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.
Conclusions: 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.