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

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

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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