Neutrophil-fibroblast crosstalk drives immunofibrosis in Crohn’s disease through IFNα pathway

Crohn’s disease (CD) is characterized by chronic inflammation and intestinal fibrosis leading to lifelong complications. However, disease pathogenesis is elusive and the therapeutic options are limited. We investigated the interaction between neutrophils and intestinal fibroblasts in the development of CD immunofibrosis. Peripheral neutrophils, enriched neutrophil extracellular traps (eNETs), serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies from CD and ulcerative colitis (UC) patients, and healthy individuals (HI), were studied. Neutrophil-targeted transcriptomics, multi-cytokine profiling and cell-based functional assays at mRNA/protein level were performed. Compared to UC, PIFs from CD patients displayed a distinct fibrotic phenotype characterized by negative Krüppel-like Factor-2 (KLF2) and increased cellular communication network factor-2 (CCN2) expression leading to collagen production. In UC and CD, PIFs-derived IL-8 appears as a culprit chemoattractant of neutrophils in the intestine, where CD neutrophils were accumulated close to fibrotic lesions. Functionally, only CD neutrophils via eNETs can induce a CD-like phenotype in HI PIFs, suggesting their fibrotic plasticity. High IFNα in serum and IFΝ-responsive signature in peripheral neutrophils were observed in CD, distinguishing it from UC. Moreover, CD serum can stimulate the release of fibrogenic eNETs from neutrophils in an IFNα-dependent manner, suggesting the priming role of IFNα in circulating neutrophils. Inhibition of eNETs or JAK signaling in neutrophils or PIFs prevented the neutrophil-mediated fibrotic effect on PIFs. Furthermore, serum IFNα and transcripts of key IFN-signaling components in neutrophils were well-correlated with CD severity. This study reveals the important role of IFNα/neutrophil/fibroblast axis in CD immunofibrosis, suggesting candidate biomarkers and putative therapeutic targets. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Greek General Secretariat for Research and Innovation (GSRI), Research & Innovation Programme CytoNET, grant MIS-5048548 and by GSRI, Regional Excellence Programme InTechThrace, grant MIS-5047285. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of the University Hospital of Alexandroupolis gave ethical approval for this work (Ref. Number 803/23-09-2019). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors