异基因造血干细胞移植患者外周血CD4~+T细胞中STAT3启动子区DNA甲基化水平与急性移植物抗宿主病的关系
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摘要
目的:探讨异基因造血干细胞移植患者外周血CD4~+T细胞中STAT3启动子区DNA甲基化水平与急性移植物抗宿主病(acute graft versus host disease,a GVHD)的关系。方法:收集行同胞全相合异基因造血干细胞移植的40例患者的血液样本,ELISA检测各组患者血清IL-10,TGF-β1,IL-17A,IL-17F等细胞因子水平;实时定量PCR检测各组患者外周血CD4+T细胞中Treg(Foxp3,CTLA4,IL-10,TGF-β1)和Th 17(RORγt,IL-17A,IL-17F)相关基因的转录水平;实时定量PCR和Western印迹检测各组患者STAT3的表达水平;亚硫酸氢盐处理后测序(bisulfite sequencing PCR,BSP)法检测各组患者STAT3基因启动子区DNA甲基化水平。结果:与未发生a GVHD患者比较,a GVHD患者血清中IL-10及TGF-β1水平明显降低,IL-17A及IL-17F水平明显升高;a GVHD患者外周血CD4+T细胞中Foxp3,CTLA4,IL-10,TGF-β1转录水平明显降低,RORγt,IL-17A,IL-17F转录水平明显升高;a GVHD患者外周血CD4+T细胞中STAT3的表达水平明显升高,STAT3启动子区DNA甲基化水平明显降低,且STAT3表达水平与其启动子区DNA甲基化水平呈明显负相关。结论:Treg/Th17的比例失衡是异基因造血干细胞移植后患者发生a GVHD的重要因素,STAT3启动子区DNA低甲基化可能介导STAT3的过度表达,参与Treg/Th 17的比例失衡。
Objective: To study the relationship between acute graft versus host disease(a GVHD) and the methylation status of the STAT3 promoter in peripheral blood CD4~+ T cells from patients after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods: We collected 40 patients who underwent allo-HSCT from HLA-identical sibling donors. Serum IL-10, TGF-β1, IL-17 A and IL-17 F levels were detected by ELISA. Foxp3 cytotoxic T-lymphocyte-associated protein 4(CTLA4), IL-10, TGF-β1, RORγt, IL-17 A and IL-17 F m RNA levels in CD4~+ T cells were measured by real-time PCR. STAT3 expression levels were detected by real-time PCR and Western blot, and promoter DNA methylation was analyzed by bisulfite sequencing PCR(BSP). Results: IL-10 and TGF-β1 levels were significantly down-regulated, while IL-17 A and IL-17 F levels were significantly up-regulated in patients with a GVHD compared with patients without a GVHD. Foxp3, CTLA4, IL-10, TGF-β1 m RNA levels were significantly down-regulated, while RORγt, IL-17 A, IL-17 F m RNA levels were significantly up-regulated in patients with a GVHD compared with patients without a GVHD. STAT3 expression was increased, while STAT3 promoter DNA was hypomethylated in patients with a GVHD compared with those without a GVHD. The STAT3 m RNA level was negatively correlated with STAT3 promoter DNA methylation. Conclusion: The imbalance of Treg/Th17 in CD4~+ T cells from patients after allo-HSCT is a key factor for triggering a GVHD, and the DNA hypomethylation of STAT3 promoter could promote its expression in CD4~+ T cells and contribute to the imbalance.
Objective: To study the relationship between acute graft versus host disease(a GVHD) and the methylation status of the STAT3 promoter in peripheral blood CD4~+ T cells from patients after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods: We collected 40 patients who underwent allo-HSCT from HLA-identical sibling donors. Serum IL-10, TGF-β1, IL-17 A and IL-17 F levels were detected by ELISA. Foxp3 cytotoxic T-lymphocyte-associated protein 4(CTLA4), IL-10, TGF-β1, RORγt, IL-17 A and IL-17 F m RNA levels in CD4~+ T cells were measured by real-time PCR. STAT3 expression levels were detected by real-time PCR and Western blot, and promoter DNA methylation was analyzed by bisulfite sequencing PCR(BSP). Results: IL-10 and TGF-β1 levels were significantly down-regulated, while IL-17 A and IL-17 F levels were significantly up-regulated in patients with a GVHD compared with patients without a GVHD. Foxp3, CTLA4, IL-10, TGF-β1 m RNA levels were significantly down-regulated, while RORγt, IL-17 A, IL-17 F m RNA levels were significantly up-regulated in patients with a GVHD compared with patients without a GVHD. STAT3 expression was increased, while STAT3 promoter DNA was hypomethylated in patients with a GVHD compared with those without a GVHD. The STAT3 m RNA level was negatively correlated with STAT3 promoter DNA methylation. Conclusion: The imbalance of Treg/Th17 in CD4~+ T cells from patients after allo-HSCT is a key factor for triggering a GVHD, and the DNA hypomethylation of STAT3 promoter could promote its expression in CD4~+ T cells and contribute to the imbalance.
引文
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[16]Ogawa C,Tone Y,Tsuda M,et al.TGF-β-mediated Foxp3 gene expression is cooperatively regulated by Stat5,Creb,and AP-1through CNS[J].J Immunol,2014,192(1):475.
[17]Solt L A,Kumar N,Nuhant P,et al.Su ppression of TH17differentiation and autoimmunity by a synthetic ROR ligand[J].Nature,2011,472(7344):491-494.
[18]Noack M,Miossec P.Th17 and regulator y T cell balance in autoimmune and inflammatory diseases[J].Autoimmun Rev,2014,13(6):668-677.
[19]Korn T,Bettelli E,Oukka M,et al.IL-17 and Th17 cells[J].Annu Rev Immunol,2009,27:485-517.
[20]Miossec P,Kolls JK.Targeting IL-17 and TH17 cells in chronic inflammation[J].Nat Rev Drug Discov,2012,11(10):763.
[21]Chen Z,Laurence A,Kanno Y,et al.Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells[J].Proc Natl Acad Sci USA,2006,103(21):8137-8142.
[22]Yang XO,Panopoulos AD,Nurieva R,et al.STAT3 regulates cytokine-mediated generation of inflammatory helper T cell[J].J Biol Chem,2007,282(13):9358-9363.
[23]Chen Z,Laurence A,O'Shea JJ.Signal transduction pathways and transcriptional regulation in the control of Th17 differentiation[J].Semin Immunol,2007,19(6):400-408.
[24]Fujino M,Li XK.Role of STAT3 in regulator y T lymphocyte plasticity during acute graft-vs.-host-disease[J].JAKSTAT,2013,2(4):e24529.
[25]Lal G,Zhang N,Van d TW,et al.Epigenetic regulation of Foxp3expression in regulatory T cells by DNA methylation[J].J Immunol,2009,182(1):259.
[2]Murata M,Nakasone H,Kanda J,et al.Clinical factors predicting the response of acute graft-versus-host disease to corticosteroid therapy:an analysis from the GVHD Working Group of the Japan Society for Hematopoietic Cell Transplantation[J].Biol Blood Marrow Transplant,2013,19(8):1183-1189.
[3]Carlson MJ,West ML,Coghill JM,et al.In vitro-differentiated TH17 cells mediate lethal acute graft-versus-host disease with severe cutaneous and pulmonary pathologic manifestations[J].Blood,2009,113(6):1365.
[4]Hoffmann P,Ermann J,Edinger M,et al.Donor-type CD4+CD25+regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation[J].J Exp Med,2002,196(3):389-399.
[5]Liu Y,Cai Y,Dai L,et al.The expression of Th17-associated cytokines in human acute graft-versus-host disease[J].Biol Blood Marrow Transplant,2013,19(10):1421-1429.
[6]Fujioka T,Tamaki H,Ikegame K,et al.Frequency of CD4+FOXP3+regulatory T-cells at early stages after HLA-mismatched allogeneic hematopoietic SCT predicts the incidence of acute GVHD[J].Bone Marrow Transplant,2013,48(6):859-864.
[7]Kane A,Deenick EK,Ma CS,et al.STAT3 is a central regulator of lymphocyte differentiation and function[J].Curr Opin Immunol,2014,28:49-57.
[8]Mathur AN,Chang HC,Zisoulis DG,et al.STAT3 and Stat4 direct development of IL-17-secreting Th cells[J].J Immunol,2007,178(8):4901-4907.
[9]Laurence A,Amarnath S,Mariotti J,et al.STAT3 transcription factor promotes instability of n Treg cells and limits generation of i Treg cells during acute murine graft-versus-host disease[J].Immunity,2012,37(2):209-222.
[10]Annalisa V.DNA methylation,epigenetics,and evolution in vertebrates:facts and challenges[J].Int J Evolut Biol,2014,2014(6):475981.
[11]Richardson BC,Patel DR.Epigenetics in 2013.DNA methylation and mi RNA:key roles in systemic autoimmunity[J].Nat Rev Rheumatol,2014,10(2):72.
[12]Richardson BC.Role of DNA methylation in the regulation of cell function:autoimmunity,aging and cancer[J].J Lab Clin Med,2002,132(8 Suppl):2401S.
[13]Wu Y,Borde M,Heissmeyer V,et al.FOXP3 controls regulatory T cell function through cooperation with NFAT[J].Cell,2006,126(2):375.
[14]Chen Z,Lin F,Gao Y,et al.FOXP3 and RORγt:Transcriptional regulation of Treg and Th17[J].Int Immuno Pharmacol,2011,11(5):536-542.
[15]Qureshi OS,Zheng Y,Nakamura K,et al.Trans-endocytosis of CD80 and CD86:a molecular basis for the cell-extrinsic function of CTLA-4[J].Science,2011,332(6029):600-603.
[16]Ogawa C,Tone Y,Tsuda M,et al.TGF-β-mediated Foxp3 gene expression is cooperatively regulated by Stat5,Creb,and AP-1through CNS[J].J Immunol,2014,192(1):475.
[17]Solt L A,Kumar N,Nuhant P,et al.Su ppression of TH17differentiation and autoimmunity by a synthetic ROR ligand[J].Nature,2011,472(7344):491-494.
[18]Noack M,Miossec P.Th17 and regulator y T cell balance in autoimmune and inflammatory diseases[J].Autoimmun Rev,2014,13(6):668-677.
[19]Korn T,Bettelli E,Oukka M,et al.IL-17 and Th17 cells[J].Annu Rev Immunol,2009,27:485-517.
[20]Miossec P,Kolls JK.Targeting IL-17 and TH17 cells in chronic inflammation[J].Nat Rev Drug Discov,2012,11(10):763.
[21]Chen Z,Laurence A,Kanno Y,et al.Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells[J].Proc Natl Acad Sci USA,2006,103(21):8137-8142.
[22]Yang XO,Panopoulos AD,Nurieva R,et al.STAT3 regulates cytokine-mediated generation of inflammatory helper T cell[J].J Biol Chem,2007,282(13):9358-9363.
[23]Chen Z,Laurence A,O'Shea JJ.Signal transduction pathways and transcriptional regulation in the control of Th17 differentiation[J].Semin Immunol,2007,19(6):400-408.
[24]Fujino M,Li XK.Role of STAT3 in regulator y T lymphocyte plasticity during acute graft-vs.-host-disease[J].JAKSTAT,2013,2(4):e24529.
[25]Lal G,Zhang N,Van d TW,et al.Epigenetic regulation of Foxp3expression in regulatory T cells by DNA methylation[J].J Immunol,2009,182(1):259.