrated rapid activation of Cdk5 in the brain during SIR. Hyperactivity of Cdk5 contributed to enhanced phosphorylation of tau and glycogen synthase kinase 3ß. Inhibition of Cdk5 with Roscovitine reduced activation of NF-κB and expression of inflammation-related genes, demonstrating the critical role of Cdk5 in regulation of gene transcription during SIR.

Evaluation of usefulness of myeloperoxidase index (MPXI) for differential diagnosis of systemic inflammatory response syndrome (SIRS) in the emergency department.

BACKGROUND: The myeloperoxidase index (MPXI) is elevated in infection. We ascertained whether MPXI might be useful in differentiation of sepsis versus non-infectious systemic inflammatory response syndrome (SIRS) in emergency department (ED). METHODS: After exclusion of patients with an age of <18 years, trauma, haematological disease and on anticancer chemotherapy, 444 consecutive cases with SIRS (sepsis: 224, 50.3%; and non-infectious SIRS: 220, 49.7%) diagnosed and treated at the ED of The Wonju Severance Christian Hospital from May 2012 to June 2012 were retrospectively reviewed. RESULTS: Median MPXI was higher in sepsis versus non-infectious SIRS (0.1 (IQR: -3.1 to 2.5) vs -1.2 (-4.1 to 1.6), respectively, p=0.020). Median white cell count, neutrophil percentage, C reactive protein level and δ neutrophil index were also higher. However, MPXI resulted as not statistically useful for differential diagnostic parameter in analysis. CONCLUSIONS: MPXI is higher in sepsis than in non-infectious SIRS. However, there is currently no evidence that the MPXI adds any additional benefit to differentiate sepsis from non-infectious SIRS in the ED. Therefore, further study will be needed.

Acid sphingomyelinase serum activity predicts mortality in intensive care unit patients after systemic inflammation: a prospective cohort study.

INTRODUCTION: Acid sphingomyelinase is involved in lipid signalling pathways and regulation of apoptosis by the generation of ceramide and plays an important role during the host response to infectious stimuli. It thus has the potential to be used as a novel diagnostic marker in the management of critically ill patients. The objective of our study was to evaluate acid sphingomyelinase serum activity (ASM) as a diagnostic and prognostic marker in a mixed intensive care unit population before, during, and after systemic inflammation. METHODS: 40 patients admitted to the intensive care unit at risk for developing systemic inflammation (defined as systemic inflammatory response syndrome plus a significant procalcitonin [PCT] increase) were included. ASM was analysed on ICU admission, before (PCT before), during (PCT peak) and after (PCT low) onset of SIRS. Patients undergoing elective surgery served as control (N = 8). Receiver-operating characteristics curves were computed. RESULTS: ASM significantly increased after surgery in the eight control patients. Patients from the intensive care unit had significantly higher ASM on admission than control patients after surgery. 19 out of 40 patients admitted to the intensive care unit developed systemic inflammation and 21 did not, with no differences in ASM between these two groups on admission. In patients with SIRS and PCT peak, ASM between admission and PCT before was not different, but further increased at PCT peak in non-survivors and was significantly higher at PCT low compared to survivors. Survivors exhibited decreased ASM at PCT peak and PCT low. Receiver operating curve analysis on discrimination of ICU mortality showed an area under the curve of 0.79 for ASM at PCT low. CONCLUSIONS: In summary, ASM was generally higher in patients admitted to the intensive care unit compared to patients undergoing uncomplicated surgery. ASM did not indicate onset of systemic inflammation. In contrast to PCT however, it remained high in non-surviving ICU patients after systemic inflammation.

Pyroptotic death storms and cytopenia.

For over two decades, we have embraced the cytokine storm theory to explain sepsis, severe sepsis and septic shock. The failure of numerous large-scale clinical trials, which aimed to treat sepsis by neutralizing inflammatory cytokines and LPS, indicates that alternative pathophysiological mechanisms are likely to account for sepsis and the associated immune suppression in patients with severe infection. Recent insights that extricate pyroptotic death from inflammatory cytokine production in vivo have highlighted a need to investigate the consequences of apoptotic and non-apoptotic death in contributing to cytopenia and immune suppression. In this review, we will focus on the biochemical and cellular mechanisms controlling pyroptosis, a Caspase-1/11 dependent form of cell death during infection.

RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome.

Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed pathways: survival-inflammation and cell death. An additional switch decides, depending on the cellular context, between caspase-dependent apoptosis and RIP kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the contribution of both cell death pathways in TNF-induced systemic inflammatory response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3 or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion of RIPK3 conferred complete protection against lethal SIRS and reduced the amounts of circulating damage-associated molecular patterns. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal ligation and puncture, underscoring the clinical relevance of RIPK kinase inhibition in sepsis and identifying components of the necroptotic pathway that are potential therapeutic targets for treatment of SIRS and sepsis.

Predictive value of serum cholinesterase for the prognosis of aged patients with systemic inflammatory response syndrome.

BACKGROUND: Some studies found that cholinesterase (ChE) can be an independent risk factor for patients with multiple organ dysfunction syndrome. To assess aged patients with systemic inflammatory response syndrome (SIRS) early and predict their prognosis, the predictive value of ChE for the prognosis of aged patients with SIRS was analyzed. METHODS: From September 2009 to September 2010, all aged patients with SIRS in the ICU of the Second Affiliated Hospital of Zhejiang University School of Medicine were retrospectively analyzed if they met inclusion criteria: patients aged ≥ 65 years and met American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference criteria for SIRS. Serum ChE, albumin, D-dimer, lactic acid and C-reactive protein (CRP) were measured, and the Acute Physiology and Chronic Health Evaluation (APACHE) II and Glasgow Coma Scale (GCS) scores were evaluated within the first 24 hours in the ICU. Fisher's exact test was used for comparison of the primary disease between the deceased group and surviving group. For comparison of study variables between the two groups, the Student's t test or Mann-Whitney U test was used. Multivariate significance was tested with binary Logistic regression analysis. RESULTS: The clinical data of 124 aged patients with SIRS were collected and analyzed. Sixty-six patients (46 male, 20 female, mean age (78.70 ± 8.08) years) who died were included in the deceased group and 58 patients (34 male, 24 female, mean age (76.02 ± 6.57) years) who survived were included in the surviving group. There were no significant differences in age, gender, APACHE II score and GCS score between the deceased group and surviving group (all P > 0.05), but there were significant differences in lactic acid (P = 0.011), D-dimer (P = 0.011), albumin (P = 0.007), CRP (P = 0.008), and ChE (P < 0.0001). The correlation analysis showed that the APACHE II score and CRP were not correlated with ChE (both P < 0.05). D-dimer and albumin were correlated with ChE (Spearman's rho correlation coefficients were -0.206 and 0.324, the corresponding P values were 0.022 and < 0.0001). Multiple Logistic regression analysis showed that age, gender, lactic acid, D-dimer, albumin, CRP, APACHE II score, and GCS score were not independent risk factors for prognosis of aged patients with SIRS, but that ChE was (P < 0.0001). The receiver operating characteristic curve of ChE had an area under the curve of 0.797 (standard error = 0.04; P < 0.0001), and a ChE of 103.00 U/L was the cut-off value with sensitivity = 0.793, specificity = 0.742. CONCLUSION: Serum ChE might be a predictive marker for the prognosis of aged patients with SIRS, with low serum ChE levels indicating poor prognosis.

Influence of neutrophil elastase inhibitor on the postoperative course in patients with esophageal cancer after transthoracic esophagectomy.

BACKGROUND/AIMS: We evaluated influence of sivelestat sodium hydrate in the clinical course after transthoracic esophagectomy. METHODOLOGY: Forty-two consecutive patients with esophageal cancer underwent transthoracic esophagectomy. Twenty-two patients were treated with sivelestat (sivelestat group) and twenty patients were untreated (control group). Sivelestat (0.2 mg/(kg·h)-1) was continuously administered for 5 days since ICU admission. Postoperative morbidity, duration of systemic inflammatory response syndrome (SIRS) and mechanical ventilation, and the time to refilling were examined. The level of serum white blood cells (WBC), C-reactive protein (CRP), aspartate transaminase (AST), alanine aminotransferase (ALT), total bilirubin (TB) and the PaO2/FiO2 ratio were measured. RESULTS: The durations of SIRS and mechanical ventilation and the time to refilling were significantly shorter in the sivelestat group than in the control group. Postoperative changes in the serum WBC, CRP, AST and ALT levels were not significantly different between the groups; however, serum TB level was significantly lower in the sivelestat group than in the control group. The PaO2/FiO2 ratio at postoperative day 3 was significantly higher in the former than in the latter. CONCLUSIONS: We demonstrated that sivelestat might contribute to the improvement of acute lung injury, hyperbilirubinemia and postoperative circulatory failure.

Increased myeloperoxidase enzyme activity in plasma is an indicator of inflammation and onset of sepsis.

INTRODUCTION: Circulating lipopolysaccharides released from bacteria may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO enzyme activity in plasma of critically ill patients and to check the hypothesis that these concentrations in plasma would be higher in sepsis and systemic inflammatory conditions, as neutrophils release their contents before proliferating in response to stress. MATERIAL AND METHODS: Blood samples were collected from 105 critically ill patients admitted to the intensive care unit, consisting of those with systemic inflammatory response syndrome (n = 42), sepsis (n = 37), and septic shock (n = 26). Plasma MPO enzyme activity was determined by o-dianisidine-H(2)O(2) method, modified for 96-well plates. RESULTS: The plasma MPO enzyme activity in sepsis patients was significantly higher than that in the control group (mean, 2.4 ± 1.8 in sepsis and 1.86 ± 1.2 nmol per milligram protein per 10 minutes in systemic inflammatory response syndrome vs 0.32 ± 0.11 nmol per milligram protein per 10 minutes in healthy controls). Mean plasma lactate levels in sepsis (7.8 ± 1.2 mmol/L) and shock patients (9.5 ± 1.2 mmol/L) and cytokines like tumor necrosis factor-α, interleukin-8, and interleukin-1ß were simultaneously evaluated to establish onset of inflammation and sepsis. These results show that neutrophil activation occurring during inflammation and sepsis could be detected by plasma MPO concentration. CONCLUSION: The plasma MPO concentrations may be a marker of the neutrophil proliferation and severity of inflammation.

Phosphoinositide-3 kinase gamma activity contributes to sepsis and organ damage by altering neutrophil recruitment.

RATIONALE: Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase γ (PI3Kγ) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. OBJECTIVES: This study aimed to determine the specific role of the kinase activity of PI3Kγ in the pathogenesis of sepsis and multiorgan damage. METHODS: PI3Kγ wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation-induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3Kγ inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. MEASUREMENTS AND MAIN RESULTS: Both genetic and pharmaceutical PI3Kγ kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3Kγ pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. CONCLUSIONS: This study establishes PI3Kγ as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

CO2 abdominal insufflation decreases local and systemic inflammatory response in experimental acute pancreatitis.

OBJECTIVES: Acute pancreatitis (AP) is a serious disease that is amplified by an associated systemic inflammatory response. We investigated the effect of CO2 pneumoperitoneum on the local and systemic inflammatory response in AP. METHODS: Acute pancreatitis was induced in Wistar rats by 5% taurocholate intraductal injection. Carbon dioxide pneumoperitoneum was applied for 30 minutes before the induction of AP. Inflammatory parameters were evaluated in the peritoneum (ascites, cell number, and tumor necrosis factor alpha [TNF-alpha]), serum (amylase, TNF-alpha, interleukin-6 [IL-6], and IL-10), pancreas (myeloperoxidase [MPO] activity, cyclo-oxygenase 2 and inducible nitric oxide synthase expression, and histological diagnosis), liver, and lung (mitochondria dysfunction and MPO activity). RESULTS: Abdominal insufflation with CO2 before induction of AP caused a significant decrease in ascites volume, cells, and TNF-alpha in the peritoneal cavity and in serum TNF-alpha and IL-6 but not IL-10 levels. In the pancreas, this treatment reduced MPO activity, acinar and fat necrosis, and the expression of inducible nitric oxide synthase and cyclo-oxygenase 2. There were no significant differences on serum amylase levels, liver mitochondrial function, and pulmonary MPO between groups. CONCLUSIONS: Our data demonstrated that CO2 pneumoperitoneum reduced pancreatic inflammation and attenuated systemic inflammatory response in AP. This article suggests that CO2 pneumoperitoneum plays a critical role on the better outcome in patients undergoing laparoscopic pancreatic surgery.

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.