Project Summary Perianal fistulizing Crohn’s disease is a debilitating phenotype of Crohn’s disease (CD) involving the rectum. It is often refractory to treatment and is associated with severe disability and poor quality of life. Fistulizing CD disproportionately affects African Americans (AA), presenting with much higher prevalence, and more severe with destructive pathology. Besides fistulizing CD often requires a more aggressive combination of medical and surgical intervention than does luminal disease. Several lines of evidence suggest that interactions between the rectal epithelium, the microbiome, and the immune system, drive patent-specific pathogenesis of perianal fistula, but there is very little known about these at the molecular and cellular level, particularly with ancestral differences. The complexity of perianal fistula involving communication between microbes and multiple immune and non- immune cell types provides a strong rationale for a need for deep molecular characterization of the disease. One hypothesis is that different biological mechanisms due in part to divergent genomic architecture between African- and European-ancestry patients contributes to the disparity in health outcomes. Consequently, integrative genomic comparisons utilizing state-of-the-art single cell gene expression profiling of rectal biopsies and of peripheral blood immune cells, will be used to investigate the nature of pathological mechanisms of perianal fistulizing CD including specific comparison of African and European ancestry patient groups. Building on preliminary transcriptome profiling of the whole mucosa between these ancestry groups that implicates differences in metabolic and inflammatory signaling, and taking advantage of the emerging Gut Cell Atlas, our profiling of 120 cases will establish which cellular and gene expression features associate with remission, stabilization, or progression of disease. Aim 1 is to biopsy rectum and take blood samples from 120 patients at presentation, and again at follow-up, also with microbiome sampling (rectal wash as well as stool), supported by deep clinical phenotyping. Two ancestral populations (European and AA) are equally powered for comparison studies. Aim 2 is to use single cell RNA sequencing to profile changes in the relative abundance of each of dozens of key cell types, and to identify key genes that are consistent biomarkers of the three therapeutic response states within each cell type. Advanced bioinformatics approaches will be used to characterize the network of cellular interactions, and to link the genetic regulation of gene expression to genome-wide association studies of CD by identifying cell-type specific eQTL. Subsequently, in Aim 3, mechanistic dissection of specific pathways will be pursued with in vitro cultures of patient-derived intestinal organoids to assess patient specific responses when stimulated by relevant molecules. The findings will both illuminate cellular and molecular me...