Paneth cell-specific TCF4 down-regulation potentiates alcohol-induced dysbiosis of gut microbiota and tissue injury at the gut-liver axis

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Introduction: Alcohol misuse results in hepatitis, pancreatitis, and neurodegenerative disorders. Alcohol consumption alters gut microbiota and increases gut permeability, leading to endotoxemia and multiple organ damage. However, the cellular and molecular mechanisms of alcohol-induced microbiota dysbiosis and barrier dysfunction remain poorly defined. Antimicrobial peptides such as α-defensins, secreted by the Paneth cells, regulate gut microbiota homeostasis. We previously reported that ethanol (EtOH) feeding reduces α-defensin expression in mice. α-Defensin expression is regulated by Wnt signaling and T-cell factor 4 (TCF4). We investigated the role of Paneth cell TCF4 in alcohol-induced microbiota dysbiosis and tissue injury at the gut-liver axis. Materials and methods: TCF4fl/wt (WT) and TCF4fl/wt-Defa6Cre (KD; heterozygous Paneth cell-specific knockout of TCF4) mice were fed a Lieber-DiCarli liquid diet with or without EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, & 6% 1wk) (EF). Control groups were pair-fed (PF) isocaloric EtOH-free diet. Intestinal permeability was measured by the vascular-to-luminal flux of FITC-inulin (6 kDa) in vivo, and tight junction (TJ) and adherens junction (AJ) integrity were assessed by confocal microscopy for occludin, ZO-1, E-cadherin, and β-catenin. Paneth cell function and mucosal inflammation were assessed by RT-PCR for defensins and cytokines. Gut microbiota was analyzed by 16S rRNA sequencing of colonic flushing. Plasma lipopolysaccharide (LPS) and cytokines were measured to evaluate endotoxemia and systemic inflammation. Liver damage was assessed by measuring plasma AST, liver triglyceride, and cytokine mRNA. Results and conclusion: EtOH reduced TCF4 expression in the ileum in both WT and KD mice; lowest TCF4 expression was observed in KD-EF mice. Similarly, the expression of DEFA5 and DEFA6 was reduced by EtOH in WT and KD mice, with the lowest expression in KD-EF mice. EtOH-induced mucosal permeability was associated with reduced staining for TJ and AJ proteins, indicating the mucosal barrier dysfunction. EtOH increased intestinal mRNA for IL-1β, IL-6, TNFα, and MCP-1, indicating the mucosal inflammatory response. EtOH-induced gut permeability, TJ/AJ disruption, and mucosal inflammation were significantly higher in KD-EF mice than in WT-EF mice. Shannon index analysis indicated that EtOH reduced α-diversity of microbiota in KD but not in WT mice. Firmicutes: Bacteriodetes ratio and the abundance of Verrucomicrobia were reduced by EtOH in WT but not in KD mice. The abundance of Bacillus was increased by EtOH both in WT and KD mice; however, this effect was more severe in KD mice. EtOH-induced plasma LPS and cytokine elevations were higher in KD mice than in WT mice. Elevation of plasma AST, liver triglyceride, and liver cytokine mRNA indicated EtOH-induced liver damage; the damage was more severe in KD mice. These data indicate that Paneth cell TCF4 downregulation exacerbates alcohol-induced microbiota dysbiosis, gut barrier dysfunction, endotoxemia, systemic inflammation, and liver damage; suggesting that Paneth cell Wnt signal downregulation plays a crucial role in alcohol-associated organ damage. NIH/NIAA - R01AA012307, AA029270; Veterans Administration - IO1BX003014. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.