What question did this study set out to answer?

This research aims to understand the immune mechanisms underlying perianal fistulas in Crohn’s disease and their prognostic implications.

January 23, 2026

DOP076Spatially and functionally uncoupled cytokine responses differentially activate wound healing and keratinisation responses in a prognostically relevant manner in perianal fistulising Crohn’s disease

Key Points

This research aims to understand the immune mechanisms underlying perianal fistulas in Crohn’s disease and their prognostic implications.
Performed bulk RNA-sequencing on fistula and rectal biopsies from patients.
Conducted single-cell RNA-sequencing on CD45+ fistula tract cells.
Utilized spatial transcriptomics to analyze immune microenvironments in specimens.
Assessed wound repair mechanisms using cytokine-treated organoids and JAK inhibitors.
Dysregulation of extracellular matrix and epithelial-to-mesenchymal transition observed in both fistula types.
Perianal Crohn's disease showed an enriched immune microenvironment with several cytokines compared to benign cryptoglandular fistulas.
Cytokine responses were spatially uncoupled, influencing different aspects of wound healing and keratinisation.
Higher levels of IFNγ correlated with more severe fistula presentations, while keratinisation signified a better prognosis.

Abstract

Abstract Background Perianal fistulas are a severe complication of Crohn’s disease (CD) affecting up to 40% of patients. Their aetiology remains poorly understood, and new insights into immune mechanisms are needed. Methods Fistula tract biopsies, tract-derived cells, and rectal biopsies were obtained from patients with perianal CD (pCD) and cryptoglandular fistulas (CGF), a benign, repairable fistula type occurring without CD. Bulk RNA-sequencing (RNA-seq), single-cell (scRNA-seq), and spatial transcriptomics (ST) were used to map the immune microenvironment. RNA-seq profiled fistula (n = 26) and rectal biopsies (n = 22). scRNA-seq was performed on CD45+ fistula tract cells (n = 19), and ST on pCD proctectomy specimens (n = 3) using the CosMx SMI 6000-plex panel. Healthy control and pCD cytokine-treated organoids (n = 4) ± upadacitinib were used to assess wound repair mechanisms and therapeutic potential. Results Both fistula types showed dysregulation of the extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT). However, pCD exhibited a denser immune microenvironment, enriched for multiple cytokines (e.g. IL1β, IL12, IL18, TNFα, interferon-γ (IFNγ)), chemokines and TNF superfamily members (e.g. TNFSF4, TNFSF9, TNFSF15) compared to CGF. Wound-healing pathways diverged, with a shift towards injury-related inflammatory responses in pCD, such as the “burn wound response”, whereas CGF healing was dominated by “keratinisation”. Cytokine-treated organoids revealed TNFα/IFNγ drove inflammatory healing, while IL22 regulated EMT and keratinisation. These responses were spatially uncoupled: IFNγ-responsive cells localised to niches with mononuclear phagocytes and B-cells, TNFα-responsive cells to stromal compartments, and IL22 transcripts to keratinocytes and the epithelium. RNA-seq from an independent pCD cohort suggested these molecular changes were prognostically relevant. IFNγ enrichment correlated with fistula severity defined via the TOpClass classification system, whereas keratinisation was associated with a favourable prognosis. Integration of single-cell data with in-silico drug screening identified MAP kinase (p38, MEK), cyclin-dependent kinase 9 (CDK9), and JAK inhibitors as potential therapies. Proof-of-concept experiments validated our predictive modelling, showing that JAK1 inhibitors potently inhibited pathogenic cell populations isolated from the tract and the burn wound healing pathway in our organoid system. Conclusion Our data identify spatial and functional compartmentalisation of cytokine responses in fistula tracts, which may be clinically and prognostically relevant in pCD. As well as informing prognosis, high-dimensional molecular profiling of fistula tracts can be harnessed to pinpoint novel therapeutic approaches. Conflict of interest: Dr. Constable, Laura: No conflict of interest Cozzetto, Domenico: No conflict of interest Hanna, Luke: No conflict of interest Abdurahiman, Saeed: No conflict of interest Iqbal, Nusrat: No conflict of interest Anandabaskaran, Sulak: European Crohn’s Colitis Organisation Research Grant in 2022 and has received conference attendance support from AbbVie, Johnson & Johnson, Dr Falk and Ferring. Martinez, Adriana: No conflict of interest Kudo, Hiromi: No conflict of interest Lambie, Nathalie: No conflict of interest Rekopoulou, Katerina: No conflict of interest Panneer Selvam, Neha: No conflict of interest Matthews, Nik: No conflict of interest Goldin, Rob: No conflict of interest Sebastian, Shaji: Grant: Takeda, Tillots pharma, Biogen, Pfizer, Abbvie, Johnson & Johnson, Olympus -Odin Vision. Personal Fees: Tillots, Johnson & Johnson, Olympus Odin Vision, AbbVie, Takeda, Merck, Pharmacosmos, Amgen, Eli Lilly, BMS, Odin Vision. Non-financial Support: Tillots, Takeda, AbbVie, Celltrion, Johnson & Johnson, Eli Lilly, Alphasigma, Ferring Pharma. Verstockt, Bram: Research support from: AbbVie, Biora Therapeutics, Celltrion, Landos, Pfizer, Sanofi, Sossei Heptares/Nxera and Takeda. Speaker’s fees from: Abbvie, Agomab, Alfasigma, Biogen, Bristol Myers Squibb, Celltrion, Eli Lily, Falk, Ferring, Galapagos, Materia Prima, Johnson and Johnson, Pfizer, Sandoz, Takeda, Tillots Pharma, Truvion and Viatris. Consultancy fees from: Abbvie, Alfasigma, Alimentiv, Anaptys Bio, Applied Strategic, Astrazeneca, Atheneum, BenevolentAI, Biora Therapeutics, Boxer Capital, Bristol Myers Squibb, Domain Therapeutics, Eli Lily, Galapagos, Guidepont, Landos, Merck, Mirador Therapeutics, Mylan, Nxera, Inotrem, Ipsos, Johnson and Johnson, Pfizer, Sandoz, Sanofi, Santa Ana Bio, Sapphire Therapeutics, Sosei Heptares, Takeda, Tillots Pharma and Viatris. Stock options: Vagustim and Thethis Pharma. Hart, Ailsa: Grant: Takeda. Personal Fees: Abbvie, Amgen, Arena, AZ, Falk, Celltrion, Eli Lilly, Ferring, Genentech/ Roche, GSK, Pfizer, Takeda, Napp, Pharmacosmos, Janssen (J&J), Bristol-Myers Squibb, Gilead, Galapagos, Alfasigma Tozer, Philip: Personal Fees: Takeda - speakers fees, member of Inspire, and advisory boards. Ferring - speakers fees. Falk - speakers fees. Tillott’s - speakers fees. J&J - speakers fees. Abbvie - speakers fees Powell, Nick: Grant: Takeda, BMS, Pfizer, Astra-Zeneca. Personal Fees: Abbvie, Abivax, Allergan, Astra-Zeneca, Bristol-Myers Squibb, Celgene, Celltrion, Dr Falk Pharma UK Ltd, Ferring, Galapagos, GSK, Janssen, MSD, Roche, Pfizer, Sobi, Takeda, Tillotts.

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