Potential Role of Epithelial Protein Disulphide Isomerases in Crohn’s Disease Fibrosis

Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD) characterized by an accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive extracellular matrix. In in-vitro experiments, this myofibroblastic differentiation is elicited by a whole series of factors among which transforming growth factor β1 (TGF-β1) seems to play a key role. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: We performed a pilot proteomic study comparing the proteome of surface epithelium isolated by laser-capture microdissection in normal and fibrotic zones of resected ileal CD strictures (13 zones collected in 5 patients). The pro-fibrotic role of selected epithelial proteins was investigated through in-vitro experiments using HT-29 epithelial cells and a CCD-18Co fibroblast to myofibroblast differentiation model. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress proteins increase in the epithelium of CD ileal fibrotic strictures, including Anterior gradient protein 2 homolog (AGR2), Protein disulphide isomerase A6 (PDIA6) and Endoplasmic reticulum resident protein 44 (ERP44) which are 3 protein disulphide isomerases. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2, PDIA6, ERP44 as well as TGF β1 intracellular expression and their secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin (Tm), led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. The application of blocking agents for AGR2, PDIA6, ERP44 or TGF β1 in the supernatant of these Tm pre conditioned HT-29 cells, attenuated the myofibroblastic differentiation induced by this supernatant, suggesting a pro-fibrotic role of these secreted epithelial proteins. Conclusions: The development of CD fibrotic strictures may involve ER stress in epithelial cells, releasing a whole set of proteins into their environment, including AGR2, PDIA6, ERP44 as well as TGF-β1, which could exercise a pro-fibrotic role through a paracrine action