PROJECT SUMMARY/ABSTRACT Inflammatory Bowel Disease (IBD) and its subtype Crohn’s disease (CD) arise due to a loss of tolerance to environmental antigens in genetically susceptible individuals. Longitudinal analysis has identified an inverse correlation between rates of communicable parasitic diseases and IBD, a phenomenon termed the “hygiene hypothesis.” Tuft cells have recently been identified as a critical component for sensing and responding to parasitic infections. Our lab has recently identified that tuft cells may be heterogeneous in their lineage and function in microbiome response, and specific populations may be critical for mitigating inflammation in ileal inflammatory disease. Here, we propose innovative experimental and computational approaches to dissect the heterogeneity of tuft cell lineage, structure, and function as they relate to regulating immune responses in in vivo models of CD. In aim 1, we will conduct single-cell sequencing, high resolution microscopy, and functional secretory assays to understand lineage-structure-function relationships in different tuft cell populations in mouse and enteroid models. In aim 2, we utilize multiplex imaging, Cytometry Time-of-Flight (CyTOF), and cytokine profiling to probe the downstream tuft-type 2 response network by utilizing several chemical and genetic models of pathway ablation. Through our findings, we aim to make significant contributions to the current understanding of tuft cell biology and epithelial-microbiome crosstalk in the intestine. Ultimately, this research will allow us to diagnose, stratify, and, ultimately, treat patients with IBD and expand the pool of targets in this complex, multifactorial disease.