Background and Aims Based on genetics and natural history, Crohn’s disease can be separated into two entities, an ileal and a colonic disease. Protein-based approaches are needed to elucidate whether such subphenotypes are related to distinct pathophysiological processes. Methods The proteome of ulcer edges was compared with that of paired control tissue samples [n = 32 biopsies] by differential proteomics in the ileum and the colon of Crohn’s disease patients [n = 16]. The results were analysed using a hypothesis-driven approach [based on the literature] and a hypothesis-free approach [pathway enrichment analyses] to determine common and segment-specific pathophysiological processes associated with ileal and colonic CD ulcer edges. To confirm the involvement of a key pathway highlighted by proteomics, two proteins were also studied by immunochemistry. Results In the ileum and the colon, 4428 and 5204 proteins, respectively, were identified and quantified. Ileal and colonic ulcer edges differed in having a distinct distribution of proteins associated with epithelial–mesenchymal transition, neutrophil degranulation, and ribosomes. Ileal and colonic ulcer edges were similarly characterized by an increase in the proteins implicated in the endoplasmic reticulum protein-processing pathway and a decrease in mitochondrial proteins. Immunochemistry confirmed the presence of endoplasmic reticulum stress in the mucosa of ileal and colonic ulcer edges. Conclusion This study provides protein-based evidence for partially distinct pathophysiological processes being associated with ileal and colonic ulcer edges in Crohn’s disease patients. This could constitute a first step toward the development of gut segment–specific diagnostic markers and therapeutics.

Background and aims Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an accumulation of fibroblasts differentiating into myofibroblasts secreting excessive extracellular matrix. 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). Proteins of interests were validated by immunohistochemistry (IHC) in ileal and colonic samples of stricturing CD (n=44), pure inflammatory CD (n=29) and control (n=40) subjects. The pro-fibrotic role of one selected epithelial protein 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) and Binding-immunoglobulin protein (BiP). This was confirmed by IHC. In HT-29 cells, tunicamycin-induced ER stress triggered AGR2 intracellular expression and its secretion. Supernatant of these HT-29 cells, pre-conditioned by tunicamycin, led to a myofibroblastic differentiation when applied on CCD-18Co fibroblasts. By using recombinant protein and blocking agent for AGR2, we demonstrated that the secretion of this protein by epithelial cells can play a role in the myofibroblastic differentiation. Conclusions The development of CD fibrotic strictures could involve epithelial ER stress and particularly the secretion of AGR2.

Summary Background Ileal and colonic Crohn's disease seem to be two separate entities. Aims To describe the main physiological distinctions between the small and the large intestine and to analyse the differences between ileal and colonic Crohn's disease. Methods The relevant literature was critically examined and synthesised. Results The small and large intestine have fundamental distinctions (anatomy, cellular populations, immune defence, microbiota). The differences between ileal and colonic Crohn's disease are highlighted by a heterogeneous body of evidence including clinical features (natural history of the disease, efficacy of treatments, and monitoring), epidemiological data (smoking status, age, gender) and biological data (genetics, microbiota, immunity, mesenteric fat). However, the contribution of these factors to disease location remains poorly understood. Conclusion The classification of ileal and colonic Crohn's disease as distinct subphenotypes is well supported by the literature. Understanding of these differences could be exploited to develop more individualised patient care.

Background Evolution of Crohn's disease (CD) is often marked by fibrostenotic complications and available biotherapies cannot prevent or treat intestinal fibrosis. The pathophysiological mechanisms of intestinal fibrosis are multiple and poorly understood. The intestinal epithelium probably plays a key role. The endoplasmic reticulum stress (ERS) is involved in the pathophysiology of IBD and in fibrosis. Protein disulfide isomerases (PDIs) take part in the ER stress response, but their precise roles and associations with CD remain poorly characterized. We investigated the distribution of selected PDIs within the ileum and colon of adult and pediatric patients with inflammatory or stenosing CD. Methods Tissues from 72 pediatric and 47 adult CDs, and 26 pediatric and 48 adult patients without IBD were included from 4 tertiary hospitals. The degree of fibrosis and inflammatory infiltrate were evaluated. The distribution of 4 PDIs (AGR2, BiP, PDIA6, ERP44) within the tissues was studied by immunohistochemistry (IHC) using a semiquantitative scoring scale (0 to 4). Statistical tests used were ANOVA or Kruskal-Wallis (with post hoc test), T-Student and Mann-Whitney. Results The clinical characteristics of the patients included are described in Table 1. The PDIs provide an IHC signal which was mainly epithelial. The distribution of PDIs differed according to the segment studied (colon or ileum), the age of CD onset, location in the epithelium (surface, median crypt, or bottom of crypt), and the normal, inflammatory, and/or fibrotic features of the tissues. In the ileum of adult and pediatric CDs, the distribution of AGR2 was significantly higher in the epithelium adjacent to fibro-inflammatory tissues (p<0.01). In pediatric cases, AGR2 increased significantly with the fibrosis grade within the surface epithelium of the ileum (Fig. 1). The distributions of the other PDIs (BiP, ERP44 and PDIA6) were rather influenced by the inflammatory infiltrates and varied in adult and pediatric CD. The distribution of BiP was significantly higher in tissues with acute and/or chronic inflammation and independent from fibrosis. In adults, the distribution of PDIA6 in the colonic epithelium was significantly higher in CD cases compared to non-CD cases. Conclusion Our results suggest that the studied PDIs may have different roles in the ERS response within the intestinal epithelium in adult and pediatric CD. These PDIs need to be functionally explored further to better understand their specific involvement in inflammation and fibrosis in CD. The increase of AGR2 in fibro-inflammatory tissues, not observed with other PDIs, suggests a specific link between AGR2 and intestinal fibrosis.

Background Intestinal fibrosis is a complication of Crohn’s disease (CD) characterised by myofibroblasts and extracellular matrix accumulation within the submucosa and smooth muscles, leading to bowel strictures. No medical treatment exists to treat or reverse intestinal fibrosis leading often to surgical resection. The potential role of intestinal epithelium in the fibrotic process remains poorly defined. Methods We performed a pilot study on ileal fibrostricturing CD surgical samples (n = 5), comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones. Confirmation of the specific protein increases was obtained by immunohistochemistry in colonic and ileal samples of CD (n = 44) compared with healthy subjects (n = 40), as well as in intestinal epithelial cell line under induced endoplasmic reticulum (ER) stress. A model of fibroblast to myofibroblast differentiation induction was also challenged using preconditioned media of intestinal epithelial cells after a pulsed ER stress. Results Label-free proteomics revealed high ER stress in the epithelium surrounding fibrotic bowel wall, involving anterior gradient protein 2 homolog (AGR2) and 78 kDA glucose-regulated protein (BiP). Confirmation of both proteins increase was obtained by immunohistochemistry. ER stress induction in intestinal epithelial cells was associated with an intracellular increase of AGR2, BiP and ER stress markers as sXPB1 and CHOP. AGR2 was also detected in the culture medium of these epithelial cells and myofibroblast differentiation was obtained using this culture medium. Conclusion The increase of ER stress proteins observed in fibrostenosing tissues together with these preliminary evidences of fibroblast to myofibrobast differentiation obtained by paracrine action of intestinal epithelial cell preconditioned to ER stress induction suggest a role of epithelial ER stress in Crohn’s disease intestinal fibrosis.