Tina Raselli

Alterations in Enterohepatic Fgf15 Signaling and Changes in Bile Acid Composition Depend on Localization of Murine Intestinal Inflammation.

Background:Fibroblast growth factor (FGF) 15/19 is part of the gut-liver crosstalk accounting for bile acid (BA) metabolism regulation. Dysregulation of fibroblast growth factor 15/19 signaling is observed in different pathological conditions, for example, in gastrointestinal diseases such as inflammatory bowel disease (IBD). To understand the molecular bases, we analyzed the enterohepatic regulation of Fgf15-mediated pathway in 2 different inflammatory bowel disease mouse models. Methods:Target genes of the BA-farnesoid-X-receptor (Fxr)-Ffg15 axis were quantified by RT-PCR or western blotting in gut and liver of dextran sulfate sodium (DSS)-treated and IL10(−/−) mice. Serum Fgf15 levels were analyzed by ELISA. Biliary and fecal BA composition was differentiated by HPLC-MS/MS. Results:Dextran sulfate sodium-treated mice with ileum-sparing colitis showed higher Fgf15 serum levels. In contrast, IL10(−/−) mice with ileitis had a trend toward decreased Fgf15 serum levels compared with controls and increased expression of Asbt as a negative Fxr-target gene. In hepatic tissue of both models, no histological changes, but higher interleukin 6 (IL-6) mRNA expression and down-regulation of Fxr and Cytochrom P450 7a1 mRNA expression were observed. Fibroblast growth factor receptor 4 up-regulation was in line with higher Fgf15 serum levels in dextran sulfate sodium-treated mice. A distinct fecal BA profile was observed in both models with significantly higher levels of taurine-conjugated BA in particular tauro-&bgr;-muricholic acid in IL10(−/−) mice. Conclusions:Ileum-sparing colitis is characterized by activation of Fxr-Fgf15 signaling with higher expression of Fxr-target gene Fgf15, whereas ileal inflammation showed no signs of Fxr-Fgf15 activation. Abundance of BA such as T-&bgr;-MCA may be important for intestinal Fxr activation in mice.

Deficiency of Protein Tyrosine Phosphatase Non-Receptor Type 2 in Intestinal Epithelial Cells Has No Appreciable Impact on Dextran Sulphate Sodium Colitis Severity But Promotes Wound Healing.

Background/Aims: The protein tyrosine phosphatase non-receptor type 2 (PTPN2) is known to mediate susceptibility to inflammatory bowel diseases. Cell culture experiments suggest that PTPN2 influences barrier function, autophagy and secretion of pro-inflammatory cytokines. PTPN2 knockout mice die a few weeks after birth due to systemic inflammation, emphasizing the importance of this phosphatase in inflammatory processes. The aim of this study was to investigate the role of PTPN2 in colon epithelial cells by performing dextran sulphate sodium (DSS)-induced colitis in PTPN2xVilCre mice. Methods: Acute colitis was induced by administering 2.5 or 2% DSS for 7 days and chronic colitis by 4 cycles of treatment using 1% DSS. Body weight of mice was measured regularly and colonoscopy was done at the end of the experiments. Mice were sacrificed afterwards and colon specimens were obtained for H&E staining. For analysis of wound healing, mechanical wounds were introduced during endoscopy and wound closure assessed by daily colonoscopy. Results: Although colonoscopy and weight development suggested changes in colitis severity, the lack of any influence of PTPN2 deficiency on histological scoring for inflammation severity after acute or chronic DSS colitis indicates that colitis severity is not influenced by epithelial-specific loss of PTPN2. Chronic colitis induced the development of aberrant crypt foci more frequently in PTPN2xVilCre mice compared to their wild type littermates. On the other hand, loss of PTPN2-induced enhanced epithelial cell proliferation and promoted wound closure. Conclusions: Loss of PTPN2 in intestinal epithelial cells (IECs) has no significant influence on inflammation in DSS colitis. Obviously, loss of PTPN2 in IECs can be compensated in vivo, thereby suppressing a phenotype. This lack of a colitis-phenotype might be due to enhanced epithelial cell proliferation and subsequent increased wound-healing capacity of the epithelial layer.

A cell type-specific role of protein tyrosine phosphatase non-receptor type 2 in regulating ER stress signalling.

Background/Aims: Genetic polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) have been associated with inflammatory bowel disease (IBD). A recent study demonstrated that PTPN2 regulates ER stress signalling in pancreatic β-cells. Therefore, we investigated whether PTPN2 regulates ER stress pathways, apoptosis and cytokine secretion in human intestinal epithelial cells (IECs) and monocytes. Methods: THP-1 and HT-29 IECs were stimulated with 2 µg/ml tunicamycin (TNM) for the indicated periods of time. For knockdown experiments, cells were transfected using a mixture of three different PTPN2-specific siRNA oligonucleotides. Cell lysates were analysed by Western blot and real-time PCR. Cytokine secretion was studied by ELISA measurements of cell culture supernatant. Results: TNM treatment reduced PTPN2 protein levels in HT-29 IECs and THP-1 monocytes. Knockdown of PTPN2 in THP-1 monocytes led to an exaggerated induction of phospho-eIF2α, enhanced PARP cleavage, indicative of apoptosis, and attenuated IL-8 and TNF secretion upon TNM stimulation. In HT-29 cells PTPN2 deficiency caused reduced phosphorylation of eIF2α and PARP cleavage under ER stress conditions. Conclusions: Whereas the knockdown of PTPN2 made THP-1 cells more susceptible to ER stress, PTPN2 deficiency reduced ER stress responses in HT-29 IECs. This suggests that PTPN2 regulates adaptation to ER stress in a cell type-specific manner.

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.

Intestinal Activation of pH-Sensing Receptor OGR1 [GPR68] Contributes to Fibrogenesis

Background and Aims pH-sensing ovarian cancer G-protein coupled receptor-1 [OGR1/GPR68] is regulated by key inflammatory cytokines. Patients suffering from inflammatory bowel diseases [IBDs] express increased mucosal levels of OGR1 compared with non-IBD controls. pH-sensing may be relevant for progression of fibrosis, as extracellular acidification leads to fibroblast activation and extracellular matrix remodelling. We aimed to determine OGR1 expression in fibrotic lesions in the intestine of Crohns disease [CD] patients, and the effect of Ogr1 deficiency in fibrogenesis. Methods Human fibrotic and non-fibrotic terminal ileum was obtained from CD patients undergoing ileocaecal resection due to stenosis. Gene expression of fibrosis markers and pH-sensing receptors was analysed. For the initiation of fibrosis in vivo, spontaneous colitis by Il10-/-, dextran sodium sulfate [DSS]-induced chronic colitis and the heterotopic intestinal transplantation model were used. Results Increased expression of fibrosis markers was accompanied by an increase in OGR1 [2.71 ± 0.69 vs 1.18 ± 0.03, p = 0.016] in fibrosis-affected human terminal ileum, compared with the non-fibrotic resection margin. Positive correlation between OGR1 expression and pro-fibrotic cytokines [TGFB1 and CTGF] and pro-collagens was observed. The heterotopic animal model for intestinal fibrosis transplanted with terminal ileum from Ogr1-/- mice showed a decrease in mRNA expression of fibrosis markers as well as a decrease in collagen layer thickness and hydroxyproline compared with grafts from wild-type mice. Conclusions OGR1 expression was correlated with increased expression levels of pro-fibrotic genes and collagen deposition. Ogr1 deficiency was associated with a decrease in fibrosis formation. Targeting OGR1 may be a potential new treatment option for IBD-associated 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.