Annika Wyss

The EBI2-oxysterol axis promotes the development of intestinal lymphoid structures and colitis

The gene encoding for Epstein-Barr virus-induced G-protein coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied. We found increased mRNA expression of EBI2 and oxysterol synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated extraintestinal levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2-/- mice in the IL-10 colitis model. The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2-/- mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures. In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis.

The oxysterol synthesizing enzyme CH25H contributes to the development of intestinal fibrosis

Intestinal fibrosis and stenosis are common complications of Crohn’s disease (CD), frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidized cholesterol derivatives, with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase (CH25H) converts cholesterol to 25-hydroxycholesterol (25-HC), which modulates immune responses and oxidative stress. In human intestinal samples from CD patients we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme using the sodium dextran sulfate (DSS)-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis.

Mo1685 Role of EBI2 and Oxysterols in the Development of Intestinal Lymphoid Structures and Colon Inflammation

Orexins (orexin-A and orexin-B) are hypothalamic peptides involved in the sleep/wake control which interact with two GPCR sub-types, OX1R and OX2R. We have demonstrated that OX1R was highly expressed in digestive cancer cell lines derived from colon, pancreas and liver cancers (1). Our immunohistochemistry studies indicate that OX1R was also detected in 100 % of human Inflammatory Bowel Disease (IBD) epithelia including Crohns disease (21 samples) and Ulcerative Colitis (UC, 20 samples). We have investigated the role of orexin A (OxA) on acute inflammation in two UC mice models including chemically mice treated with Dextran Sulfate Sodium (DSS) and EXCY2 mice genetically invalidated for IL10 and Nox1 genes (IL10-/and Nox1-/-) which develop spontaneous UC-like disease (2). The daily intraperitoneal injection of OxA (0,22 μmol/Kg) in orally DSS-treated mice ameliorates the Disease Activity Index scored by measuring weight, length of colon, diarrhea and blood presence in the stool as compared to untreated mice. Moreover, OxA improves also histologic aspect of colon epithelium. The cytokinic profile analysis revealed that OxA reduces the pro-inflammatory cytokines secretion such as TNFα, IL6, IL8 homolog and IL1B in DSS-induced colitis mice colon extracts. In contrast, OxA has no effect on INFγ, IL10, and IL12 cytokine secretions. The recent development of a new spontaneous UC-like mouse model EXCY2, is ideally suited to study the OxA effect on spontaneous colitis. Indeed, EXCY2 mice develop a spontaneous colitis at 6/7 weeks of age with an upwards gradient from the rectum and reproduce all molecular characteristics seen in UC including, loss of goblet cells, deregulation of endoplasmic reticulum stress, tobacco protective effect ... Intraperitoneal injection (2/week) of OxA (0,22 μmol/Kg) during 21 days alleviated severe colitis in 10 week-old EXCY2 mice, OxA-treated EXCY2 mice exhibited a normal colonic mucosa (general crypts aspect, crypt size, presence of goblet cells, absence of immune cells infiltration) as compared to vehicle treated EXCY2 mice. These data indicate that OxA 1) exerts an original anti-inflammatory properties in DSS-treated mouse model; 2) and strongly protects from spontaneous colitis developed in EXCY2 mice model and trigger mucosal healing certainly by controlling the different pathways involved in the onset of UC. In conclusion, the system orexins/OX1R may represents an innovative and effective target in the treatment of UC. 1Voisin et al., Cancer research 2011, 71:3341-51 2Treton et al., PLOS One 2014, 9:e101669

Sa1716 Role of EBI2 and Oxysterols in the Pathogenesis of Fatty Liver Disease

A S L D A b st ra ct s score). Comparison of the means was done with paired t-tests, with a p value of 0.05 to be considered statistically significant. Results: For needle biopsy (n=30), the average SP from PVAs were 50.2 ± 21.0% and 55.7±23.3% respectively, compared to 31.7±9.2% and 35.4±10.8% from the CIA, which was significantly different (p=0.0000). There was more difference between pathologists than CIA conducted by 2 investigators (mean difference 5.5, p=0.029) (Figure1). There was also significant difference in NAS scores between the pathologists (by NAS score of 1 on average, 4 versus 5, se 0.25, p=0.004). For wedges, the average SP from PVA was 48.5±26.7% and 51.149.8±26.3% respectively, comparing to that from CIA 31.8±10.2%. SP was consistently higher with PVA than CIA, especially for >33% steatosis. There is no significant difference in NAS scores between the pathologists (3.3 vs 3.5, p=0.46). The CIA analysis resulted in similar NAS scores when combined with either one of the pathologist readings for the needle and wedge biopsies respectively (Figure1). After substituting the SP with CIA results, more than half of the samples that were diagnosed with definite NASH were changed to either borderline or no NASH. Conclusion: The concordance of steatosis quantifications by pathologists is lower than by CIA. Pathologists tend to overestimate the severity of steatosis, with ensuing higher NAS score, which could result in over-diagnosis of NASH. There is a need for the objective standardization of the histological quantification of liver steatosis. The inter-observer variability for the quantification of fatty liver and diagnosis of NASH can be improved by the implementation of a CIA.