Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation de Vallière, Cheryl ; Wang, Yu ; Eloranta, Jyrki J ; Vidal, Solange ; Clay, Ieuan ; Spalinger, Marianne R ; Tcymbarevich, Irina ; Terhalle, Anne ; Ludwig, Marie-Gabrielle ; Suply, Thomas ; Fried, Michael ; Kullak-Ublick, Gerd A ; Frey-Wagner, Isabelle ; Scharl, Michael ; Seuwen, Klaus ; Wagner, Carsten A ; Rogler, Gerhard Abstract: BACKGROUND A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflam- mation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa. METHODS OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1 and Ogr1 (WT) resi- dential peritoneal macrophages. RESULTS Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-฀B inhibitors. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1/Il-10 mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways. CONCLUSIONS OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-฀B inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological rolefor pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation. DOI: https://doi.org/10.1097/MIB.0000000000000375 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-111122 Journal Article Accepted Version Originally published at: de Vallière, Cheryl; Wang, Yu; Eloranta, Jyrki J; Vidal, Solange; Clay, Ieuan; Spalinger, Marianne R; Tcymbarevich, Irina; Terhalle, Anne; Ludwig, Marie-Gabrielle; Suply, Thomas; Fried, Michael; Kullak- Ublick, Gerd A; Frey-Wagner, Isabelle; Scharl, Michael; Seuwen, Klaus; Wagner, Carsten A; Rogler, Gerhard (2015). G Protein-coupled pH-sensing Receptor OGR1 Is a Regulator of Intestinal Inflammation. Inflammatory Bowel Diseases, 21(6):1269-1281. DOI: https://doi.org/10.1097/MIB.0000000000000375 Manuscript I The G protein-coupled pH-sensing receptor OGR1 is a regulator of intestinal inflammation Cheryl de Vallière1*, Yu Wang1,2*, Jyrki J. Eloranta3 , Solange Vidal4, Ieuan Clay4, Marianne R. Spalinger1, Irina Tcymbarevich1, Anne Terhalle1, Marie-Gabrielle Ludwig4, Thomas Suply4, Michael Fried1, Gerd A. Kullak-Ublick3, Isabelle Frey-Wagner1, Michael Scharl1, Klaus Seuwen4, Carsten A. Wagner2, Gerhard Rogler1 1Division of Gastroenterology and Hepatology, University Hospital Zürich, Switzerland, 2Institute of Physiology, University of Zürich, Switzerland, 3Department of Clinical Pharmacology and Toxicology, University Hospital Zürich, Switzerland, 4Novartis Institutes for Biomedical Research, Basel, Switzerland Corresponding author: Gerhard Rogler, MD, PhD Division of Gastroenterology and Hepatology University Hospital Zürich Rämistrasse 100 8091 Zürich Switzerland Tel. +41-(0)44-255-9477 E-mail: [email protected] * Cheryl de Vallière and Yu Wang contributed equally to this work. 46 Manuscript I ABSTRACT Objective: A novel family of proton sensing G-protein coupled receptors (GPCRs), including OGR1, GPR4, and TDAG8 was identified to be important for physiological pH homeostasis and inflammation. Thus we determined the function of proton-sensing OGR1 in the intestinal mucosa. Design: OGR1 expression in colonic tissues was investigated in controls and IBD patients. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied over 200 days. Microarray profiling was performed using Ogr1-/- and Ogr1+/+ (WT) residential peritoneal macrophages. Results: IBD patients expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant up-regulation of OGR1 expression, which could be reversed by the presence of NF- -Meier survival analysis showed a significantly delayed onset and progressionκB inhibitors. of rectal Kaplan prolapse in female Ogr1-/-// Il-10-/- mice. These mice displayed significantly less rectal prolapses. Up-regulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell adhesion gene pathways. Conclusion: OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF- OGR1 expression by TNF. OGR1 deficiency protectsκB frominhibition spontaneous reverses inflammation the induction in theof Il-10 KO model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation. 47 Manuscript I Key Words: GPCR; pH-sensing receptors; OGR1; IBD; microarrays; animal model Abbreviations: CD, Crohn's disease; DSS, dextran sulphate sodium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPCR, G protein coupled receptor; GPR4, G protein coupled receptor 4; GPR65, G protein coupled receptor 65; GPR68, G protein coupled receptor 68; H&E, hematoxylin and eosin; HPRT, Hypoxanthine-guanine phosphoribosyltransferase; IBD, inflammatory bowel disease; IFN- IL, interleukin; MPO, myeloperoxidase; OGR1, ovarian cancer G-protein coupled receptor 1; OR, odds ratio; PBS, phosphate buffered saline; RT-PCR, reverseγ, interferon transcription gamma; polymerase chain reaction; TDAG8, T-cell death associated gene 8; TNF, tumour necrosis factor; UC, Ulcerative colitis. Grants: This work was supported by a collaborative grant from the Zürich Center for Integrative Human Physiology (ZIHP) to CW and GR, research grants from the Swiss National Science Foundation to GR (Grant No.314730-153380) and the Swiss IBD Cohort (Grant No 3347CO-108792). The funding institutions had no role in the study design, in the collection, analysis and interpretation of data and in the writing of the manuscript. Competing interests: The authors declare no competing interests. Author contributions: CdV performed experiments, analyzed the data and wrote the first draft of the manuscript; YW, SV, IC, MRS, IT, AT, M-GL, TS, performed experiments and were involved in data analysis, MF, GAK-U, IF-W, MS, JJE, KS, CAW, GR conceived, designed and supervised the study and respective experiments. All authors wrote, corrected and approved the manuscript. 48 Manuscript I INTRODUCTION The mechanisms involved in the maintenance of mucosal homeostasis are important in our understanding of the pathophysiology of inflammatory bowel disease (IBD). Both formsinflammation of the thatdisease, is associated Crohn’s withdisease extracellular (CD) and acidification ulcerative ofcolitis mucosal (UC), tissue. give Mucosal rise to inflammation is interpreted as a local response to tissue damage and microbial invasion. A number of studies suggest that an acidic environment affects the progression and resolution of inflammation.1-3 Inflammation has been attributed to an increase in local proton concentration and lactate production4 and subsequent pro-inflammatory cytokine production, such as tumour necrosis factor (TNF), interleukin-6 (IL-6), interferon-gamma (IFN- -1-beta (IL- 5, 6 7-10 andγ) anti-TNF and interleukin targeted therapies1β). are TNF successful is one of thein characterizingboth CD and cytokinesUC. Activated in IBD macrophages, which are key cellular mediators of acute and chronic inflammation, are primary producers of TNF11. TNF activates the nuclear transcription factor kappa B (NF- one of the key regulators in chronic mucosal inflammation.12, 13 κB), G-protein-coupled receptors (GPCRs), cell-surface molecules involved in signal transduction, are targeted by key inflammatory cytokines.14 The ovarian cancer G-protein coupled receptor 1 (OGR1) family of receptors, which include OGR1, G protein coupled receptor 4 (GPR4), and T-cell death associated gene (TDAG8), sense extracellular protons through histidine residues located on the extracellular region of the receptors, resulting in the modification of a variety of cell functions.15, 16 Early signaling pathways of pH- sensing receptors triggered by acidification include phospholipase C activation, inositol trisphosphate formation and subsequent Ca2+ release15 or cyclic adenosine monophosphate (cAMP) production.17, 18 The increase of intracellular calcium influx and accumulation of cAMP has been shown to regulate a vast range of cellular responses. Moreover, OGR1 and TDAG8 are alleged to act in opposition in a regulatory manner, either enhancing or inhibiting