The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation Abstract Background and Aims During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. Methods The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. Results Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. Conclusions Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD. G-protein coupled receptor, pH receptors, GPR4, IBD, animal model 1. Introduction A local acidification in the gut lumen as well as in the mucosa has been observed during intestinal inflammation and implicated in the pathogenesis and progression of inflammatory bowel disease [IBD]. Fallingborg et al. reported that intraluminal colonic pH values in the proximal parts of the colon were significnatly lower in patients with active ulcerative colitis [UC] than in normal subjects [lowest values 2.3, 2.9, and 3.4].1 Nugent and coworkers also reported a decrease of colonic luminal pH values to less than 5.5 in two out of six patients with active UC.2 Also in patients with active Crohn’s disease [CD], three out of four CD patients investigated had decreased pH values in the proximal colon [pH 5.3] and distal colon [pH 5.3] as compared with normal controls [pH 6.8].2 Although there is still some controversy about the range of the intestinal luminal pH in IBD patients,3,4 it is widely accepted that inflammation is accompanied by tissue acidification due to hypoxia and excessive production and insufficient elimination of glycolytic metabolites. This indicates that luminal and tissue pH is decreased during active IBD. The [patho]physiological impact of these observations, however, has remained incompletely understood to date. G protein-coupled receptors [GPCR] play an important role in regulating intestinal functions and have been implicated in the development and course of IBD.5,6 Only recently, we found that the GPCR OGR1 [ovarian cancer G-protein-coupled receptor 1, GPR68] plays a role in IBD and that genetic deletion of OGR1 partially prevents the development of colitis in the IL-10 deficient IBD mouse model.7 The effects of OGR1 on intestinal function and inflammation may involve regulation of the intestinal barrier function 8. OGR1 belongs to the same family of proton-activated G protein-coupled receptors as GPR4.9–11 A third family member is the T-cell death-associated gene 8 [TDAG8, GPR65].9–11 Accumulating evidence indicates that members of this family of GPCRs, namely GPR4, OGR1, and TDAG8, are activated by protons upon a decrease of pH. At pH 7.6 the receptors are almost silent, whereas at pH 6.8 they are fully activated and thus may play a crucial role in pH homeostasis.9,11 GPR4 activation is transduced via the Gas pathway, followed by intracellular cAMP accumulation.9,11 Half-maximal activation of cAMP formation by GPR4 expressed in HEK293 cells occurred at pH 7.55.11 Limited studies exist on the relationship between GPR4 and tumour development or regulation of metabolic acidosis in the kidney.12–18 The function of all three proton-activated receptors has been linked to inflammatory processes in various tissues19 but the role of GPR4 in IBD is currently unknown. Gpr4 mRNA was found to be widely expressed in a variety of tissues including small intestine, colon, and spleen, and localised to─among other cell types─endothelial cells throughout the body. In endothelial cells, activation of GPR4 by extracellular acidification stimulates proinflammatory pathways and molecules involved in the adhesion of monocytes including CXCL2, CCL20, VCAM1, and SELE20,21, 37. Since GPR4 is expressed along the small and large intestine, we hypothesised that this proton-activated receptor might be involved in sensing local pH changes and may participate in the pathophysiology of IBD. Therefore, we examined the role of GPR4 in two murine models of colitis in vivo, the dextran sulphate sodium [DSS]-induced chronic colitis model and the spontaneous colitis model in IL-10 deficient animals. Collectively, these data demonstrate that absence of GPR4 is associated with ameliorated colitis, indicating that pH sensing plays an important role in the pathophysiology of IBD. 2. Methods 2.1. Human colonic biopsies Human colon biopsies were collected from patients during colonoscopy performed at the Division of Gastroenterology and Hepatology, University Hospital Zurich [Switzerland]. CD patients [eight with severe, seven with moderate inflammation and 14 in remission] and UC patients [five with severe and three with moderate inflammation] underwent colonoscopy for assessment of inflammation. Biopsies from patients with colitis were taken from inflamed areas. The control biopsies [17 controls] were from subjects undergoing colonoscopy for screening for colorectal cancer. The protocol for the study was approved by the local Cantonal Ethics Committee Zurich, Switzerland. 2.2. Induction of chronic colitis with DSS Gpr4-/- mice [BALB/c and C57BL/6 background] were provided by Thomas Suply and Klaus Seuwen, Novartis, Basel.13,22Gpr4-/- mice [C57BL/6] were bred to Il10-/- mice [C57BL/6]23–25 with the goal to generate Gpr4-/- /II-10-/- mice. All transgenic strains were bred in the standard animal facility of the Institute of Physiology, University of Zurich. Animal experiments were performed in the Zurich Integrative Rodent Physiology [ZIRP] core facility according to the guidelines of the Swiss animal welfare law and approved by the Cantonal Veterinary Office Zurich, Switzerland. Three experiments [two experiments on a BALB/c and one on a C57/BL6 background] were performed with DSS [MP Biomedicals, LLC, Solon, OH, USA] induced chronic colitis. Female mice at the age of 10–13 weeks and with a body weight around 20 g were used in the experiment. Chronic colitis was induced in wild-type and Gpr4-/- mice with four cycles of 3 % DSS in drinking water for 7 days followed by 10 days of regular drinking water. After the last cycle, all animals were allowed to recover for 5 weeks and subsequently sacrificed for sample collection. Mice on water served as controls throughout the experiments. In the spontaneous IL-10 deficient colitis model, the onset and development of inflammatory markers, colitis, and rectal prolapses were monitored over 200 days and data were analysed using Kaplan-Meier analysis [log rank Mantel-Cox test]. For the evaluation by histology, flow cytometry, and for the determination of cytokine [mRNA] expression profiles, some mice were sacrificed by cervical dislocation at 80 days of age. For all experiments, wild-type littermates were used. Histological analysis was performed as described previously.26–28 The sections were stained with haematoxylin and eosin [H&E] and scored by two independent researchers in a blinded fashion. Data for the DSS colitis model originate from one round of experiments with mice with the identical genetic background [littermates], but the other two rounds of experiments yielded qualitatively similar results. 2.3. Genomic DNA extraction and genotyping DNA extraction was done according to standard NaOH digestion. The polymerase chain reactions [PCR reactions] used for GPR4 genotyping were set up with following oligonucleotides: 5’-atgggatcggccattgaacaa-3’ [TS426], 5’-tcatcctgatcgacaagacc-3’ [TS427], 5’- gctgccatgtggactctcga-3’ [TS428], 5’-caggaaggcgatgctgatat-3’ [TS429]. TS426-TS427 is specific for neo [479 bps], and TS428-TS429 is specific for the GPR4 allele [302 bps]. Il-10-/- mice were screened with the following primers: forward 5’-GTGGGTGCAGTTATTGTCTTCCCG-3’ [oIMR0086], reverse 5’-GCCTTCAGTATA AAA GGGGGACC-3’. 2.4. Assessment of colonoscopy score in mice Mucosal damage was assessed by the murine endoscopic index of colitis severity [MEICS] as described previously.27–30 Animals were anaesthetised intraperitoneally with 90–120 mg of ketamine [Narketan 10%, Vétoquinol AG, Bern, Switzerland] and 8 mg of xylazine [Rompun 2%, Bayer, Zurich, Switzerland] per kg body weight and examined by colonoscopy [Karl Storz Tele Pack Pal 20043020, Karl Storz Endoskope, Tuttlingen, Germany]. 2.5. Myeloperoxidase[] activity assay Myeloperoxidase [MPO] activity was calculated by photoabsorbance, as previously described.27,28 Briefly, colon tissues were homogenised in 50 mM phosphate-buffered saline [PBS, pH 6.0] with 0.5% hexadecyltrimethylammonium bromide [H-5882, Sigma]. After performing three cycles of freeze-and-thaw, 20 µl of the homogenates’ supernatant were mixed with 280 µl of 0.02% dianisidine [D-3252, Sigma] solution. After 20 min incubation at room temperature, absorbance was measured at 460 nm. Protein concentration of the colon tissue supernatant was determined by Bradford protein assay [Bio-Rad]. MPO activity was calculated as mean absorbance/incubation time/protein concentration. 2.6. RNA extraction and quantitative real-time PCR Total RNA was extracted from colon and mesenteric lymph nodes tissue using the Qiagen RNeasy Mini Kit at a Qiacube workstation [Qiagen, Hilden, Germany] according to the manufacturer’s instructions.27,28 cDNA was prepared from adjusted RNA samples [2 µg/20 µl reaction] using the TaqMan High Capacity Reverse Transcriptase Reagent Kit [Applied Biosystems, Forster City, CA, USA]. Thermocycling conditions for reverse transcription were set at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 s [TGradient thermocycler, Biomera, Goettingen, Germany]. Semi-quantitative real-time [RT]-PCR Taqman assays [7900 Fast Real Time PCR system, Applied Biosystems; Forster City, CA, USA] were performed under the following cycling conditions: 20 s at 95°C, then 45 cycles of 95°C for 3 s and 60°C for 30 s with the TaqMan Fast Universal Mastermix. TaqMan assay probes for GPR4, TDAG8, OGR1, iNOS, IL-10, TNF-α, IFN-γ, IL-6, IL-18, MCP-1 [Life Technologies/ABI, Forster City, CA, USA] were used [Supplementary Table 1, available as Supplementary data at ECCO-JCC online]. RNA samples from individual animals were run in triplicates including a negative control [without cDNA]. The comparative ΔCt method was applied to determine the quantity of the cytokines relative to the endogenous control Gapdh [mouse GAPDH, Mm03302249_g1, Reporter = VIC and Quencher = MGB] and a reference sample. The relative quantification value was expressed and shown as 2−ΔCt. 2.7. RNA in situ hybridization [RNAscope] and immunohistochemistry Il-10-/- [C57BL/6] and Gpr4-/- mice [C57BL/6] were used to examine where Gpr4 mRNA is expressed in the murine proximal colon. C57BL/6 wild-type mice were used as a wild-type reference [n = 3 per strain]. Proximal colon of isoflurane-anesthetised mice was harvested and incubated for 24 h in 4% paraformaldehyde [PFA]/PBS. The PFA/PBS solution was replaced by 10% sucrose in PBS up to the tissue sinking to the bottom of the container. This step was repeated with 20% and 30% sucrose solutions. Colon rings were cut and embedded in Optimal Cutting Temperature [OCT]; 5-μm sections were prepared on Superfrost microscope slides [Thermo Fisher Scientific, Braunschweig, Germany] and kept for up to 1 week at -80°C. The RNA in situ hybridisation was performed using the the RNAscope 2.5 HD assay, Red, and the RNAscope 2.0 HD detection kit, Brown [Advanced Cell Diagnostics, Hayward, CA, USA] following the manufacturer’s protocol. Briefly, slides were rehydrated in PBS and were incubated with pretreatment solutions at recommended temperatures. Four signal amplification steps were performed at 40°C and two additional steps at room temperature with the appropriate solutions and probes designed and provided by the supplier [Advanced Cell Diagnostics, Hayward, CA, USA]. The fifth amplification step was extended from 30 min to 1 h in order to enhance the chromogenic signal. Detection of chromogenic signal was performed for 10 min using the specific reagents for the Red or Brown kit. RNA in situ hybridisation with the RNAscope 2.5 HD assay Red was followed by immunohistochemistry for cluster of differentiation 31 [CD31 or PECAM1], an endothelial marker, or immunofluorescence for α-Smooth Muscle Actin [α-SMA] or F4/80, a macrophage marker. Colon rings subjected to the combination of in situ hybridisation for Gpr4 with immunofluorescence for F4/80 and αSMA, were incubated for 75 min at room temperature either with 1:10 rat anti-mouse F4/80 [MCA497R, Bio-Rad, Cressier, Switzerland] or with 1:50 rabbit anti-αSMA [Ab5694, Abcam, Cambridge, UK]. Next slides were washed twice in hypertonic PBS [18 g NaCl/l] and once in normal PBS, for 5 min at each step. Secondary antibodies were added to the sections for 1 h at room temperature. Sections stained for F4/80 were combined with 1:500 goat anti-rat IgG [H+L] cross-absorbed secondary antibody, Alexa Fluor 488 [A11006, Thermo Fisher Scientific, Braunschweig, Germany] and sections stained for αSMA were combined with 1:500 donkey anti-rabbit IgG H&L, Alexa Fluor® 647 [ab150075, Abcam, Cambridge, UK]. Slides were washed twice in hypertonic PBS and once in normal PBS, for 5 min at each step, and mounted with Dako glycergel mounting medium [Dako, Switzerland]. Colon rings subjected to the combination of in situ hybridisation for Gpr4 with immunohistochemistry for CD31, were incubated with Avidin/Biotin blocking reagents [Avidin/Biotin blocking kit, Vector Laboratories, Burlingame, CA, USA] and washed with PBS as described by the manufacturer. Next the tissue was incubated with 1:7 rat anti-mouse CD31 [550274, BD Pharmingen, USA] overnight at 4°C, washed twice with PBS and incubated at room temperature for 30 min with 1:500 secondary antibody Biotin-SP-conjugated donkey anti-rat IgG [H+L] [Jackson ImmunoResearch, West Grove, PA, USA]. The slides were washed twice in PBS and the immunohistochemical staining was obtained by incubating the colon sections for 30 min with Vecstatin Elite ABC reagent [Vector Laboratories, Burlingame, CA, USA]. ABC solution was washed twice with PBS and replaced by DAB solution for 5 min [prepared as described by the manufacturer, Vector Laboratories, Burlingame, CA, USA]. After washing for 5 min in water, slides were counterstained with haematoxylin I and the slides were mounted with VectaMount Mounting Medium HT-5000 [Vector Laboratories, Burlingame, CA, USA]. Slides subjected to RNA in situ hybridisation with RNAscope 2.0 HD detection kit; Brown were counterstained directly after the detection of the chromogenic signal. 2.8. Preparation of lamina propria lymphocytes[] Lamina propria lymphocytes [LPLs] were isolated from Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice at 80 days of age, following a modified protocol by Weigmann.31 Briefly, the dissected colon was washed with Ca+- and Mg+-free PBS. The tissue was cut and incubated in medium containing 20 mM EDTA [Sigma-Aldrich] for 30 min at 37°C under shaking. LPLs were isolated from the lamina propria by enzymatic digestion [in DMEM medium containing 300 U/ml collagenase type I, 2 mg/ml hyaluronidase, 0.3 mg/ml DNase and 5 mM CaCl2.2H2O] for 15 min at 37°C under shaking. The LPLs were purified by discontinuous Ficoll density-gradient centrifugation. 2.9. Flow cytometric analysis [FACS] Single cell suspensions from lamina propria [LP] of mice were prepared as described above and stained for analysis by flow cytometry using PBS containing 4% fetal calf serum and 2.5 mM EDTA. At least 0.5 × 106 LP cells/well were stained at 4°C in the dark with the following fluorochrome-labelled monoclonal antibodies [all from BD Biosciences]: α-CD3, α-CD4, α-CD8, α-CD25, α-CD45.2, and α-CD161 [NK1.1]. Viable cells were acquired on a FACS Canto II [BD Biosciences] and analysed using FlowJo software [TriStar Inc]. 2.10. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 [Version 5.04, GraphPad Software Inc., San Diego, CA, US] and SPSS [8.0 for Windows, SPSS Inc., Chicago, IL, US]. Groups of data were compared between genotypes using the nonparametric Mann-Whitney U-test or Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test. All data were expressed as the means ± standard error of the mean [SEM]. Probabilities [p, two tailed] of p < 0.05 were considered statistically significant. Body weight comparison was performed using ‘General Linear Model, repeated measures’, and the full factorial model with type III sum of squares method.32 The ‘General Linear Model, repeated measures’ integrated both ‘individual deviation of daily body weight’ and ‘difference in genotype groups’ into the analysis to avoid systemic bias.32 For prolapse rate comparison studies, statistical differences between genotypes were calculated by the chi square test with Fisher’s exact test [exact significance, two-sided] and risk estimate test from the contingency table. The prolapse survival analysis was performed using Kaplan-Meier prolapse-free survival analysis [log-rank Mantel-Cox test] and estimated median prolapse-free survival time. 3. Results 3.1. IBD patients show enhanced GPR4 mRNA expression compared with healthy controls According to the National Center for Biotechnology Information [NCBI] Gene Expression Omnibus [GEO] profile and Gene database [http://www.ncbi.nlm.nih.gov/sites/geo]33 and the BioGPS database of the Scripps Research Institute [http://biogps.org][eg GEO profile data set GDS3113/181558, GDS1096/211266_s_at, GDS1096/206236_at],34 the human small intestine and colon express GRP4 at moderate levels [Supplementary Figure 1, available as Supplementary data at ECCO-JCC online]. GPR4 mRNA expression in the colon of healthy subjects and IBD patients was confirmed by RT-qPCR. GPR4 expression was significantly higher in UC [n = 8] and CD [n = 29] patients compared with healthy controls [n = 17], at 3.9-fold [p < 0.01] and 4.2-fold [p < 0.001], respectively Figure 1A]. These results suggest that GPR4 may play a role during inflammation. Figure 1. View largeDownload slide GPR4 mRNA detection in colonic tissue from humans and mice. [A] GPR4 mRNA was detected in colonic biopsies from controls [normal subjects], and patients with ulcerative colitis [UC], or Crohn’s disease [CD] by real-time polymerase chain reaction [RT-PCR] Taqman assays. A minimum of five patients per group was tested for quantification. [B] Gpr4, Tdag8, and Ogr1 mRNA detection in colonic tissues from wild-type mice or Gpr4-/- mice with water or dextran sodium sulphate [DSS]-induced chronic colitis. Groups of data were compared between the control group and different individual groups using the non-parametric Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.01,** p < 0.001.*** Figure 1. View largeDownload slide GPR4 mRNA detection in colonic tissue from humans and mice. [A] GPR4 mRNA was detected in colonic biopsies from controls [normal subjects], and patients with ulcerative colitis [UC], or Crohn’s disease [CD] by real-time polymerase chain reaction [RT-PCR] Taqman assays. A minimum of five patients per group was tested for quantification. [B] Gpr4, Tdag8, and Ogr1 mRNA detection in colonic tissues from wild-type mice or Gpr4-/- mice with water or dextran sodium sulphate [DSS]-induced chronic colitis. Groups of data were compared between the control group and different individual groups using the non-parametric Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.01,** p < 0.001.*** 3.2. TDAG8 and OGR1 do not depend on GPR4 during DSS colitis in- mice In wild-type mice, DSS-induced chronic colitis caused no upregulation of Gpr4 mRNA expression in colonic tissue. Similar results for the two other members of the pH receptor family, TDAG8 and OGR1, were found upon administration of DSS in both Gpr4+/+ and Gpr4-/- mice [Figure 1B], which indicated that TDAG8 and OGR1 were not upregulated on the mRNA level due to the lack of GPR4 in vivo. 3.3. Lack of GPR4 reduces inflammation in the chronic DSS model, with ameliorated body weight recovery Since GPR4 is expressed in human inflamed colonic tissue and proton-activated receptors have been linked to inflammatory diseases, we tested the impact of genetic deletion of GPR4 on the severity of chronic colitis in the DSS model. A total of 22 DSS-treated Gpr4+/+ [16 BALB and six C57BL/6] mice and 18 DSS-treated Gpr4-/- [11 BALB and seven C57BL/6] mice in three independent experiments were compared with five Gpr4+/+ and six Gpr4-/- control mice [C57BL/6] receiving normal water. Compared with Gpr4+/+ mice, Gpr4-/- mice showed less reduction in body weight upon DSS treatment [Figure 2A]. Gpr4-/- mice lost clearly less body weight [on Day 62: 2.7% vs Gpr4+/+ + DSS mice: -1.5%] and from Day 62 to Day 83 showed an enhanced ability to regain weight [p < 0.05, Figure 2A]. All three independent experiments showed similar results: Gpr4-/- mice recovered with higher body weights, indicating that GPR4 deficiency ameliorated inflammation-associated body weight changes (p < 0.05 for BALB [experiments 1 and 2] and p < 0.01 ** for C57BL/6). Figure 2. View large Download slide View large Download slide Body weight loss analysis and histological assessment of colonic inflammation. [A] Gpr4-/- mice, compared with Gpr4+/+ mice, showed less relative body weight loss during dextran sodium sulphate [DSS]-induced chronic colitis. After four cycles of DSS treatment [lasting 22 days], Gpr4-/- mice exhibited clearly reduced gain of body weight [F = 2.980, p < 0.05 *] than Gpr4+/+ mice. The body weight changes are expressed as relative change of body weight in % relative to Day 0. Histology scores were analysed to assess the epithelial damage [B] and leukocyte infiltration [C], indicating less severe inflammation in Gpr4-/- mice with DSS. The right panel shows a representative histological section from wild-type or Gpr4-/- mice treated with DSS, scale bar 100 μm. Data are representative of three independent experiments each with 6–8 female mice/group. Figure 2. View large Download slide View large Download slide Body weight loss analysis and histological assessment of colonic inflammation. [A] Gpr4-/- mice, compared with Gpr4+/+ mice, showed less relative body weight loss during dextran sodium sulphate [DSS]-induced chronic colitis. After four cycles of DSS treatment [lasting 22 days], Gpr4-/- mice exhibited clearly reduced gain of body weight [F = 2.980, p < 0.05 *] than Gpr4+/+ mice. The body weight changes are expressed as relative change of body weight in % relative to Day 0. Histology scores were analysed to assess the epithelial damage [B] and leukocyte infiltration [C], indicating less severe inflammation in Gpr4-/- mice with DSS. The right panel shows a representative histological section from wild-type or Gpr4-/- mice treated with DSS, scale bar 100 μm. Data are representative of three independent experiments each with 6–8 female mice/group. Colonic specimens from all groups were analysed for severity of inflammation by histological scoring by two blinded experts as described previously.8 The histological score was significantly lower in Gpr4-/- mice with DSS-induced chronic colitis [Figure 2B, C; and Supplementary Figure 2, available as Supplementary data at ECCO-JCC online] compared with wild-type controls [2.3 ± 0.38 vs. 4.9 ± 0.81; p < 0.001] [Supplementary Figure 1A, available as Supplementary data at ECCO-JCC online]. DSS-treated Gpr4-/- mice had lower scores for both epithelial injury and leukocyte infiltration [p < 0.001 each] [Figure 2B and C]. All three experiments showed consistent results independent of genetic background. In contrast, DSS-treated Gpr4-/- mice had slightly shorter colon lengths and a higher endoscopic MEICS score [p < 0.05, Supplementary Figure 3A and 3B, available as Supplementary data at ECCO-JCC online]. GPR4 deficiency did not influence MPO activity or spleen weight upon colitis induction [Supplementary Figure 3C and D]. No differences in the cytokine expression profiles of iNOS, IL-10, TNF-α, IL-6, or MCP-1 from colon and mesenteric lymph nodes of DSS-induced colitis Gpr4+/+ and Gpr4-/- mice were observed [Figure 3; and Supplementary Figure 3E]. Only IFN-γ mRNA expression in colon samples was higher in Gpr4-/- mice treated with DSS compared with wild-type mice receiving DSS [Figure 3]. Figure 3. View largeDownload slide Assessment of the colitis severity during dextran sodium sulphate [DSS]induced chronic colitis. The mRNA expression levels of iNOS, IL-10, TNF-α, IFN-γ, IL-6, and MCP-1 in colon from Gpr4-/- mice and Gpr4+/+ mice with or without administration of DSS were not changed between genotypes for the same treatment. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0.01.** p < 0.001.*** Data are representative of three independent experiments. Figure 3. View largeDownload slide Assessment of the colitis severity during dextran sodium sulphate [DSS]induced chronic colitis. The mRNA expression levels of iNOS, IL-10, TNF-α, IFN-γ, IL-6, and MCP-1 in colon from Gpr4-/- mice and Gpr4+/+ mice with or without administration of DSS were not changed between genotypes for the same treatment. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0.01.** p < 0.001.*** Data are representative of three independent experiments. 3.4. Spontaneous colitis in the IL-10 KO mouse is attenuated by GPR4 deficiency The impact of GPR4 in colitis development was further assessed in the spontaneous colitis model in IL10-deficient animals. All mice were maintained in the same animal housing room and all experiments were carried out during the same time period. The occurrence of rectal prolapse as a sign of spontaneous colitis in Il-10-/- mice was monitored for 200 days. No prolapses were observed in control Gpr4+/+ /Il-10+/+ mice in the breeding colonies for 200 days [n > 100 for each gender]. In comparison with Gpr4+/+ /Il-10-/- mice, both female and male Gpr4-/- /Il-10-/- mice, had a significantly lower rectal prolapse incidence (female: 6.9%, n = 29 vs 66.7%, n = 12, p < 0.001, odds ratios of Gpr4-/-/Gpr4+/+ = 0.037 [95% CL 0.006–0.241]; male: 24.4%, n = 41 vs 52.0%, n = 25, p = 0.033, odds ratios = 0.298 [95% CL 0.103–0.859]; chi square test with Fisher’s exact test/two-sided). Kaplan-Meier prolapse-free survival analysis showed a significantly delayed onset of rectal prolapse in Gpr4-/- /Il-10-/- mice as compared with Gpr4+/+ /Il-10-/- mice (estimated median prolapse-free survival time, female: > 200 days vs 123 days; p < 0.001; male: > 200 days vs 161 days, p = 0.007, log-rank [Mantel-Cox] test, Figure 4A; and Supplementary Figure 5A, available as Supplementary data at ECCO-JCC online). Figure 4. View largeDownload slide Development of inflammatory bowel disease [IBD] and progression to prolapse were reduced by the deletion of GPR4 from IL10-deficient mice. [A] Kaplan-Meier prolapse-free survival curve showed delayed onset and progression of prolapses in female Gpr4-/- /Il-10-/- mice relative to female Gpr4+/+ /Il-10-/- mice (estimated median prolapse-free survival time, > 200 days vs 123 days, p < 0.001,*** log-rank [Mantel-Cox] test). Black dotted lines, Gpr4-/- /Il-10-/- mice [6.9% prolapses, n = 29, female]; black solid line, Gpr4+/+ /Il-10-/- mice [66.7% prolapses, n = 12, female]; grey dotted lines, Gpr4+/+ /Il-10+/+ mice [0% prolapses, n = 31, female]. No rectal prolapse was detected in the Gpr4+/+ /Il-10+/+ mice in these breeding colonies for 200 days [> 100 mice]. Comparison of myepoperoxidase [MPO] activity in colon tissue [B], colon length [C], and relative spleen weight [D] showed attenuated colitis in female Gpr4-/- /Il-10-/- mice (not significant but relative spleen weight, p < 0.01,** Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Figure 4. View largeDownload slide Development of inflammatory bowel disease [IBD] and progression to prolapse were reduced by the deletion of GPR4 from IL10-deficient mice. [A] Kaplan-Meier prolapse-free survival curve showed delayed onset and progression of prolapses in female Gpr4-/- /Il-10-/- mice relative to female Gpr4+/+ /Il-10-/- mice (estimated median prolapse-free survival time, > 200 days vs 123 days, p < 0.001,*** log-rank [Mantel-Cox] test). Black dotted lines, Gpr4-/- /Il-10-/- mice [6.9% prolapses, n = 29, female]; black solid line, Gpr4+/+ /Il-10-/- mice [66.7% prolapses, n = 12, female]; grey dotted lines, Gpr4+/+ /Il-10+/+ mice [0% prolapses, n = 31, female]. No rectal prolapse was detected in the Gpr4+/+ /Il-10+/+ mice in these breeding colonies for 200 days [> 100 mice]. Comparison of myepoperoxidase [MPO] activity in colon tissue [B], colon length [C], and relative spleen weight [D] showed attenuated colitis in female Gpr4-/- /Il-10-/- mice (not significant but relative spleen weight, p < 0.01,** Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Figure 4B and Supplementary Figure 5B [available as Supplementary data at ECCO-JCC online] illustrate the level of granulocyte infiltration as measured by MPO activity in colon tissue of mice at 80 days of age. This age was chosen as none of the mice had developed a prolapse at this age. In Gpr4-/- /Il-10-/- male mice, MPO activity was significantly lower than that in Gpr4+/+ /Il-10-/- male mice [0.013 ± 0.068 vs 0.53 ± 0.101, p < 0.05]. A similar trend was seen in female Gpr4-/- /Il-10-/- animals [Figure 4B]. Histological scoring by two blinded investigators of 80-day old mice showed that colons from male [histological score of 1.6 ± 0.91] and female [1.6 ± 0.93] Gpr4+/+ /Il-10+/+ mice were morphologically normal. Gpr4-/- /Il-10-/- male [2.3 ± 0.68] and female [2.6 ± 1.69] mice displayed significantly less mucosal injury and infiltration as compared with Gpr4+/+ /Il-10-/- male [6.3 ± 0.45] and female [6.5 ± 1.12] mice [p < 0.05 for both]: [Figure 5A and B and Supplementary Figures 4 and 6 [available as Supplementary data at ECCO-JCC online], consistent with the prolapse ratio and prolapse-free survival analysis. Figure 5. View largeDownload slide Less histological damage in Gpr4-/- /Il-10-/- female mice. [A] The total histology scores of distal colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice at 80 days of age are shown, indicating reduced inflammation in Gpr4-/- /Il-10-/- mice [p < 0.05, Gpr4-/- /Il-10-/- compared with Gpr4+/+ /Il-10-/-]. [B] Haematoxylin and eosin [H&E]-stained sections showed the significant difference in the damage to epithelial integrity and intensity of the leukocyte infiltration into inflamed sites. Scale bar 100 μm. The total histology scores are representative for overall histology scores of distal colon [epithelial injury plus leukocyte infiltration]. Data are presented as mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0 .01, ** p < 0.001.*** Figure 5. View largeDownload slide Less histological damage in Gpr4-/- /Il-10-/- female mice. [A] The total histology scores of distal colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice at 80 days of age are shown, indicating reduced inflammation in Gpr4-/- /Il-10-/- mice [p < 0.05, Gpr4-/- /Il-10-/- compared with Gpr4+/+ /Il-10-/-]. [B] Haematoxylin and eosin [H&E]-stained sections showed the significant difference in the damage to epithelial integrity and intensity of the leukocyte infiltration into inflamed sites. Scale bar 100 μm. The total histology scores are representative for overall histology scores of distal colon [epithelial injury plus leukocyte infiltration]. Data are presented as mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0 .01, ** p < 0.001.*** 3.5. Reduction of mucosal CD4+ T helper cell infiltrate upon Gpr4 deficiency Spontaneous colitis in IL-10 deficient mice is mediated by Th1 and Th17 cell infiltration.35,36 In order to identify cellular players underlying the reduced colitis in Gpr4-/- mice, we subsequently analysed cellular infiltrates in the lamina propria by flow cytometry. As shown in Figure 6 and Supplementary Figure 6 [available as Supplementary data at ECCO-JCC online], the percentage of total CD4+ cells and the CD4+ to CD8+ ratios in the lamina propria were significantly higher in Gpr4+/+ /Il-10-/- as compared with wild-type controls [p < 0.01, p < 0.001, and p < 0.001]. Gpr4+/+ /Il-10-/- mice had significantly higher counts of CD4+ cells, but not of CD8+ cells in the lamina propria. The percentage of CD4+ in CD3+ was significantly lower in Gpr4-/- /Il10-/- [p < 0.05, Figure 6] whereas no differences of regulatory T cells, natural killer cells, total CD45+ cells, monocytes/macrophages or neutrophils in LPLs composition were observed among the three groups [Supplementary Table 2, available as Supplementary data at ECCO-JCC online]. Figure 6. View largeDownload slide Suppression of IFN-γ-producing CD4+ T helper cells in Gpr4-/- /Il-10-/- mice. Lamina propria leukocytes [LPLs] were isolated from the colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/,- and Gpr4+/+ /Il-10+/+ mice, stained to identify subpopulations, and were analysed by flow cytometry. LPL profiles from flow cytometry analysis demonstrated that ablation of GPR4 suppressed accumulation of CD4+ [T helper] cells, mainly Th1 cells, but not CD8+ [T cytotoxic] cells: quantification of CD4+ T cells, percentage of CD4+ within CD3+ T cells, CD4+/CD8+ ratio, quantification of CD8+ T cells, and percentage of CD8+ within CD3+ T cells. The difference between Gpr4-/- /Il-10-/,- and Gpr4+/+ /Il-10-/- did not reach statistical significance. Representative flow cytometry data of more than five qualitatively similar experiments are shown; isolated LPLs from three female mice were pooled in each group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** Figure 6. View largeDownload slide Suppression of IFN-γ-producing CD4+ T helper cells in Gpr4-/- /Il-10-/- mice. Lamina propria leukocytes [LPLs] were isolated from the colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/,- and Gpr4+/+ /Il-10+/+ mice, stained to identify subpopulations, and were analysed by flow cytometry. LPL profiles from flow cytometry analysis demonstrated that ablation of GPR4 suppressed accumulation of CD4+ [T helper] cells, mainly Th1 cells, but not CD8+ [T cytotoxic] cells: quantification of CD4+ T cells, percentage of CD4+ within CD3+ T cells, CD4+/CD8+ ratio, quantification of CD8+ T cells, and percentage of CD8+ within CD3+ T cells. The difference between Gpr4-/- /Il-10-/,- and Gpr4+/+ /Il-10-/- did not reach statistical significance. Representative flow cytometry data of more than five qualitatively similar experiments are shown; isolated LPLs from three female mice were pooled in each group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** 3.6. GPR4 modulates expression of factors involved in inflammation We further characterised mRNA expression of cytokines and other factors involved in cell adhesion and shown to be regulated by GPR4 37 in colon tissue and mesenteric lymph nodes, using age-matched female [Figure 7; and Supplementary Figure 8, available as Supplementary data at ECCO-JCC online] and male [Supplementary Figure 9, [available as Supplementary data at ECCO-JCC online] mice at 80 days of age. As shown in Figure 7 iNOS, IFN-γ, MCP-1, CXCL1, and CXCL2 mRNA expression was significantly lower in the colon of Gpr4-/- /Il-10-/- mice [p < 0.05], which reconfirmed reduced Th1-cell infiltrates in mice lacking GPR4. Furthermore, Gpr4-/- /Il-10-/- mice showed a trend for lower mRNA expression of IL-6, SELE, and VCAM1 as compared with Gpr4+/+ /Il-10-/- mice [Figure 7; and Supplementary Figure 8]. Male mice had similar patterns of changes in colon [Supplementary Figure 9]. However, in lymph nodes from female and male mice no clear differences could be detected [Supplementary Figure 8B]. Figure 7. View largeDownload slide Analysis of mRNA expression profiles of cytokines in colon from Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice. The mRNA expression profiles of iNOS, IFN-γ, IL-6, MCP-1, CXCL1, and CXCL1 were analysed by semi-quantitative real-time quantitative polymerase chain reaction [RT-qPCR] in colon tissue from of all three female strains [Gpr4-/- /Il-10-/-, Gpr4+/+ /I-10-/-, and Gpr4+/+ /Il-10+/+ mice]. Th1 associated IFN-γ expression was significantly lower in colon of female Gpr4-/- /IL-10-/- mice compared with female Gpr4+/+ /Il-10-/- mice (p < 0.05,* Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Data are presented as relative expression normalised to the housekeeping gene GAPDH, n = 6–9 mice per group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** Figure 7. View largeDownload slide Analysis of mRNA expression profiles of cytokines in colon from Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice. The mRNA expression profiles of iNOS, IFN-γ, IL-6, MCP-1, CXCL1, and CXCL1 were analysed by semi-quantitative real-time quantitative polymerase chain reaction [RT-qPCR] in colon tissue from of all three female strains [Gpr4-/- /Il-10-/-, Gpr4+/+ /I-10-/-, and Gpr4+/+ /Il-10+/+ mice]. Th1 associated IFN-γ expression was significantly lower in colon of female Gpr4-/- /IL-10-/- mice compared with female Gpr4+/+ /Il-10-/- mice (p < 0.05,* Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Data are presented as relative expression normalised to the housekeeping gene GAPDH, n = 6–9 mice per group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** 3.7. Localisation of Gpr4 mRNA in colon tissue Last, we performed chromogenic RNA in situ hybridisation of Gpr4 mRNA [RNAscope] in the proximal colon to examine where GPR4 may act to modulate inflammation. The tissue viability and assay quality were tested with the positive control peptidyl-prolyl cis-trans isomerase B [Ppib], and a probe for the bacterial gene dihydrodipicolinatereductase [Dapb] [data not shown] was used as an additional negative control to Gpr4-/-. Wild-type and Il10-/- mice displayed chromogenic signals in the lamina propria and muscularis [Figure 8; and Supplementary Figure 10, available as Supplementary data at ECCO-JCC online], whereas the signal was absent from Gpr4-/- colon [Supplementary Figure 11, available as Supplementary data at ECCO-JCC online]. Gpr4 mRNA-related signal was prominent in cells lining small vessels, consistent with the localisation of Gpr4 in endothelial cells. Signal intensity appeared to be higher in colon from Il10-/- mice and was also found in cells clustering in the interstitium. Co-staining with CD31, a marker of endothelial cells, demonstrated partial co-localisation of the Gpr4 signal with CD31. However, cells in the muscularis as well as clusters of interstitial cells, particularly abundant in colon from Il10-/- mice, were positive for Gpr4 and negative for CD31 [Figure 8]. Further co-localisation studies demonstrated partial co-localisation of Gpr4 mRNA with α-smooth muscle actin, particularly seen in the muscularis layer [Figure 9A-D]. Moreover, co-stainings with F4/80, a marker for macrophages, detected some macrophages with positive staining for Gpr4 mRNA [Figure 9E-H] in the colon of both wild-type [WT] and Il10-/- mice. Figure 8. View largeDownload slide Localisation of Gpr4 mRNA in endothelial cells in the colon from Gpr4+/+ and Il10-/- mice. Chromogenic in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. Sections were also labelled with antibodies against CD31, a marker of endothelial cells, showing partial co-localisation with Gpr4. [A-C] Wild-type colon, [D-F] colon from Il10-/-. Arrows indicate representative areas with Gpr4 mRNA expression. Scale bar 50 μm. Inserts show higher magnifications. Figure 8. View largeDownload slide Localisation of Gpr4 mRNA in endothelial cells in the colon from Gpr4+/+ and Il10-/- mice. Chromogenic in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. Sections were also labelled with antibodies against CD31, a marker of endothelial cells, showing partial co-localisation with Gpr4. [A-C] Wild-type colon, [D-F] colon from Il10-/-. Arrows indicate representative areas with Gpr4 mRNA expression. Scale bar 50 μm. Inserts show higher magnifications. Figure 9. View largeDownload slide Localisation of Gpr4 mRNA in smooth muscle cells and macrophages in the colon from Gpr4+/+ and Il10-/- mice. Fluorescent in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. [A-D] Sections were also labelled with antibodies against α-smooth muscle actin [green], a marker of smooth muscle cells showing partial co-localisation with Gpr4. [E-H] Co-staining for F4/80 [green], a marker for macrophages, detected staining of some macrophages, see insert. Nuclei were stained with 4',6-diamidino-2-phenylindole [DAPI] [blue]. Scale bar 50 μm. Inserts show higher magnifications. Figure 9. View largeDownload slide Localisation of Gpr4 mRNA in smooth muscle cells and macrophages in the colon from Gpr4+/+ and Il10-/- mice. Fluorescent in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. [A-D] Sections were also labelled with antibodies against α-smooth muscle actin [green], a marker of smooth muscle cells showing partial co-localisation with Gpr4. [E-H] Co-staining for F4/80 [green], a marker for macrophages, detected staining of some macrophages, see insert. Nuclei were stained with 4',6-diamidino-2-phenylindole [DAPI] [blue]. Scale bar 50 μm. Inserts show higher magnifications. 4. Discussion This is the first detailed study on the [patho-]physiological function of the proton-activated G-protein coupled receptor GPR4 in the intestine and its impact on chronic intestinal inflammation. We demonstrate that GPR4 deletion protects against murine experimental colitis, in both DSS-induced chronic colitis and the spontaneous colitis observed in IL-10 deficient mice. We show that GPR4 mRNA is expressed in the muscularis and lamina propria of colon and is strongly upregulated in the colons of IBD patients. The localisation based on mRNA in situ hybridisation is consistent with reports suggesting a predominant expression of GPR4 in endothelial cells.38 However, also smooth muscle cells in the muscularis and some macrophages showed expression of Gpr4. In the chronic DSS colitis model,35,36,39,40Gpr4-/- mice lost less body weight and had lower histology scores compared with wild-type littermates, indicating less severe inflammation. In the spontaneous IL-10 deficient colitis model,23–25 lack of GPR4 significantly delayed onset and progression of rectal prolapses. As IL-10 is a well-known anti-inflammatory cytokine and suppressive for Th1 cells and macrophages,41 IL-10 knockout mice are thought to have a Th1-cell driven disease.35,36 Consistent with reduced Th1 cell infiltrates in Gpr4-/-/Il-10-/- mice, a significantly lower IFN-γ expression was found. Also, iNOS, MCP-1, CXCL1, and CXCL2 were reduced in the absence of GPR4. Further, flow cytometry analysis demonstrated that GPR4 knockout mice exhibit reduced infiltration of CD4+ T cells into the colon. The anti-inflammatory effect of GPR4 deficiency seemed not to be mediated by a regulatory T cell-dependent mechanism, as no significant differences in regulatory T cell numbers were observed between groups. However, we cannot exclude a functional change of regulatory T cells in GPR4-deficient animals. Therefore, activation of GPR4 is likely to exacerbate intestinal inflammation. Based on the expression of GPR4 in endothelial cells, the fact that GPR4 in a pH-dependent fashion triggers expression of pathways involved in cell adhesion and inflammation in endothelial cells, and that endothelial cells can recruit and regulate inflammatory cells, we hypothesise that GPR4 may play a role in modulating inflammation in IBD. The downstream signals may involve, among others, the IFN-γ pathway. IFN-γ aggravates inflammation by increasing iNOS expression, activating macrophages and natural killer cells, favouring Th1 cell immune responses, and inducing apoptosis.42,43 Also, Gpr4-positive macrophages may contribute to inflammation. The role of Gpr4 in smooth muscle cells, however, remains elusive at this point. The excessive production of glycolytic metabolites in inflamed tissue causes the accumulation of protons, which may further activate GPR4, contributing to a positive feedback loop which may lead to a vicious cycle. Thus blockade of GPR4 may be a promising new target for IBD treatment. In vitro stimulation of GPR4 caused upregulation of many transcripts involved in inflammatory processes.37 Recent data published on Gpr4-/- mice support the deleterious role of GPR4 activation during inflammation. Reduced immune responses and attenuated airway hyper-responsiveness were observed in GPR4-deficient mice following ovalbumin exposure.44 This was accompanied by a reduction in the number of eosinophils in broncho-alveolar lavage fluid.45 In the cigarette smoke-induced chronic obstructive bowel disease [COPD] mouse model, Gpr4-/- mice had accelerated elimination of airway inflammation and enhanced neutrophil clearance.45 In patients with systemic sclerosis, expression of GPR4 correlates with the severity of lung disease.47 Similarly, a study published during the preparation of this manuscript describes a role of GPR4 in aggravating the response to an acute DSS-mediated colonic chemical insult.38 Yang et al. found evidence that acidosis/GPR4 signalling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway.20 The activation of GPR4 by acidosis upregulated the expression of multiple adhesion molecules such as SELE, VCAM-1, and ICAM-1 in vitro and increased the adhesiveness of human umbilical vein endothelial cells [HUVECs] expressing endogenous GPR4. These adhesion molecules are involved in the binding of leukocytes.20 In our studies, Gpr4-/-/Il-10-/- mice expressed lower mRNA levels of iNOS, TNF-α, IFN-γ, IL-6, MCP-1, CXCL2, CXCL1, SELE, and VCAM-1 which at least in part is in agreement with the mentioned in vitro observations. A regulation of the NF-κB pathway by GPR4-dependent signalling has been postulated,37 further supporting our findings. In summary, our results demonstrate that GPR4- deficiency protects from experimental colitis in two different mouse models, indicating an important pathophysiological role for the proton-activated receptor during the pathogenesis of mucosal inflammation. Future research needs to address the role of GPR4 in human IBD. Based on our mouse data, GPR4 may become a promising novel target for pharmacological IBD therapy. Funding This work was supported by a collaborative grant for the Zurich Center for Integrative Human Physiology [ZIHP] to AWr, OB, GR, and CAW, and by grants from the Swiss National Science Foundation to CAW [31003A_155959/1] and GR [153380 and 148422]. PHIS has been a recipient of a fellowship from the IKPP Kidney C H under the European Unions Seventh Framework Programme for Research, Technological Development and Demonstration under the grant agreement no 608847, and Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq] grant number 205625/2014-2. Conflict of Interest All authors declare that they have no conflict of interests that influenced design, performance, analysis, or interpretation of experiments. Author Contributions YW, CdeV, IL, PHIS, SG, AG, HM, KL, LW, MH, CK, KT performed experiments; YW, CdeV, IL, SG, AG, AW, KL, LW, MH, CK, OB, IF-W, GR, CAW analysed data; OB, IF-Wagner, GR, CAW planned experiments; OB, GR, CAW obtained funding for the project; YW, GR, CAW wrote the manuscript; and all authors read and approved the manuscript. Supplementary Data Supplementary data are available at ECCO-JCC online. Acknowledgments We thank Prof. Dr Burkhardt Seifert and Dr Sarah R. Haile [Division of Biostatistics, University of Zurich] for the statistics support and advice. We also thank Dr Klaus Seuwen, Novartis Institutes for BioMedical Research [NIBR], Basel, Switzerland, for his critical comments and valuable suggestions as well as for providing the Gpr4 KO mice. 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Abstract

Abstract Background and Aims During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. Methods The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. Results Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. Conclusions Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD. G-protein coupled receptor, pH receptors, GPR4, IBD, animal model 1. Introduction A local acidification in the gut lumen as well as in the mucosa has been observed during intestinal inflammation and implicated in the pathogenesis and progression of inflammatory bowel disease [IBD]. Fallingborg et al. reported that intraluminal colonic pH values in the proximal parts of the colon were significnatly lower in patients with active ulcerative colitis [UC] than in normal subjects [lowest values 2.3, 2.9, and 3.4].1 Nugent and coworkers also reported a decrease of colonic luminal pH values to less than 5.5 in two out of six patients with active UC.2 Also in patients with active Crohn’s disease [CD], three out of four CD patients investigated had decreased pH values in the proximal colon [pH 5.3] and distal colon [pH 5.3] as compared with normal controls [pH 6.8].2 Although there is still some controversy about the range of the intestinal luminal pH in IBD patients,3,4 it is widely accepted that inflammation is accompanied by tissue acidification due to hypoxia and excessive production and insufficient elimination of glycolytic metabolites. This indicates that luminal and tissue pH is decreased during active IBD. The [patho]physiological impact of these observations, however, has remained incompletely understood to date. G protein-coupled receptors [GPCR] play an important role in regulating intestinal functions and have been implicated in the development and course of IBD.5,6 Only recently, we found that the GPCR OGR1 [ovarian cancer G-protein-coupled receptor 1, GPR68] plays a role in IBD and that genetic deletion of OGR1 partially prevents the development of colitis in the IL-10 deficient IBD mouse model.7 The effects of OGR1 on intestinal function and inflammation may involve regulation of the intestinal barrier function 8. OGR1 belongs to the same family of proton-activated G protein-coupled receptors as GPR4.9–11 A third family member is the T-cell death-associated gene 8 [TDAG8, GPR65].9–11 Accumulating evidence indicates that members of this family of GPCRs, namely GPR4, OGR1, and TDAG8, are activated by protons upon a decrease of pH. At pH 7.6 the receptors are almost silent, whereas at pH 6.8 they are fully activated and thus may play a crucial role in pH homeostasis.9,11 GPR4 activation is transduced via the Gas pathway, followed by intracellular cAMP accumulation.9,11 Half-maximal activation of cAMP formation by GPR4 expressed in HEK293 cells occurred at pH 7.55.11 Limited studies exist on the relationship between GPR4 and tumour development or regulation of metabolic acidosis in the kidney.12–18 The function of all three proton-activated receptors has been linked to inflammatory processes in various tissues19 but the role of GPR4 in IBD is currently unknown. Gpr4 mRNA was found to be widely expressed in a variety of tissues including small intestine, colon, and spleen, and localised to─among other cell types─endothelial cells throughout the body. In endothelial cells, activation of GPR4 by extracellular acidification stimulates proinflammatory pathways and molecules involved in the adhesion of monocytes including CXCL2, CCL20, VCAM1, and SELE20,21, 37. Since GPR4 is expressed along the small and large intestine, we hypothesised that this proton-activated receptor might be involved in sensing local pH changes and may participate in the pathophysiology of IBD. Therefore, we examined the role of GPR4 in two murine models of colitis in vivo, the dextran sulphate sodium [DSS]-induced chronic colitis model and the spontaneous colitis model in IL-10 deficient animals. Collectively, these data demonstrate that absence of GPR4 is associated with ameliorated colitis, indicating that pH sensing plays an important role in the pathophysiology of IBD. 2. Methods 2.1. Human colonic biopsies Human colon biopsies were collected from patients during colonoscopy performed at the Division of Gastroenterology and Hepatology, University Hospital Zurich [Switzerland]. CD patients [eight with severe, seven with moderate inflammation and 14 in remission] and UC patients [five with severe and three with moderate inflammation] underwent colonoscopy for assessment of inflammation. Biopsies from patients with colitis were taken from inflamed areas. The control biopsies [17 controls] were from subjects undergoing colonoscopy for screening for colorectal cancer. The protocol for the study was approved by the local Cantonal Ethics Committee Zurich, Switzerland. 2.2. Induction of chronic colitis with DSS Gpr4-/- mice [BALB/c and C57BL/6 background] were provided by Thomas Suply and Klaus Seuwen, Novartis, Basel.13,22Gpr4-/- mice [C57BL/6] were bred to Il10-/- mice [C57BL/6]23–25 with the goal to generate Gpr4-/- /II-10-/- mice. All transgenic strains were bred in the standard animal facility of the Institute of Physiology, University of Zurich. Animal experiments were performed in the Zurich Integrative Rodent Physiology [ZIRP] core facility according to the guidelines of the Swiss animal welfare law and approved by the Cantonal Veterinary Office Zurich, Switzerland. Three experiments [two experiments on a BALB/c and one on a C57/BL6 background] were performed with DSS [MP Biomedicals, LLC, Solon, OH, USA] induced chronic colitis. Female mice at the age of 10–13 weeks and with a body weight around 20 g were used in the experiment. Chronic colitis was induced in wild-type and Gpr4-/- mice with four cycles of 3 % DSS in drinking water for 7 days followed by 10 days of regular drinking water. After the last cycle, all animals were allowed to recover for 5 weeks and subsequently sacrificed for sample collection. Mice on water served as controls throughout the experiments. In the spontaneous IL-10 deficient colitis model, the onset and development of inflammatory markers, colitis, and rectal prolapses were monitored over 200 days and data were analysed using Kaplan-Meier analysis [log rank Mantel-Cox test]. For the evaluation by histology, flow cytometry, and for the determination of cytokine [mRNA] expression profiles, some mice were sacrificed by cervical dislocation at 80 days of age. For all experiments, wild-type littermates were used. Histological analysis was performed as described previously.26–28 The sections were stained with haematoxylin and eosin [H&E] and scored by two independent researchers in a blinded fashion. Data for the DSS colitis model originate from one round of experiments with mice with the identical genetic background [littermates], but the other two rounds of experiments yielded qualitatively similar results. 2.3. Genomic DNA extraction and genotyping DNA extraction was done according to standard NaOH digestion. The polymerase chain reactions [PCR reactions] used for GPR4 genotyping were set up with following oligonucleotides: 5’-atgggatcggccattgaacaa-3’ [TS426], 5’-tcatcctgatcgacaagacc-3’ [TS427], 5’- gctgccatgtggactctcga-3’ [TS428], 5’-caggaaggcgatgctgatat-3’ [TS429]. TS426-TS427 is specific for neo [479 bps], and TS428-TS429 is specific for the GPR4 allele [302 bps]. Il-10-/- mice were screened with the following primers: forward 5’-GTGGGTGCAGTTATTGTCTTCCCG-3’ [oIMR0086], reverse 5’-GCCTTCAGTATA AAA GGGGGACC-3’. 2.4. Assessment of colonoscopy score in mice Mucosal damage was assessed by the murine endoscopic index of colitis severity [MEICS] as described previously.27–30 Animals were anaesthetised intraperitoneally with 90–120 mg of ketamine [Narketan 10%, Vétoquinol AG, Bern, Switzerland] and 8 mg of xylazine [Rompun 2%, Bayer, Zurich, Switzerland] per kg body weight and examined by colonoscopy [Karl Storz Tele Pack Pal 20043020, Karl Storz Endoskope, Tuttlingen, Germany]. 2.5. Myeloperoxidase[] activity assay Myeloperoxidase [MPO] activity was calculated by photoabsorbance, as previously described.27,28 Briefly, colon tissues were homogenised in 50 mM phosphate-buffered saline [PBS, pH 6.0] with 0.5% hexadecyltrimethylammonium bromide [H-5882, Sigma]. After performing three cycles of freeze-and-thaw, 20 µl of the homogenates’ supernatant were mixed with 280 µl of 0.02% dianisidine [D-3252, Sigma] solution. After 20 min incubation at room temperature, absorbance was measured at 460 nm. Protein concentration of the colon tissue supernatant was determined by Bradford protein assay [Bio-Rad]. MPO activity was calculated as mean absorbance/incubation time/protein concentration. 2.6. RNA extraction and quantitative real-time PCR Total RNA was extracted from colon and mesenteric lymph nodes tissue using the Qiagen RNeasy Mini Kit at a Qiacube workstation [Qiagen, Hilden, Germany] according to the manufacturer’s instructions.27,28 cDNA was prepared from adjusted RNA samples [2 µg/20 µl reaction] using the TaqMan High Capacity Reverse Transcriptase Reagent Kit [Applied Biosystems, Forster City, CA, USA]. Thermocycling conditions for reverse transcription were set at 25°C for 10 min, 37°C for 120 min, and 85°C for 5 s [TGradient thermocycler, Biomera, Goettingen, Germany]. Semi-quantitative real-time [RT]-PCR Taqman assays [7900 Fast Real Time PCR system, Applied Biosystems; Forster City, CA, USA] were performed under the following cycling conditions: 20 s at 95°C, then 45 cycles of 95°C for 3 s and 60°C for 30 s with the TaqMan Fast Universal Mastermix. TaqMan assay probes for GPR4, TDAG8, OGR1, iNOS, IL-10, TNF-α, IFN-γ, IL-6, IL-18, MCP-1 [Life Technologies/ABI, Forster City, CA, USA] were used [Supplementary Table 1, available as Supplementary data at ECCO-JCC online]. RNA samples from individual animals were run in triplicates including a negative control [without cDNA]. The comparative ΔCt method was applied to determine the quantity of the cytokines relative to the endogenous control Gapdh [mouse GAPDH, Mm03302249_g1, Reporter = VIC and Quencher = MGB] and a reference sample. The relative quantification value was expressed and shown as 2−ΔCt. 2.7. RNA in situ hybridization [RNAscope] and immunohistochemistry Il-10-/- [C57BL/6] and Gpr4-/- mice [C57BL/6] were used to examine where Gpr4 mRNA is expressed in the murine proximal colon. C57BL/6 wild-type mice were used as a wild-type reference [n = 3 per strain]. Proximal colon of isoflurane-anesthetised mice was harvested and incubated for 24 h in 4% paraformaldehyde [PFA]/PBS. The PFA/PBS solution was replaced by 10% sucrose in PBS up to the tissue sinking to the bottom of the container. This step was repeated with 20% and 30% sucrose solutions. Colon rings were cut and embedded in Optimal Cutting Temperature [OCT]; 5-μm sections were prepared on Superfrost microscope slides [Thermo Fisher Scientific, Braunschweig, Germany] and kept for up to 1 week at -80°C. The RNA in situ hybridisation was performed using the the RNAscope 2.5 HD assay, Red, and the RNAscope 2.0 HD detection kit, Brown [Advanced Cell Diagnostics, Hayward, CA, USA] following the manufacturer’s protocol. Briefly, slides were rehydrated in PBS and were incubated with pretreatment solutions at recommended temperatures. Four signal amplification steps were performed at 40°C and two additional steps at room temperature with the appropriate solutions and probes designed and provided by the supplier [Advanced Cell Diagnostics, Hayward, CA, USA]. The fifth amplification step was extended from 30 min to 1 h in order to enhance the chromogenic signal. Detection of chromogenic signal was performed for 10 min using the specific reagents for the Red or Brown kit. RNA in situ hybridisation with the RNAscope 2.5 HD assay Red was followed by immunohistochemistry for cluster of differentiation 31 [CD31 or PECAM1], an endothelial marker, or immunofluorescence for α-Smooth Muscle Actin [α-SMA] or F4/80, a macrophage marker. Colon rings subjected to the combination of in situ hybridisation for Gpr4 with immunofluorescence for F4/80 and αSMA, were incubated for 75 min at room temperature either with 1:10 rat anti-mouse F4/80 [MCA497R, Bio-Rad, Cressier, Switzerland] or with 1:50 rabbit anti-αSMA [Ab5694, Abcam, Cambridge, UK]. Next slides were washed twice in hypertonic PBS [18 g NaCl/l] and once in normal PBS, for 5 min at each step. Secondary antibodies were added to the sections for 1 h at room temperature. Sections stained for F4/80 were combined with 1:500 goat anti-rat IgG [H+L] cross-absorbed secondary antibody, Alexa Fluor 488 [A11006, Thermo Fisher Scientific, Braunschweig, Germany] and sections stained for αSMA were combined with 1:500 donkey anti-rabbit IgG H&L, Alexa Fluor® 647 [ab150075, Abcam, Cambridge, UK]. Slides were washed twice in hypertonic PBS and once in normal PBS, for 5 min at each step, and mounted with Dako glycergel mounting medium [Dako, Switzerland]. Colon rings subjected to the combination of in situ hybridisation for Gpr4 with immunohistochemistry for CD31, were incubated with Avidin/Biotin blocking reagents [Avidin/Biotin blocking kit, Vector Laboratories, Burlingame, CA, USA] and washed with PBS as described by the manufacturer. Next the tissue was incubated with 1:7 rat anti-mouse CD31 [550274, BD Pharmingen, USA] overnight at 4°C, washed twice with PBS and incubated at room temperature for 30 min with 1:500 secondary antibody Biotin-SP-conjugated donkey anti-rat IgG [H+L] [Jackson ImmunoResearch, West Grove, PA, USA]. The slides were washed twice in PBS and the immunohistochemical staining was obtained by incubating the colon sections for 30 min with Vecstatin Elite ABC reagent [Vector Laboratories, Burlingame, CA, USA]. ABC solution was washed twice with PBS and replaced by DAB solution for 5 min [prepared as described by the manufacturer, Vector Laboratories, Burlingame, CA, USA]. After washing for 5 min in water, slides were counterstained with haematoxylin I and the slides were mounted with VectaMount Mounting Medium HT-5000 [Vector Laboratories, Burlingame, CA, USA]. Slides subjected to RNA in situ hybridisation with RNAscope 2.0 HD detection kit; Brown were counterstained directly after the detection of the chromogenic signal. 2.8. Preparation of lamina propria lymphocytes[] Lamina propria lymphocytes [LPLs] were isolated from Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice at 80 days of age, following a modified protocol by Weigmann.31 Briefly, the dissected colon was washed with Ca+- and Mg+-free PBS. The tissue was cut and incubated in medium containing 20 mM EDTA [Sigma-Aldrich] for 30 min at 37°C under shaking. LPLs were isolated from the lamina propria by enzymatic digestion [in DMEM medium containing 300 U/ml collagenase type I, 2 mg/ml hyaluronidase, 0.3 mg/ml DNase and 5 mM CaCl2.2H2O] for 15 min at 37°C under shaking. The LPLs were purified by discontinuous Ficoll density-gradient centrifugation. 2.9. Flow cytometric analysis [FACS] Single cell suspensions from lamina propria [LP] of mice were prepared as described above and stained for analysis by flow cytometry using PBS containing 4% fetal calf serum and 2.5 mM EDTA. At least 0.5 × 106 LP cells/well were stained at 4°C in the dark with the following fluorochrome-labelled monoclonal antibodies [all from BD Biosciences]: α-CD3, α-CD4, α-CD8, α-CD25, α-CD45.2, and α-CD161 [NK1.1]. Viable cells were acquired on a FACS Canto II [BD Biosciences] and analysed using FlowJo software [TriStar Inc]. 2.10. Statistical analysis Statistical analyses were performed using GraphPad Prism 5 [Version 5.04, GraphPad Software Inc., San Diego, CA, US] and SPSS [8.0 for Windows, SPSS Inc., Chicago, IL, US]. Groups of data were compared between genotypes using the nonparametric Mann-Whitney U-test or Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test. All data were expressed as the means ± standard error of the mean [SEM]. Probabilities [p, two tailed] of p < 0.05 were considered statistically significant. Body weight comparison was performed using ‘General Linear Model, repeated measures’, and the full factorial model with type III sum of squares method.32 The ‘General Linear Model, repeated measures’ integrated both ‘individual deviation of daily body weight’ and ‘difference in genotype groups’ into the analysis to avoid systemic bias.32 For prolapse rate comparison studies, statistical differences between genotypes were calculated by the chi square test with Fisher’s exact test [exact significance, two-sided] and risk estimate test from the contingency table. The prolapse survival analysis was performed using Kaplan-Meier prolapse-free survival analysis [log-rank Mantel-Cox test] and estimated median prolapse-free survival time. 3. Results 3.1. IBD patients show enhanced GPR4 mRNA expression compared with healthy controls According to the National Center for Biotechnology Information [NCBI] Gene Expression Omnibus [GEO] profile and Gene database [http://www.ncbi.nlm.nih.gov/sites/geo]33 and the BioGPS database of the Scripps Research Institute [http://biogps.org][eg GEO profile data set GDS3113/181558, GDS1096/211266_s_at, GDS1096/206236_at],34 the human small intestine and colon express GRP4 at moderate levels [Supplementary Figure 1, available as Supplementary data at ECCO-JCC online]. GPR4 mRNA expression in the colon of healthy subjects and IBD patients was confirmed by RT-qPCR. GPR4 expression was significantly higher in UC [n = 8] and CD [n = 29] patients compared with healthy controls [n = 17], at 3.9-fold [p < 0.01] and 4.2-fold [p < 0.001], respectively Figure 1A]. These results suggest that GPR4 may play a role during inflammation. Figure 1. View largeDownload slide GPR4 mRNA detection in colonic tissue from humans and mice. [A] GPR4 mRNA was detected in colonic biopsies from controls [normal subjects], and patients with ulcerative colitis [UC], or Crohn’s disease [CD] by real-time polymerase chain reaction [RT-PCR] Taqman assays. A minimum of five patients per group was tested for quantification. [B] Gpr4, Tdag8, and Ogr1 mRNA detection in colonic tissues from wild-type mice or Gpr4-/- mice with water or dextran sodium sulphate [DSS]-induced chronic colitis. Groups of data were compared between the control group and different individual groups using the non-parametric Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.01,** p < 0.001.*** Figure 1. View largeDownload slide GPR4 mRNA detection in colonic tissue from humans and mice. [A] GPR4 mRNA was detected in colonic biopsies from controls [normal subjects], and patients with ulcerative colitis [UC], or Crohn’s disease [CD] by real-time polymerase chain reaction [RT-PCR] Taqman assays. A minimum of five patients per group was tested for quantification. [B] Gpr4, Tdag8, and Ogr1 mRNA detection in colonic tissues from wild-type mice or Gpr4-/- mice with water or dextran sodium sulphate [DSS]-induced chronic colitis. Groups of data were compared between the control group and different individual groups using the non-parametric Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.01,** p < 0.001.*** 3.2. TDAG8 and OGR1 do not depend on GPR4 during DSS colitis in- mice In wild-type mice, DSS-induced chronic colitis caused no upregulation of Gpr4 mRNA expression in colonic tissue. Similar results for the two other members of the pH receptor family, TDAG8 and OGR1, were found upon administration of DSS in both Gpr4+/+ and Gpr4-/- mice [Figure 1B], which indicated that TDAG8 and OGR1 were not upregulated on the mRNA level due to the lack of GPR4 in vivo. 3.3. Lack of GPR4 reduces inflammation in the chronic DSS model, with ameliorated body weight recovery Since GPR4 is expressed in human inflamed colonic tissue and proton-activated receptors have been linked to inflammatory diseases, we tested the impact of genetic deletion of GPR4 on the severity of chronic colitis in the DSS model. A total of 22 DSS-treated Gpr4+/+ [16 BALB and six C57BL/6] mice and 18 DSS-treated Gpr4-/- [11 BALB and seven C57BL/6] mice in three independent experiments were compared with five Gpr4+/+ and six Gpr4-/- control mice [C57BL/6] receiving normal water. Compared with Gpr4+/+ mice, Gpr4-/- mice showed less reduction in body weight upon DSS treatment [Figure 2A]. Gpr4-/- mice lost clearly less body weight [on Day 62: 2.7% vs Gpr4+/+ + DSS mice: -1.5%] and from Day 62 to Day 83 showed an enhanced ability to regain weight [p < 0.05, Figure 2A]. All three independent experiments showed similar results: Gpr4-/- mice recovered with higher body weights, indicating that GPR4 deficiency ameliorated inflammation-associated body weight changes (p < 0.05 for BALB [experiments 1 and 2] and p < 0.01 ** for C57BL/6). Figure 2. View large Download slide View large Download slide Body weight loss analysis and histological assessment of colonic inflammation. [A] Gpr4-/- mice, compared with Gpr4+/+ mice, showed less relative body weight loss during dextran sodium sulphate [DSS]-induced chronic colitis. After four cycles of DSS treatment [lasting 22 days], Gpr4-/- mice exhibited clearly reduced gain of body weight [F = 2.980, p < 0.05 *] than Gpr4+/+ mice. The body weight changes are expressed as relative change of body weight in % relative to Day 0. Histology scores were analysed to assess the epithelial damage [B] and leukocyte infiltration [C], indicating less severe inflammation in Gpr4-/- mice with DSS. The right panel shows a representative histological section from wild-type or Gpr4-/- mice treated with DSS, scale bar 100 μm. Data are representative of three independent experiments each with 6–8 female mice/group. Figure 2. View large Download slide View large Download slide Body weight loss analysis and histological assessment of colonic inflammation. [A] Gpr4-/- mice, compared with Gpr4+/+ mice, showed less relative body weight loss during dextran sodium sulphate [DSS]-induced chronic colitis. After four cycles of DSS treatment [lasting 22 days], Gpr4-/- mice exhibited clearly reduced gain of body weight [F = 2.980, p < 0.05 *] than Gpr4+/+ mice. The body weight changes are expressed as relative change of body weight in % relative to Day 0. Histology scores were analysed to assess the epithelial damage [B] and leukocyte infiltration [C], indicating less severe inflammation in Gpr4-/- mice with DSS. The right panel shows a representative histological section from wild-type or Gpr4-/- mice treated with DSS, scale bar 100 μm. Data are representative of three independent experiments each with 6–8 female mice/group. Colonic specimens from all groups were analysed for severity of inflammation by histological scoring by two blinded experts as described previously.8 The histological score was significantly lower in Gpr4-/- mice with DSS-induced chronic colitis [Figure 2B, C; and Supplementary Figure 2, available as Supplementary data at ECCO-JCC online] compared with wild-type controls [2.3 ± 0.38 vs. 4.9 ± 0.81; p < 0.001] [Supplementary Figure 1A, available as Supplementary data at ECCO-JCC online]. DSS-treated Gpr4-/- mice had lower scores for both epithelial injury and leukocyte infiltration [p < 0.001 each] [Figure 2B and C]. All three experiments showed consistent results independent of genetic background. In contrast, DSS-treated Gpr4-/- mice had slightly shorter colon lengths and a higher endoscopic MEICS score [p < 0.05, Supplementary Figure 3A and 3B, available as Supplementary data at ECCO-JCC online]. GPR4 deficiency did not influence MPO activity or spleen weight upon colitis induction [Supplementary Figure 3C and D]. No differences in the cytokine expression profiles of iNOS, IL-10, TNF-α, IL-6, or MCP-1 from colon and mesenteric lymph nodes of DSS-induced colitis Gpr4+/+ and Gpr4-/- mice were observed [Figure 3; and Supplementary Figure 3E]. Only IFN-γ mRNA expression in colon samples was higher in Gpr4-/- mice treated with DSS compared with wild-type mice receiving DSS [Figure 3]. Figure 3. View largeDownload slide Assessment of the colitis severity during dextran sodium sulphate [DSS]induced chronic colitis. The mRNA expression levels of iNOS, IL-10, TNF-α, IFN-γ, IL-6, and MCP-1 in colon from Gpr4-/- mice and Gpr4+/+ mice with or without administration of DSS were not changed between genotypes for the same treatment. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0.01.** p < 0.001.*** Data are representative of three independent experiments. Figure 3. View largeDownload slide Assessment of the colitis severity during dextran sodium sulphate [DSS]induced chronic colitis. The mRNA expression levels of iNOS, IL-10, TNF-α, IFN-γ, IL-6, and MCP-1 in colon from Gpr4-/- mice and Gpr4+/+ mice with or without administration of DSS were not changed between genotypes for the same treatment. For quantification, values are mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0.01.** p < 0.001.*** Data are representative of three independent experiments. 3.4. Spontaneous colitis in the IL-10 KO mouse is attenuated by GPR4 deficiency The impact of GPR4 in colitis development was further assessed in the spontaneous colitis model in IL10-deficient animals. All mice were maintained in the same animal housing room and all experiments were carried out during the same time period. The occurrence of rectal prolapse as a sign of spontaneous colitis in Il-10-/- mice was monitored for 200 days. No prolapses were observed in control Gpr4+/+ /Il-10+/+ mice in the breeding colonies for 200 days [n > 100 for each gender]. In comparison with Gpr4+/+ /Il-10-/- mice, both female and male Gpr4-/- /Il-10-/- mice, had a significantly lower rectal prolapse incidence (female: 6.9%, n = 29 vs 66.7%, n = 12, p < 0.001, odds ratios of Gpr4-/-/Gpr4+/+ = 0.037 [95% CL 0.006–0.241]; male: 24.4%, n = 41 vs 52.0%, n = 25, p = 0.033, odds ratios = 0.298 [95% CL 0.103–0.859]; chi square test with Fisher’s exact test/two-sided). Kaplan-Meier prolapse-free survival analysis showed a significantly delayed onset of rectal prolapse in Gpr4-/- /Il-10-/- mice as compared with Gpr4+/+ /Il-10-/- mice (estimated median prolapse-free survival time, female: > 200 days vs 123 days; p < 0.001; male: > 200 days vs 161 days, p = 0.007, log-rank [Mantel-Cox] test, Figure 4A; and Supplementary Figure 5A, available as Supplementary data at ECCO-JCC online). Figure 4. View largeDownload slide Development of inflammatory bowel disease [IBD] and progression to prolapse were reduced by the deletion of GPR4 from IL10-deficient mice. [A] Kaplan-Meier prolapse-free survival curve showed delayed onset and progression of prolapses in female Gpr4-/- /Il-10-/- mice relative to female Gpr4+/+ /Il-10-/- mice (estimated median prolapse-free survival time, > 200 days vs 123 days, p < 0.001,*** log-rank [Mantel-Cox] test). Black dotted lines, Gpr4-/- /Il-10-/- mice [6.9% prolapses, n = 29, female]; black solid line, Gpr4+/+ /Il-10-/- mice [66.7% prolapses, n = 12, female]; grey dotted lines, Gpr4+/+ /Il-10+/+ mice [0% prolapses, n = 31, female]. No rectal prolapse was detected in the Gpr4+/+ /Il-10+/+ mice in these breeding colonies for 200 days [> 100 mice]. Comparison of myepoperoxidase [MPO] activity in colon tissue [B], colon length [C], and relative spleen weight [D] showed attenuated colitis in female Gpr4-/- /Il-10-/- mice (not significant but relative spleen weight, p < 0.01,** Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Figure 4. View largeDownload slide Development of inflammatory bowel disease [IBD] and progression to prolapse were reduced by the deletion of GPR4 from IL10-deficient mice. [A] Kaplan-Meier prolapse-free survival curve showed delayed onset and progression of prolapses in female Gpr4-/- /Il-10-/- mice relative to female Gpr4+/+ /Il-10-/- mice (estimated median prolapse-free survival time, > 200 days vs 123 days, p < 0.001,*** log-rank [Mantel-Cox] test). Black dotted lines, Gpr4-/- /Il-10-/- mice [6.9% prolapses, n = 29, female]; black solid line, Gpr4+/+ /Il-10-/- mice [66.7% prolapses, n = 12, female]; grey dotted lines, Gpr4+/+ /Il-10+/+ mice [0% prolapses, n = 31, female]. No rectal prolapse was detected in the Gpr4+/+ /Il-10+/+ mice in these breeding colonies for 200 days [> 100 mice]. Comparison of myepoperoxidase [MPO] activity in colon tissue [B], colon length [C], and relative spleen weight [D] showed attenuated colitis in female Gpr4-/- /Il-10-/- mice (not significant but relative spleen weight, p < 0.01,** Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Figure 4B and Supplementary Figure 5B [available as Supplementary data at ECCO-JCC online] illustrate the level of granulocyte infiltration as measured by MPO activity in colon tissue of mice at 80 days of age. This age was chosen as none of the mice had developed a prolapse at this age. In Gpr4-/- /Il-10-/- male mice, MPO activity was significantly lower than that in Gpr4+/+ /Il-10-/- male mice [0.013 ± 0.068 vs 0.53 ± 0.101, p < 0.05]. A similar trend was seen in female Gpr4-/- /Il-10-/- animals [Figure 4B]. Histological scoring by two blinded investigators of 80-day old mice showed that colons from male [histological score of 1.6 ± 0.91] and female [1.6 ± 0.93] Gpr4+/+ /Il-10+/+ mice were morphologically normal. Gpr4-/- /Il-10-/- male [2.3 ± 0.68] and female [2.6 ± 1.69] mice displayed significantly less mucosal injury and infiltration as compared with Gpr4+/+ /Il-10-/- male [6.3 ± 0.45] and female [6.5 ± 1.12] mice [p < 0.05 for both]: [Figure 5A and B and Supplementary Figures 4 and 6 [available as Supplementary data at ECCO-JCC online], consistent with the prolapse ratio and prolapse-free survival analysis. Figure 5. View largeDownload slide Less histological damage in Gpr4-/- /Il-10-/- female mice. [A] The total histology scores of distal colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice at 80 days of age are shown, indicating reduced inflammation in Gpr4-/- /Il-10-/- mice [p < 0.05, Gpr4-/- /Il-10-/- compared with Gpr4+/+ /Il-10-/-]. [B] Haematoxylin and eosin [H&E]-stained sections showed the significant difference in the damage to epithelial integrity and intensity of the leukocyte infiltration into inflamed sites. Scale bar 100 μm. The total histology scores are representative for overall histology scores of distal colon [epithelial injury plus leukocyte infiltration]. Data are presented as mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0 .01, ** p < 0.001.*** Figure 5. View largeDownload slide Less histological damage in Gpr4-/- /Il-10-/- female mice. [A] The total histology scores of distal colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice at 80 days of age are shown, indicating reduced inflammation in Gpr4-/- /Il-10-/- mice [p < 0.05, Gpr4-/- /Il-10-/- compared with Gpr4+/+ /Il-10-/-]. [B] Haematoxylin and eosin [H&E]-stained sections showed the significant difference in the damage to epithelial integrity and intensity of the leukocyte infiltration into inflamed sites. Scale bar 100 μm. The total histology scores are representative for overall histology scores of distal colon [epithelial injury plus leukocyte infiltration]. Data are presented as mean ± standard error of the mean [SEM]; n ≥ 5 per group; p < 0.05,* p < 0 .01, ** p < 0.001.*** 3.5. Reduction of mucosal CD4+ T helper cell infiltrate upon Gpr4 deficiency Spontaneous colitis in IL-10 deficient mice is mediated by Th1 and Th17 cell infiltration.35,36 In order to identify cellular players underlying the reduced colitis in Gpr4-/- mice, we subsequently analysed cellular infiltrates in the lamina propria by flow cytometry. As shown in Figure 6 and Supplementary Figure 6 [available as Supplementary data at ECCO-JCC online], the percentage of total CD4+ cells and the CD4+ to CD8+ ratios in the lamina propria were significantly higher in Gpr4+/+ /Il-10-/- as compared with wild-type controls [p < 0.01, p < 0.001, and p < 0.001]. Gpr4+/+ /Il-10-/- mice had significantly higher counts of CD4+ cells, but not of CD8+ cells in the lamina propria. The percentage of CD4+ in CD3+ was significantly lower in Gpr4-/- /Il10-/- [p < 0.05, Figure 6] whereas no differences of regulatory T cells, natural killer cells, total CD45+ cells, monocytes/macrophages or neutrophils in LPLs composition were observed among the three groups [Supplementary Table 2, available as Supplementary data at ECCO-JCC online]. Figure 6. View largeDownload slide Suppression of IFN-γ-producing CD4+ T helper cells in Gpr4-/- /Il-10-/- mice. Lamina propria leukocytes [LPLs] were isolated from the colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/,- and Gpr4+/+ /Il-10+/+ mice, stained to identify subpopulations, and were analysed by flow cytometry. LPL profiles from flow cytometry analysis demonstrated that ablation of GPR4 suppressed accumulation of CD4+ [T helper] cells, mainly Th1 cells, but not CD8+ [T cytotoxic] cells: quantification of CD4+ T cells, percentage of CD4+ within CD3+ T cells, CD4+/CD8+ ratio, quantification of CD8+ T cells, and percentage of CD8+ within CD3+ T cells. The difference between Gpr4-/- /Il-10-/,- and Gpr4+/+ /Il-10-/- did not reach statistical significance. Representative flow cytometry data of more than five qualitatively similar experiments are shown; isolated LPLs from three female mice were pooled in each group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** Figure 6. View largeDownload slide Suppression of IFN-γ-producing CD4+ T helper cells in Gpr4-/- /Il-10-/- mice. Lamina propria leukocytes [LPLs] were isolated from the colon of female Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/,- and Gpr4+/+ /Il-10+/+ mice, stained to identify subpopulations, and were analysed by flow cytometry. LPL profiles from flow cytometry analysis demonstrated that ablation of GPR4 suppressed accumulation of CD4+ [T helper] cells, mainly Th1 cells, but not CD8+ [T cytotoxic] cells: quantification of CD4+ T cells, percentage of CD4+ within CD3+ T cells, CD4+/CD8+ ratio, quantification of CD8+ T cells, and percentage of CD8+ within CD3+ T cells. The difference between Gpr4-/- /Il-10-/,- and Gpr4+/+ /Il-10-/- did not reach statistical significance. Representative flow cytometry data of more than five qualitatively similar experiments are shown; isolated LPLs from three female mice were pooled in each group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** 3.6. GPR4 modulates expression of factors involved in inflammation We further characterised mRNA expression of cytokines and other factors involved in cell adhesion and shown to be regulated by GPR4 37 in colon tissue and mesenteric lymph nodes, using age-matched female [Figure 7; and Supplementary Figure 8, available as Supplementary data at ECCO-JCC online] and male [Supplementary Figure 9, [available as Supplementary data at ECCO-JCC online] mice at 80 days of age. As shown in Figure 7 iNOS, IFN-γ, MCP-1, CXCL1, and CXCL2 mRNA expression was significantly lower in the colon of Gpr4-/- /Il-10-/- mice [p < 0.05], which reconfirmed reduced Th1-cell infiltrates in mice lacking GPR4. Furthermore, Gpr4-/- /Il-10-/- mice showed a trend for lower mRNA expression of IL-6, SELE, and VCAM1 as compared with Gpr4+/+ /Il-10-/- mice [Figure 7; and Supplementary Figure 8]. Male mice had similar patterns of changes in colon [Supplementary Figure 9]. However, in lymph nodes from female and male mice no clear differences could be detected [Supplementary Figure 8B]. Figure 7. View largeDownload slide Analysis of mRNA expression profiles of cytokines in colon from Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice. The mRNA expression profiles of iNOS, IFN-γ, IL-6, MCP-1, CXCL1, and CXCL1 were analysed by semi-quantitative real-time quantitative polymerase chain reaction [RT-qPCR] in colon tissue from of all three female strains [Gpr4-/- /Il-10-/-, Gpr4+/+ /I-10-/-, and Gpr4+/+ /Il-10+/+ mice]. Th1 associated IFN-γ expression was significantly lower in colon of female Gpr4-/- /IL-10-/- mice compared with female Gpr4+/+ /Il-10-/- mice (p < 0.05,* Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Data are presented as relative expression normalised to the housekeeping gene GAPDH, n = 6–9 mice per group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** Figure 7. View largeDownload slide Analysis of mRNA expression profiles of cytokines in colon from Gpr4-/- /Il-10-/-, Gpr4+/+ /Il-10-/-, and Gpr4+/+ /Il-10+/+ mice. The mRNA expression profiles of iNOS, IFN-γ, IL-6, MCP-1, CXCL1, and CXCL1 were analysed by semi-quantitative real-time quantitative polymerase chain reaction [RT-qPCR] in colon tissue from of all three female strains [Gpr4-/- /Il-10-/-, Gpr4+/+ /I-10-/-, and Gpr4+/+ /Il-10+/+ mice]. Th1 associated IFN-γ expression was significantly lower in colon of female Gpr4-/- /IL-10-/- mice compared with female Gpr4+/+ /Il-10-/- mice (p < 0.05,* Kruskal-Wallis one-way analysis of variance [ANOVA] followed by Dunn’s multiple-comparison test). Data are presented as relative expression normalised to the housekeeping gene GAPDH, n = 6–9 mice per group. Data are presented as mean ± standard error of the mean [SEM]; p < 0.05,* p < 0.01,** p < 0.001.*** 3.7. Localisation of Gpr4 mRNA in colon tissue Last, we performed chromogenic RNA in situ hybridisation of Gpr4 mRNA [RNAscope] in the proximal colon to examine where GPR4 may act to modulate inflammation. The tissue viability and assay quality were tested with the positive control peptidyl-prolyl cis-trans isomerase B [Ppib], and a probe for the bacterial gene dihydrodipicolinatereductase [Dapb] [data not shown] was used as an additional negative control to Gpr4-/-. Wild-type and Il10-/- mice displayed chromogenic signals in the lamina propria and muscularis [Figure 8; and Supplementary Figure 10, available as Supplementary data at ECCO-JCC online], whereas the signal was absent from Gpr4-/- colon [Supplementary Figure 11, available as Supplementary data at ECCO-JCC online]. Gpr4 mRNA-related signal was prominent in cells lining small vessels, consistent with the localisation of Gpr4 in endothelial cells. Signal intensity appeared to be higher in colon from Il10-/- mice and was also found in cells clustering in the interstitium. Co-staining with CD31, a marker of endothelial cells, demonstrated partial co-localisation of the Gpr4 signal with CD31. However, cells in the muscularis as well as clusters of interstitial cells, particularly abundant in colon from Il10-/- mice, were positive for Gpr4 and negative for CD31 [Figure 8]. Further co-localisation studies demonstrated partial co-localisation of Gpr4 mRNA with α-smooth muscle actin, particularly seen in the muscularis layer [Figure 9A-D]. Moreover, co-stainings with F4/80, a marker for macrophages, detected some macrophages with positive staining for Gpr4 mRNA [Figure 9E-H] in the colon of both wild-type [WT] and Il10-/- mice. Figure 8. View largeDownload slide Localisation of Gpr4 mRNA in endothelial cells in the colon from Gpr4+/+ and Il10-/- mice. Chromogenic in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. Sections were also labelled with antibodies against CD31, a marker of endothelial cells, showing partial co-localisation with Gpr4. [A-C] Wild-type colon, [D-F] colon from Il10-/-. Arrows indicate representative areas with Gpr4 mRNA expression. Scale bar 50 μm. Inserts show higher magnifications. Figure 8. View largeDownload slide Localisation of Gpr4 mRNA in endothelial cells in the colon from Gpr4+/+ and Il10-/- mice. Chromogenic in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. Sections were also labelled with antibodies against CD31, a marker of endothelial cells, showing partial co-localisation with Gpr4. [A-C] Wild-type colon, [D-F] colon from Il10-/-. Arrows indicate representative areas with Gpr4 mRNA expression. Scale bar 50 μm. Inserts show higher magnifications. Figure 9. View largeDownload slide Localisation of Gpr4 mRNA in smooth muscle cells and macrophages in the colon from Gpr4+/+ and Il10-/- mice. Fluorescent in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. [A-D] Sections were also labelled with antibodies against α-smooth muscle actin [green], a marker of smooth muscle cells showing partial co-localisation with Gpr4. [E-H] Co-staining for F4/80 [green], a marker for macrophages, detected staining of some macrophages, see insert. Nuclei were stained with 4',6-diamidino-2-phenylindole [DAPI] [blue]. Scale bar 50 μm. Inserts show higher magnifications. Figure 9. View largeDownload slide Localisation of Gpr4 mRNA in smooth muscle cells and macrophages in the colon from Gpr4+/+ and Il10-/- mice. Fluorescent in situ hybridisation of Gpr4 mRNA [red dots] in murine proximal colon using RNAscope. [A-D] Sections were also labelled with antibodies against α-smooth muscle actin [green], a marker of smooth muscle cells showing partial co-localisation with Gpr4. [E-H] Co-staining for F4/80 [green], a marker for macrophages, detected staining of some macrophages, see insert. Nuclei were stained with 4',6-diamidino-2-phenylindole [DAPI] [blue]. Scale bar 50 μm. Inserts show higher magnifications. 4. Discussion This is the first detailed study on the [patho-]physiological function of the proton-activated G-protein coupled receptor GPR4 in the intestine and its impact on chronic intestinal inflammation. We demonstrate that GPR4 deletion protects against murine experimental colitis, in both DSS-induced chronic colitis and the spontaneous colitis observed in IL-10 deficient mice. We show that GPR4 mRNA is expressed in the muscularis and lamina propria of colon and is strongly upregulated in the colons of IBD patients. The localisation based on mRNA in situ hybridisation is consistent with reports suggesting a predominant expression of GPR4 in endothelial cells.38 However, also smooth muscle cells in the muscularis and some macrophages showed expression of Gpr4. In the chronic DSS colitis model,35,36,39,40Gpr4-/- mice lost less body weight and had lower histology scores compared with wild-type littermates, indicating less severe inflammation. In the spontaneous IL-10 deficient colitis model,23–25 lack of GPR4 significantly delayed onset and progression of rectal prolapses. As IL-10 is a well-known anti-inflammatory cytokine and suppressive for Th1 cells and macrophages,41 IL-10 knockout mice are thought to have a Th1-cell driven disease.35,36 Consistent with reduced Th1 cell infiltrates in Gpr4-/-/Il-10-/- mice, a significantly lower IFN-γ expression was found. Also, iNOS, MCP-1, CXCL1, and CXCL2 were reduced in the absence of GPR4. Further, flow cytometry analysis demonstrated that GPR4 knockout mice exhibit reduced infiltration of CD4+ T cells into the colon. The anti-inflammatory effect of GPR4 deficiency seemed not to be mediated by a regulatory T cell-dependent mechanism, as no significant differences in regulatory T cell numbers were observed between groups. However, we cannot exclude a functional change of regulatory T cells in GPR4-deficient animals. Therefore, activation of GPR4 is likely to exacerbate intestinal inflammation. Based on the expression of GPR4 in endothelial cells, the fact that GPR4 in a pH-dependent fashion triggers expression of pathways involved in cell adhesion and inflammation in endothelial cells, and that endothelial cells can recruit and regulate inflammatory cells, we hypothesise that GPR4 may play a role in modulating inflammation in IBD. The downstream signals may involve, among others, the IFN-γ pathway. IFN-γ aggravates inflammation by increasing iNOS expression, activating macrophages and natural killer cells, favouring Th1 cell immune responses, and inducing apoptosis.42,43 Also, Gpr4-positive macrophages may contribute to inflammation. The role of Gpr4 in smooth muscle cells, however, remains elusive at this point. The excessive production of glycolytic metabolites in inflamed tissue causes the accumulation of protons, which may further activate GPR4, contributing to a positive feedback loop which may lead to a vicious cycle. Thus blockade of GPR4 may be a promising new target for IBD treatment. In vitro stimulation of GPR4 caused upregulation of many transcripts involved in inflammatory processes.37 Recent data published on Gpr4-/- mice support the deleterious role of GPR4 activation during inflammation. Reduced immune responses and attenuated airway hyper-responsiveness were observed in GPR4-deficient mice following ovalbumin exposure.44 This was accompanied by a reduction in the number of eosinophils in broncho-alveolar lavage fluid.45 In the cigarette smoke-induced chronic obstructive bowel disease [COPD] mouse model, Gpr4-/- mice had accelerated elimination of airway inflammation and enhanced neutrophil clearance.45 In patients with systemic sclerosis, expression of GPR4 correlates with the severity of lung disease.47 Similarly, a study published during the preparation of this manuscript describes a role of GPR4 in aggravating the response to an acute DSS-mediated colonic chemical insult.38 Yang et al. found evidence that acidosis/GPR4 signalling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway.20 The activation of GPR4 by acidosis upregulated the expression of multiple adhesion molecules such as SELE, VCAM-1, and ICAM-1 in vitro and increased the adhesiveness of human umbilical vein endothelial cells [HUVECs] expressing endogenous GPR4. These adhesion molecules are involved in the binding of leukocytes.20 In our studies, Gpr4-/-/Il-10-/- mice expressed lower mRNA levels of iNOS, TNF-α, IFN-γ, IL-6, MCP-1, CXCL2, CXCL1, SELE, and VCAM-1 which at least in part is in agreement with the mentioned in vitro observations. A regulation of the NF-κB pathway by GPR4-dependent signalling has been postulated,37 further supporting our findings. In summary, our results demonstrate that GPR4- deficiency protects from experimental colitis in two different mouse models, indicating an important pathophysiological role for the proton-activated receptor during the pathogenesis of mucosal inflammation. Future research needs to address the role of GPR4 in human IBD. Based on our mouse data, GPR4 may become a promising novel target for pharmacological IBD therapy. Funding This work was supported by a collaborative grant for the Zurich Center for Integrative Human Physiology [ZIHP] to AWr, OB, GR, and CAW, and by grants from the Swiss National Science Foundation to CAW [31003A_155959/1] and GR [153380 and 148422]. PHIS has been a recipient of a fellowship from the IKPP Kidney C H under the European Unions Seventh Framework Programme for Research, Technological Development and Demonstration under the grant agreement no 608847, and Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq] grant number 205625/2014-2. Conflict of Interest All authors declare that they have no conflict of interests that influenced design, performance, analysis, or interpretation of experiments. Author Contributions YW, CdeV, IL, PHIS, SG, AG, HM, KL, LW, MH, CK, KT performed experiments; YW, CdeV, IL, SG, AG, AW, KL, LW, MH, CK, OB, IF-W, GR, CAW analysed data; OB, IF-Wagner, GR, CAW planned experiments; OB, GR, CAW obtained funding for the project; YW, GR, CAW wrote the manuscript; and all authors read and approved the manuscript. Supplementary Data Supplementary data are available at ECCO-JCC online. Acknowledgments We thank Prof. Dr Burkhardt Seifert and Dr Sarah R. Haile [Division of Biostatistics, University of Zurich] for the statistics support and advice. We also thank Dr Klaus Seuwen, Novartis Institutes for BioMedical Research [NIBR], Basel, Switzerland, for his critical comments and valuable suggestions as well as for providing the Gpr4 KO mice. 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Journal of Crohn's and ColitisOxford University Press

Published: Mar 1, 2018

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