The roles of intestinal epithelial AhR in alcohol-induced barrier dysfunction and liver injury

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PROJECT SUMMARY Throughout the COVID-19 pandemic, alcohol sales, use and related hospitalizations have dramatically increased, disproportionately affecting minority communities. Alcohol impairs intestinal barrier function through changes in tight junction protein expression. Alcohol-induced intestinal permeability leads to endotoxin from gut microbes leaking out and traveling through portal circulation to prime the liver for injury. The aryl hydrocarbon receptor (AhR), a ubiquitously expressed transcription factor involved in the clearance of xenobiotics and maintenance of immune cell function, has been implicated in the maintenance of intestinal barrier function. AhR activation with dietary ligands has been shown to improve intestinal barrier function in models of inflammation. Furthermore, alcohol-induced gut permeability and liver injury have been shown to be partially attenuated with AhR ligands. However, these limited studies have not examined the role of cell specific AhR signaling in the maintenance of intestinal barrier function in response to alcohol. Our preliminary data demonstrate that the combination of intestinal epithelial specific (IEC) AhR knockout and treatment with the AhR ligand β- naphthoflavone (BNF) provides protection against alcohol induced gut permeability and liver injury. We observed i) decrease in ethanol-induced endotoxemia (intestinal permeability marker), ii) decrease in the induction of serum alanine aminotransferase (ALT; hepatic injury marker), and iii) induction of AhR target genes in ileal mucosa. Previous studies have shown that intestinal epithelial AhR regulates availability of dietary AhR ligands to immune cells. Therefore, we hypothesize that the absence of intestinal epithelial AhR increases the availability of AhR ligands to intestinal immune cells, leading to an increase in immune AhR signaling, and IL-10 and IL-22 mediated improvement in ethanol induced intestinal barrier permeability and subsequent protection to liver injury. The studies proposed in Specific Aim 1 will elucidate the mechanisms of intestinal epithelial and immune cell specific AhR on alcohol mediated gut permeability—with focus on differences between the small and large intestines—and liver injury using intestinal epithelial and immune cell specific AhR knockout mice. These studies will also delineate the ethanol induced changes in microbial metabolites and their role in AhR activation by reporter assays. Specific Aim 2 will utilize state-of-the- art intestinal epithelial enteroid-immune cell co-cultures to study the crosstalk between IECs and immune cells in mediating alcohol induced gut permeability. Overall, the successful completion of the proposed work could have profound implications for the treatment of intestinal barrier contributing liver injury such as in alcoholic liver disease. All experiments will consider variations that may arise from sex differences, with attention to statistics and reproducibility. The proposed studies will provide comprehensive training for the applicant and identify novel therapeutic targets to aid intestinal barrier function and allow for intervention in alcohol use disorder.