Study On Effect Of IL-22 On Fas Expression In Oligodendrocytes And Foxp3 Expression In T Cells And Signal Pathway In Multiple Sclerosis

Part I:Expression of IL-22 and its relationship with Foxp3 and Ikaros in patients with multiple sclerosisObjective:To investigate the expression level of IL-22,Foxp3 and Ikaros in patients with MS,and the relationships among them.Methods:The peripheral blood serum of MS patients and healthy volunteers were collected.Lymphocyte separating medium Ficoll density gradient centrifugation was used to separate peripheral blood mononuclear cells(PBMC)and CD4+T cells were separated and purified by magnetic cell sorting method.Th22 and Treg in PBMC of MS patients and normal volunteers was analysed by flow cytometry.IL-22 in serum was determined by ELISA.Expression of Foxp3 and Ikaros in PBMC of MS patients were determined by real time PCR.Results:In MS patients,CD4+CCR4+CCR6+CCR10+IL-22+T cells which is Th22 was increased,and the proportion of CD4+CD25+Foxp3+ cells(Treg)was decreased compared with the normal volunteers in PBMC.The serum IL-22 level was increased in MS patients.The expression of Foxp3 and Ikaros was suppressed in PBMC of MS patients.Conclusions:In MS patients,the high expression of IL-22 regulated the expression of Foxp3 in MS.Part II:Study on the expression of IL-22 and Fas in brain of experimental autoimmune encephalomyelitis animal model Objective:To investigate the expression of IL-22,Treg and Fas in experimental autoimmune encephalomyelitis(EAE)mouse model,and the relationship among IL-22,Treg and Fas.Methods:In this study,MOG35-55 combined with pertussis toxin(PTX)was used to immune C57BL/6 mice to prepare EAE mouse model.Monitoring the change of body weight and clinical symptom scoring in mice model identify if EAE model was preparedsuccessfully.Expression of IL-22 in serum of EAE animals and normal control mice was detected by ELISA method.Treg in peripheral cells were analyzed by flow cytometry.Expression level of Fas in mice brain tissue was assayed by real-time quantitative PCR and western blot.Results:The weight of EAE mice was significantly decreased and with neurological dysfunction such as slow action,quadriplegia,even death.The model was prepared successfully.Compared to normal control mice,IL-22 in serum of EAE model was increased,while percentage of Treg was decreased in peripheral blood.It was showed that Fas in brain tissue of EAE animals was enhanced.The expression level of IL-22 was positively correlated with the expression of Fas in brain tissue,but negatively correlated with the proportion of T regulatory cells in peripheral blood.Conclusion:IL-22 may modulate EAE diseaseby regulating of Foxp3 and Fas expression.Part III:Study on effect of IL-22 on Fas expression inoligodendrocytes and Foxp3 expression in T cells andsignal pathway in multiple sclerosisObjective:To investigate the relationship among IL-22,Treg and Fas,and to explore their roles in MS development,further investigate the signal pathway that IL-22 regulated Foxp3 and Fas and the relationships of the three proteins.Methods:the prokaryotic expression plasmid of IL-22 was constructed and the recombinant IL-22 protein was purified.When B104 cells and CG4 cells were co cultured,CG4 cells candifferentiate into oligodendrocytes.Primary oligodendrocytes in mouse embryonic brain tissue were obtained.IL-22 protein was used to treat these two cells.The cells were harvested and intracellular phosphorylation of MSK1(Thr581),MSK1,phosphorylation of NF-?B p65(Ser468),Fas were detected by western blot.Real time quantitative PCR was used to detect the expression of Fas and Ikaros.PBMC were collected from subjects,and expression of p65,PTEN,PI3K,AKT,FoxO1,Ikaros and Foxp3 were determined in order to further verify the effect of IL-22 on Foxp3.Normal CD4+T cells were treated with IL-22 protein in vitro,and the expression levels of p65,PTEN,PI3K,AKT,FoxO1,Ikaros and FOXP3 were detected.To verify the function of Treg,the Treg cells of normal mice and EAE mice were separated by magnetic bead sorting,and the cells were counted,co-cultured with the primary oligodendrocytes pretreated with IL-22,and the expression of Fas was detected after harvesting oligodendrocytes.Results:The Recombinant plasmid PET20b-IL-22(human/mouse)was constructed successfully,and the recombinant IL-22 protein was prepared.IL-22 promotes the expression of p-MSK1,p-P65 and Fas in glial cells.IL-22 can promote the expression of p-P65 and p-AKT in T cells,and inhibit the expression of PTEN,FoxO1 and Ikaros,Foxp3.Cells co-culture experiments showed that Treg cells could reverse Fas expression induced IL-22 in oligodendrocyte.Conclusions:IL-22 regulates the expression of Fas by activating the MSK-NF?B signaling pathway in oligodendrocytes.And IL-22 regulates the expression of Foxp3 by activating the NFr?B-PTEN-AKT signaling pathway in T cells.Treg cells could reverse Fas expression induced IL-22 in oligodendrocyte.Part IV:Effects and mechanisms of artemisinin on microglia in experimental autoimmune encephalomyelitisObjective:To study effect of inflammasome on EAE and function of artemisinin on microglia cells in EAE.Methods:In vitro,artemisinin was used to treat mouse microglia cell line BV-2 cells and IL-1p expression was detected by ELISA.Primary microglia cells were treated with artemisinin and then co-cultured with T cells by magnetic activated cell sorting(MACS),cytokins in T cells and Treg was determined by FCM.In EAE model,the mice were treated with artemisinin and glibenclamide,respectively.Western blot was used to detect NLRP3 expression and FCM was used to determine IL-10 and TNF-a expression and T cells and Treg cells percentage in brain tissue,Results:Artemisinin could effectively inhibit IL-1? in BV-2 cells.The IL-10 and Treg expression in T cells co-cultured with the mouse primary microglia cells were increased.In vivo,compared to control group and Glibenclamide group,IL-10 was increased and TNF-? was decreased in artemisinin group.NLRP3 was declined in artemisinin group.Treg percentage was enhanced in artemisinin group.Conclusion:Artemisinin could significantly decreased inflammatory reaction in EAE and could be a potencial clinical drug to treat MS.