Spatially Resolved Insights Into Fistulating Crohn’s Disease Pathogenesis - scRNA-Seq

Crohn's disease often presents with fistulae, abnormal tunnels connecting the intestine to the skin or other organs. Despite the profoundly adverse impact of fistulae on patient morbidity, the molecular basis of their formation remains unclear, largely due to the challenge of capturing intact fistula tracts and their inherent heterogeneity. Here, we construct an unbiased, subcellular-resolution spatial expression atlas of 62 intestinal fistulae spanning diverse anatomical locations. We describe fistula-associated epithelial, immune and stromal cell states, revealing abnormal zonation of growth factors and morphogens linked to establishment of tunnelling anatomy. We identified fistula-associated stromal (FAS) fibroblasts assembled in concentric layers, forming a proliferative, lumen-adjacent zone (LAZ) underlying surface neutrophil and macrophage-rich granulation tissue, followed by active lesion core FAS cells all encircled by quiescent, pro-fibrotic outer zone (FOZ) fibroblasts. We mapped fistula tract ECM architecture and demonstrated FAS populations locate to different collagen structures, exhibiting functional properties spanning proliferation, migration and active ECM remodelling, to dense collagen deposition and fibrosis. We define niches supporting epithelialization of fistula tunnels and a putative precursor FAS population detected at the base of ulcers in non-penetrating Crohn’s. This precursor population co-localises with inflammatory as opposed to pro-fibrotic SPP1+ macrophages and fails to induce developmental transcription factors, observed in FAS populations located in fistula tracts. Our study demonstrates that common molecular pathways and cellular niches underpin fistulae arising at different intestinal locations and reveal the cellular protagonists of fistula establishment and persistence. Our resource will inform development of model systems and interventions that mitigate aberrant fibroblast activity while preserving their regenerative properties in Crohn’s.