Abstract Background Although extracellular matrix deposition was long considered the main cause of stenosis in Crohn’s disease (CD) strictures, newer data indicate human intestinal smooth muscle cell (HIMC) hyperplasia as the primary driver. We hypothesized that factors secreted from the submucosa (SM) stimulate HIMC proliferation. Methods Histopathological slides from 50 stricturing CD patients (3 sections per patient: non-inflamed CDni, inflamed CDi, and stricture CDs), were analyzed. A new full thickness single-nuclei RNA sequencing (snRNAseq) atlas was generated (n = 13; CDni, CDi, and CDs) and non-IBD (NL, n = 15) patients. Conditioned media from human intestinal SM from NL, CDni, CDi and CDs, and supernatants from human intestinal myofibroblasts (HIMF) cultured with TGFβ1 were generated and tested for effect on HIMC proliferation. Proteomic analysis of the tissue conditioned media was performed. The proliferative effect of GREM1 on HIMCs was assessed. Results Histopathologic analysis demonstrated progressive wall thickening (from CDni to CDs, p 0.0001), predominantly driven by a 45% increase in the muscularis propria (MP), with greater thickening of the inner MP (IMP) versus outer MP(28.1% vs 16.9%). Correlation was noted between the degree of SM fibrosis and IMP thickness(p 0.001). A brand new snRNAseq atlas with 903,460 nuclei revealed fibroblasts as major signal-senders in CDs. Increased fibroblast-HIMC interactions were noted in CDs and cell-cell interaction analysis revealed GREM1 as a molecular candidate. HIMF and fibroblasts are a major source for GREM1 with an increased expression in CDs. Expression of GREM1 was the highest in the stromal compartment and increased from NL to CDs. We identified five distinct fibroblasts subpopulations, all showing increased GREM1 expression from NL to CDs, with the most prominent upregulation observed in the ADAMTS16 subset. Conditioned media from CDs SM induced robust proliferation of HIMC relative to CDni (EdU count: 3.31-fold, p = 0.0133). Proteomic analysis revealed upregulation of multiple soluble SM factors in CDs, namely, stanniocalcin 1 (STC1), regenerating islet-derived protein 4 (REG4), and GREM1. Supernatants of TGF-β1 activated HIMFs, but not of activated lamina propria mononuclear cells, increased HIMC proliferation (EdU count: 2.75-fold, p = 0.01). This suggests direct HIMF-HIMC interaction. Finally, recombinant GREM1, but not STC1 or REG4, stimulate HIMC proliferation (EdU count: 6.6-fold, p = 0.0008). Conclusion IMP thickening is a major contributor to CDs stenosis. Multiomics analysis reveals fibroblast-derived GREM1 as a SM-derived factor driving HIMC proliferation. This represents a novel and targetable mechanism for CD fibrostenotic strictures. Conflict of interest: Dr. Veisman, Ido: No conflict of interest Massey, William: No conflict of interest Prasad, Ankita: No conflict of interest Banerjee, Suhanti: No conflict of interest Czarnecki, Doug: No conflict of interest Chauhan, Gaurav: No conflict of interest Mukherjee, Pranab: No conflict of interest West, Gail: No conflict of interest Jatana, Samreen: No conflict of interest Braga Neto, Manuel: No conflict of interest Liu, Weiwei: No conflict of interest Gordon, Ilyssa: No conflict of interest Rieder, Florian: Personal Fees: Adiso, Adnovate, Agomab, Allergan, AbbVie, Arena, Astra Zeneca, Boehringer-Ingelheim, Celgene/BMS, Celltrion, CDISC, Celsius, Cowen, Ferring, Galapagos, Galmed, Genentech, Gilead, Gossamer, Granite, Guidepoint, Helmsley, Horizon Therapeutics, Image Analysis Limited, Index Pharma, Landos, Jannsen, Koutif, Mestag, Metacrine, Mopac, Morphic, Organovo, Origo, Palisade, Pfizer, Pliant, Prometheus Biosciences, Receptos, RedX, Roche, Samsung, Sanofi, Surmodics, Surrozen, Takeda, Techlab, Teva, Theravance, Thetis, UCB, Ysios, 89Bio
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