Abstract Refractory rectal fistulae are a major unmet medical need and disproportionately impact African- American Crohn's disease patients for unknown reasons. The mainstay of therapy is anti-TNF monoclonal antibodies; however, durable, fisula-free remission is achieved in only a minority of patients. In this supplemental proposal, we hypothesize that the major effect players that modulate susceptibility to perianal fistula include rectal epithelial cells and newly migrated monocytes. Given the markedly distinct contributions for major effect, innate immune loci between European (NOD2, IL23R, autophagy), Far East Asian (TNFSF15) and African- American (PTGER4, LACC1, FCGR2A) Crohn's disease, combined with the marked induction of the monokine CCL2 in inflamed rectum, in Aim 1, we seek to explore macrophage intrinsic differences between African-American and European ancestry people driven by hypoxia and muramyl dipeptide (MDP) stimulation. Once cell culture conditions are optimized to maximize reproducibility and inter-individual variability, we will perform bulk RNASeq and Olink analyses, and potentially scRNASeq. We seek to discover new, potentially rare, macrophage subtypes that may reside within unique, chronic inflammatory milieus that may drive perianal fistula. In Aim 2, we will compare IL-17A vs. IL-17C effects on epithelial cells and with macrophage co- culture. Key readouts will be epithelial gap closure capacity with different levels of differentiation, and transcripts modulating epithelial-mesenchymal transition (EMT). Finally, in Aim 3, we will perform genetic association and transcriptome analyses in order to prioritize additional contributing cell types driving severe rectal fistulae. We anticipate that blockade of anti-IL12/23 in anti-TNF non-responders may not be as successful in salvaging responses compared to with ileal Crohn's disease. However, if protective cell types and molecules can be identified, local therapies to prevent peri-rectal fistulae formation acting directly on epithelial cells may be developed.