Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice.

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The "multiple hits hypothesis" suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.We used mice with wildtype, or littermates with CH25H-/-, EBI2-/-, or CYP7B1-/- genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve = 0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H-/-, EBI2-/-, or CYP7B1-/-) were observed.In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 - oxysterol axis to the intestinal microbiota was detectable in the current study.

Elevated oxysterol levels in human and mouse livers reflect nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.

ß6 -integrin serves as a novel serum tumor marker for colorectal carcinoma.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify ß6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.

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 ß-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 oxysterol receptor GPR183 in inflammatory bowel diseases.

Immune cell trafficking is an important mechanism for the pathogenesis of inflammatory bowel disease (IBD). The oxysterol receptor GPR183 and its ligands, dihydroxylated oxysterols, can mediate positioning of immune cells including innate lymphoid cells. GPR183 has been mapped to an IBD risk locus, however another gene, Ubac2 is encoded on the reverse strand and associated with Behçet's disease, therefore the role of GPR183 as a genetic risk factor requires validation. GPR183 and production of its oxysterol ligands are up-regulated in human IBD and murine colitis. Gpr183 inactivation reduced severity of colitis in group 3 innate lymphoid cells-dependent colitis and in IL-10 colitis but not in dextran sodium sulphate colitis. Irrespectively, Gpr183 knockout strongly reduced accumulation of intestinal lymphoid tissue in health and all colitis models. In conclusion, genetic, translational and experimental studies implicate GPR183 in IBD pathogenesis and GPR183-dependent cell migration might be a therapeutic drug target for IBD. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

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 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.

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 Crohn's 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.

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-ß-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-ß-MCA may be important for intestinal Fxr activation in mice.

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1ß, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1ß secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1ß release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1ß. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1ß levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.