Tumor Necrosis Factor Receptor 1 Regulates Colonic Mesenchymal Cell Diversity and the Epithelial Stem Cell Niche

BACKGROUND & AIMS: Tumor necrosis factor receptor 1 regulates intestinal epithelial survival and repair, yet its role in the colonic mesenchyme remains unclear. We investigated how mesenchymal tumor necrosis factor receptor 1 signaling controls stromal cell identity, stem cell niche function, and epithelial homeostasis. METHODS: mice were analyzed using immunostaining, Western blotting, bulk and single-cell RNA sequencing, and organoid-mesenchymal coculture systems. Functional studies included wound healing assays, recombinant R-spondin 3 rescue, and integrin A6 blockade. RESULTS: Tumor necrosis factor receptor 1 deletion altered mesenchymal composition, reducing platelet derived growth factor receptor α expression while increasing α smooth muscle actin expression in vivo and in vitro. Single-cell RNA sequencing demonstrated loss of mesenchymal heterogeneity following tumor necrosis factor receptor 1 deletion, including depletion of a distinct tumor necrosis factor- and interferon-responsive mesenchymal subpopulation enriched for Mmp9, Cd200, Traf1, and Tnfrsf9. Transcriptomic profiling revealed downregulation of stem cell niche factors including Rspo3, Wnt2b, and Fgf2, alongside increased extracellular matrix and proliferative signaling pathways, particularly integrin A6, phosphoinositide 3-kinase, mitogen-activated protein kinase, and Ras-associated protein 1 signaling. Mesenchymal tumor necrosis factor receptor 1 deletion impaired epithelial proliferation and reduced Lgr5+ and Ephb3+ intestinal stem cell expression in vivo. In organoid coculture systems, tumor necrosis factor receptor 1-deficient mesenchymal cells suppressed epithelial stem cell marker expression and altered colonoid morphology, effects which were rescued by recombinant R-spondin 3. Integrin A6 blockade restored platelet derived growth factor receptor α and R-spondin 3 expression and normalized proliferative signaling and migration in tumor necrosis factor receptor 1-deficient myofibroblasts. Similarly, tumor necrosis factor receptor 1-dependent regulation of integrin A6, platelet derived growth factor receptor α, and R-spondin 3 was observed in human colonic myofibroblasts. CONCLUSIONS: Mesenchymal tumor necrosis factor receptor 1 preserves colonic mesenchymal diversity, sustains R-spondin 3-dependent stem cell niche signaling, and maintains intestinal stem cell homeostasis.