GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

Abstract Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.