Background: Inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are typically associated with a decrease in local pH. Genome-wide association studies (GWAS) revealed a strong genetic impact on IBD, identifying over 200 non-overlapping single-nucleotide polymorphism (SNP) genetic risk loci for IBD. G-protein-coupled receptor (GPCR) T-cell death associated gene 8 (TDAG8 or GPR65) has been identified found to be a genetic risk gene factor for IBD in recent GWASs. TDAG8 belongs a family of proton-sensing GPCRs, which consists of OGR1, TDAG8 and GPR4. Therefore, we aim to investigate the role of TDAG8 in a murine IBD models.
Methods: Chronic colitis was induced in WT and TDAG8-/- mice with 4 cycles of 2% DSS in drinking water for 7 days followed by 10 days of regular drinking water. Colon specimens were obtained for haemalaum and eosin, mRNA, Immunohistochemistry (IHC). Peritoneal macrophages (MΦs) from WT and TDAG8-/- mice were isolated. Cells were treated with pH 6.8 serum free RPMI to activate TDAG8 using pH 7.6 as negative controls. After 24 h RNA was collected and transferred for sequencing.
Results: In the chronic colitis model weight changes, colonoscopy scores, colon lengths and spleen weights, MPO activity and histological scores did not show any statistically significant differences between WT and TDAG8-/-. In DSS challenged mice mRNA expression of IFNγ, TNFα, IL6, iNOS was increased in the TDAG8-/- group. No significant differences for mRNA expression of IL17a, Gata3, Foxp3 and RORc were detected. IHC staining revealed that DSS-treated TDAG8-/- specimens showed increased immunoreactivity of MΦ marker F4/80 compared to WT and water controls. Protein staining of T cell marker CD3 showed no difference between WT and TDAG8-/- mice. Interestingly, mRNA and protein expression of OGR1 were downregulated in TDAG8-/- colon tissue. To further examine the role of TDAG8 in MΦs, we performed RNA-sequencing after pH shift from pH 7.6 to pH 6.8. Pathways in mouse MΦs, mediated by TDAG8, were positively enriched for regulation of lymphocyte and leukocyte activation, apoptosis, M1 regulation. Conversely, pathways in TDAG8-deficient MΦs were upregulated for cytokine production involved in inflammatory response and M2 regulation. Moreover, expression of OGR1 was significantly downregulated in TDAG8-/- MΦs.
Conclusions: Although, TDAG8 does not play an important role in murine chronic colitis model, it seems to be relevant in the inflammatory response of macrophages.