AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS IS ESSENTIAL FOR I3C-MEDIATED PROTECTION AGAINST COLITIS

Abstract

Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease that affects the colon and is characterized by localized tissue injury and disruption of the intestinal epithelial barrier. Pathophysiology of UC includes genetic predisposition, epithelial barrier defects, dysregulated immune responses, microbial dysbiosis, and environmental factors. Several therapeutic interventions for colitis have been identified, but many current treatments have various negative side effects, which is alarming given the incidence of UC is increasing around the world. Thus a search for safe therapeutic strategies to control the pathogenesis of colitis is still greatly needed. Previous studies from our laboratory reported that indole-3-carbinol (I3C), a natural plant product and ligand for the aryl hydrocarbon receptor (AhR), exhibited protective effects against colitis through innate lymphoid type 3 (ILC3) cells in an interleukin-22 (IL-22) dependent manner. The aim of this study was to ascertain the possible role of AhR in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. Using in vivo mouse studies and in vitro primary CEC organoid cultures, we investigated how key intestinal regulatory mediators (e.g. mucins and anti-microbial peptides, or AMPs) were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis. Results showed that AV mice had a higher disease score and increased inflammation in the colon when compared to wild type (WT) or littermate (LM) controls. In addition, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. This suggested that the IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice when compared to wild type (WT) or littermate (LM) controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids from WT and AV mice validated these results. Collectively, these data showed that AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.