Elucidating mechanisms of fibrosis associated with the Crohns disease-associated pathogenic variant in the metal transporter ZIP8

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PROJECT ABSTRACT Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis, affect 1 in 200 Americans. Anti-tumor necrosis factor (TNF)-alpha therapies remain the cornerstone of therapy for patients with IBD, but approximately 40% of patients will experience primary or secondary loss of response, and “anti- TNF-experienced” patients are less likely to respond to all subsequent therapies in current clinical practice. Single-cell RNA sequencing has uncovered key cellular signatures associated with anti-TNF refractoriness that strongly implicate signaling between inflammatory macrophages and activated fibroblasts. There remains a major gap in our understanding of the patient-specific factors that predispose to this aberrant macrophage- fibroblast activation, how it links to anti-TNF unresponsiveness, and the potential to modulate it to change to disease course and treatment response. Our work has focused on the functional implications of a pathogenic variant in a metal transporter, ZIP8 A391T, that dysregulates manganese homeostasis and is associated with complicated (stricturing and penetrating) Crohn’s disease. In studying ZIP8 391-Thr in a mouse model (Zip8 393T-knock-in (KI)), we have shown increased susceptibility to colitis, but even more interestingly, we have observed enhanced fibrosis. These mice also exhibit marked induction of Il-11, a pro-inflammatory and pro- fibrotic cytokine that is a hallmark of aberrant signaling between inflammatory macrophages and activated fibroblasts in patients with Crohn’s disease – particularly anti-TNF refractory disease. We therefore hypothesize that study of aberrant macrophage-fibroblast signaling is a key feature of disease pathogenesis in the Zip8 393T-KI mice. Establishing the underlying disease mechanisms are important because (1) up to 25% of patients with Crohn’s disease carry ZIP8 391-Thr in some populations and (2) the Zip8 393T-KI could serve as a novel translational model for mechanistic studies, particularly related to anti-TNF non-response. We will study this hypothesis in two aims: In Aim 1, we will determine if Zip8 393T-KI perturbs macrophage innate immune responses; in Aim 2, we will compare fibroblast activation induced by Zip8 393T-KI vs. WT Zip8 macrophages. The long-term goal is to establish if ZIP8 391-Thr genotype has clinical implications for the prevention of and treatment of patients with Crohn’s disease and use study of ZIP8 391-Thr-related pathology as key opportunity to elucidate the role of Mn homeostasis in human disease. This R03 application builds from a K08 that established the altered Mn homeostasis and fibroinflammatory phenotype in colitis in Zip8 393T-KI mice. This application with strong translational relevance will provide critical mechanistic insight of the ZIP8 genotypic effect on macrophage-fibroblast signaling and the interaction with Mn homeostasis to prioritize therapeutic targets, including STAT3 or IL-11 inhibition, and patient studies as part of an R01 application.