BACKGROUND: Neutrophil extracellular traps (NETs) consist of DNA filaments and cytoplasmic protein granules, extruded by neutrophils after PAD4-dependent activation. NETs release occurs early during inflammation in various immune-related diseases, further aggravating tissue injury and contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in Crohn’s disease (CD).

METHODS: NETs and activated fibroblasts were labelled by multiplex immunofluorescence staining on unaffected, inflamed and fibrotic ileum derived from patients with stricturing CD undergoing ileocaecal resection. Intestinal fibroblasts isolated from unaffected CD ileum were co-cultured with NETs for 24 hours to analyse their transcriptome and quantify collagen released with SIRCOL®. Fibroblast migratory activity was investigated with a scratch test. Immunofluorescence intensity of collagen and fibroblast activation protein (FAP) was quantified using the Operetta® Analysis System. Transfection of fibroblasts with NF-kB-luciferase reporter plasmid was performed to evaluate the TLR2/NF-kB pathway, and a specific TLR2 inhibitor (TL2-C29) tested. Finally, mice with selective deletion of PAD4 in neutrophils (PAD4^fl/flMRP8Cre⁺) were subjected to chronic Dextran Sulphate Sodium (DSS) colitis to assess the role of PAD4-mediated NETosis in intestinal fibrosis development.

RESULTS: We found spatial tissue distribution of clusters of NETs adjacent to FAP⁺ fibroblasts in ileal ulcerations in patients with active CD. Transcriptomics demonstrated upregulation of various profibrotic processes in fibroblasts stimulated with NETs, including TGFβ-receptor signalling pathway, positive regulation of collagen metabolic process, and TLR-signalling pathways. This correlated with increased proliferation rate (p<0.0001), slower wound healing capability (p<0.0001) and higher collagen release in the medium (p<0.01) in the NET-treated group, as well as higher expression of collagens and FAP. Among TLR genes, TLR2 was found upregulated after stimulation with NETs (p=0.02). Transfection data showed significant upregulation (p<0.05) of NF-kB in the NETs-treated group, whereas its expression and soluble collagen release decreased with the addition of the TLR2 antagonist TL2-C29. In line, a significantly lower amount of collagen deposition was observed in the colon of PAD4^fl/flMRP8Cre⁺mice subjected to chronic DSS colitis, as well as modulation of MMP2/TIMP2 balance and reduced fibroblast activation.

CONCLUSION: NETs may represent an early trigger of intestinal fibroblast activation via the TLR2/NF-kB axis. As NETs are early players during inflammation, blocking PAD4 may reduce inflammation and thus fibrogenesis in CD.

Andrea Galli

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