Tu1825 CAP-D3 Promotes Bacterial Clearance in Human Intestinal Epithelial Cells Through Repression of Amino Acid Transporters

Homolog-1 (LRH-1, also known as NR5A2) is expressed at the base of the intestinal crypts where the ISCs reside.This nuclear receptor interacts with the WNT/β-catenin pathway, drives intestinal corticosteroid production, and when absent exacerbates experimental colitis.We are investigating the role of LRH-1 in ISCs with respect to cell renewal and epithelial barrier function with the aim of developing a relevant intestinal tissue model system amenable to drug discovery.Methods: To evaluate the contributions of LRH-1 to intestinal growth and epithelial integrity, we employed the intestinal organoid system utilizing mice harboring conditional LRH-1 knockout in ISCs (Lrh isc ) and intestinal epithelium (Lrh vil ).Lrh1 knockout was achieved by activation of an inducible Cre driver under control of the Lgr5 or Villin promoter for Lrh isc and Lrh vil , respectively.Following in vitro Lrh1 knockout, the Lrh isc and Lrh vil organoids were evaluated for growth using EdU incorporation, Ki-67 staining, and observation of the crypt budding rate.To explore the effects of LRH-1 loss on epithelial integrity, immunohistochemistry and a dextran exclusion assay were used to examine the architecture and function of tight junctions, respectively.Results: LRH-1 ISC knockout reduces the rate of crypt proliferation and slows ISC-driven epithelial turnover as evidenced by diminished EdU incorporation and proliferative antigen Ki-67 staining.Epithelial tight junctions are distorted with LRH-1 knockout, resulting in increased epithelial permeability as demonstrated by dextran exclusion.Staining for tight junction protein Zona Occludens-1 suggests disordered tight junction formation upon LRH-1 knockout.Moreover, intestinal organoids deficient in LRH-1 expression are more susceptible to TNF-α-mediated inflammatory epithelial damage.Conclusions: LRH-1 knockout from ISCs and intestinal epithelium results in reduced epithelial cell growth, compromises epithelial integrity, and an enhanced susceptibility to inflammatory damage.Our results suggest LRH-1 is a viable target for novel, epithelium-targeted IBD therapy.Additionally, the intestinal organoid system facilitates functional studies of epithelial integrity and may serve as a tool for evaluating and developing synthesis LRH-1 agonists.