异基因造血干细胞移植术后CD4~+T细胞中缺乏SIRT1诱发急性移植物抗宿主病
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摘要
目的:研究异基因造血干细胞移植患者外周血CD4~+T细胞中SIRT1基因表达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40例行同胞全相合异基因造血干细胞移植患者的血液样本,采用实时定量PCR和Western印迹检测各组患者SIRT1的表达水平;采用Western印迹检测各组患者STAT3乙酰化及磷酸化水平;采用免疫共沉淀和Western印迹检测各组患者SIRT1和STAT3结合水平;a GVHD患者CD4~+T细胞过表达SIRT1后采用Western印迹检测STAT3乙酰化及磷酸化水平,采用实时定量PCR检测Th17相关基因RORγt,IL-17A,IL-17F的表达。结果:与未发生a GVHD患者比较,a GVHD患者外周血CD4~+T细胞中SIRT1表达水平明显降低;STAT3表达水平、乙酰化及磷酸化水平均明显升高,且STAT3乙酰化与磷酸化水平呈明显正相关(r=0.69,P<0.01);STAT3与SIRT1结合显著减少。a GVHD患者外周血CD4~+T细胞中过表达SIRT1后,STAT3乙酰化及磷酸化水平均明显降低,RORγt,IL-17A,IL-17F的表达明显减少(P<0.05)。结论:CD4~+T细胞中SIRT1表达不足是异基因造血干细胞移植术后患者STAT3高度乙酰化及磷酸化,介导Th17的相关基因表达升高,进而诱发a GVHD的重要因素。
Objective: To study the relationship between acute graft-versus-host disease(aG VHD) and the SIRT1 expression in peripheral blood CD4~+T cells from patients after allogeneic hematopoieticstem cell transplantation(allo-HSCT).Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen(HLA)-identical sibling donors. SIRT1 expression level in CD4~+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4~+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4~+T cells was analyzed by coimmunoprecipitation and Western blot. Over-expression of SIRT1 in aG VHD CD4~+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The m RNA levels of RORγt, IL-17 A, IL-17 F related to Th17 were detected by real-time PCR.Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with a GVHD compared with patients without a GVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation(r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4~+T cells from patients with aG VHD compared with patients without aG VHD. After SIRT1 over-expression in aG VHD CD4~+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17 A, and IL-17 F related to Th17 were significantly down-regulated(P<0.05).Conclusion: SIRT1 deficiency in CD4~+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aG VHD after allogeneic hematopoietic stem cell transplantation.
Objective: To study the relationship between acute graft-versus-host disease(aG VHD) and the SIRT1 expression in peripheral blood CD4~+T cells from patients after allogeneic hematopoieticstem cell transplantation(allo-HSCT).Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen(HLA)-identical sibling donors. SIRT1 expression level in CD4~+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4~+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4~+T cells was analyzed by coimmunoprecipitation and Western blot. Over-expression of SIRT1 in aG VHD CD4~+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The m RNA levels of RORγt, IL-17 A, IL-17 F related to Th17 were detected by real-time PCR.Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with a GVHD compared with patients without a GVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation(r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4~+T cells from patients with aG VHD compared with patients without aG VHD. After SIRT1 over-expression in aG VHD CD4~+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17 A, and IL-17 F related to Th17 were significantly down-regulated(P<0.05).Conclusion: SIRT1 deficiency in CD4~+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aG VHD after allogeneic hematopoietic stem cell transplantation.
引文
[1]Kong S,Yeung P,Fang D.The class III histone deacetylase sirtuin 1in immune suppression and its therapeutic potential in rheumatoid arthritis[J].J Genet Genomics,2013,40(7):347-354.
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[10]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.
[11]Noack M,Miossec P.Th17 and regulator y T cell balance in autoimmune and inflammatory diseases[J].Autoimmun Rev,2014,13(6):668-677.
[12]Nishihara M,Ogura H,Ueda N,et al.IL-6-gp130-STAT3 in T cells directs the development of IL-17+Th with a minimum effect on that of Treg in the steady state[J].Int Immunol,2007,19(6):695-702.
[13]Xu Y,Li L,Chen Y,et al.Role of HMGB1 in regulation of STAT3expression in CD4(+)T cells from patients with a GVHD after allogeneic hematopoietic stem cell transplantation[J].Clin Immunol,2015,161(2):278-283.
[14]Qin H,Kim HJ,Kim JY,et al.Activation of signal transducer and activator of transcription 3 through a phosphomimetic serine 727promotes prostate tumorigenesis independent of tyrosine 705phosphorylation[J].Cancer Res,2008,68(19):7736-7741.
[15]Betts BC,Sagatys EM,Veerapathran A,et al.CD4+T cell STAT3phosphorylation precedes acute GVHD,and subsequent Th17 tissue invasion correlates with GVHD severity and therapeutic response[J].J Leukoc Biol,2015,97(4):807-819.
[16]Yuan ZL,Guan YJ,Chatterjee D,et al.Stat3 dimerization regulated by reversible acetylation of a single lysine residue[J].Science,2005,307(5707):269-273.
[17]Qi Q,Yang Z.Regulation and function of signal transducer and activator of transcription 3[J].World J Biol Chem,2014,5(2):231-239.
[18]Nie Y,Erion DM,Yuan Z,et al.STAT3 inhibition of gluconeogenesis is downregulated by Sir T1[J].Nat Cell Biol,2009,11(4):492-500.
[19]Gao R,Chen J,Hu Y,et al.Sirt1 deletion leads to enhanced inflammation and aggravates endotoxin-induced acute kidney injury[J].PLo S One,2014,9(6):e98909.
[20]Sestito R,Madonna S,Scarponi C,et al.STAT3-dependent effects of IL-22 in human keratinocytes are counterregulated by sirtuin 1through a direct inhibition of STAT3 acetylation[J].FASEB J,2011,25(3):916-927.
[21]Scuto A,Kirschbaum M,Buettner R,et al.SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45Gby repressing the binding of NF-kappa B/STAT3 complex to its promoter in malignant lymphoid cells[J].Cell Death Dis,2013,4:e635.
[22]Sethi G,Chatterjee S,Rajendran P,et al.Inhibition of STAT3dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo[J].Mol Cancer,2014,13:66.
[23]徐雅靖,张媛媛,陈焱,等.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发生的关系[J].中南大学学报(医学版),2017,42(8):911-918.XU Yajing,ZHANG Yuanyuan,CHEN Yan,et al.Relationship between the methylation status of STAT3 promoter DNA in peripheral blood CD4+T cells from patients after allo-HSCT and a GVHD[J].Journal of Central South University.Medical Science,2017,42(8):911-918.
[24]Zhao M,Liu S,Luo S,et al.DNA methylation and m RNA and micro RNA expression of SLE CD4+T cells correlate with disease phenotype[J].J Autoimmun,2014,54:127-136.
[25]Xie Z,Chang C,Zhou Z.Molecular mechanisms in autoimmune type 1 diabetes:a critical review[J].Clin Rev Allergy Immunol,2014,47(2):174-192.
[26]Elmali N,Baysal O,Harma A,et al.Effects of resveratrol in inflammatory arthritis[J].Inflammation,2007,30(1/2):1-6.
[27]Lee SM,Yang H,Tartar DM,et al.Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1diabetes[J].Diabetologia,2011,54(5):1136-1146.
[28]Tian J,Chen J,Gao J,et al.Resveratrol inhibits TNF-alpha-induced IL-1beta,MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of PI3kinase/Akt pathway[J].Rheumatol Int,2013,33(7):1829-1835.
[2]Yang H,Bi Y,Xue L,et al.Multifaceted modulation of SIRT1 in cancer and inflammation[J].Crit Rev Oncog,2015,20(1/2):49-64.
[3]Liu G,Bi Y,Shen B,et al.SIRT1 limits the function and fate of myeloid-derived suppressor cells in tumors by orchestrating HIF-1alpha-dependent glycolysis[J].Cancer Res,2014,74(3):727-737.
[4]Zhang J,Lee SM,Shannon S,et al.The type III histone deacetylase Sirt1 is essential for maintenance of T cell tolerance in mice[J].JClin Invest,2009,119(10):3048-3058.
[5]Gao B,Kong Q,Kemp K,et al.Analysis of sirtuin 1 expression reveals a molecular explanation of IL-2-mediated reversal of T-cell tolerance[J].Proc Natl Acad Sci USA,2012,109(3):899-904.
[6]Kong S,Kim SJ,Sandal B,et al.The type III histone deacetylase Sirt1protein suppresses p300-mediated histone H3 lysine 56 acetylation at Bclaf1 promoter to inhibit T cell activation[J].J Biol Chem,2011,286(19):16967-16975.
[7]Sequeira J,Boily G,Bazinet S,et al.sirt1-null mice develop an autoimmune-like condition[J].Exp Cell Res,2008,314(16):3069-3074.
[8]Zou T,Yang Y,Xia F,et al.Resveratrol inhibits CD4+T cell activation by enhancing the expression and activity of Sirt1[J].PLo S One,2013,8(9):e75139.
[9]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):1365-1374.
[10]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.
[11]Noack M,Miossec P.Th17 and regulator y T cell balance in autoimmune and inflammatory diseases[J].Autoimmun Rev,2014,13(6):668-677.
[12]Nishihara M,Ogura H,Ueda N,et al.IL-6-gp130-STAT3 in T cells directs the development of IL-17+Th with a minimum effect on that of Treg in the steady state[J].Int Immunol,2007,19(6):695-702.
[13]Xu Y,Li L,Chen Y,et al.Role of HMGB1 in regulation of STAT3expression in CD4(+)T cells from patients with a GVHD after allogeneic hematopoietic stem cell transplantation[J].Clin Immunol,2015,161(2):278-283.
[14]Qin H,Kim HJ,Kim JY,et al.Activation of signal transducer and activator of transcription 3 through a phosphomimetic serine 727promotes prostate tumorigenesis independent of tyrosine 705phosphorylation[J].Cancer Res,2008,68(19):7736-7741.
[15]Betts BC,Sagatys EM,Veerapathran A,et al.CD4+T cell STAT3phosphorylation precedes acute GVHD,and subsequent Th17 tissue invasion correlates with GVHD severity and therapeutic response[J].J Leukoc Biol,2015,97(4):807-819.
[16]Yuan ZL,Guan YJ,Chatterjee D,et al.Stat3 dimerization regulated by reversible acetylation of a single lysine residue[J].Science,2005,307(5707):269-273.
[17]Qi Q,Yang Z.Regulation and function of signal transducer and activator of transcription 3[J].World J Biol Chem,2014,5(2):231-239.
[18]Nie Y,Erion DM,Yuan Z,et al.STAT3 inhibition of gluconeogenesis is downregulated by Sir T1[J].Nat Cell Biol,2009,11(4):492-500.
[19]Gao R,Chen J,Hu Y,et al.Sirt1 deletion leads to enhanced inflammation and aggravates endotoxin-induced acute kidney injury[J].PLo S One,2014,9(6):e98909.
[20]Sestito R,Madonna S,Scarponi C,et al.STAT3-dependent effects of IL-22 in human keratinocytes are counterregulated by sirtuin 1through a direct inhibition of STAT3 acetylation[J].FASEB J,2011,25(3):916-927.
[21]Scuto A,Kirschbaum M,Buettner R,et al.SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45Gby repressing the binding of NF-kappa B/STAT3 complex to its promoter in malignant lymphoid cells[J].Cell Death Dis,2013,4:e635.
[22]Sethi G,Chatterjee S,Rajendran P,et al.Inhibition of STAT3dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo[J].Mol Cancer,2014,13:66.
[23]徐雅靖,张媛媛,陈焱,等.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发生的关系[J].中南大学学报(医学版),2017,42(8):911-918.XU Yajing,ZHANG Yuanyuan,CHEN Yan,et al.Relationship between the methylation status of STAT3 promoter DNA in peripheral blood CD4+T cells from patients after allo-HSCT and a GVHD[J].Journal of Central South University.Medical Science,2017,42(8):911-918.
[24]Zhao M,Liu S,Luo S,et al.DNA methylation and m RNA and micro RNA expression of SLE CD4+T cells correlate with disease phenotype[J].J Autoimmun,2014,54:127-136.
[25]Xie Z,Chang C,Zhou Z.Molecular mechanisms in autoimmune type 1 diabetes:a critical review[J].Clin Rev Allergy Immunol,2014,47(2):174-192.
[26]Elmali N,Baysal O,Harma A,et al.Effects of resveratrol in inflammatory arthritis[J].Inflammation,2007,30(1/2):1-6.
[27]Lee SM,Yang H,Tartar DM,et al.Prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type 1diabetes[J].Diabetologia,2011,54(5):1136-1146.
[28]Tian J,Chen J,Gao J,et al.Resveratrol inhibits TNF-alpha-induced IL-1beta,MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of PI3kinase/Akt pathway[J].Rheumatol Int,2013,33(7):1829-1835.