Genetic Regulation of Tissue Fibrosis in Human Intestinal Organoids

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The long-term goal of our research program is to define genomic and microbial mechanisms driving disease complications in Crohn's Disease (CD). Persistent rates of stricturing complications requiring surgery despite increased use of anti-TNFα therapy increase the urgency of this work. Interactions between intestinal epithelial cells, myeloid cells, and myofibroblasts regulate the balance between wound healing and fibrosis in CD. The overall objective of this project will be to develop a novel human intestinal organoid (HIO) model system to test genetic mechanisms regulating tissue inflammatory responses and collagen production. Our preliminary data suggests a critical role for myofibroblast extrinsic reactive oxygen species (ROS) production by the intestinal epithelial DUOX2 and neutrophil NOX2 NADPH oxidases in regulating production of epithelial and myeloid inflammatory cytokines including TGFβ which promote myofibroblast activation and extra-cellular matrix production. We found that loss-of-function genetic variants in DUOX2 were associated with three-fold lower rates of stricturing in CD, while loss-of-function genetic variants in the CYBA, NCF1, NCF2, and NCF4 genes comprising the NOX2 complex were associated with three-fold higher rates of stricturing. Analysis of CD patient ileal biopsies defined a gene expression program encoding epithelial and myeloid inflammatory signals driving myofibroblast activation and collagen production in those who later developed strictures. A novel perturbagen bioinformatics approach prioritized small molecules likely to reverse the pro-fibrotic gene expression signature. We have established inducible pluripotent stem cell (iPSC)-derived human intestinal organoids and neutrophil- like cells from CD patients with and without DUOX2 and NCF1 mutations, and confirmed variation in ROS production. This will provide a novel model system to test genetic regulation of tissue fibrosis. We hypothesize that genetic variation in NADPH oxidase function regulates ROS production and epithelial and myeloid inflammatory signals driving myofibroblast activation and extra-cellular matrix production. We will test this hypothesis in the following Aims: In Aim 1 we will differentiate isogenic iPSC with and without DUOX2 mutations into human intestinal organoids. We will test the effects of microbial products upon organoid ROS and inflammatory cytokine production, collagen content, and tissue stiffness. We will validate findings using gene expression and collagen content data derived from CD patient ileal biopsy and surgical resection samples. In Aim 2 we will differentiate isogenic iPSC with and without NCF1 mutations into neutrophil-like cells and human intestinal organoids. We will determine the effects of neutrophil products and small molecules upon organoid ROS and inflammatory cytokine production, collagen content, and tissue stiffness. We will validate findings using CD patient neutrophils and ileal samples. These studies will address the goals of this Program Announcement to utilize identifiable human specimens to delineate candidate gene functions in disease states, and to more precisely define the pathophysiologic mechanisms that lead to end-organ injury.