OGR1 (GPR68) and TDAG8 (GPR65) Have Antagonistic Effects in Models of Colonic Inflammation.

G-protein-coupled receptors (GPRs), including pro-inflammatory ovarian cancer GPR1 (OGR1/GPR68) and anti-inflammatory T cell death-associated gene 8 (TDAG8/GPR65), are involved in pH sensing and linked to inflammatory bowel disease (IBD). OGR1 and TDAG8 show opposite effects. To determine which effect is predominant or physiologically more relevant, we deleted both receptors in models of intestinal inflammation. Combined Ogr1 and Tdag8 deficiency was assessed in spontaneous and acute murine colitis models. Disease severity was assessed using clinical scores. Colon samples were analyzed using quantitative polymerase chain reaction (qPCR) and flow cytometry (FACS). In acute colitis, Ogr1-deficient mice showed significantly decreased clinical scores compared with wildtype (WT) mice, while Tdag8-deficient mice and double knockout (KO) mice presented similar scores to WT. In Il-10-spontaneous colitis, Ogr1-deficient mice presented significantly decreased, and Tdag8-deficient mice had increased inflammation. In the Il10-/- × Ogr1-/- × Tdag8-/- triple KO mice, inflammation was significantly decreased compared with Tdag8-/-. Absence of Ogr1 reduced pro-inflammatory cytokines in Tdag8-deficient mice. Tdag8-/- had significantly more IFNγ+ T-lymphocytes and IL-23 T-helper cells in the colon compared with WT. The absence of OGR1 significantly alleviates the intestinal damage mediated by the lack of functional TDAG8. Both OGR1 and TDAG8 represent potential new targets for therapeutic intervention.

Deletion of Smad7 Ameliorates Intestinal Inflammation and Contributes to Fibrosis.

BACKGROUND: Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of transforming growth factor (TGF)-ß compared with non-IBD controls. SMAD7 negatively regulates TGF-ß signaling. An earlier study aiming to target Smad7 showed a lack of clinical benefit. It remains unknown whether inhibition of SMAD7 is beneficial in specific settings of IBD. We evaluated the effect of Smad7 deficiency on inflammation, fibrogenesis, and wound healing. METHODS: For the initiation of fibrosis in Smad7-/- (Smad7Δex-I) CD-1 mice, the dextran sodium sulfate-induced chronic colitis model and the heterotopic transplantation model of fibrosis were used. Wound closure of fibroblasts from Smad7-/- mice was determined using culture inserts and electric cell-substrate impedance sensing in vitro. RESULTS: In dextran sodium sulfate-induced chronic colitis, Smad7 deficiency was associated with ameliorated inflammation, as evidenced by decreased clinical score, histological score, and myeloperoxidase activity. Absence of SMAD7 decreased T-cell accumulation in colonic tissue and tumor necrosis factor (TNF) mRNA expression levels. Smad7-/- mice showed a significant increase in hydroxyproline and collagen content, as well as ColIVa1 mRNA expression. Wild type mice transplanted with terminal ileum from Smad7-/- mice in the heterotopic animal model for intestinal fibrosis showed a significant increase in collagen content and protein expression of α-smooth muscle actin. CONCLUSIONS: Smad7 deficiency is associated with a decrease in intestinal inflammation and an increase in fibrosis. Targeting SMAD7 constitutes a potential new treatment option for IBD; progression of disease-associated fibrosis should be considered.

We evaluated the effect of Smad7 deficiency on inflammation and fibrogenesis. Smad7 deficiency was associated with ameliorated inflammation and increased collagen deposition. When targeting Smad7 as therapeutic strategy in IBD, potential initiation or aggravation of fibrosis should be considered.

pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis.

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD.

New Therapeutic Approach for Intestinal Fibrosis Through Inhibition of pH-Sensing Receptor GPR4.

BACKGROUND: Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of pH-sensing receptors compared with non-IBD controls. Acidification leads to angiogenesis and extracellular matrix remodeling. We aimed to determine the expression of pH-sensing G protein-coupled receptor 4 (GPR4) in fibrotic lesions in Crohn's disease (CD) patients. We further evaluated the effect of deficiency in Gpr4 or its pharmacologic inhibition. METHODS: Paired samples from fibrotic and nonfibrotic terminal ileum were obtained from CD patients undergoing ileocaecal resection. The effects of Gpr4 deficiency were assessed in the spontaneous Il-10-/- and the chronic dextran sodium sulfate (DSS) murine colitis model. The effects of Gpr4 deficiency and a GPR4 antagonist (39c) were assessed in the heterotopic intestinal transplantation model. RESULTS: In human terminal ileum, increased expression of fibrosis markers was accompanied by an increase in GPR4 expression. A positive correlation between the expression of procollagens and GPR4 was observed. In murine disease models, Gpr4 deficiency was associated with a decrease in angiogenesis and fibrogenesis evidenced by decreased vessel length and expression of Edn, Vegfα, and procollagens. The heterotopic animal model for intestinal fibrosis, transplanted with terminal ileum from Gpr4-/- mice, revealed a decrease in mRNA expression of fibrosis markers and a decrease in collagen content and layer thickness compared with grafts from wild type mice. The GPR4 antagonist decreased collagen deposition. The GPR4 expression was also observed in human and murine intestinal fibroblasts. The GPR4 inhibition reduced markers of fibroblast activation stimulated by low pH, notably Acta2 and cTgf. CONCLUSIONS: Expression of GPR4 positively correlates with the expression of profibrotic genes and collagen. Deficiency of Gpr4 is associated with a decrease in angiogenesis and fibrogenesis. The GPR4 antagonist decreases collagen deposition. Targeting GPR4 with specific inhibitors may constitute a new treatment option for IBD-associated fibrosis.

A Novel OGR1 (GPR68) Inhibitor Attenuates Inflammation in Murine Models of Colitis.

BACKGROUND AND AIMS: Local extracellular acidification is associated with several conditions, such as ischemia, cancer, metabolic disease, respiratory diseases, and inflammatory bowel disease (IBD). Several recent studies reported a link between IBD and a family of pH-sensing G protein-coupled receptors. Our previous studies point to an essential role for OGR1 (GPR68) in the modulation of intestinal inflammation and fibrosis. In the current study, we evaluated the effects of a novel OGR1 inhibitor in murine models of colitis. METHODS: The effects of a novel small-molecule OGR1 inhibitor were assessed in the acute and chronic dextran sulfate sodium (DSS) murine models of colitis. Macroscopic disease indicators of intestinal inflammation were evaluated, and epithelial damage and immune cell infiltration and proliferation were assessed by immunohistochemistry. RESULTS: The OGR1 inhibitor ameliorated clinical parameters in acute and chronic DSS-induced colitis. In mice treated with the OGR1 inhibitor, endoscopy showed no thickening and normal vascularity, while fibrin was not detected. Histopathological findings revealed a decrease in severity of colonic inflammation in the OGR1 inhibitor group when compared to vehicle-DSS controls. In OGR1 inhibitor-treated mice, staining for the macrophage marker F4/80 and cellular proliferation marker Ki-67 revealed a reduction of infiltrating macrophages and slightly enhanced cell proliferation, respectively. This was accompanied by a reduction in pro-inflammatory cytokines, TNF and IL-6, and the fibrosis marker TGF-ß1. CONCLUSION: This is the first report providing evidence that a pharmacological inhibition of OGR1 has a therapeutic effect in murine colitis models. Our data suggest that targeting proton-sensing OGR1 using specific small-molecule inhibitors may be a novel therapeutic approach for the treatment of IBD.