Dietary Supplementation With Nonfermentable Fiber Alters the Gut Microbiota and Confers Protection in Murine Models of Sepsis.
ABSTRACT: Links between microbial alterations and systemic inflammation have been demonstrated in chronic disease, but little is known about these interactions during acute inflammation. This study investigates the effect of dietary supplementation with cellulose, a nonfermentable fiber, on the gut microbiota, inflammatory markers, and survival in two murine models of sepsis.Prospective experimental study.University laboratory.Six-week-old male C57BL/6 wild-type mice.Mice were assigned to low-fiber, normal-fiber, or high-fiber diets with or without antibiotics for 2 weeks and then subjected to sepsis by cecal ligation and puncture or endotoxin injection. Fecal samples were collected for microbiota analyses before and after dietary interventions.Mice that received a high-fiber diet demonstrated increased survival after cecal ligation and puncture relative to mice receiving low-fiber or normal-fiber diets. The survival benefit was associated with decreased serum concentration of pro-inflammatory cytokines, reduced neutrophil infiltration in the lungs, and diminished hepatic inflammation. The high-fiber diet also increased survival after endotoxin injection. Bacterial 16S ribosomal RNA gene sequences from each sample were amplified, sequenced, and analyzed. Fiber supplementation yielded an increase in relative abundance of the genera Akkermansia and Lachnospiraceae, taxa commonly associated with metabolic health. Administration of antibiotics to mice on the high-fiber diet negated the enrichment of Akkermansia species and the survival benefit after cecal ligation and puncture.Dietary supplementation with cellulose offers a microbe-mediated survival advantage in murine models of sepsis. Improved understanding of the link between diet, the microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis.
Project description:The gut plays a vital role in critical illness, and alterations in the gut structure and function have been reported in endotoxemia and sepsis models. Previously, we have demonstrated a novel link between the diet-induced alteration of the gut microbiome with cellulose and improved outcomes in sepsis. As compared to mice receiving basal fiber (BF) diet, mice that were fed a non-fermentable high fiber (HF) diet demonstrated significant improvement in survival and decreased organ injury in both cecal-ligation and puncture (CLP) and endotoxin sepsis models. To understand if the benefit conferred by HF diet extends to the gut structure and function, we hypothesized that HF diet would be associated with a reduction in sepsis-induced gut epithelial loss and permeability in mice. We demonstrate that the use of dietary cellulose decreased LPS-mediated intestinal hyperpermeability and protected the gut from apoptosis. Furthermore, we noted a significant increase in epithelial cell proliferation, as evidenced by an increase in the percentage of bromodeoxyuridine-positive cells in HF fed mice as compared to BF fed mice. Thus, the use of HF diet is a simple and effective tool that confers benefit in a murine model of sepsis, and understanding the intricate relationship between the epithelial barrier, gut microbiota, and diet will open-up additional therapeutic avenues for the treatment of gut dysfunction in critical illness.
Project description:OBJECTIVES:Our goal was to "reverse translate" the human response to surgical sepsis into the mouse by modifying a widely adopted murine intra-abdominal sepsis model to engender a phenotype that conforms to current sepsis definitions and follows the most recent expert recommendations for animal preclinical sepsis research. Furthermore, we aimed to create a model that allows the study of aging on the long-term host response to sepsis. DESIGN:Experimental study. SETTING:Research laboratory. SUBJECTS:Young (3-5 mo) and old (18-22 mo) C57BL/6j mice. INTERVENTIONS:Mice received no intervention or were subjected to polymicrobial sepsis with cecal ligation and puncture followed by fluid resuscitation, analgesia, and antibiotics. Subsets of mice received daily chronic stress after cecal ligation and puncture for 14 days. Additionally, modifications were made to ensure that "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" recommendations were followed. MEASUREMENTS AND MAIN RESULTS:Old mice exhibited increased mortality following both cecal ligation and puncture and cecal ligation and puncture + daily chronic stress when compared with young mice. Old mice developed marked hepatic and/or renal dysfunction, supported by elevations in plasma aspartate aminotransferase, blood urea nitrogen, and creatinine, 8 and 24 hours following cecal ligation and puncture. Similar to human sepsis, old mice demonstrated low-grade systemic inflammation 14 days after cecal ligation and puncture + daily chronic stress and evidence of immunosuppression, as determined by increased serum concentrations of multiple pro- and anti-inflammatory cytokines and chemokines when compared with young septic mice. In addition, old mice demonstrated expansion of myeloid-derived suppressor cell populations and sustained weight loss following cecal ligation and puncture + daily chronic stress, again similar to the human condition. CONCLUSIONS:The results indicate that this murine cecal ligation and puncture + daily chronic stress model of surgical sepsis in old mice adhered to current Minimum Quality Threshold in Pre-Clinical Sepsis Studies guidelines and met Sepsis-3 criteria. In addition, it effectively created a state of persistent inflammation, immunosuppression, and weight loss, thought to be a key aspect of chronic sepsis pathobiology and increasingly more prevalent after human sepsis.
Project description:Malignancy increases sepsis incidence 10-fold and elevates sepsis-associated mortality. Advances in treatment have improved survival of cancer patients shortly after sepsis, but there is a paucity of information on how sepsis impacts cancer growth, development, and prognosis. To test this, cecal ligation and puncture surgery was performed on B16 melanoma-bearing mice to show that sepsis has detrimental effects in hosts with advanced tumors, leading to increased mortality. Surprisingly, mice experiencing cecal ligation and puncture-induced sepsis earlier during tumor development exhibited CD8 T cell-dependent attenuation of tumor growth. Sepsis-resistant CD8 tumor-infiltrating T cells showed increased in vivo activation, effector IFN-? cytokine production, proliferation, and expression of activation/inhibitory PD-1/LAG-3 receptors because of a sepsis-induced liberation of tumor Ags. Sepsis-reinvigorated CD8 tumor-infiltrating T cells were also amenable to (anti-PD-L1/LAG-3) checkpoint blockade therapy, further prolonging cancer-associated survival in sepsis survivors. Thus, sepsis has the capacity to improve tumor-specific CD8 T cell responses, leading to better cancer prognosis and increased survival.
Project description:OBJECTIVE:Sepsis-induced multi-organ failure continues to have a high mortality. The liver is an organ central to the disease pathogenesis. The objective of this study was to identify the liver proteins that change in abundance with sepsis and subsequently identify new drug targets. DESIGN:Proteomic discovery study and drug target validation. For the proteomics study, three biological replicate mice were used per group. SETTING:Research institute laboratory. SUBJECTS:Three-month-old C57BL/6 mice. INTERVENTIONS:We used a mouse model of sepsis based on cecal ligation and puncture, but with fluid and antibiotic resuscitation. Liver proteins that changed in abundance were identified by difference in gel electrophoresis. We compared liver proteins from 6-hr post-cecal ligation and puncture to sham-operated mice ("early proteins") and 24-hr post-cecal ligation and puncture with 6-hr post-cecal ligation and puncture ("late proteins"). Proteins that changed in abundance were identified by tandem mass spectrometry. We then inhibited the receptor for one protein and determined the effect on sepsis-induced organ dysfunction. RESULTS:The liver proteins that changed in abundance after sepsis had a range of functions such as acute phase response, coagulation, endoplasmic reticulum stress, oxidative stress, apoptosis, mitochondrial electron transfer proteins, and nitric oxide metabolism. We found that cyclophilin increased in abundance after cecal ligation and puncture. When the receptor for this protein, CD147, was inhibited, sepsis-induced renal dysfunction was reduced. There was also a significant reduction in serum cytokine production when CD147 was inhibited. CONCLUSION:By applying proteomics to a clinically relevant mouse model of sepsis, we identified a number of novel proteins that changed in abundance. The inhibition of the receptor for one of these proteins, cyclophilin, attenuated sepsis-induced acute renal failure. The application of proteomics to sepsis research can facilitate the discovery of new therapeutic targets.
Project description:Multiple organ dysfunction induced by sepsis often involves kidney injury. Extracellular histones released in response to damage-associated molecular patterns are known to facilitate sepsis-induced organ dysfunction. Recombinant human soluble thrombomodulin (rhTM) and its lectin-like domain (D1) exert anti-inflammatory effects and neutralize damage-associated molecular patterns. However, the effects of rhTM and D1 on extracellular histone H3 levels and kidney injury remain poorly understood. Our purpose was to investigate the association between extracellular histone H3 levels and kidney injury, and to clarify the effects of rhTM and D1 on extracellular histone H3 levels, kidney injury, and survival in sepsis-induced rats. Rats in whom sepsis was induced via cecal ligation and puncture were used in this study. Histone H3 levels, histopathology of the kidneys, and the survival rate of rats at 24 h after cecal ligation and puncture were investigated. Histone H3 levels increased over time following cecal ligation and puncture. Histopathological analyses indicated that the distribution of degeneration foci among tubular epithelial cells of the kidney and levels of histone H3 increased simultaneously. Administration of rhTM and D1 significantly reduced histone H3 levels compared with that in the vehicle-treated group and improved kidney injury. The survival rates of rats in rhTM- and D1-treated groups were significantly higher than that in the vehicle-treated group. The results of this study indicated that rhTM and its D1 similarly reduce elevated histone H3 levels, thereby reducing acute kidney injury. Our findings also proposed that rhTM and D1 show potential as new treatment strategies for sepsis combined with acute kidney injury.
Project description:Peroxisome proliferator-activated receptor-? is significantly down-regulated in circulating leukocytes from children with sepsis. Peroxisome proliferator-activated receptor-? null (Ppara) mice have greater mortality than wild-type mice when subjected to sepsis by cecal ligation and puncture. We sought to characterize the role of peroxisome proliferator-activated receptor-? in sepsis and to identify the mechanism whereby peroxisome proliferator-activated receptor-? confers a survival advantage.Prospective randomized preclinical study.Laboratory investigation.Male C57Bl/6J and Ppara mice (B6.129S4-Ppara/J), aged 12-16 weeks.Bone marrow chimeric mice were generated and subjected to cecal ligation and puncture. Survival was measured for 7 days. Separate groups of nontransplanted mice underwent cecal ligation and puncture and were euthanized 24 hours later for plasma and tissue analyses.Ppara mice had dramatically reduced survival compared with wild-type mice irrespective of the peroxisome proliferator-activated receptor-? status of the bone marrow they received (3% vs 63%; p < 0.0001). No difference in survival was observed between Ppara mice that received wild-type versus Ppara marrow or in wild-type mice receiving wild-type versus Ppara marrow. In septic, nontransplanted mice at 24 hours, Ppara mice had elevated cardiac troponin levels compared with wild-type mice. Cardiac histologic injury scores were greater in Ppara versus wild-type mice. Expression of transcription factors and enzymes related to fatty acid oxidation in the heart were profoundly down-regulated in both wild-type and Ppara mice, but more so in the Ppara mice.Peroxisome proliferator-activated receptor-? expression in nonhematopoietic tissues plays a critical role in determining clinical outcome in experimental polymicrobial sepsis and is more important to survival in sepsis than hematopoietic peroxisome proliferator-activated receptor-? expression. Cardiac injury due to inadequate energy production from fatty acid substrate is a probable mechanism of decreased survival in Ppara mice. These results suggest that altered peroxisome proliferator-activated receptor-?-mediated cellular metabolism may play an important role in sepsis-related end-organ injury and dysfunction, especially in the heart.
Project description:Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood.Septic stress induces glucocorticoids production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI)-null mice, which are completely deficient in inducible glucocorticoids in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture induced profound thymocyte apoptosis in SR-BI(+/+) mice, but no thymocyte apoptosis in SR-BI(-/-) mice because of lack of inducible glucocorticoids. Unexpectedly, supplementation of glucocorticoids only partly restored thymocyte apoptosis in SR-BI(-/-) mice. We demonstrated that high-density lipoprotein (HDL) is a critical modulator for thymocyte apoptosis. SR-BI(+/+) HDL significantly enhanced glucocorticoid-induced thymocyte apoptosis, but SR-BI(-/-) HDL had no such activity. Further study revealed that SR-BI(+/+) HDL modulates glucocorticoid-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI(-/-) HDL loses such regulatory activity. To understand why SR-BI(-/-) HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI(-/-) HDL compared with SR-BI(+/+) HDL. Normalization of unesterified cholesterol in SR-BI(-/-) HDL by probucol administration or lecithin cholesteryl acyltransferase expression restored glucocorticoid-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI(+/+) HDL rendered SR-BI(+/+) HDL dysfunctional. Using lckCre-GR(fl/fl) mice in which thymocytes lack cecal ligation and puncture-induced thymocyte apoptosis, we showed that lckCre-GR(fl/fl) mice were significantly more susceptible to cecal ligation and puncture-induced septic death than GR(fl/fl) control mice, suggesting that glucocorticoid-induced thymocyte apoptosis is required for protection against sepsis.The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL.
Project description:Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release.We investigated the contribution of calpain in eliciting tissue damage during sepsis.To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor.In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy.These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.
Project description:The mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway is an essential component of innate immunity necessary for mediating proinflammatory responses in the setting of sepsis. We previously demonstrated that the mitogen-activated protein kinase 1/2 inhibitor trametinib prevents endotoxin-induced renal injury in mice. We therefore assessed efficacy of trametinib in a more clinically relevant experimental model of sepsis.Controlled in vivo laboratory study.University animal research laboratory.Male C57BL/6 mice.Mice were subjected to cecal ligation and puncture to induce sepsis or underwent sham operation as controls. Six hours after cecal ligation and puncture, mice were randomized to four experimental groups as follows: 1) sham control; 2) sham control + trametinib (1?mg/kg, IP); 3) cecal ligation and puncture; and 4) cecal ligation and puncture + trametinib. All animals received buprenorphine (0.05?mg/kg, SC) and imipenem/cilastatin (14?mg/kg, SC) in 1.5?mL of warm saline (40?mL/kg) at the 6-hour time point. Mice were euthanized at 18 hours after induction of cecal ligation and puncture.Trametinib inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase signaling 6 hours after cecal ligation and puncture attenuated increases in circulating proinflammatory cytokines (tumor necrosis factor-?, interleukin-1?, interleukin-6, and granulocyte macrophage colony-stimulating factor) and hypothermia at 18 hours. Trametinib also attenuated multiple organ injury as determined by serum creatinine, alanine aminotransferase, lactate dehydrogenase, and creatine kinase. At the organ level, trametinib completely restored peritubular capillary perfusion in the kidney. Restoration of microvascular perfusion was associated with reduced messenger RNA expression of well-characterized markers of proximal tubule injury. mitogen-activated protein kinase/extracellular signal-regulated kinase blockade attenuated cecal ligation and puncture-mediated up-regulation of cytokines (tumor necrosis factor-?, interleukin-1?) and restored interleukin-6 to control levels in the renal cortex, indicating the protective effects on the proximal tubule occur primarily through modulation of the proinflammatory response in sepsis.These data reveal that the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor trametinib attenuates systemic inflammation and multiple organ damage in a clinically relevant model of sepsis. Because trametinib has been safely used in humans, we propose that this drug might represent a translatable approach to limit organ injury in septic patients.
Project description:RATIONALE:BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. OBJECTIVE:We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. METHODS AND RESULTS:The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture-induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture-induced sepsis. CONCLUSIONS:Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.