TRAF-6调控TGF-β1-Smad2/Smad3信号通路在Graves病免疫发病机制中的作用
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摘要
目的:探讨肿瘤坏死因子受体相关因子-6(TRAF-6)是否在Graves病(GD)的发生过程中发挥免疫耐受作用及其可能的机制。方法:纳入Graves病初诊患者(GD) 30例,GD缓解期患者(e GD) 30例及健康人群(NC) 28例。提取外周血单个核细胞(PBMCs),分别采用实时定量PCR检测维甲酸相关孤儿受体(RORγt)、叉头蛋白P3(Foxp3)、白介素-2(IL-2)、TRAF-6 mRNA的表达,Western blot检测p-Smad2/Smad3、总Smad2/Smad3、TRAF-6蛋白的表达,ELISA检测血浆中转化生长因子-β1(TGF-β1)、IL-2、IL-17A、IL-10蛋白的表达。结果:①与NC组相比,GD组中IL-2的mRNA和蛋白、Foxp3 mRNA和IL-10蛋白的表达水平均减少,而RORγt mRNA和IL-17A蛋白的表达水平均增加,以上结果差异均有统计学意义(P<0. 05)。②与NC组相比,GD组中TGF-β1蛋白的表达水平降低,差异有统计学意义(P<0. 05); GD组中p-Smad2/Smad3的蛋白表达较NC组有上升趋势(P>0. 05)。③GD组中的TRAF-6 mRNA与NC组比较表达降低,差异有统计学意义(P<0. 05); TRAF-6蛋白表达较NC组有下降趋势(P>0. 05)。④e GD组中TRAF-6 mRNA的表达水平较GD组增加,差异有统计学意义(P<0. 05),较NC组差异无统计学意义(P>0. 05);其余指标在e GD组与GD组、e GD组和NC组之间的差异均没有统计学意义(P>0. 05)。结论:GD患者中TRAF-6低表达不足以抑制TGF-β1介导的Smad2/Smad3磷酸化,导致增多的Smad2/Smad3磷酸化蛋白下调IL-2表达,进而诱导Th17细胞的分化,同时抑制Treg细胞的分化,使机体不能维持免疫耐受,促进GD的发生。
Objective: To investigate whether tumor necrosis factor receptor-associated factor-6( TRAF-6) exerts immune tolerance during the development of Graves' disease( GD) and its possible mechanism. Methods: 30 GD newly diagnosed patients( GD group),30 GD remission patients( e GD group) and 28 healthy subjects( NC group) were recruited in this study. Peripheral blood mononuclear cells( PBMCs) were extracted and the mRNA expression of retinoid-related orphan receptor gamma t( RORγt),forkhead box protein p3( Foxp3),interleukine-2( IL-2) and TRAF-6 were detected by real-time quantitative PCR( RT-PCR). TRAF-6,pSmad2,p-Smad3,and total Smad2/3 protein expression were determined by Western blot. ELISA was used to detect the expression of transforming growth factor-β1( TGF-β1),IL-2,IL-17 A and IL-10 in plasma. Results: ①The mRNA and protein expression of IL-2,Foxp3 mRNA and IL-10 protein expression in the GD group were significantly lower than those in the NC group,while RORγt mRNA and IL-17 protein expression were significantly higher than those in the NC group( P<0. 05). ②The TGF-β1 protein level in the GD group was significantly lower than that in the NC group( P <0. 05). The protein expression of p-Smad/Smad3 was higher in the GD group than those in the NC group( P>0. 05). ③The expression of TRAF-6 mRNA in GD group was significantly lower than that in NC group( P<0. 05). The expression of TRAF-6 protein was lower than NC group( P>0. 05). ④The expression level of TRAF-6 mRNA in the e GD group was significantly higher than that in the GD group( P<0. 05),but no significant difference was found between the e GD group and the NC group( P > 0. 05). There was no significant difference in other indexes between e GD group and GD group,and between e GD group and NC group( P>0. 05). Conclusion: TRAF-6 may play an immunotolerant role in the development of GD. Low expression of TRAF-6 may be not sufficient to inhibit the phosphorylation of Smad2/3 by TGF-β1,while increased phosphorylation of Smad2/3 down-regulate the expression of IL-2,inducing the differentiation of Th17 cells and inhibiting the differentiation of Treg cells,which makes it impossible for the host to maintain immune tolerance and promote the development of GD.
Objective: To investigate whether tumor necrosis factor receptor-associated factor-6( TRAF-6) exerts immune tolerance during the development of Graves' disease( GD) and its possible mechanism. Methods: 30 GD newly diagnosed patients( GD group),30 GD remission patients( e GD group) and 28 healthy subjects( NC group) were recruited in this study. Peripheral blood mononuclear cells( PBMCs) were extracted and the mRNA expression of retinoid-related orphan receptor gamma t( RORγt),forkhead box protein p3( Foxp3),interleukine-2( IL-2) and TRAF-6 were detected by real-time quantitative PCR( RT-PCR). TRAF-6,pSmad2,p-Smad3,and total Smad2/3 protein expression were determined by Western blot. ELISA was used to detect the expression of transforming growth factor-β1( TGF-β1),IL-2,IL-17 A and IL-10 in plasma. Results: ①The mRNA and protein expression of IL-2,Foxp3 mRNA and IL-10 protein expression in the GD group were significantly lower than those in the NC group,while RORγt mRNA and IL-17 protein expression were significantly higher than those in the NC group( P<0. 05). ②The TGF-β1 protein level in the GD group was significantly lower than that in the NC group( P <0. 05). The protein expression of p-Smad/Smad3 was higher in the GD group than those in the NC group( P>0. 05). ③The expression of TRAF-6 mRNA in GD group was significantly lower than that in NC group( P<0. 05). The expression of TRAF-6 protein was lower than NC group( P>0. 05). ④The expression level of TRAF-6 mRNA in the e GD group was significantly higher than that in the GD group( P<0. 05),but no significant difference was found between the e GD group and the NC group( P > 0. 05). There was no significant difference in other indexes between e GD group and GD group,and between e GD group and NC group( P>0. 05). Conclusion: TRAF-6 may play an immunotolerant role in the development of GD. Low expression of TRAF-6 may be not sufficient to inhibit the phosphorylation of Smad2/3 by TGF-β1,while increased phosphorylation of Smad2/3 down-regulate the expression of IL-2,inducing the differentiation of Th17 cells and inhibiting the differentiation of Treg cells,which makes it impossible for the host to maintain immune tolerance and promote the development of GD.
引文
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[25]杨渝,刘纯.亚硒酸钠对Graves病患者外周血中Treg细胞的影响[J].中国免疫学杂志,2015,31(6):814-817.Yang Y,Liu C. Effect of sodium selenite on Treg cells in peripheral blood mononuclear cells in patients with Graves'disease[J]. Chin J Immunol,2015,31(6):814-817.
[26] Laurence A,Tato CM,Davidson TS,et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation[J]. Immunity,2007,26(3):371-381.
[27]谢克俭,张琦,夏玉祥,等.动态观察131I治疗对Graves病患者血清细胞因子水平的影响[J].中国免疫学杂志,2006,22(10):956-958.Xie KJ,Zhang Q,Xia YX,et al. Dynamic study on the influence of the serum cytokine levels in patients with Graves'disease before and after treated with131I[J]. Chin J Immunol,2006,22(10):956-958.
[28]王建国,史春云,丑广程,等. Graves病患者血清TLR4、NF-κB和Treg/Th17相关细胞因子表达变化及意义[J].中国现代医学杂志,2017,27(24):58-61.Wang JG,Shi CY,Chou GC,et al. Involvement of circulating TLR4,NF-κB and Treg/Th17 related cytokines in Graves'disease[J]. China J Modern Med,2017,27(24):58-61.
[29]李红林,高美华,郑云会,等.细胞因子IFN-γ、IL-6、IL-17和TGF-β1在Graves病发病中的作用[J].中国免疫学杂志,2015,31(2):253-256.Li HL,Gao MH,Zheng YH,et al. Study on levels of IFN-γ,IL-6,IL-17 and TGF-β1 in patients with Graves'disease[J]. Chin J Immunol,2015,31(2):253-256.
[30] Chang H,Brown CW,Matzuk MM. Genetic analysis of the mammalian transforming growth factor-beta superfamily[J].Endocr Rev,2002,23(6):787-823.
[31] Shi Y,Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus[J]. Cell,2003,113(6):685-700.
[32] Chen YG. Endocytic regulation of TGF-beta signaling[J]. Cell Res,2009,19(1):58-70.
[33] Huse M,Muir TW,Xu L,et al. The TGF beta receptor activation process:an inhibitor-to substrate-binding switch[J]. Mol Cell,2001,8(3):671-682.
[34] Mu Y,Gudey SK,Landstrom M. Non-Smad signaling pathways[J]. Cell Tissue Res,2012,347(1):11-20.
[2] Zheng L,Ye P,Liu C. The role of the IL-23/IL-17 axis in the pathogenesis of Graves'disease[J]. Endocr J,2013,60(5):591-597.
[3] Belkaid Y. Regulatory T cells and infection:a dangerous necessity[J]. Nat Rev Immunol,2007,7(11):875-888.
[4] Workman CJ,Szymczak-Workman AL,Collison LW,et al. The development and function of regulatory T cells[J]. Cell Mol Life Sci,2009,66(16):2603-2622.
[5] Shevach EM. Mechanisms of Foxp3+T regulatory cell-mediated suppression[J]. Immunity,2009,30(5):636-645.
[6] Josefowicz SZ,Lu LF, Rudensky AY. Regulatory T cells:mechanisms of differentiation and function[J]. Ann Rev Immunol,2012,30(1):531-564.
[7] Quaresma JA,Esteves PC,de Sousa Aarao TL,et al. Apoptotic activity and Treg cells in tissue lesions of patients with leprosy[J].Microb Pathog,2014,76:84-88.
[8] Li C,Yuan J,Zhu YF,et al. Imbalance of Th17/Treg in different subtypes of autoimmune thyroid diseases[J]. Cell Physiol Biochem,2016,40(1-2):245-252.
[9] Kleczynska W,Jakiela B,Plutecka H,et al. Imbalance between Th17 and regulatory T-cells in systemic lupus erythematosus[J].Folia Histochem Cytobiol,2011,49(4):646-653.
[10] Zeng C,Shi X,Zhang B,et al. The imbalance of Th17/Th1/Tregs in patients with type 2 diabetes:relationship with metabolic factors and complications[J]. J Mol Med(Berl),2012,90(2):175-186.
[11] Fainboim L,Arruvito L. Mechanisms involved in the expansion of Tregs during pregnancy:role of IL-2/STAT5 signalling[J]. J Reprod Immunol,2011,88(2):93-98.
[12] Setoguchi R,Hori S,Takahashi T,et al. Homeostatic maintenance of natural Foxp3(+)CD25(+)CD4(+)regulatory T cells by interleukin(IL)-2 and induction of autoimmune disease by IL-2neutralization[J]. J Exp Med,2005,201(5):723-735.
[13] de la Rosa M,Rutz S,Dorninger H,et al. Interleukin-2 is essential for CD4+CD25+regulatory T cell function[J]. Eur J Immunol,2004,34(9):2480-2488.
[14] Li MO,Wan YY,Sanjabi S,et al. Transforming growth factor-beta regulation of immune responses[J]. Ann Rev Immunol,2006,24:99-146.
[15] Fontenot JD,Rasmussen JP,Gavin MA,et al. A function for interleukin 2 in Foxp3-expressing regulatory T cells[J]. Nat Immunol,2005,6(11):1142-11451.
[16]耿明霞,马杰,何峰容,等. Graves病患者血清细胞因子水平和甲状腺功能指标的检测分析[J].微循环学杂志,2005,15(3):27-28.Geng MX,Ma J,He FR,et al. Study of serum cytokine levels and thyroid function in patients with Graves'disease[J]. Chin J Microcirculation,2005,15(3):27-28.
[17] Han D,Walsh MC,Cejas PJ,et al. Dendritic cell expression of the signaling molecule TRAF6 is critical for gut microbiota-dependent immune tolerance[J]. Immunity,2013,38(6):1211-1222.
[18] Han D,Walsh MC,Kim KS,et al. Dendritic cell expression of the signaling molecule TRAF6 is required for immune tolerance in the lung[J]. Int Immunol,2017,29(2):71-78.
[19] King CG,Kobayashi T,Cejas PJ,et al. TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis[J]. Nat Med,2006,12(9):1088-1092.
[20] Cejas PJ,Walsh MC,Pearce EL,et al. TRAF6 inhibits Th17 differentiation and TGF-beta-mediated suppression of IL-2[J].Blood,2010,115(23):4750-4757.
[21] Muto G,Kotani H,Kondo T,et al. TRAF6 is essential for maintenance of regulatory T cells that suppress Th2 type autoimmunity[J]. PLo S One,2013,8(9):e74639.
[22] Govinden R,Bhoola KD. Genealogy,expression,and cellular function of transforming growth factor-beta[J]. Pharmacol Ther,2003,98(2):257-265.
[23] Guo A,Tan Y,Liu C,et al. MST-4 and TRAF-6 expression in the peripheral blood mononuclear cells of patients with Graves'disease and its significance[J]. BMC Endocr Disord,2017,17(1):11.
[24]滕卫平,曾正陪,李光伟,等.中国甲状腺疾病诊治指南[M].北京:中华医学会内分泌学分会,2007:47.Teng WP,Zeng ZP,Li GW,et al. Guidelines for the diagnosis and treatment of thyroid diseases in China[M]. Beijing endocrinology branch of Chinese medical association,2007:47.
[25]杨渝,刘纯.亚硒酸钠对Graves病患者外周血中Treg细胞的影响[J].中国免疫学杂志,2015,31(6):814-817.Yang Y,Liu C. Effect of sodium selenite on Treg cells in peripheral blood mononuclear cells in patients with Graves'disease[J]. Chin J Immunol,2015,31(6):814-817.
[26] Laurence A,Tato CM,Davidson TS,et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation[J]. Immunity,2007,26(3):371-381.
[27]谢克俭,张琦,夏玉祥,等.动态观察131I治疗对Graves病患者血清细胞因子水平的影响[J].中国免疫学杂志,2006,22(10):956-958.Xie KJ,Zhang Q,Xia YX,et al. Dynamic study on the influence of the serum cytokine levels in patients with Graves'disease before and after treated with131I[J]. Chin J Immunol,2006,22(10):956-958.
[28]王建国,史春云,丑广程,等. Graves病患者血清TLR4、NF-κB和Treg/Th17相关细胞因子表达变化及意义[J].中国现代医学杂志,2017,27(24):58-61.Wang JG,Shi CY,Chou GC,et al. Involvement of circulating TLR4,NF-κB and Treg/Th17 related cytokines in Graves'disease[J]. China J Modern Med,2017,27(24):58-61.
[29]李红林,高美华,郑云会,等.细胞因子IFN-γ、IL-6、IL-17和TGF-β1在Graves病发病中的作用[J].中国免疫学杂志,2015,31(2):253-256.Li HL,Gao MH,Zheng YH,et al. Study on levels of IFN-γ,IL-6,IL-17 and TGF-β1 in patients with Graves'disease[J]. Chin J Immunol,2015,31(2):253-256.
[30] Chang H,Brown CW,Matzuk MM. Genetic analysis of the mammalian transforming growth factor-beta superfamily[J].Endocr Rev,2002,23(6):787-823.
[31] Shi Y,Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus[J]. Cell,2003,113(6):685-700.
[32] Chen YG. Endocytic regulation of TGF-beta signaling[J]. Cell Res,2009,19(1):58-70.
[33] Huse M,Muir TW,Xu L,et al. The TGF beta receptor activation process:an inhibitor-to substrate-binding switch[J]. Mol Cell,2001,8(3):671-682.
[34] Mu Y,Gudey SK,Landstrom M. Non-Smad signaling pathways[J]. Cell Tissue Res,2012,347(1):11-20.