Activation of mucosal insulin receptor exacerbates intestinal inflammation by promoting tissue resident memory T cells differentiation through EZH2.

BACKGROUND: Inflammatory Bowel Diseases (IBD), an autoimmune disease characterised by abnormal intestinal immunity, are related to vital morbidity around the world. However, therapeutic agents for IBD have not achieved desired benefit. Exploring new therapeutic targets for IBD, especially based on its abnormally intestinal immunity, could alleviate the flare-up and worsening of IBD. Tissue resident memory T cells (TRM) are core of multiple autoimmune diseases, including IBD. However, the mechanism of TRM differentiation remains to be investigated. METHODS: The alterations in mRNA and lncRNA profile of intestinal intraepithelial lymphocytes (IELs), the largest component of intestinal TRM, were analyzed in DSS-induced chronic colitis. Based on it, we examined the function of rectal insulin instillation in a dextran sodium sulfate (DSS) induced chronic colitis. Furthermore, we investigated the downstream-target of the insulin pathway-EZH2 and the crucial role of EZH2 in intestinal tissue resident memory T cell differentiation by utilizing EZH2fl/flCD4cre mice. RESULTS: Insulin receptor (INSR) expression was found to be significantly reduced. Activation of mucosal insulin pathway by rectal insulin instillation exacerbated colitis by disrupting IELs subgroups and up-regulating TNF-ɑ and IL-17 expression. Rectal insulin instillation promoted EZH2 expression and EZH2 inhibition alleviated chronic colitis. EZH2fl/flCD4cre mice restored the normal IEL subgroups and suppressed TNF-ɑ and IL-17 expression, exhibiting alleviated colitis. IELs from EZH2fl/flCD4cre mice exhibit significant changes in TRM related phenotype. CD4+TRM was significantly increased in chronic colitis and decreased in EZH2fl/flCD4cre mice. CONCLUSION: Insulin receptor of intestinal mucosal T-cells could promote intestinal TRM differentiation via EZH2. Our discoveries suggest that therapies targeting colonic INSR and EZH2 could be potential treatment for IBD based on its regulatory effects on TRM. Insulin receptor inhibitors rather than insulin should be applied during colitis-active phase. In addition, EZH2 shows to be a downstream signal of the insulin pathway and EZH2 inhibitor could alleviating intestinal inflammation. However, the critical role of EZH2 in TRM differentiation restricts the anti-tumor effects of EZH2 inhibitor in vivo.

Effects of fecal stream deprivation on human intestinal barrier after loop ileostomy.

BACKGROUND AND AIM: Intestinal homeostasis is closely associated with the normal intestinal luminal physiological environment. Temporary loop ileostomy changes the intestinal structure and diverts the fecal stream, thereby disturbing the intestinal environment. This study aimed to clarify the changing situation of the human intestinal mucosa barrier in the absence of a fecal stream after loop ileostomy. METHODS: We obtained paired samples from the fed (fecal stream maintained) and unfed (no fecal stream) portions of the loop ileostomy and subjected these samples to RNA sequencing. We also determined transepithelial electrical resistance. The mucus layer thickness and content of MUC2, tight junction proteins, and common antimicrobial peptides in ileum mucosa were studied. RESULTS: Transcriptome data revealed that genes associated with enhancing the intestinal barrier function of the unfed ileum were significantly decreased and genes associated with immune defense response were significantly increased. The transepithelial electrical resistance was lower and the mucus layer thickness was thinner in the unfed ileal mucosa than in the fed ileum. The MUC2, Occludin, and zonula occludens 1 content was lower in the unfed ileum than in the fed ileum. α-Defensin 5, α-defensin 6, and lysozyme content was higher in the unfed ileum than in the enterally fed ileum. CONCLUSION: Intestinal barrier function is weakened after long-term fecal diversion, but antimicrobiota defense function is strengthened. Thus, the intestinal mucosa barrier adopts an alternative stable state during fecal diversion, which may explain the clinical paucity of cases of enterogenic infection caused by loop ileostomy.

Antifungal Treatment Aggravates Sepsis through the Elimination of Intestinal Fungi.

Prophylactic antifungal therapy is widely adopted clinically for critical patients and effective in reducing the morbidity of invasive fungal infection and improves outcomes of those diagnosed patients; however, it is not associated with higher overall survival. As intestinal commensal fungi play a fundamental role in the host immune response in health and disease, we propose that antifungal therapy may eliminate intestinal fungi and aggravate another critical syndrome, sepsis. Here, with murine sepsis model, we found that antifungal therapy with fluconazole dismissed intestinal fungal burden and aggravated endotoxin-induced but no gram-positive bacteria-induced sepsis. Nevertheless, antifungal therapy did not exert its detrimental effect on germ-free mice. Moreover, colonizing more commensal fungi in the mouse intestine or administration of fungal cell wall component mannan protected the mice from endotoxin-induced sepsis. On the molecular level, we demonstrated that antifungal therapy aggravated endotoxin sepsis through promoting Gasdermin D cleavage in the distal small intestine. Intestinal colonization with commensal fungi inhibited Gasdermin D cleavage in response to lipopolysaccharide challenge. These findings show that intestinal fungi inhibit Gasdermin D-mediated pyroptosis and protect the mice from endotoxin-induced sepsis. This study demonstrates the protective role of intestinal fungi in the pathogenesis of endotoxin-induced sepsis in the laboratory. It will undoubtedly prompt us to study the relationship between antifungal therapy and sepsis in critical patients who are susceptible to endotoxin-induced sepsis in the future.

Interleukin-28A maintains the intestinal epithelial barrier function through regulation of claudin-1.

BACKGROUND: Interleukin-28A (IL-28A or interferon-λ2) is reported to maintain intestinal mucosal homeostasis. However, the effects and mechanisms of IL-28A on intestinal ischemia reperfusion (I/R) have not yet been studied. METHODS: Adult C57BL/6 mice were randomly divided into three groups: sham, I/R, and I/R+IL-28A (n=5 in each group). The I/R+IL-28A group mice were injected with recombinant mouse IL-28A 12 hours before the operation. Mice were sacrificed 6 hours after reperfusion. The mucosal permeability was investigated, and histology analyses were performed. Additionally, a hypoxic Caco-2 cell culture model was established. Fludarabine was used to inhibit phosphorylated signal transducer and activator of transcription 1 (pSTAT1). The expression of IL-28A, tight junctions (TJs), and pSTAT1 was assessed by western blot, immunohistochemical (IHC) staining, or immunofluorescence staining. Epithelial permeability was measured by transepithelial electrical resistance (TER). RESULTS: The expression of IL-28A was decreased in intestinal lamina propria in the I/R group compared with the control group. Administration of IL-28A significantly alleviated the I/R-induced increase in intestinal permeability and tissue damage. Treatment with IL-28A significantly attenuated intestinal I/R-induced disruption of TJ proteins, including zonula occludens-1 (ZO-1), occludin, and claudin-1. In vitro, IL-28A treatment reversed the decrease in TER of Caco-2 monolayers exposed to hypoxic environments. IL-28A led to the activation of STAT1 and the upregulation of claudin-1 expression both in vivo and in vitro. Also, inhibiting phosphorylation of STAT1 reversed the effects of IL-28A on the expression and distribution of claudin-1 in Caco-2 cells. CONCLUSIONS: Intestinal epithelial barrier dysfunction caused by intestinal I/R is ameliorated by IL-28A via the regulation of claudin-1.

Intestinal epithelial cells related lncRNA and mRNA expression profiles in dextran sulphate sodium-induced colitis.

Intestinal epithelial barrier damage caused by intestinal epithelial cells (IECs) dysfunction plays a crucial role in the pathogenesis and development of inflammatory bowel disease (IBD). Recently, some studies have suggested the emerging role of long non-coding RNAs (lncRNAs) in IBD. The aim of this study was to reveal lncRNAs and mRNA expression profiles in IECs from a mouse model of colitis and to expand our understanding in the intestinal epithelial barrier regulation. IECs from the colons of wild-type mice and dextran sulphate sodium (DSS)-induced mice were isolated for high-throughput RNA-sequencing. A total of 254 up-regulated and 1013 down-regulated mRNAs and 542 up-regulated and 766 down-regulated lncRNAs were detected in the DSS group compared with the Control group. Four mRNAs and six lncRNAs were validated by real-time quantitative PCR. Function analysis showed that dysregulated mRNAs participated in TLR7 signalling pathway, IL-1 receptor activity, BMP receptor binding and IL-17 signalling pathway. Furthermore, the possibility of indirect interactions between differentially expressed mRNAs and lncRNAs was illustrated by the competing endogenous RNA (ceRNA) network. LncRNA ENSMUST00000128026 was predicted to bind to mmu-miR-6899-3p, regulating Dnmbp expression. LncRNA NONMMUT143162.1 was predicted to competitively bind to mmu-miR-6899-3p, regulating Tnip3 expression. Finally, the protein-protein interaction (PPI) network analysis was constructed with 311 nodes and 563 edges. And the highest connectivity degrees were Mmp9, Fpr2 and Ccl3. These results provide novel insights into the functions of lncRNAs and mRNAs involved in the regulation of the intestinal epithelial barrier.

SCFAs induce autophagy in intestinal epithelial cells and relieve colitis by stabilizing HIF-1α.

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of barrier integrity during colonic mucosal injury. Previous works have shown that the absence of autophagy is implicated in the development of inflammatory bowel disease (IBD). Additionally, changes in bacterial profiles in the gut are intimately associated with IBD. Although HIF-1α, autophagy, microbiota, and their metabolites are all involved in the pathogenesis of IBD, their roles are not known. In this study, we investigated the relationship between HIF-1α and autophagy in healthy and inflammatory states using transgenic mice, colitis models, and cell culture models. We confirmed that the absence of intestinal epithelial HIF-1α changed the composition of the intestinal microbes and increased the susceptibility of mice to dextran sodium sulfate (DSS)-induced colitis. In addition, autophagy levels in the intestinal epithelial cells (IECs) were significantly reduced in IEC-specific HIF-1α-deficient (HIF-1α∆IEC) mice. Moreover, in the cell culture models, butyrate treatment significantly increased autophagy in HT29 cells under normal conditions, whereas butyrate had little effect on autophagy after HIF-1α ablation. Furthermore, in the DSS-induced colitis model, butyrate administration relieved the colonic injury and suppressed inflammation in Cre-/HIF-1α- (HIF-1αloxP/loxP) mice. However, the butyrate-mediated protection against colonic injury was considerably diminished in the HIF-1α∆IEC mice. These results show that HIF-1α, autophagy, and intestinal microbes are essential for the maintenance of intestinal homeostasis. Butyrate can alleviate DSS-induced colitis by regulating autophagy via HIF-1α. These insights may have important implications for the development of therapeutic strategies for IBD. KEY MESSAGES: • The absence of intestinal epithelial HIF-1α leads to downregulation of autophagy in mice. • The absence of intestinal epithelial HIF-1α exacerbates DSS-induced colitis. • Short-chain fatty acids (SCFAs) can alleviate DSS-induced colitis by regulating autophagy via HIF-1α.

Intestinal Epithelial Cells-Derived Hypoxia-Inducible Factor-1α Is Essential for the Homeostasis of Intestinal Intraepithelial Lymphocytes.

Hif-1α is a master regulator which involved in the transcriptional regulation of anti-inflammatory or cellular responding to hypoxia. Previous work shows that the absence of Hif-1α results in the destruction of intestinal epithelial cell (IEC) and abnormalities of intestinal barrier function. However, we know very little about other functions of Hif-1α on intestinal intraepithelial lymphocyte (IEL). Therefore, we generated a transgenic mouse (Hif1-α ΔIEC mice), which was knocked out Hif1-α specifically in IECs, to study the effect of Hif1-α on IEL. IELs were isolated from the small intestine and colon of mice, respectively, and examined by flow cytometry and quantitative real-time PCR. All the cytokines expression was detected by quantitative real-time PCR. The NSAID enteropathy was induced by gavaged with 5 mg/kg indomethacin and the experimental colitis was induced by administration of 2.5% DSS. We found that the number of IELs is increased in Hif1-α ΔIEC mice. It is showed that knockout of Hif1-α in IECs led to significant changes in IEL phenotype, including a marked decline in the CD8αα+ and TCRγδ+ population. The reduction of CD8αα+ IELs is accompanied by increased apoptosis, decreased proliferation and weakened migration in Hif1-α ΔIEC mice. Moreover, absence of intestinal epithelial Hif1-α markedly changed the population of IELs in NSAID-induced small intestinal injury and increased susceptibility to dextran sulfate sodium-induced colitis. In summary, our results first time demonstrate that IEC-derived Hif1-α is essential for maintaining IELs homeostasis and intestinal microbiota.

AhR activation protects intestinal epithelial barrier function through regulation of Par-6.

The Par complex (Par-6/Par-3/aPKC) plays a key role in the maintenance of the intestinal barrier function through the regulation of epithelial junction formation. The aryl hydrocarbon receptor (AhR) has been shown to be an important regulator for intestinal homeostasis. In this study, we investigated the role of the AhR activation on the regulation of Par complex. AhR activation by 6-formylindolo (3,2-b) carbazole (FICZ) represses the abnormal expression of the Par complex in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In T84 cells, overexpression of Par-6 causes intestinal barrier dysfunction. Lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction and increase in Par-6 expression was prevented by AhR activation. However, FICZ did not alter the expression of Par-3 or aPKC. Furthermore, AhR activation alleviated LPS-induced increase of Par-6 through repressing the expression of activating protein-2γ (Ap-2γ). These results reveal the protective effects of AhR activation on LPS induced disruption of intestinal epithelial barrier function through suppressing the expression of Par-6 expression. Our findings provide novel insights into the protective role of AhR in intestinal barrier function.