P003 Activation of endoplasmic reticulum stress in the intestinal mucosa of Crohn’s disease patients
A. Coope*1, J. D. Botezelli2, M. L. S. Ayrizono1, L. A. Velloso3, R. F. Leal1
1University of Campinas, Coloproctology Unit, Surgery Department, Campinas, Brazil, 2University of Campinas, Campinas, Brazil, 3University of Campinas, Laboratory of Cell Signalling, Internal Medicine Department, Campinas, Brazil
The pathogenesis of Crohn’s disease (CD) is still complex and under investigation. The prevalence of CD is increasing worldwide, and it arises as a complex interplay between genetic and environmental components. CD is characterised by a transmural granulomatous inflammation, which can affect any part of the gastrointestinal tract, especially the ileum and/or colon. Evidences suggest a link between CD with endoplasmic reticulum (ER) stress. ER organelle is crucial for synthesis, folding, and processing of secreted and membrane proteins and lipid. The accumulation of unfolded proteins in the ER lumen activates the unfolded protein response (UPR), which resolves the protein-folding defect and restores ER homeostasis. A genetic deletion of XBP1 in intestinal epithelial cells resulted in enteritis and Paneth cell impairment, common features of CD. Here we tested the hypothesis that ER stress plays a role in the pathophysiology of CD and is induced in the intestinal mucosa of CD patients.
To test the hypothesis, surgical specimens were collected from CD patients with active disease (CD group) and intestinal biopsies from patients with no endoscopic alterations (CTR group). ER stress was evaluated by the analyses of important UPR markers by immunoblotting and qPCR assays. This study was performed in accordance with the Declaration of Helsinki and was approved by the institutional ethics committee of the University of Campinas.
We first evaluated the ER stress signalling IRE1/sXBP1, the ER transmembrane sensor protein: IRE1 is responsible to induce splicing of XBP1 (sXPB1). Our results show an increased expression of sXBP1 in the intestinal mucosa of CD patients compared with controls (p < 0.018). The second ER stress signalling investigated was PERK/EIF2alpha pathway; PERK protein once activated phosphorylate and inhibits the EIF2alpha. Here we show that PERK gene expression is increased in CD group (p < 0,025), as well as EIF2alpha protein expression (p < 0,0031) and pEIF2alpha/EIF2alpha ratio, demonstrating that this pathway is activated in intestine of CD patients. By qPCR we observed an increase in the cleaved/activated form of ATF6 protein in the intestinal mucosa of CD patients (p < 0.0327); however, this increase does not translate in protein content augmentation. We also observed increased expression of genes related to ER stress activation, such as ATF3 (p < 0.001), DNAJC3 (p < 0.0394), and the chaperones GRP94 (p < 0.0318) and GRP78 (p < 0.046). No differences were observed in CALR and STC2 expression.
Our results demonstrate the activation of the 3 branches of ER stress in the intestinal mucosa of CD patients. Thus, ER stress is an important mechanism of the inflammatory process in CD and may be an attractive therapeutic target.