GPR65 Promotes Intestinal Mucosal Th1 And Th17 Cell Differentiation And Gut Inflammation Via Downregulating NUAK2

Inflammatory bowel diseases（IBD）are chronic remittent inflammatory disease that occurs repeatedly in the gastrointestinal tract,including Crohn’s disease（CD）and ulcerative colitis（UC）.The etiopathogenesis is diversity and has not been fully elucidated.It is mainly related to abnormal immune cells responses in intestinal mucosa caused by genetic susceptibility,dietary habits,environmental factors,intestinal flora disorder and persistent intestinal infection.Abnormal responses of large number of immune cells,especially CD4⁺T cells,exist in the inflamed intestinal mucosa of patients with IBD.G protein-coupled receptor 65（GPR65）is a seven-transmembrane protein located in the cell membrane.As a susceptibility gene for IBD,it has been shown to promote the pathogenicity of Th17 cells and induce T cell apoptosis.However,the potential role of GPR65 in the regulation of CD4⁺T cell immune response in the pathogenesis of IBD is not fully understood.Here,we detected the expression level of GPR65 in IBD patients,and the effect of GPR65knockout in mouse CD4⁺T cells on the development of intestinal inflammation.Meanwhile,we investigated the specific regulatory target gene of GPR65,and explored its regulation of Th1 and Th17 cell immune response in intestinal mucosa of IBD.Part 1Aims:To explore the expression of GPR65 in gut mucosa and PB-CD4⁺T cells of IBD patients.Moreover,compare GPR65 expression in inflamed and normal gut mucosa of the same,and that analyze the reasons for the differential expression of GPR65.Methods:The endoscopic biopsies were collected from 87 active CD patients（A-CD）,31 CD patients in remission（R-CD）,77 active UC patients（A-UC）,35 UC patients in remission（R-UC）,and 64 healthy controls（HC）.PB-CD4⁺T cells were collected from 46 A-CD,38 R-CD,50 A-UC,21 R-UC,and 46 HC.The total RNAs were extracted and GPR65 expression was detected by q RT-PCR.The frequency of GPR65⁺cells were detected in PB-CD4⁺T cells by flow cytometric analysis.Various cytokines were co-culture with PB-CD4⁺T cells for 72 hours,and then GPR65expression was determined by flow cytometric analysis.Results:GPR65 expression was increased in inflamed gut mucosa and PB-CD4⁺T cells of active IBD patients,and its expression was positively correlated with disease activity.In addition,TNF-αcould upregulate GPR65 expression in PB-CD4⁺T cells in caspase-3/8 manner.Meanwhile,GPR65 expression was increased in Th1 and Th17cells by contrast with Th0 cells.Conclusions:The increased GPR65 expression in the active IBD patients plays a key role in the etiopathogenesis of diseases.Anti-TNF-αtreatment inhibited GPR65expression in CD4⁺T cells and intestinal mucosa.Part 2Aims:To investigated whether GPR65 influences the differentiation of IBD CD4⁺T cell.Methods:Isolation of PB-CD4⁺T cells from active IBD patients and healthy controls,these cells were transfected with lentivirus expressing GPR65（LV-GPR65）,GPR65sh RNA（LV-SHGPR65）and negative control（LV-NC）,respectively.After 5 days,the transfected cells were collected for q RT-PCR and flow cytometric analysis,and the supernatants were obtained for ELISA.Results:LV-GPR65 transfected CD4⁺T cells showed higher expression of IL-17A,RORC,IFN-γ,TBET,and TNF-α.Neither Foxp3,IL-10,IL-4 nor GATA3 expression was influenced in LV-GPR65 or LV-sh GPR65 transfected CD4⁺T cells.Conclusions:Overexpression of GPR65 promotes Th1 and Th17 cell differentiation,but does not affect Th2 and Treg cells.Part 3Aims:To investigate the effect of GPR65 deficiency in CD4⁺T cell in mice intestinal mucosal inflammation.Methods:Gpr65^ΔCD4 and Gpr65^flx/flx mice were constructed by CRISPR/Cas9technique.GPR65^ΔCD4 and Gpr65^flx/flx mice induced acute colitis by TNBS rectally and chronic colitis in Rag1^-/-mice reconstituted with CD45RB^highCD4⁺T cells.The mice colon tissues were collected for flow cytometric analysis,and RNA analysis,respectively.Results:IL-17A-,IFN-γ-and TNF-α-expressing CD4⁺T cells were decreased in LPMCs of Gpr65^ΔCD4 mice and Rag1^-/-mice reconstituted with Gpr65^ΔCD4CD45RB^highCD4⁺T cells by flow cytometric analysis.Consistently,the m RNA level of Ifn-γ,Tbx21,Il-17a,Rorc and Tnf-αwas also reduced in the colonic tissues of TNBS-induced colitic Gpr65^ΔCD4 mice and Rag1^-/-mice reconstituted with Gpr65^ΔCD4CD45RB^highCD4⁺T cells.Conclusions:GPR65 deficiency in CD4⁺T cells suppresses mice intestinal mucosal inflammation by inhibiting Th1 and Th17 cell immune response.Part 4Aims:To investigate the target gene of GPR65 and its expression in IBD patients.Methods:The splenic CD4⁺T cells were isolated by flow cytometry from Gpr65^ΔCD4 and Gpr65^flx/flx mice,and that analyzed by RNA sequencing.The endoscopic biopsies were collected from 44 A-CD,24 R-CD,43 A-UC,19 R-UC,and28 HC.PB-CD4⁺T cells were collected from 17 A-CD,12 R-CD,25 A-UC,11 R-UC,and 22 HC.The total RNAs were extracted and GPR65 expression was detected by q RT-PCR.Results:RNA sequencing found the expression of some proinflammatory genes was decreased in Gpr65^ΔCD4 CD4⁺T cells.Nuak2 expression was increased in Gpr65^ΔCD4 CD4⁺T cells and negatively correlated with Gpr65 expression.Additionally,NUAK2 expression was upregulated in LV-sh GPR65 transfected CD4⁺T cells and decreased in Th1,Th17,and Tregs.Meanwhile,the expression of NUAK2was decreased in inflamed gut mucosa and PB-CD4⁺T cells of active IBD patients.Anti-TNF-αtreatment promoted NUAK2 expression in intestinal mucosa.Conclusions:GPR65 inhibits the expression of NUAK2,which is involved in Th1and Th17 cellular immune responses.Part 5Aims:To probe into the mechanism of GPR65 regulating NUAK2 expression.Methods:PB-CD4⁺T cells were collected from 16 A-CD,10 A-UC,and 14 HC.The protein levels of PKA-C-Raf-ERK1/2-LKB1-NUAK2 were detected in splenic CD4⁺T cells of Gpr65^ΔCD4 and Gpr65^flx/flx mice by western blot.c AMP production was assessed by ELISA.Splenic CD4⁺T cells of Gpr65^ΔCD4 and Gpr65^flx/flx mice were transfected with LV-sh Nuak2,the total RNAs were extracted and related gene expression was detected by q RT-PCR.Results:C-Raf,ERK1/2,and LKB1 m RNA level was decreased in PB-CD4⁺T cells of active IBD patients.In the splenic CD4⁺T cells of Gpr65^ΔCD4 mice,the production of c AMP and PKA was decreased,but the protein levels of C-Raf,P-C-Raf,ERK1/2,ERK1/2,LKB1,and NUAK2 were increased.In addition,the expression of Tbx21,Rorc,Ifn-γ,Tnf-α（11）and Il-17a was upregulated in LV-sh Nuak2 transfected CD4⁺T cells.Conclusions:GPR65 deletion suppresses Th1 and Th17 cell immune response through the C-Raf-EKR1/2-LKB1-NUAK2 signaling pathway.SummaryOur data suggest that GPR65 expression is increased in inflamed gut tissues and PB-CD4⁺T cells of active IBD patients.TNF-αupregulates GPR65 expression in a caspase-3/8-dependent manner,and that anti-TNF-αtreatment suppresses GPR65expression in CD4⁺T cells and intestinal mucosa.Overexpression of GPR65 promote the differentiation of Th1 and Th17 cells,and deletion of GPR65 on CD4⁺T cells can alleviate the occurrence of acute and chronic colitis in mice by inhibiting the Th1 and Th17 cells immune responses.Additionally,GPR65 deficiency promote CD4⁺T cells to express NUAK2 via the c AMP-PKA-C-Raf-ERK1/2-LKB1 pathway,and restrict Th1 and Th17 cell differentiation and intestinal mucosal inflammation.