Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock.

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

The RIPK3 Scaffold Regulates Lung Inflammation During Pseudomonas Aeruginosa Pneumonia.

RIPK3 (receptor-interacting protein kinase 3) activity triggers cell death via necroptosis, whereas scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or killed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality after Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.

Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle.

Cell production and death are tightly regulated in the rapidly renewing gut epithelium, with proliferation confined to crypts and apoptosis occurring in villi and crypts. This study sought to determine how stress alters these compartmentalized processes. Wild-type mice made septic via cecal ligation and puncture had decreased crypt proliferation and increased crypt and villus apoptosis. Fabpi-TAg mice expressing large T-antigen solely in villi had ectopic enterocyte proliferation with increased villus apoptosis in unmanipulated animals. Septic fabpi-TAg mice had an unexpected increase in villus proliferation compared with unmanipulated littermates, whereas crypt proliferation was decreased. Cell cycle regulators cyclin D1 and cyclin D2 were decreased in jejunal tissue in septic transgenic mice. In contrast, villus and crypt apoptosis were increased in septic fabpi-TAg mice. To examine the relationship between apoptosis and proliferation in a compartment-specific manner, fabpi-TAg mice were crossed with fabpl-Bcl-2 mice, resulting in expression of both genes in the villus but Bcl-2 alone in the crypt. Septic bi-transgenic animals had decreased crypt apoptosis but had a paradoxical increase in villus apoptosis compared with septic fabpi-TAg mice, associated with decreased proliferation in both compartments. Thus, sepsis unmasks compartment-specific proliferative and apoptotic regulation that is not present under homeostatic conditions.-Lyons, J. D., Klingensmith, N. J., Otani, S., Mittal, R., Liang, Z., Ford, M. L., Coopersmith, C. M. Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle.

CHRONIC ETHANOL USE WORSENS GUT PERMEABILITY AND ALTERS TIGHT JUNCTION EXPRESSION IN A MURINE SEPSIS MODEL.

ABSTRACT: Alcohol use disorder is associated with increased mortality in septic patients. Murine studies demonstrate that ethanol/sepsis is associated with changes in gut integrity. This study examined intestinal permeability after ethanol/sepsis and investigated mechanisms responsible for alterations in barrier function. Mice were randomized to drink either 20% ethanol or water for 12 weeks and then were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability was disproportionately increased in ethanol/septic mice via the pore, leak, and unrestricted pathways. Consistent with increased permeability in the leak pathway, jejunal myosin light chain (MLC) kinase (MLCK) expression and the ratio of phospho-MLC to total MLC were both increased in ethanol/CLP. Gut permeability was altered in MLCK -/- mice in water/CLP; however, permeability was not different between WT and MLCK -/- mice in ethanol/CLP. Similarly, jejunal IL-1ß levels were decreased while systemic IL-6 levels were increased in MLCK -/- mice in water/CLP but no differences were identified in ethanol/CLP. While we have previously shown that mortality is improved in MLCK -/- mice after water/CLP, mortality was significantly worse in MLCK -/- mice after ethanol/CLP. Consistent with an increase in the pore pathway, claudin 4 levels were also selectively decreased in ethanol/CLP WT mice. Furthermore, mRNA expression of jejunal TNF and IFN-γ were both significantly increased in ethanol/CLP. The frequency of CD4 + cells expressing TNF and IL-17A and the frequency of CD8 + cells expressing IFN-γ in Peyer's Patches were also increased in ethanol/CLP. Thus, there is an ethanol-specific worsening of gut barrier function after CLP that impacts all pathways of intestinal permeability, mediated, in part, via changes to the tight junction. Differences in the host response in the setting of chronic alcohol use may play a role in future precision medicine approaches toward the treatment of sepsis.

MYOSIN LIGHT CHAIN KINASE DELETION WORSENS LUNG PERMEABILITY AND INCREASES MORTALITY IN PNEUMONIA-INDUCED SEPSIS.

ABSTRACT: Increased epithelial permeability in sepsis is mediated via disruptions in tight junctions, which are closely associated with the perijunctional actin-myosin ring. Genetic deletion of myosin light chain kinase (MLCK) reverses sepsis-induced intestinal hyperpermeability and improves survival in a murine model of intra-abdominal sepsis. In an attempt to determine the generalizability of these findings, this study measured the impact of MLCK deletion on survival and potential associated mechanisms following pneumonia-induced sepsis. MLCK -/- and wild-type mice underwent intratracheal injection of Pseudomonas aeruginosa . Unexpectedly, survival was significantly worse in MLCK -/- mice than wild-type mice. This was associated with increased permeability to Evans blue dye in bronchoalveolar lavage fluid but not in tissue homogenate, suggesting increased alveolar epithelial leak. In addition, bacterial burden was increased in bronchoalveolar lavage fluid. Cytokine array using whole-lung homogenate demonstrated increases in multiple proinflammatory and anti-inflammatory cytokines in knockout mice. These local pulmonary changes were associated with systemic inflammation with increased serum levels of IL-6 and IL-10 and a marked increase in bacteremia in MLCK -/- mice. Increased numbers of both bulk and memory CD4 + T cells were identified in the spleens of knockout mice, with increased early and late activation. These results demonstrate that genetic deletion of MLCK unexpectedly increases mortality in pulmonary sepsis, associated with worsened alveolar epithelial leak and both local and systemic inflammation. This suggests that caution is required in targeting MLCK for therapeutic gain in sepsis.

Outcome prediction in sepsis combined use of genetic polymorphisms - A study in Japanese population.

Genetic polymorphisms have recently been found to be related to clinical outcome in septic patients. The present study investigated to evaluate the influence of genetic polymorphisms in Japanese septic patients on clinical outcome and whether use of genetic polymorphisms as predictors would enable more accurate prediction of outcome. Effects of 16 genetic polymorphisms related to pro-inflammatory mediators and conventional demographic/clinical parameters (age, sex, past medical history, and APACHE II score) on ICU mortality as well as disease severity during ICU stay were examined in the septic patients (n=123) admitted to the ICU between October 2001 and November 2007 by multivariable logistic regression analysis. ICU mortality was significantly associated with TNF -308GA, IL1ß -31CT/TT, and APACHE II score. Receiver-operating characteristics (ROC) analysis demonstrated that, compared with APACHE II score alone (ROC-AUC=0.68), use of APACHE II score and two genetic parameters (TNF -308 and IL1ß -31) enabled more accurate prediction of ICU mortality (ROC-AUC=0.80). Significant association of two genetic polymorphisms, TNF -308 and IL1ß -31, with ICU mortality was observed in septic patients. In addition, combined use of these genetic parameters with APACHE II score may enable more accurate prediction of outcome in septic patients.

Case Report: Lifesaving Hemostasis With Resuscitative Endovascular Balloon Occlusion of the Aorta in a Patient With Cardiac Arrest Caused by Upper Gastrointestinal Hemorrhage.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) is performed to treat hemorrhagic shock, whose cause is located below the diaphragm. However, its use in patients with gastrointestinal hemorrhage is relatively rare. The 45-year-old man with a history of dilated cardiomyopathy had experienced epigastric discomfort and had an episode of presyncope. On his presentation, the patient's blood pressure was 82/64 mmHg, heart rate 140/min, and consciousness level GCS E4V5M6. Hemodynamics stabilized rapidly with a transfusion that was administered on an emergency basis, and a blood sample only showed mild anemia (Hb, 11.5 g/dL). The patient was admitted to investigating the presyncope episode, and the planned endoscopy was scheduled the following day. The patient had an episode of presyncope soon and was found in hemorrhagic shock resulting from a duodenal ulcer rapidly deteriorated to cardiac arrest. Although a spontaneous heartbeat was restored with cardiopulmonary resuscitation, the patient's hemodynamics were unstable despite the emergency blood transfusion administered by pumping. Consequently, a REBOA device was placed, resuscitation was continued, and hemostasis was achieved by vascular embolization for the gastroduodenal artery. The patient was subsequently discharged without complications. However, there is no established evidence regarding the REBOA use in upper gastrointestinal hemorrhage, and the investigations that have been reported have been limited. Further, one recent research suggests that appropriate patient selection and early use may improve survival in these life-threatening cases. As was seen in the present case, REBOA can effectively treat upper gastrointestinal hemorrhage by temporarily stabilizing hemodynamics and enabling a hemostatic procedure to be quickly performed during that time. This report also demonstrated the hemodynamics during the combination of intermittent and partial REBOA to avoid the complications of ischemic or reperfusion injury of the intestines or lower extremities.

TIGIT modulates sepsis-induced immune dysregulation in mice with preexisting malignancy.

TIGIT is a recently identified coinhibitory receptor that is upregulated in the setting of cancer and functionally contributes to the impairment of antitumor immunity. However, its role during sepsis is unknown. Because patients with cancer are 10 times more likely to die of sepsis than previously healthy (PH) patients with sepsis, we interrogated the role of TIGIT during sepsis in the context of preexistent malignancy. PH mice or cancer (CA) mice inoculated with lung carcinoma cells were made septic by cecal ligation and puncture (CLP). We found that sepsis induced TIGIT upregulation predominantly on Tregs and NK cells in both PH and CA mice. Anti-TIGIT Ab improved the 7-d survival of CA septic mice but not PH mice after CLP. Treatment of CA septic animals but not PH septic animals with anti-TIGIT mAb significantly reversed sepsis-induced loss of CD4+ T cells, CD8+ T cells, Foxp3+ Treg, and CD19+ B cells in the spleen, which was the result of decreased caspase-3+ apoptotic cells. In sum, we found that anti-TIGIT Ab reversed sepsis-induced T cell apoptosis in CA septic mice and led to a significant survival benefit, suggesting its use as a potential immunotherapy to improve outcomes in septic patients with cancer.

Overexpression of BCL-2 in the Intestinal Epithelium Prevents Sepsis-Induced Gut Barrier Dysfunction via Altering Tight Junction Protein Expression.

Sepsis induces both intestinal hyperpermeability and epithelial apoptosis. While each has been implicated in mediating sepsis mortality, the relationship between these two processes is unclear. We hypothesized that preventing intestinal apoptosis would prevent gut barrier dysfunction. To test this hypothesis, transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in the gut epithelium (Fabpl-Bcl-2 mice) and wild-type (WT) mice were subjected to sham laparotomy or cecal ligation and puncture and orally gavaged with fluorescein isothiocyanate conjugated-dextran (FD-4) 5 h before sacrifice. Serum FD-4 concentration was assayed to measure intestinal permeability, and jejunal tight junctions were assayed for mRNA and protein expression. Baseline FD-4 concentration was similar between WT and Fabpl-Bcl-2 mice. Intestinal permeability increased 6, 12, 24, and 48 h following sepsis in WT mice; however, FD-4 concentration was significantly lower at each timepoint in Fabpl-Bcl-2 mice. In addition, there were no statistically significant changes in permeability between septic and sham transgenic mice. Intestinal mRNA expression of claudin 3, claudin 5, and occludin was lower in septic Fabpl-Bcl-2 mice, while claudin 4 mRNA levels were higher in Fabpl-Bcl-2 mice. In contrast, no differences were detected in claudins 2, 7, 15, JAM-A, or ZO-1. Protein levels followed the same trend for all tight junction mediators different between WT and Fabpl-Bcl-2 mice except occludin was significantly higher in transgenic mice. Together these results demonstrate that decreasing intestinal epithelial apoptosis prevents hyperpermeability following sepsis via tight junction alterations which may be at least partially responsible for improved survival conferred by Bcl-2 overexpression.

The effects of body temperature control on cytokine production in a rat model of ventilator-induced lung injury.

BACKGROUND AND PURPOSE: Injurious ventilation with high peak inspiratory pressure (PIP) is known to cause systemic inflammatory response through cytokine production. This study was performed to examine whether body temperature could regulate cytokine production in ventilator-induced lung injury (VILI) model. METHODS: After performing anesthesia, tracheostomy, and catheter insertion, rats were ventilated with 17cmH(2)O of PIP in the low-pressure (LP) group or 35cmH(2)O in the high-pressure (HP) group. Then, each group was divided into three subgroups; hyperthermia (39 degrees C), normothermia (37 degrees C), and hypothermia (34 degrees C) group. Six groups were observed for 6h. RESULTS: Plasma levels of pro-inflammatory cytokines, TNF-a and IL-6 at 1h after the start of observation were highest in 39 degrees C-HP group and were lowest in 34 degrees C-HP group. Furthermore, sustained high plasma levels of IL-6 were observed only in 39 degrees C-HP group. In contrast, plasma levels of anti-inflammatory cytokine, IL-10 at 1h were highest in 34 degrees C-HP group, and lowest in 39 degrees C-HP group. CONCLUSION: The body temperature significantly affects cytokine production in a model of VILI. Body temperature control may be a potentially effective therapeutic modality to regulate cytokine production in VILI.

Gut integrity in critical illness.

BACKGROUND: The gut is hypothesized to be the "motor" of critical illness. Under basal conditions, the gut plays a crucial role in the maintenance of health. However, in critical illness, all elements of the gut are injured, potentially worsening multiple organ dysfunction syndrome. MAIN BODY: Under basal conditions, the intestinal epithelium absorbs nutrients and plays a critical role as the first-line protection against pathogenic microbes and as the central coordinator of mucosal immunity. In contrast, each element of the gut is impacted in critical illness. In the epithelium, apoptosis increases, proliferation decreases, and migration slows. In addition, gut barrier function is worsened via alterations to the tight junction, resulting in intestinal hyperpermeability. This is associated with damage to the mucus that separates the contents of the intestinal lumen from the epithelium. Finally, the microbiome of the intestine is converted into a pathobiome, with an increase in disease-promoting bacteria and induction of virulence factors in commensal bacteria. Toxic factors can then leave the intestine via both portal blood flow and mesenteric lymph to cause distant organ damage. CONCLUSION: The gut plays a complex role in both health and critical illness. Here, we review gut integrity in both health and illness and highlight potential strategies for targeting the intestine for therapeutic gain in the intensive care unit.

Critical illness and the role of the microbiome.

The number of microbes living within the intestinal lumen is similar to the number of all cells of human origin in the host. Although historically little attention has been paid to the massive microbial community residing inside each of us, the last few years have witnessed an explosion of information related to the role of the microbiome in the maintenance of health and in the pathogenesis of disease. Here, we review data suggesting that the microbiome is converted into a pathobiome in critical illness and potential strategies for targeting the microbiome for therapeutic gain in the intensive care unit.

Sepsis erodes CD8+ memory T cell-protective immunity against an EBV homolog in a 2B4-dependent manner.

Epstein-Barr virus (EBV) reactivation commonly occurs following sepsis, but the mechanisms underlying this are unknown. We utilized a murine EBV homolog (gHV) and the cecal ligation and puncture model of polymicrobial sepsis to study the impact of sepsis on gHV reactivation and CD8+ T cell immune surveillance following a septic insult. We observed a significant increase in the frequency of gHV-infected germinal center B cells on day 7 following sepsis. This increase in viral load was associated with a concomitant significant decrease in the frequencies of gHV-specific CD8+ T cells, as measured by class I MHC tetramers corresponding to the immunodominant viral epitopes. Phenotypic analysis revealed an increased frequency of gHV-specific CD8+ T cells expressing the 2B4 coinhibitory receptor in septic animals compared with sham controls. We sought to interrogate the role of 2B4 in modulating the gHV-specific CD8+ T cell response during sepsis. Results indicated that in the absence of 2B4, gHV-specific CD8+ T cell populations were maintained during sepsis, and gHV viral load was unchanged in 2B4-/- septic animals relative to 2B4-/- sham controls. WT CD8+ T cells upregulated PD-1 during sepsis, whereas 2B4-/- CD8+ T cells did not. Finally, adoptive transfer studies revealed a T cell-intrinsic effect of 2B4 coinhibition on virus-specific CD8+ T cells and gHV viral load during sepsis. These data demonstrate that sepsis-induced immune dysregulation erodes antigen-specific CD8+ responses against a latent viral infection and suggest that blockade of 2B4 may better maintain protective immunity against EBV in the context of sepsis.

Chronic Alcohol Ingestion Worsens Survival and Alters Gut Epithelial Apoptosis and CD8+ T Cell Function After Pseudomonas Aeruginosa Pneumonia-Induced Sepsis.

Mortality is higher in septic patients with a history of alcohol use disorder than in septic patients without a history of chronic alcohol usage. We have previously described a model of chronic alcohol ingestion followed by sepsis from cecal ligation and puncture in which alcohol-fed septic mice have higher mortality than water-fed septic mice, associated with altered gut integrity and increased production of TNF and IFNγ by splenic CD4 T cells without alterations in CD8 T cell function. The purpose of this study was to determine whether this represents a common host response to the combination of alcohol and sepsis by creating a new model in which mice with chronic alcohol ingestion were subjected to a different model of sepsis. C57Bl/6 mice were randomized to receive either alcohol or water for 12 weeks and then subjected to Pseudomonas aeruginosa pneumonia. Mice were sacrificed either 24 hours after the onset of sepsis or followed for survival. Alcohol-fed septic mice had significantly higher 7-day mortality than water-fed septic mice (96% vs 58%). This was associated with a 5-fold increase in intestinal apoptosis in alcohol-fed septic animals, accompanied by an increase in the pro-apoptotic protein Bax. Serum IL-6 levels were higher and IL-2 levels were lower in alcohol-fed septic mice. In contrast, CD8 T cell frequency was lower in alcohol-fed mice than water-fed septic mice, associated with increased production of IFNγ and TNF in stimulated splenocytes. No significant differences were noted in CD4 T cells, lung injury or bacteremia. Mice with chronic alcohol ingestion thus have increased mortality regardless of their septic insult, associated with changes in both the gut and the immune system.

Increased attrition of memory T cells during sepsis requires 2B4.

Recent seminal studies have revealed that laboratory mice differ from adult humans with regard to the frequency, number, and distribution of memory T cells. Because our data show that memory T cells are more susceptible to sepsis-induced death than naive T cells, in this study we developed a model in which mice possess a memory T cell compartment more similar to that of adult humans, to better study immune responses during sepsis in the more physiologically relevant context of high frequencies of memory T cells. Using this model, we found that CD44hi memory T cells significantly upregulated the coinhibitory molecule 2B4 during sepsis, and 2B4+ memory T cells coexpressed markers of both activation and exhaustion. Genetic deficiency in 2B4 resulted in decreased mortality during sepsis. Mechanistically, this decreased mortality was associated with reduced caspase-3/7+ apoptotic T cells in 2B4-/- relative to WT, septic hosts. These results were corroborated by analysis of PBMCs isolated from human patients with sepsis, which showed increased frequencies of caspase-3/7+ apoptotic cells among 2B4+ relative to 2B4- T cells. Thus, 2B4 plays a critical role in sepsis-induced apoptosis in both murine memory T cells and those isolated from human patients with sepsis.

Clinical application of cytokine-related gene polymorphism analysis using a newly developed DNA chip in critically ill patients.

OBJECTIVES: To investigate the usefulness of analysis of single nucleotide polymorphism (SNP) using a newly developed DNA chip assay involving single base extension(SBE) and subsequent hybridization in cytokine-related genes in critical care patients. DESIGN AND METHODS: Genotyping was performed in 76 ICU patients admitted to the ICU. First, the DNA samples from 58 patients were subjected to PCR and SBE conditioning for DNA. Second, another 18 patients were subjected to genotyping for SNPs in IL-6 -596G/A, -572C/G, -174G/C, TNF-alpha -308G/A, -238G/A, IL-1beta -511C/T and -31T/C by both TaqMan and DNA chip method, and by DNA direct sequencing prospectively. RESULTS: First, PCR and SBE condition were established with initial sample sets, which were consistent with results by TaqMan method. Second, no difference was observed between two assay methods in prospective validation set. CONCLUSIONS: The genotyping assay using the new chip was developed and its usefulness was confirmed.