Abstract Background Postoperative recurrence and complications remain major challenges in inflammatory bowel disease (IBD), yet the molecular determinants driving these outcomes are poorly understood. We hypothesized that residual “priming factors” within resection margins act as a pathological memory that promotes disease recurrence. This study aimed to spatially and temporally resolve the molecular architecture of intestinal fibrosis and to identify predictive biomarkers of postoperative complications. Methods Intestinal resections from CD patients and controls were analyzed using spatial transcriptomics (10x Visium) and integrated with single-cell RNA-seq data (scIBD dataset). Tissue regions were annotated by pathologists, and clustering, pseudotime, and spatial deconvolution analyses were performed using Seurat, Harmony, SPATA2, and Monocle3 pipelines. Clinical history and follow-up data were also collected. Results Spatially resolved transcriptomics identified 21 transcriptionally distinct clusters across healthy, inflamed, and fibrotic regions. Fibrosis was primarily confined to the mesenchymal compartment, with fibroblasts transitioning toward inflammatory and reticular phenotypes. Pseudotime analysis revealed distinct mesenchymal trajectories: stromal cells evolved into differentiated fibroblasts, while myofibroblasts and transitional stroma gave rise to reticular fibroblasts and mesodermal populations, characteristic of deep and superficial fibrosis, respectively. No evidence of epithelial-to-mesenchymal transition was observed. Notably, by comparing patients in remission with those experiencing postoperative recurrence, we found a differential enrichment of immune cell subsets. Memory and naïve B cells were enriched in the lymphoid aggregates of remission patients, whereas plasma cells were increased in granulation tissue. Moreover, regulatory T cells (Tregs) were more abundant in lymphoid aggregates from remission samples compared to those from patients with postoperative recurrence. Conclusion Our spatial and temporal analyses demonstrate that intestinal fibrosis in CD originates predominantly from mesenchymal transitions rather than epithelial remodeling. In addition, the enrichment of B cells and Tregs in resection margins from remission patients suggests that immune-regulatory networks are crucial for maintaining disease quiescence. These findings provide a molecular framework for predictive modeling of postoperative recurrence and identify novel cellular crosstalk and mechanisms as potential therapeutic targets. Funded by the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS. Project code: PNRR-MCNT2-2023-12377779; CUP master: C43C24000410007. Conflict of interest: Massimino, Luca: No conflict of interest Parigi, Tommaso Lorenzo: No conflict of interest Fiorio, Francesco: No conflict of interest Larranaga, Leire: No conflict of interest Versiglia, Alice: No conflict of interest Spanò, Salvatore: No conflict of interest Mino, Sara: No conflict of interest Bugatti, Mattia: No conflict of interest Frontali, Alice: No conflict of interest Scarfò, Federico: No conflict of interest Vignali, Andrea: No conflict of interest Municchi, Andrea: No conflict of interest Villanacci, Vincenzo: No conflict of interest Albarello, Luca: No conflict of interest Ponzoni, Maurilio: No conflict of interest Danese, Silvio: Personal Fees: AbbVie, Alimentiv, Allergan, Amgen, Applied Molecular Transport, AstraZeneca, Athos Therapeutics, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Dr Falk Pharma, Eli Lilly, Enthera, Ferring Pharmaceuticals Inc., Gilead, Hospira, Inotrem, Janssen, Johnson & Johnson, Morphic, MSD, Mundipharma, Mylan, Pfizer, Roche, Sandoz, Sublimity Therapeutics, Takeda, Teladoc Health, TiGenix, UCB Inc., Vial, Vifor Lecture fees from Abbvie, Amgen, Ferring Pharmaceuticals Inc., Gilead, Janssen, Mylan, Pfizer, Takeda Ungaro, Federica: No conflict of interest Sileri, Pierpaolo: No conflict of interest
Massimino et al. (Thu,) studied this question.