Th17 Differentiation Are Regulated By S1P-S1P1 Signal In CD4SP Thymocytes Of EAMG Rat

Background and objectiveAs one of the most widely expressed receptors in the G protein-coupled receptor superfamily,Sphingosine-1-phosphate receptor 1(S1P1)is highly expressed in immune organs,lungs,brain and lowly expressed in the kidney in adult.Immunohistochemical staining of mouse thymus with anti-S1P1 antibody revealed that S1P1 is mainly expressed on the surface of thymocytesin the medulla region of the thymus.Thymocytes in the medulla region of the thymus are mainly single positive cells,Michael A et al.thought that the egress of single positive T cells from the thymus was mediated by S1P1 which can specifically recognize and bind S1 P.The expression of S1P1 is regulated by the transcription factor Forkhead Box transcription O1(FoxO1).In mammals,FoxO1 is highly expressed on mature T and B lymphocytes and can regulate differentiation and function of T cells.In the early stages of single positive development of thymocytes,elevated FOXO1 causes expression of S1P1 until thymocytes mature and migrate out of the thymus.The the RNA and protein levels of FoxO1 and S1P1 in the thymus of Myasthenia gravis(MG)patients were higher than the normal control group in the previous study of the MG,suggesting that FoxO1 may be related to the occurrence and development of MG through regulating S1P1 expression.The homeostasis of mature thymocytesbetweenintrathymusandperipheral T cell poolscan maintain immune function.In certain pathological and autoimmune,homeostasis is disrupted by S1P/S1P1-mediated abnormal thymocyte output.Myasthenia gravis is an acetylcholine receptor(AChR)autoantibody-mediated and T-cell dependent autoimmune disease.Thymus dysfunction and development of MG has close ralation.After partial MG patients were treated with thymusectomy,the symptoms were significantly reduced.By detecting S1P1 mRNA levels in thymus tissue of MG patients and normal control groups,it was found that S1P1 mRNA level in MG thymus tissue was significantly increased.Immunohistochemical results of thymus tissue showed that S1P1 protein expression level was significantly higher in MG thymus than in normal control group.S1P1 not only mediates the output of mature thymocytes,but also can affect the differentiation and maturation of thymocytes through G protein-mediated signaling pathways in the studies of functions of S1P/S1P1 in the non-migration of T cells.In S1P1 transgenic mice,high levels of activated transcription factors such as c-maf,JunB,and Gata3 lead to higher expression of IL-4 and lower expression of IFN-γwhich promotes the differentiation of naive T cells into Th2 cells,high levels of activated transcription factors can also induce Th17 production by IL-6,and high levels of activated transcription factors negatively regulate the differentiation and development of Treg cells in the thymus by activating the Akt-mTOR pathway.This study was based on the EAMG rat model and aimed to study the relationship between S1P1 and FoxO1 and the effect of S1P1 on Th17 differentiation and the expression of IL-17 and other cytokines in CD4+CD8-SP thymocytes.Methods1.EAMG rat : 6-8 weeks female Lewis rats were immunized 3 times with multi-site injections of AchR polypeptide subcutaneously(dorsal skin and lower limb footpads).Blood serum was taken from the eyelids and anti-AchR antibodies were measured by ELISA,and anti-AchR-positive rats were selected for experimentatio2.The thymocytes and lymph node T cells of EAMG rats were isolated,and the number of thymocytes subsets and T lymphocyte subsets and the positive rate of S1P1 and FoxO1 of these subsets were detected by flow cytometry;the expression of S1P1 and FoxO1 were analyzed by immunohistochemical staining in thymus andlymph node.3.Dendritic cells were induced from bone marrow cells isolated from rat bone marrow with IL-4 and GM-CSF;CD4+CD8-thymocytes without Treg(CD4+CD8-CD25+CD127+/-)were sorted by flow cytometry and after co-cultured with induced Dendritic cellsfor 7 days,S1 P or FTY720 was added for 6 hours,and the expression of cytokines was detected by flow cytometry.4.Jurkat cells were infected with construct FoxO1 expression lentivirus and interfere lentivirusfor 24 hours,the effects of FoxO1 on S1P1 expression was detected by fluorescence quantitative PCR,WB and flow cytometry.Results1.After Lewis rats were immunized 3 times with AchR polypeptides,the antibody anti-AchR titers in the EAMG group were significantly higher than those in the control group(P<0.05),accompaningwith a decrease in weight and a increase in functional scores,which demonstratedthe establishment of an EAMG rat model.2.In the thymus of EAMG rats,CD4-CD8-cells,CD4+CD8+cells and SP cells(CD4+CD8-cells,CD4-CD8+ cells)were no significant change,P>0.05;in CD4+CD8-SP thymocytes,Treg cells(CD4+CD8-CD25+CD127+/-thymocytes)were not significantly altered(P>0.05),Th17 cells(CD4+CD8-IL-17+ thymocytes)significantly increased(P<0.05).S1P1+ CD4-CD8-thymocytes and S1P1+CD4+CD8-thymocytes increased(P < 0.05),S1P1+CD4+CD8+thymocytes and S1P1+CD4-CD8+ thymocytes did not change significantly(S1P1+CD4+CD8+thymocytes did not change significantly(P>0.05);in CD4+CD8-SP thymocytes,there were no significant changes in S1P1+Treg cells and S1P1+Th17cells(P>0.05).FoxO1+CD4-CD8-thymocytes and FoxO1+CD4+CD8-thymocytes increased(P<0.05).In EAMG rat lymph nodes,there was no significant change in CD4+ T cells and CD8+ T cells,the two subpopulations expressed S1P1 and FoxO1;in CD4+ T cells,Treg cells decreased(P<0.05),and Th17 cells had no significant changes.Immunohistochemical staining of thymus and lymph nodes in model rats also demonstrated that S1P1 is mainly expressed in the medulla of EAMG rats.3.After CD7+CD8-thymocytes were co-cultured with dendritic cells for 7 days,IL-17 and IL-22 were significantly increased after S1 P or stimulation for 6 h(P<0.05).4.After Jurkat cells were infected with FoxO1-lentiviruses for 120 h,in FoxO1-overexpression group,the mRNA expression levels and the proteins expression levels of FoxO1,KLF2,S1P1,and CD62 L were significantly increased(P<0.05),the proteins expression levels of FoxO1,FoxO1-p,and KLF2 were increased(P<0.05),the S1P1+ cells and CD62L+ cells ratios were increased(P<0.05),there was no significant change in CCR7+ cells and CD69+ cells(P>0.05);in FoxO1-interference group,the mRNA expression levels and the proteins expression levels of FoxO1,KLF2,S1P1,and CD62 L were significantly decreased(P<0.05),the proteins expression levels of FoxO1,FoxO1-p,and KLF2 were decreased(P<0.05),the S1P1+ cells ratios were increased(P<0.05),but the S1P1+ cells ratios were decreased(P<0.05)at 72 h.ConclusionIn EAMG rat CD4+SP thymocytes,S1P1 can promote the differentiation of Th17 cells and the expression of IL-17 and other inflammatory cytokines by binding to its ligand S1P;the expression of S1P1 are regulated by FoxO1.