MULTIMODAL SINGLE CELL ANALYSES REVEAL PATHOGENIC FIBROBLASTS IN PERIANAL FISTULA

Background: Perianal fistulae are highly morbid complications of Crohn’s disease (CD) with a cumulative incidence of 20-25%; despite demonstrated efficacy of infliximab treatment, estimates of complete resolution are only 20-30%. Ultimately, 20% of patients will require proctectomy. Across adult and pediatric populations, black CD patients are more likely to have colon-only CD, perianal fistula (OR 1.7-2.47, RR: 2.63) and progression to complex disease.
Methods: We performed single cell RNA sequencing (scRNAseq) of paired rectum and colon biopsies taken from equal African and European ancestry patients (n=14, 130,961 cells) with biopsies taken from two patients undergoing complete proctectomy, allowing for direct fistulous tract biopsies; single-nucleus RNAseq + ATAC (10X Multiome) was executed on a subset of n=6 patients (99,085 nuclei). A parallel cohort of healthy donors (n=8) and CD patients (n=11) with equal ancestry representation was recruited for peripheral blood mononuclear cell studies. We isolated CD14+ monocytes and differentiated sans microbial stimuli for 14 days; image analysis, quantitative Reverse Transcription-PCR, and Luminex immunoassays were performed.
Results: Myeloid and stromal cells were subset and re-clustered with the same cell types from the entire cohort of perianal fistula patients; n=20,704 cells were distributed among n=14 granular clusters. CHI3L1 was a top differentially expressed gene (DEG) (fold change=3.51, p-adj<1E-300) between stromal cells of fistula vs. colonic and rectal tissue origin. CHI3L1hi fibroblasts from fistulous tracts disproportionately expressed a destructive gene module (e.g. matrix metalloproteinases) and markers of macrophage activation. After two weeks’ differentiation in culture, isolated CD14+ monocytes differentially assumed a spindle-like morphology (p<0.001), expressed CHI3L1 (p=0.0145), and secreted CHI3L1 (p<0.001) in CD patients of African ancestry compared to European ancestry. Single cell gene regulatory network analyses focused on IBD-associated transcription factors confirmed cell-specific transcription factor enrichment (e.g. GATA2 in mast cells) and a broad array of predicted transcription factors in fibroblasts, including AP-1 (FOS, JUN). Multiome ATAC analysis of the CHI3L1 promoter revealed n=33 footprints bound in peaks from fibroblasts, over 60% of which were transcription factors belonging to the AP-1 family.
Conclusions: We present the first scRNAseq analysis of direct ex-vivo fistula tracts and identify CHI3L1 expressing fibroblasts that expressed common myeloid and known fibroblast destructive gene modules. Epigenetic control of inflammatory memory by AP-1 (JUN, FOS; PMID: 34320411) in a chronic inflammation context represents a promising mechanism of sustained injury in fistula by aberrantly differentiated innate immune cells.