Genetic and Small Molecule Regulation of Mechanisms of Crohn’s Disease Stricture Formation

The long-term goal of our research program is to define mechanisms regulating treatment responses and disease complications in Crohn's Disease (CD). Persistent rates of ileal strictures requiring surgery despite widespread use of anti-TNF therapy increase the urgency of this work. Interactions between intestinal epithelial cells (IEC), macrophages, and fibroblasts mediate fibrostenosis in CD. The IEC NADPH oxidase DUOX2 produces reactive oxygen species (ROS) to control mucosal microbes, while also regulating cellular metabolism and repair via redox signaling. The phagocyte NADPH oxidase NOX2 complex produces ROS to kill pathogens, while constraining inflammasome activation and excessive cytokine production. We found that loss-of-function genetic variants in DUOX2, and loss-of-function genetic variants in CYBA, NCF1, NCF2, and NCF4 comprising the NOX2 complex, were associated with higher rates of strictures. This suggested a novel redox signaling mechanism constraining epithelial and macrophage dependent myofibroblast activation and extra-cellular matrix (ECM) production. Our perturbagen bioinformatic analysis of the treatment naïve CD ileal transciptome prioritized small molecules likely to inhibit inflammatory macrophage and fibroblast function and prevent strictures.The overall objective of this project will be to utilize a novel induced pluripotent stem cell (iPSC) derived macrophage:human intestinal organoid co-culture system to test mechanisms by which DUOX2 and NOX2 gene variants and candidate small molecules control intestinal epithelial cell and macrophage mitochondrial function and inflammatory signals regulating tissue fibrosis. We hypothesize that DUOX2 and NOX2 redox signaling regulates mitochondrial complex I assembly and function, thereby constraining mitochondrial ROS dependent cytokines driving tissue fibrosis. Small molecules and monoclonal antibodies will ameliorate pro-fibrotic effects of DUOX2 and NOX2 gene variants. We will test this hypothesis in the following Aims: Aim 1. Define mechanisms of tissue fibrosis in DUOX2var HIO. We will test DUOX2var regulation of epithelial redox signaling, protein kinase A activity, and mitochondrial complex I respiration, and associated mitochondrial ROS dependent TGFB secretion and HIO collagen production. The ability of candidate small molecules and TGFB blockade to prevent pro-fibrotic effects of bacterial products upon DUOX2var HIO will be determined. Aim 2. Define pro-fibrotic mechanisms of NOX2var macrophages. We will test NOX2var regulation of macrophage redox signaling, protein kinase A activity, and mitochondrial complex I respiration, and associated mitochondrial ROS dependent TL1A secretion and HIO collagen production. The ability of small molecules and TL1A blockade to prevent pro-fibrotic effects of NOX2var macrophages upon isogenic HIOs will be determined. These studies will advance precision medicine, by utilizing a platform we have developed to screen candidate small molecules in patient-derived organoids with clinically important genotypes.