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DOP85 The role of fibroblasts in the pathogenesis of Crohn’s disease-associated fistulas and in mesenchymal stem cell therapy

R.S. Bruckner1, M.C. Barnhoorn1, H. Mei2, S.M. Kiełbasa2, T.J. Harrijvan1, S.K. Hakuno1, J.J. van der Reijden1, A.E. van der Meulen-de Jong1, L.J.A.C. Hawinkels1

1Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands, 2Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands

Background

Perianal fistulas are a severe and frequent complication in Crohn’s disease (CD) patients, significantly affecting their quality of life. High recurrence rates, incomplete fistula healing and non-responding patients make the treatment challenging. Despite some novel insights, current knowledge about the pathogenesis of fistula formation is still limited. Fibroblasts are abundantly present in CD fistulas and were recently reported to regulate Th1 cell activity in inflammatory bowel disease (IBD) (Beswick EJ et al. 2018). We hypothesised that fibroblasts act as the key drivers of fistula pathogenesis by regulating inflammatory cell recruitment.

Methods

Fibroblasts were isolated from curettage material of perianal CD fistulas, non-CD-associated fistulas, and the normal colon (obtained at surgery for colorectal cancer at least 10 cm away from the primary tumour). For all experiments, fibroblast populations were identified by qPCR and flow cytometry analysis first. Then gene expression profiling via mRNA sequencing of cultured fistula- and colon-derived fibroblasts was performed. The characterisation was complemented by flow cytometry analysis. Furthermore, the interaction of fistula-derived fibroblasts with regulatory T cells (Tregs) was studied in co-culture experiments.

Results

mRNA sequencing analysis revealed no significant differences in gene expression between perianal CD fistulas and non-CD-associated fistulas. However, comparing fistula-derived samples to fibroblasts derived from the normal colon, we found over 6000 significantly differentially expressed genes. Among the 20 most significant differentially expressed genes between CD fistulas and colon fibroblasts were genes related to epithelial-mesenchymal transition, cell migration or the NF-κB signalling pathway. Furthermore, co-culture experiments with CD fistula-derived fibroblasts, revealed increased proliferation rates of CD25+FoxP3+ Treg cells. In addition, more CD4+ T cells differentiated into Treg cells after exposure to CD fistula fibroblasts.

Conclusion

Our data indicate a pathogenic role of fibroblasts located in the fistula tract. Their gene expression profile markedly differed from fibroblasts derived from the normal part of the colon. Based on our data, there is no difference between CD-associated and non-CD-associated fistula fibroblasts. CD fistula-derived fibroblasts seem to increase both the proliferation of Treg cells, and the differentiation of CD4+ T cells into Treg cells, indicating an important immunoregulatory role of fibroblasts. Ongoing experiments should reveal which cytokines and chemokines are involved in this process.

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