Vieujean, S.(1,2);Hu, S.(2);Bequet, E.(2,3);Salée, C.(2);Massot, C.(2);Bletard, N.(4);Pierre, N.(2);Quesada Calvo, F.(2);Baiwir, D.(5);Mazzucchelli, G.(6);De Pauw, E.(6);Coimbra Marques, C.(7);Delvenne, P.(4);Edouard, L.(1,2);Meuwis, M.A.(2);
(1)CHU Liège- Sart Tilman, Department of Gastroenterology, Liège, Belgium;(2)University of Liège, Laboratory of Translational Gastroenterology, Liège, Belgium;(3)University Hospital of Liège, Division of Hepato-Gastroenterology- Department of Paediatrics, Liège, Belgium;(4)University Hospital CHU of Liège, Pathological Anatomy and Cytology, Liège, Belgium;(5)University of Liège, GIGA Proteomics Facility, Liège, Belgium;(6)University of Liège, MolSys Research Unit- Laboratory of Mass Spectrometry, Liège, Belgium;(7)University Hospital CHU of Liège, Abdominal Surgery Department, Liège, Belgium
Background
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.
Conclusion
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.
