The profibrogenic role of neutrophil extracellular traps in stenotic Crohn's disease: a new antifibrotic target

BACKGROUND: Neutrophil extracellular traps (NETs) are structures of DNA filaments and protein granules, extruded by neutrophils after insults in a PAD4-dependent manner. In autoimmune events, their release occurs early during inflammation, further aggravating tissue injury and presumably contributing to fibrosis. Our aim was to investigate the potential profibrotic effect of NETs in stenotic Crohn’s disease (CD). METHODS: Immunofluorescence (IF) for PAD4, NETs markers and fibroblast activation protein (FAP) was performed on resected ileum derived from patients with stricturing CD. Human intestinal fibroblasts (HIF) were extracted from unaffected CD ileum. A CCD-18Co fibroblast line was used as confirmation. In vitro co-cultures of subconfluent HIF with NETs were performed for 24 hours, and RNA extracted for sequencing. Soluble collagen released in culture medium was quantified with SIRCOL®. Migratory activity was investigated with scratch test. IF intensity of collagen and FAP was quantified using Operetta®. Transfection of CCD-18co cells with NF-kB-luciferase reported plasmid was performed to evaluate the TLR2/NF-kB pathway. Specific inhibitors (C29 and CAPE) were used to block TLR2 and NF-kB activity, respectively. Finally, a chronic DSS mice model of intestinal fibrosis with selective PAD4 knock-out in neutrophils (PAD4fl/flMRP8Cre+) was used as confirmation. Picrosirius red staining and SIRCOL® were used to quantify the collagen amount after sacrifice. RESULTS: IF on inflamed ileum showed clusters of NETs close to FAP+ fibroblasts, suggesting in vivo interactions. Transcriptomics demonstrated an upregulation of profibrotic genes and toll-like pathways (p<0.05) in the group of HIF stimulated with NETs. Increased proliferation rate, slower wound healing ability and higher collagen release in the medium were observed in NETs group, as well as higher IF expression of collagens and FAP. Transfection showed significant upregulation (p=0.015) of NF-kB in NETs group, whereas its expression and soluble collagen release decreased using C29 and CAPE. Accordingly, phospho-NF-kB and MyD88 proteins were increased in NETs group. A significantly lower amount of collagen was measured in the colon of PAD4-knocked out mice both with Picrosirius red (p=0.04) and SIRCOL® (p=0.008), as well as reduced FAP+ fibroblasts at IF. CONCLUSION: NETs may represent an early trigger of fibroblast activation in the intestine via the TLR2/NF-kB axis. As NETs are also early players during inflammation, blocking PAD4 might improve both inflammation and fibrogenesis in CD.