JQ1 as a BRD4 Inhibitor Blocks Inflammatory Pyroptosis-Related Acute Colon Injury Induced by LPS

Endotoxemia is a severe inflammation response induced by infection especially bacterial endotoxin translocation, which severely increases mortality in combination with acute colon injury. Bromodomain-containing protein 4 (BRD4) is an important Bromo and Extra-Terminal (BET) protein to participate in inflammatory responses. However, it is still unknown about the specific connection between BRD4 and inflammation-related pyroptosis in endotoxemia colon. Here, through evaluating the mucous morphology and the expression of tight junction proteins such as occludin and ZO1, we found the upregulation of BRD4 in damaged colon with poor tight junction in an endotoxemia mouse model induced by lipopolysaccharides (LPS). Firstly, the BRD4 inhibitor JQ1 was used to effectively protect colon tight junction in endotoxemia. As detected, high levels of pro-inflammation cytokines IL6, IL1β and IL18 in endotoxemia colon were reversed by JQ1 pretreatment. In addition, JQ1 injection reduced endotoxemia-induced elevation of the phosphorylated NF κB and NLRP3/ASC/caspase 1 inflammasome complex in colon injury. Furthermore, activated pyroptosis markers gasdermins in endotoxemia colon were also blocked by JQ1 pretreatment. Together, our data indicate that BRD4 plays a critical role in regulating pyroptosis-related colon injury induced by LPS, and JQ1 as a BRD4 inhibitors can effectively protect colon from endotoxemia-induced inflammation injury.

Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute and chronic graft-versus-host disease (GVHD). The impact of obesity on allo-HSCT outcomes is poorly understood. Here, we report that obesity had a negative and selective impact on acute gut GVHD after allo-HSCT in mice with diet-induced obesity (DIO). These animals exhibited increased gut permeability, endotoxin translocation across the gut, and radiation-induced gastrointestinal damage after allo-HSCT. After allo-HSCT, both male and female DIO mouse recipients showed increased proinflammatory cytokine production and expression of the GVHD marker ST2 (IL-33R) and MHC class II molecules; they also exhibited decreased survival associated with acute severe gut GVHD. This rapid-onset, obesity-associated gut GVHD depended on donor CD4+ T cells and occurred even with a minor MHC mismatch between donor and recipient animals. Retrospective analysis of clinical cohorts receiving allo-HSCT transplants from unrelated donors revealed that recipients with a high body mass index (BMI, >30) had reduced survival and higher serum ST2 concentrations compared with nonobese transplant recipients. Assessment of both DIO mice and allo-HSCT recipients with a high BMI revealed reduced gut microbiota diversity and decreased Clostridiaceae abundance. Prophylactic antibiotic treatment protected DIO mouse recipients from endotoxin translocation across the gut and increased inflammatory cytokine production, as well as gut pathology and mortality, but did not protect against later development of chronic skin GVHD. These results suggest that obesity-induced alterations of the gut microbiota may affect GVHD after allo-HSCT in DIO mice, which could be ameliorated by prophylactic antibiotic treatment.

Endotoxin Translocation and Gut Inflammation Are Increased in Broiler Chickens Receiving an Oral Lipopolysaccharide (LPS) Bolus during Heat Stress

Lipopolysaccharides (LPS), also termed endotoxins, are the major component of the outer membrane of Gram-negative bacteria. In general, endotoxins in the intestine are considered harmless in healthy animals. However, different stressors, such as heat stress, can lead to a compromised gut barrier, resulting in endotoxin translocation. Chickens are considered to be less sensitive to the effects of LPS compared with other species, for example, humans, pigs, or calves, probably because of the lack of the functional-specific TRAM-TRIF signalling pathway (MyD88-independent). Therefore, six LPS preparations (three different strains with two different preparation methods each) were compared in murine macrophages and characterized according to their MyD88-dependent pathway activation. All tested LPS preparations induced a strong inflammatory response after 4 and 24 h on a murine macrophage cell line. However, there was a similar strong response in the gene expression profile as well as production of nitrite oxide and TNF-alpha from LPS of different strains and preparation methods. On the basis of the results of the in vitro study, one LPS preparation was chosen for the subsequent in vivo study with broilers to assess the effect of an oral LPS bolus (E. coli O55:B5 phenol extracted; 2 mg/kg b.w.) during heat stress conditions (10 h, 36 °C). The most pronounced effects were seen in broilers receiving the oral LPS bolus during heat stress conditions. The endotoxin activity in the intestine as well as the serum concentration of the 3-OH C14 (part of LPS) were increased. In addition, an increased expression of genes related to inflammation and stress response (e.g., IL-6, IL-1beta, HSP70) was observed, whereas the expression of genes associated with gut health (e.g., MUC2, FABP2) was decreased. To conclude, an increase of intestinal LPS combined with heat stress can pose a risk to animal health.

Leaky gut potentially leads to bacterial/endotoxin translocation in a rat model of non-ischemic acute kidney injury

Background: Emerging evidence indicates that there is a causal relationship between acute kidney injury (AKI) and gut barrier disruption. The aim of our study was to determine whether the translocation of gut-derived bacteria/endotoxin develops in non-ischemic AKI, and, if so, what is the mechanism behind it? Methods: SPF male Sprague-Dawley rats were randomly subjected to bilateral nephrectomy (BNx) or sham-operation and observed for 24 hours. Gut permeability was evaluated in vivo and in vitro. Serum endotoxin and bacterial loads in liver and mesenteric lymph nodes (MLN) were measured. The expression of the key tight junctions (TJs) in ileum, including zonula occluden-1(ZO-1), occludin and claudin-1 were evaluated by Western blot and immunohistochemical staining. The structure of TJs was observed using transmission electron microscopy. Apoptotic changes of ileal mucosa were evaluated by TUNEL staining and ELISA. Results: Non-ischemic AKI rats (rats subjected to BNx) demonstrated marked blunting and shortening of the gut villi. The gut mucosal permeability was increased in non-ischemic AKI rats, evidenced by the elevated serum levels of D-lactate and the increased amount of FITC-Dextran which passed through the ileum wall. Serum endotoxin was significantly elevated in non-ischemic AKI rats. Non-ischemic AKI rats had relatively higher bacterial loads in liver and MLN than sham-operated rats. For non-ischemic AKI rats, apoptosis of ileal mucosa was significantly accelerated. Neither the protein expression and the distribution pattern, nor the structure of the TJs was altered in non-ischemic AKI rats. Conclusion: Non-ischemic AKI results in profound gut barrier disruption and thus favors the subsequent episode of bacterial/endotoxin translocation. In this non-ischemic AKI model, the translocation of bacterial/endotoxin was not most likely due to a TJs mediated paracellular pathway.

Obesity-Induced Microbiome Alterations Result in Severe Gastrointestinal Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a viable treatment option for many cancers but its clinical utility is limited due to the occurrence of graft-versus-host disease (GVHD). Understanding the impact of obesity on immune function has become increasingly important in the setting of the current obesity pandemic. We report here that obesity has a negative and selective impact on acute gut GVHD. Diet-induced obese (DIO) mice exhibited increased gut permeability, endotoxin translocation and radiation-induced gastrointestinal damage. After allo-HSCT, DIO recipients across strains and sex had markedly increased pro-inflammatory cytokines (IL-6, TNF), GVHD biomarker ST2, MHC class II expression and exhibited rapid mortality associated with severe acute gut pathology. This obesity-associated lethal acute gut GVHD was dependent on donor CD4 T cells and occurred even in minor MHC mismatch strain combination in which only a delayed skin chronic GVHD resulted in lean recipients. Pro-inflammatory cytokine blockade targeting both IL-6 and TNF ameliorated obesity-associated acute gut GVHD while maintaining graft-versus-tumor (GVT) effects. Microbiome assessment of DIO mice revealed markedly reduced microbiome diversity and decreased Clostridiaceae abundance. Additionally, DIO mice had a significant increase of GVHD-associated Akkermansia muciniphila before and after allo-HSCT compared to the controls. Extended antibiotic treatment of DIO mice protected from the endotoxin translocation, cytokine storm as well as gut GVHD pathology but did not protect later development of chronic skin GVHD. These results demonstrate that obesity alters the microbiome and imparts differential effects on GVHD following allo-HSCT with decreased survival and this inferior outcome can be pre-empted by combined pro-inflammatory cytokine blockade or antibiotic pretreatment. Disclosures Pai: Roche-Genentech: Employment. Holtan:CSL Behring: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy. Ferrara:ViraCor: Consultancy; Incyte: Consultancy; Kamada: Consultancy; Mallinckrodt: Consultancy; Enlivex: Consultancy; Xenikos: Consultancy; CSL Behring: Consultancy; National Institutes of Health: Research Funding. Levine:Novartis: Honoraria; Kamada: Research Funding; bluebird bio: Consultancy; National Cancer Institute: Research Funding; Incyte: Consultancy, Research Funding; Ironwood: Honoraria; Biogen: Other: non-financial support; Viracor: Patents & Royalties: biomarker patent. Abedi:Abbie: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau; Takeda: Speakers Bureau. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Diurnal heat stress reduces pig intestinal integrity and increases endotoxin translocation

Heat stress negatively affects performance and intestinal integrity of pigs. The objective of this study was to characterize the effects of diurnal heat stress (dHS) on nursery-grower pig performance, intestinal integrity, and lipopolysaccharide (LPS) translocation. Forty-eight nursery-grower gilts, individually penned, were randomly assigned to two treatments. Twenty-four pigs were then exposed to dHS for 3 d, 6 h at 38°C and 18 h at 32°C, at 40–60% humidity. The remaining pigs were maintained under thermal neutral (TN) conditions. Changes in pig rectal temperatures (Tr), respiration rates (RR), performance, and blood parameters were evaluated. Additionally, ex vivo ileum integrity was assessed with the Ussing chamber by measuring transepithelial resistance (TER), and 4 kDa fluorescein isothiocyanate (FITC)–dextran (FD4) and FITC–LPS mucosal to serosal flux. As expected, dHS increased pig Tr and RR (P < 0.05) and reduced pig performance (P < 0.05) on the 3-d period. Compared with TN, ileum TER (P = 0.04), FITC–LPS (P < 0.001), and FD4 (P = 0.011) permeability were significantly increased due to dHS. Compared with TN pigs, dHS increased serum endotoxin by 150% (P = 0.031). Altogether, 3-d dHS significantly reduced pig performance and intestinal integrity and increased blood endotoxin concentrations.

Chronic high-dosage fish oil exacerbates gut–liver axis injury in alcoholic steatohepatitis in mice: the roles of endotoxin and IL-4 in Kupffer cell polarization imbalance

Chronic high fish oil exacerbated ALD via endotoxin translocation and interleukin-4 suppression, followed by Kupffer cell polarization imbalance.