The Impact of Peptidyl Arginine Deiminase 4-Dependent Neutrophil Extracellular Trap Formation on the Early Development of Intestinal Fibrosis in Crohn’s Disease

Abstract Background and Aims During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a peptidyl arginine deiminase 4 (PAD4)-dependent manner, aggravating tissue injury and remodeling. In this study, we investigated the potential pro-fibrotic properties and signaling of NETs in Crohn’s disease (CD). Methods NETs and activated fibroblasts were labeled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analyzed by bulk RNA sequencing to uncover cell signaling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-κB pathway was evaluated by transfection of CCD-18Co fibroblasts with an NF-κB-luciferase reporter plasmid, incorporating C29 to block TLR2 signaling. A chronic dextran sulfate sodium (DSS) mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl). Results Immunofluorescence showed spatial colocalization of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of Toll-like receptor signaling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in an NF-κB expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significant 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-κB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.