Introduction
Crohn’s disease (CD) is characterized by chronic uncontrolled inflammation which often leads to intestinal fibrosis. Up to 70% of patients with CD will develop strictures and will undergo surgery as the only available therapeutic option. However, the pathogenesis of CD fibrosis remains incompletely understood. Studies are mainly focused on mesenchymal cells, cytokines and growth factors, whereas the role of neutrophils remains elusive.1–5 Previous data by our group indicated that neutrophils may acquire a pre-fibrotic role by inducing the differentiation and activation of fibroblasts.6,7
Aims & Methods
This study aims to investigate for the first time the role of neutrophil-intestinal fibroblast interplay in the pathogenesis of CD immunofibrosis suggesting possible targets for therapeutic interventions.
Neutrophils and intestinal biopsies were obtained from treatment naive patients with moderate to severe CD (CDAI ≥220) and UC (Mayo DAI ≥6). Additionally, sex- and age-matched healthy individuals (HI), were served as controls. Intestinal tissues were characterized using immunohistochemistry and immunofluorescence. Primary intestinal fibroblasts (PIFbs) isolated from tissue specimens were also studied using real-time qPCR, confocal immunofluorescence imaging, in-cell ELISA (cytoblot) and collagen assay. Various inflammatory mediators were measured in cell cultures supernatants with ELISA and multiplex bead-based flow cytometry. The migratory capacity of neutrophils was assessed by a chemotaxis cell migration fluorometric assay.
Results
PIFbs from patients with CD, compared to UC and HI, exhibited a fibrotic phenotype that was characterized by low Krüppel-like Factor 2 (KLF2) and increased Cellular Communications Network Factor 2 (CCN2) mRNA and protein expression leading to excessive collagen production. Neutrophil-fibroblast co-existence was obvious in intestinal tissue fibrotic lesions of CD patients. In line with this, PIFbs culture supernatants from CD patients yielded high levels of IL-8 and increased chemotactic activity for HI neutrophils, which was reduced significantly upon IL-8 neutralization. Of note, compared to UC, neutrophil culture extracts from CD patients downregulated KLF2 and induced CCN2 expression in HI PIFbs acquiring a CD pre-fibrotic phenotype. This neutrophil-mediated immunofibrotic effect was abolished in-vitro by pharmaceutical inhibition of JAK/STAT signaling in PIFbs.
Conclusion
These results, provide a novel mechanistic basis for immunofibrosis in CD suggesting neutrophil/JAK/STAT/fibroblast/IL-8 molecular pathway as a candidate therapeutic target.
References
1. Zidar N, Langner C, Jerala M, Boštjančič E, Drobne D, Tomažič A. Pathology of Fibrosis in Crohn’s Disease—Contribution to Understanding Its Pathogenesis. Front Med (Lausanne). 2020;7:167. doi:10.3389/fmed.2020.00167
2. Santacroce G, Lenti MV, Di Sabatino A. Therapeutic Targeting of Intestinal Fibrosis in Crohn’s Disease. Cells. 2022;11(3):429. doi:10.3390/cells11030429
3. Wéra O, Lancellotti P, Oury C. The Dual Role of Neutrophils in Inflammatory Bowel Diseases. J Clin Med. 2016;5(12):118. doi:10.3390/jcm5120118
4. Lipson KE, Wong C, Teng Y, Spong S. CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair. 2012;5(Suppl 1):S24. doi:10.1186/1755-1536-5-S1-S24
5. Salas A, Hernandez-Rocha C, Duijvestein M, et al. JAK-STAT pathway targeting for the treatment of inflammatory bowel disease. Nat Rev Gastroenterol Hepatol. 2020;17(6):323-337. doi:10.1038/s41575-020-0273-0
6. Chrysanthopoulou A, Mitroulis I, Apostolidou E, et al. Neutrophil extracellular traps promote differentiation and function of fibroblasts. The Journal of Pathology. 2014;233(3):294-307. doi:10.1002/path.4359
7. Chrysanthopoulou A, Antoniadou C, Natsi AM, et al. Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling. Clin Immunol. 2023;247:109240. doi:10.1016/j.clim.2023.109240
Disclosure
This study was supported by the Greek General Secretariat for Research and Technology (GSRT), Research & Innovation Programme CytoNET, grant MIS-5048548 and by GSRT, Regional Excellence Programme InTechThrace, grant MIS-5047285.