Cellular complexity and crosstalk in murine TNF-dependent ileitis: Different fibroblast subsets propel spatially defined ileal inflammation through TNFR1 signalling

Crohn’s disease represents a persistent inflammatory disorder primarily affecting the terminal ileum. Through the application of single-cell RNA sequencing, we unveil the intricate cellular complexities within murine TNF-dependent ileitis, developing in Tnf ΔARE mice. Detailed immune cell analysis highlights B cell expansion, T cell effector reprogramming, and macrophage lineage shifts during inflammation. Focusing on stromal cells, we reveal a strong pro-inflammatory character, acquired by all fibroblast subsets, which exhibit complex communication patterns with the infiltrating immune and surrounding stromal cells. Interestingly, we identify that Tnf ΔARE-induced ileitis is initiated in the lamina propria via TNFR1 pathway activation in villus-associated fibroblasts (Telocytes and Pdgfralow cells). Furthermore, we unveil separate spatial subsets of fibroblasts acting as exclusive responders to TNF, each orchestrating inflammation in different intestinal layers. Additionally, manipulating the Tnfrsf1a gene exclusively in fibroblast subsets suggests that inflammation is initiated by telocytes and Pdgfralow cells, while trophocytes drive its progression. This introduces novel evidence of spatial regulation of inflammation by fibroblast subsets, inciting and advancing disease in different layers of the gut. These findings underscore the pivotal role of fibroblasts in the inception and advancement of ileitis, proposing that targeting different fibroblast populations could impede the disease development and chronicity of inflammation. ### Competing Interest Statement The authors have declared no competing interest.