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P097 Th2 cytokines are potent stimulators of pro-fibrotic responses by human intestinal subepithelial myofibroblasts

Filidou E.*1, Valatas V.2, Drygiannakis I.2, Arvanitidis K.1, Vradelis S.3, Kolios G.1

1Democritus University of Thrace, Laboratory of Pharmacology, Faculty of Medicine, Alexandroupolis, Greece 2University of Crete, Laboratory of Gastroenterology, Faculty of Medicine, Heraklion, Greece 3Democritus University of Thrace, 2nd Department of Internal Medicine of University Hospital of Alexandroupolis, Alexandroupolis, Greece

Background

In most cases, chronic inflammation precedes and triggers intestinal fibrosis in Crohn's Disease (CD). Excessive collagen deposition by subepithelial myofibroblasts (SEMFs) has a key role in fibrogenesis. Our hypothesis was that pro-inflammatory cytokines affect the expression of interleukin receptors on SEMFs and cytokine milieus reflecting alternative helper T cell (Th) polarization had a differential pro-fibrotic effect on SEMFs.

Methods

SEMFs were isolated from endoscopically-obtained colonic biopsies from healthy controls, set to culture and stimulated with recombinant IL-1α and/or TNF-α for 6h. Total RNA was extracted and interleukin receptors mRNA expression was assessed with reverse transcription quantitative (RT-q) PCR. Next, cultured SEMFs were stimulated for 6h with: a) the Th1-related cytokines TNF-α and/or IFN-γ, b) the Th2-related cytokines IL-4 and/or IL-13, c) the Th17-related cytokines IL-17 and/or IL-22 and d) the Treg-related cytokines IL-10 and/or TGF-β1. Collagen type I and type III expression was also quantitated with RT-qPCR.

Results

Unstimulated SEMFs had a basal expression of most of the studied interleukin receptors. As to Th1-related receptors, IL-1α and/or TNF-α stimulation downregulated the expression levels of some receptors (e.g. IL1R1: −0.3-fold, ±0.04, p<0.01) and upregulated others (e.g. IFNGR2: 9-fold, ±0.5, p<0.01). Moreover, IL-1α and/or TNF-α stimulation upregulated the Th2-related receptors (e.g. IL-13RA2: 68-fold, ±22, p<0.01), the Th17-related receptor IL-22R (13-fold, ±3.1, p<0.01) and the Treg-related receptors (e.g. TGFBR1: 2.5-fold, ±0.2, p<0.01). Stimulation of SEMFs with either Th1 or Th17 interleukin combinations also downregulated collagen expression (e.g. IL-17+IL-22: −0.56-fold, ±0.09, p<0.01), whereas Treg or Th2 interleukin combinations induced collagen expression (e.g. IL-4: 3.5-fold, ±0.17, p<0.01), with Th2 cytokines being the most potent.

Conclusion

Data presented suggest that SEMFs may be a dynamic crosslink between the inflammatory and the fibrotic process, as they express most of the Th-related interleukin receptors and their expression is modulated by pro-inflammatory cytokines, abundant in the inflamed mucosa of CD patients. Th2-related cytokines are the most potent stimulators of collagen production by SEMFs and thus the ones with a higher profibrotic potential.