BACKGROUND: Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered. METHODS: C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival. RESULTS: Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury. CONCLUSIONS: Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on the models used.

Background Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. Methods Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. Results In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. Conclusions Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.

While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8+ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8+ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8+ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8+ T cells and total CD4+ and CD8+ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8+ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8+ T cell functionality and differentiation.

Background Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice. Methods Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival. Results Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology. Conclusions When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and pancreatic metabolomics may help explain this differential response.

Sepsis is the leading cause of death in intensive care units in the US, and it is known that chronic alcohol use is associated with higher incidence of sepsis, longer ICU stays, and higher mortality from sepsis. Both sepsis and chronic alcohol use are associated with immune deficits such as decreased lymphocyte numbers, impaired innate immunity, delayed-type hypersensitivity reactions, and susceptibility to infections; however, understanding of specific pathways of interaction or synergy between these two states of immune dysregulation is lacking. This study therefore sought to elucidate mechanisms underlying the immune dysregulation observed during sepsis in the setting of chronic alcohol exposure. Using a murine model of chronic ethanol ingestion followed by sepsis induction via cecal ligation and puncture, we determined that while CD4+ and CD8+ T cells isolated from alcohol fed mice eventually expressed the same cellular activation markers (CD44, CD69, and CD43) and effector molecules (IFN-ã, TNF) as their water fed counterparts, there was an overall delay in the acquisition of these phenotypes. This early lag in T cell activation was associated with significantly reduced IL-2 production at a later timepoint in both the CD4+ and CD8+ T cell compartments in alcohol sepsis, as well as with a reduced accumulation of CD8dim activated effectors. Taken together, these data suggest that delayed T cell activation may result in qualitative differences in the immune response to sepsis in the setting of chronic alcohol ingestion.

In vitro studies have implicated the small heat shock protein HSPB1 in a range of physiological functions. However, its in vivo relevance is unclear as the phenotype of unstressed HSPB1−/−mice is unremarkable. To determine the impact of HSPB1 in injury, HSPB1−/−and wild type (WT) mice were subjected to cecal ligation and puncture, a model of polymicrobial sepsis. Ten-day mortality was significantly higher in HSPB1−/−mice following the onset of sepsis (65% vs. 35%). Ex vivo mechanical testing revealed that common carotid arteries from HSPB1−/−mice were more compliant than those in WT mice over pressures of 50–120 mm Hg. Septic HSPB1−/−mice also had increased peritoneal levels of IFN-γ and decreased systemic levels of IL-6 and KC. There were no differences in frequency of either splenic CD4+or CD8+T cells, nor were there differences in apoptosis in either cell type. However, splenic CD4+T cells and CD8+T cells from HSPB1−/−mice produced significantly less TNF and IL-2 following ex vivo stimulation. Systemic and local bacterial burden was similar in HSPB1−/−and WT mice. Thus while HSPB1−/−mice are uncompromised under basal conditions, HSPB1 has a critical function in vivo in sepsis, potentially mediated through alterations in arterial compliance and the immune response.

Background In the context of increasing obesity prevalence, the relationship between large visceral adipose tissue (VAT) volumes and type 2 diabetes mellitus (T2DM) is unclear. In a clinical sample of severely obese women (mean body mass index [BMI], 46 kg/m2) with fasting normoglycemia (n=40) or dysglycemia (impaired fasting glucose+diabetes; n=20), we sought to determine the usefulness of anthropometric correlates of VAT and associations with dysglycemia. Methods VAT volume was estimated using multi-slice computer tomography; anthropometric surrogates included sagittal abdominal diameter (SAD), waist circumference (WC) and BMI. Insulin sensitivity (Si), and beta-cell dysfunction, measured by insulin secretion (AIRg) and the disposition index (DI), were determined by frequently sampled intravenous glucose tolerance test. Results Compared to fasting normoglycemic women, individuals with dysglycemia had greater VAT (P<0.001) and SAD (P=0.04), but BMI, total adiposity and Si were similar. VAT was inversely associated with AIRg and DI after controlling for ancestry, Si, and total adiposity (standardized beta, −0.32 and −0.34, both P<0.05). In addition, SAD (beta=0.41, P=0.02) was found to be a better estimate of VAT volume than WC (beta=0.32, P=0.08) after controlling for covariates. Receiver operating characteristic analysis showed that VAT volume, followed by SAD, outperformed WC and BMI in identifying dysglycemic participants. Conclusions Increasing VAT is associated with beta-cell dysfunction and dysglycemia in very obese women. In the presence of severe obesity, SAD is a simple surrogate of VAT, and an indicator of glucose dysregulation.

The gut is a continuously renewing organ, with cell proliferation, migration, and death occurring rapidly under basal conditions. As the impact of critical illness on cell movement from crypt base to villus tip is poorly understood, the purpose of this study was to determine how sepsis alters enterocyte migration. Wild-type, transgenic, and knockout mice were injected with 5-bromo-2'deoxyuridine (BrdU) to label cells in S-phase before and after the onset of cecal ligation and puncture and were sacrificed at predetermined endpoints to determine distance proliferating cells migrated up the crypt-villus unit. Enterocyte migration rate was decreased from 24 to 96h after sepsis. BrdU was not detectable on villi 6 days after sham laparotomy, meaning all cells had migrated the length of the gut and been exfoliated into its lumen. However, BrdU positive cells were detectable on villi 10 days after sepsis. Multiple components of gut integrity altered enterocyte migration. Sepsis decreased crypt proliferation, which further slowed enterocyte transit as mice injected with BrdU after the onset of sepsis (decreased proliferation) had slower migration than mice injected with BrdU before the onset of sepsis (normal proliferation). Decreasing intestinal apoptosis via gut-specific overexpression of Bcl-2 prevented sepsis-induced slowing of enterocyte migration. In contrast, worsened intestinal hyperpermeability by genetic deletion of JAM-A increased enterocyte migration. Sepsis therefore significantly slows enterocyte migration, and intestinal proliferation, apoptosis and permeability all affect migration time, which can potentially be targeted both genetically and pharmacologically.

ABSTRACT: Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the peri-junctional actin-myosin ring. Genetic deletion of myosin light chain kinase (MLCK) reverses intestinal hyperpermeability and improves survival in a murine model of intra-abdominal sepsis. In an attempt to determine whether these findings could be translated using a more clinically relevant strategy, this study aimed to determine if pharmacologic inhibition of MLCK using the membrane permeant inhibitor of MLCK (PIK) improved gut barrier function and survival following sepsis. C57BL/6 mice underwent cecal ligation and puncture to induce sepsis and were then randomized to receive either PIK or vehicle. Unexpectedly, PIK significantly worsened 7-day survival following sepsis (24% vs. 62%). The three pathways of intestinal permeability were then interrogated by orally gavaging septic mice with creatinine (6Å), FD-4 (28Å), and rhodamine70 (120Å) and assaying their appearance in the bloodstream. PIK led to increased permeability in the leak pathway with higher levels of FD-4 in the bloodstream compared to septic mice given vehicle. In contrast, no differences were detected in the pore or unrestricted pathways of permeability. Examination of jejunal tight junctions for potential mechanisms underlying increased leak permeability revealed that mice that received PIK had increased phosphorylated MLC without alterations in occludin, ZO-1, or JAM-A. PIK administration was not associated with significant differences in systemic or peritoneal bacterial burden, cytokines, splenic or Peyer's Patches immune cells or intestinal integrity. These results demonstrate that pharmacologic inhibition of MLCK unexpectedly increases mortality, associated with worsened intestinal permeability through the leak pathway, and suggest caution is required in targeting the gut barrier as a potential therapy in sepsis.

The endogenous bacteria have been hypothesized to play a significant role in the pathophysiology of critical illness, although their role in sepsis is poorly understood. The purpose of this study was to determine how commensal bacteria alter the host response to sepsis. Conventional and germ free (GF) C57Bl/6 mice were subjected to Pseudomonas aeruginosa pneumonia. All GF mice died within two days while 44% of conventional mice survived for 7 days (p=0.001). Diluting the dose of bacteria 10-fold in GF mice led to similar survival in GF and conventional mice. When animals with similar mortality were assayed for intestinal integrity, GF mice had lower levels of intestinal epithelial apoptosis but similar levels of proliferation and intestinal permeability. GF mice had significantly lower levels of TNF and IL-1β in BAL fluid compared to conventional mice without changes in systemic cytokine production. Under conventional conditions, sepsis unmasks lymphocyte control of intestinal epithelial apoptosis, since sepsis induces a greater increase in gut apoptosis in Rag-1−/− mice than wild type (WT) mice. However, in a separate set of experiments, gut apoptosis was similar between septic GF Rag-1−/− mice and septic GF WT mice. These data demonstrate that the endogenous bacteria play a protective role in mediating mortality from pneumonia-induced sepsis, potentially mediated through altered intestinal apoptosis and the local pro-inflammatory response. Additionally, sepsis-induced lymphocyte-dependent increases in gut epithelial apoptosis appear to be mediated by the endogenous bacteria.