DOP83 Intestinal epithelium-specific deletion of the Aryl hydrocarbon Receptor (AhR) reduces intestinal Tight Junction permeability thereby hindering homeostatic antigen sampling and tolerogenic immune responses

Abstract Background The Aryl Hydrocarbon Receptor (AhR) plays important functions in intestinal stem cell differentiation, intestinal homeostasis, and immune regulation, however, its role in regulating intestinal tight junction (TJ) barrier and immune tolerance remains poorly understood. In this study, we assessed the role of intestinal epithelium-specific AhR deletion on the intestinal paracellular TJ barrier and its role in homeostatic intestinal antigen sampling and immune tolerance. Methods Ahr fl/fl , and AhrVil-Cre mice were maintained in a specific pathogen-free area until injected with tamoxifen (i.p) to delete the Ahr gene, and then moved to the normal housing. The AhR deletion was maintained by weekly tamoxifen injections for 3 weeks. We performed physiological assessments of the gut epithelial barrier and used flow cytometry, confocal immunofluorescence imaging, and qRT-PCR analysis were also employed to assess the effects of epithelial AhR deletion. Results Conditional gut epithelium-specific knockout of AhR (AhrΔIEC) significantly increased the colonic transepithelial resistance (TER) and reduced the paracellular flux of urea and inulin. Assessment of transcript levels in the mouse colonic tissues, after 3 weeks of AhR deletion, showed that AhrΔIEC colonic tissues had higher transcript abundance of barrier-forming proteins like Claudin-3, -4, and occludin with reduced abundance of the pore-forming claudin-2. Ultrastructural investigation of intestinal TJs of AhrΔIEC mice showed diffused, laterally extended, and collapsed TJ architecture. AhrΔIEC did not alter the baseline levels of TNF-α, IFN-γ, and IL-4 however, the transcript levels of IL-6, IL-1β, and IL-17A showed marked elevation. The AhrΔIEC colonic tissues also showed increased infiltration by CD45+ immune cells under baseline conditions with a high abundance of inflammatory dendritic cells (DCs), macrophages (MΦs), and Th17 cells with a corresponding reduction in their tolerogenic counterparts and Treg cells. The AhrΔIEC also showed reduced colonic transcript levels of IL-10, a key anti-inflammatory cytokine, and Programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), the two key proteins, involved in limiting the pro-inflammatory responses in the gut mucosa. In-vitro, AHR-/- in Caco-2 cells also reduced paracellular TJ permeability, and co-culture assays with human dendritic cells showed reduced basal to apical paracellular podia formation and antigen sampling by DCs. Conclusion Our data show that deletion of the AhR gene in the gut epithelial cells reduces the epithelial paracellular TJ permeability in-vitro and in-vivo and also highlights the role of homeostatic paracellular TJ permeability in the development of intestinal immune homeostasis.