The Impact of PAD4-dependent Neutrophil Extracellular Trap Formation on the Early Development of Intestinal Fibrosis in Crohn's Disease

  • J Crohns Colitis. 2024 Aug 10:jjae121. doi: 10.1093/ecco-jcc/jjae121.
Gabriele Dragoni  1  2  3 ,  Bo-Jun Ke  4 ,  Lucia Picariello  2 ,  Saeed Abdurahiman  4 ,  Elisabetta Ceni  2 ,  Francesca Biscu  4  5 ,  Tommaso Mello  2 ,  Simone Polvani  2 ,  Tommaso Innocenti  1  2  3 ,  Valérie Spalart  6 ,  Stefano Milani  2  3 ,  Andre D'Hoore  7 ,  Gabriele Bislenghi  7 ,  Stefano Scaringi  8 ,  Bram Verstockt  1  9 ,  Gert De Hertogh  10 ,  Kimberly Martinod  6 ,  Andrea Galli  2  3 ,  Gianluca Matteoli  4 ,  Séverine Vermeire  1  9
Affiliations
  • 1. IBD Leuven, Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Chronic Diseases and Metabolism, Leuven, Belgium.
  • 2. Gastroenterology Research Unit, Department of Experimental and Clinical Biochemical Sciences "Mario Serio", University of Florence, Florence, Italy.
  • 3. IBD Referral Center, Department of Gastroenterology, Careggi University Hospital, Florence, Italy.
  • 4. Laboratory for Mucosal Immunology, Translational Research Center for Gastrointestinal Disorders, Department of Chronic Diseases, Metabolism and Ageing, KU Leuven, Leuven, Belgium.
  • 5. Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom.
  • 6. Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium.
  • 7. Department of Abdominal Surgery, University Hospitals Leuven, KU Leuven, Leuven, Belgium.
  • 8. Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy.
  • 9. University Hospitals Leuven, Department of Gastroenterology and Hepatology, KU Leuven, Leuven, Belgium.
  • 10. Laboratory of Translational Cell and Tissue Research, Department of Imaging and Pathology, Faculty of Medicine, KU Leuven, Belgium.
PMID: 39126198 DOI: 10.1093/ecco-jcc/jjae121
Abstract

Background and aims: During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a PAD4-dependent manner, aggravating tissue injury and remodelling. In this study, we investigated the potential pro-fibrotic properties and signalling of NETs in Crohn's disease (CD).

Methods: NETs and activated fibroblasts were labelled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analysed by bulk RNA-sequencing to uncover cell signalling pathways, and by high-throughput imaging to assess Collagen production and migratory activity. Consequentially, TLR2/NF-kB pathway was evaluated by transfection of CCD-18Co fibroblasts with NF-kB-luciferase reporter plasmid, incorporating C29 to block TLR2 signalling. A chronic DSS mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl).

Results: Immunofluorescence showed spatial co-localisation of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of TLR-signalling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased Collagen release. Transfection experiments indicated a substantial increase in NF-kB expression with NETs, whereas C29 led to decreased expression and release of Collagen. In line, a significantly reduction in Collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis.

Conclusions: NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-kB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.

Keywords
Crohn’s disease; intestinal fibrosis; neutrophil extracellular traps.
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