类风湿性关节炎患者外周血Th17细胞表达水平及其临床相关性
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摘要
背景:Th17是RA发病中的重要效应性细胞,其细胞因子产物IL-17是RA关节局部炎症和骨质破化的重要上游因子。研究示,RA患者的滑膜Th17和IL-17表达量增高,并随治疗降低。但是,对Th17的研究多着重于滑膜组织局部,而对外周血Th17细胞表达水平及其临床相关性研究尚为不足;研究方法以免疫组化染色为主,而流式细胞学研究相对缺乏。
目的:评价RA患者的Th17外周表达及其临床相关性;评价DMARDs和生物制剂治疗对Th17外周表达的影响,及Th17表达水平和疗效的关系。
方法:20名活动性RA患者,分别接受DMARDs±激素治疗(n=10)和阿达木单抗+DMARDs治疗(n=10)。在DMARDs治疗第0周和第12周,阿达木单抗治疗第0周、12周和24周,分别采集外周血进行实验室检测,包括:流式细胞学检测PBMCCD4(+)IL-17(+)细胞;RT-PCR测定PBMC IL-17、RORc、FOXP3基因表达;ELISA测定IL-17、IL-6血清水平。同时设年龄性别匹配的正常人对照(n=10)。
结果:流式细胞染色示,RA患者PBMC CD4(+)IL-17(+)细胞数量和MFI相对于正常人显著性上调,并与临床疾病活动程度呈显著性正相关;在治疗第12周,RA患者PBMCCD4(+)IL-17(+)细胞数量较基线水平显著性下调,其中DMARDs组Th17细胞基线表达、阿达木单抗治疗组治疗期间平均Th17表达与临床疗效呈显著性地负相关(p＜0.05)。RT-PCR示,PBMC FOXP3 mRNA表达水平在RA患者中显著性下调并与临床疾病活动呈显著性负相关,而IL-17 mRNA表达水平则与临床疾病活动呈显著性正相关;在治疗第12周,FOXP3表达水平显著性上调(p＜0.05)。ELISA示RA患者相对于正常人血清IL-6水平升高,治疗导致血清IL-6的显著性降低(p＜0.05)。
结论:RA患者外周血Th17细胞数量、IL-6水平上调,FOXP3 mRNA表达水平下调。Th17数量及IL-17 mRNA表达水平与病情活动度显著性正相关,而FOXP3 mRNA表达水平和疾病活动度显著性负相关。DMARDs和阿达木单抗治疗导致外周血Th17细胞数量和IL-6血清水平下调,FOXP3 mRNA表达上调,提示治疗可能通过对Th17细胞和Treg细胞的调节起作用。对于接受DMARDs治疗的患者,基线PBMC Th17数量是预后的预测因子,而对于接受阿达木单抗治疗的患者,这种预测作用受到削弱,可能与其阻断了TNF-α对Th17细胞的正反馈激活作用有关。但是,在阿达木单抗治疗期间的平均Th17细胞表达水平和疗效负相关,提示高水平的Th17细胞表达可能是影响生物制剂疗效的因素之一,而针对Th17的生物靶向治疗也许可以作为RA治疗的新方向。
Background.Th17 is a newly defined subgroup of CD4(+)effecor T cells,and is considered to be one of the key T cell lineages in the development of a large scale of autoimmune diseases,including rheumatoid arthritis.Th17 is characteristic by its secretion of specific spectrum of cytokines,including IL-17,which is a important upstream cytokine in the chronic inflammation and bone destruction of RA.It has been demonstrated that both Th17 and its cytokines are up regulated in RA synovial tissue, and treatment commits their down regulation.However,these researches are mostly focused on Th17 expression in synovial fluid and tissue,and there is still limited understanding of its peripheral expression,and its correlation with clinical symptoms and effects of treatment.
Objective.To assess the peripheral expression of Th17 cells in RA patients and its clinical correlations.Investigate into the effects of DMARDs or anti-TNF-αtherapy on Th17 expression,and the correlation of its expression with clinical disease remission. Methods.20 RA patients with active disease were separately treated with DMARDs(n=10) and adalimumab(n=10).Blood samples were taken for laboratory analysis at week 0 and week 12 of treatment for DMARDs treated group,and week0, week12 and week24 of adalimumab treated group.Laboratory methods used include the following:flowcytometry for detection of PBMC CD4(+)IL-17(+) cells and their MFI; RT-PCR for semi-quantitatively determination of mRNA expression of IL-17,RORc and FOXP3;ELISA detection of serum IL-17 and IL-6.Same tests were run on age and sex marched normal controls(n=10).
Results.Flowcytometry.PBMC CD4(+)IL-17(+)T cell was significantly up regulated in RA patients,and positively correlated with disease activity.PBMC Th17 cell count decreased significantly by week 12 of treatment.The baseline Th17 level of DMARDs treated group and the mean Th17 level during Adalimumab treatment were significantly negatively correlated with clinical remission(p＜0.05).RT-PCR.PBMC FOXP3 mRNA expression was significantly down regulated in RA patients,and negatively correlated with disease activity,and IL-17 gene expression was significantly positively correlated with ESR. FOXP3 mRNA expression was significantly up regulated by week 12 of treatment(p＜0.05). ELISA.Serum IL-6 level was significantly up regulated in RA patients,and decreased significantly with treatment(p＜0.O5).
Conclusion.The PBMC Th17 cell count and IL-6 serum level were significantly up regulated in RA patients,and there was significant down regulation of FOXP3 mRNA expression.PBMC Th17 count and IL-17 mRNA expression positively correlated with disease activity,and FOXP3 gene expression had a negatively correlation.DMARDs and Adalimumab treatment decreased PBMC Th17 cell count and serum IL-6 level significantly,and increased FOXP3 expression,indicating that treatment might be effected through Th17 and Treg modulation.For DMARDs treated patients,baseline Th17 cell count was a predictive factor of disease remission with treatment,and for the Adalimumab treated group,this predictive effect is weakened,which might be attributed to that blockade of TNF-αalso blocked its positive feedback effects on Th17 cells.However,mean Th17 expression during adalimumab treatment was negatively correlated with disease remission,indicating that high level of Th17 cells might impact on the effectiveness of anti-TNF-αtherapy,and molecular targeting towards Th17 might be the new direction of RA treatment.
目的:评价RA患者的Th17外周表达及其临床相关性;评价DMARDs和生物制剂治疗对Th17外周表达的影响,及Th17表达水平和疗效的关系。
方法:20名活动性RA患者,分别接受DMARDs±激素治疗(n=10)和阿达木单抗+DMARDs治疗(n=10)。在DMARDs治疗第0周和第12周,阿达木单抗治疗第0周、12周和24周,分别采集外周血进行实验室检测,包括:流式细胞学检测PBMCCD4(+)IL-17(+)细胞;RT-PCR测定PBMC IL-17、RORc、FOXP3基因表达;ELISA测定IL-17、IL-6血清水平。同时设年龄性别匹配的正常人对照(n=10)。
结果:流式细胞染色示,RA患者PBMC CD4(+)IL-17(+)细胞数量和MFI相对于正常人显著性上调,并与临床疾病活动程度呈显著性正相关;在治疗第12周,RA患者PBMCCD4(+)IL-17(+)细胞数量较基线水平显著性下调,其中DMARDs组Th17细胞基线表达、阿达木单抗治疗组治疗期间平均Th17表达与临床疗效呈显著性地负相关(p＜0.05)。RT-PCR示,PBMC FOXP3 mRNA表达水平在RA患者中显著性下调并与临床疾病活动呈显著性负相关,而IL-17 mRNA表达水平则与临床疾病活动呈显著性正相关;在治疗第12周,FOXP3表达水平显著性上调(p＜0.05)。ELISA示RA患者相对于正常人血清IL-6水平升高,治疗导致血清IL-6的显著性降低(p＜0.05)。
结论:RA患者外周血Th17细胞数量、IL-6水平上调,FOXP3 mRNA表达水平下调。Th17数量及IL-17 mRNA表达水平与病情活动度显著性正相关,而FOXP3 mRNA表达水平和疾病活动度显著性负相关。DMARDs和阿达木单抗治疗导致外周血Th17细胞数量和IL-6血清水平下调,FOXP3 mRNA表达上调,提示治疗可能通过对Th17细胞和Treg细胞的调节起作用。对于接受DMARDs治疗的患者,基线PBMC Th17数量是预后的预测因子,而对于接受阿达木单抗治疗的患者,这种预测作用受到削弱,可能与其阻断了TNF-α对Th17细胞的正反馈激活作用有关。但是,在阿达木单抗治疗期间的平均Th17细胞表达水平和疗效负相关,提示高水平的Th17细胞表达可能是影响生物制剂疗效的因素之一,而针对Th17的生物靶向治疗也许可以作为RA治疗的新方向。
Background.Th17 is a newly defined subgroup of CD4(+)effecor T cells,and is considered to be one of the key T cell lineages in the development of a large scale of autoimmune diseases,including rheumatoid arthritis.Th17 is characteristic by its secretion of specific spectrum of cytokines,including IL-17,which is a important upstream cytokine in the chronic inflammation and bone destruction of RA.It has been demonstrated that both Th17 and its cytokines are up regulated in RA synovial tissue, and treatment commits their down regulation.However,these researches are mostly focused on Th17 expression in synovial fluid and tissue,and there is still limited understanding of its peripheral expression,and its correlation with clinical symptoms and effects of treatment.
Objective.To assess the peripheral expression of Th17 cells in RA patients and its clinical correlations.Investigate into the effects of DMARDs or anti-TNF-αtherapy on Th17 expression,and the correlation of its expression with clinical disease remission. Methods.20 RA patients with active disease were separately treated with DMARDs(n=10) and adalimumab(n=10).Blood samples were taken for laboratory analysis at week 0 and week 12 of treatment for DMARDs treated group,and week0, week12 and week24 of adalimumab treated group.Laboratory methods used include the following:flowcytometry for detection of PBMC CD4(+)IL-17(+) cells and their MFI; RT-PCR for semi-quantitatively determination of mRNA expression of IL-17,RORc and FOXP3;ELISA detection of serum IL-17 and IL-6.Same tests were run on age and sex marched normal controls(n=10).
Results.Flowcytometry.PBMC CD4(+)IL-17(+)T cell was significantly up regulated in RA patients,and positively correlated with disease activity.PBMC Th17 cell count decreased significantly by week 12 of treatment.The baseline Th17 level of DMARDs treated group and the mean Th17 level during Adalimumab treatment were significantly negatively correlated with clinical remission(p＜0.05).RT-PCR.PBMC FOXP3 mRNA expression was significantly down regulated in RA patients,and negatively correlated with disease activity,and IL-17 gene expression was significantly positively correlated with ESR. FOXP3 mRNA expression was significantly up regulated by week 12 of treatment(p＜0.05). ELISA.Serum IL-6 level was significantly up regulated in RA patients,and decreased significantly with treatment(p＜0.O5).
Conclusion.The PBMC Th17 cell count and IL-6 serum level were significantly up regulated in RA patients,and there was significant down regulation of FOXP3 mRNA expression.PBMC Th17 count and IL-17 mRNA expression positively correlated with disease activity,and FOXP3 gene expression had a negatively correlation.DMARDs and Adalimumab treatment decreased PBMC Th17 cell count and serum IL-6 level significantly,and increased FOXP3 expression,indicating that treatment might be effected through Th17 and Treg modulation.For DMARDs treated patients,baseline Th17 cell count was a predictive factor of disease remission with treatment,and for the Adalimumab treated group,this predictive effect is weakened,which might be attributed to that blockade of TNF-αalso blocked its positive feedback effects on Th17 cells.However,mean Th17 expression during adalimumab treatment was negatively correlated with disease remission,indicating that high level of Th17 cells might impact on the effectiveness of anti-TNF-αtherapy,and molecular targeting towards Th17 might be the new direction of RA treatment.
引文
[1]Mosmann, T.R. et al. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7,145-173 (1989).
[2] Abbas, A.K. etal. Functional diversity of helper T lymphocytes. Nature 383, 787-793 (1996).
[3]Weaver C.T. et al. Thl7: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 24, 677-88(2006).
[4] Lundy S.K. et al. Cells of the synovium in rheumatoid arthritis. T lymphocytes. Arthritis Res Ther. 9, 202(2007).
[5] Sutton C, et al. A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J Exp Med. 203, 1685-91(2006).
[6] Lubberts E. IL-17/TH17 targeting: on the road to prevent chronic destructive arthritis? Cytokine. 41, 84-91(2007).
[7] Zaba L. C. et al. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Thl7 responses. J Exp Med. 204, 3183-94(2007)
[8] Kuligowska M. et al. Role of interleukin-17 in cartilage and bone destruction in rheumatoid arthritis. Ortop Traumatol Rehabil. 6, 235-41(2006).
[2] Abbas, A.K. etal. Functional diversity of helper T lymphocytes. Nature 383, 787-793 (1996).
[3]Weaver C.T. et al. Thl7: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 24, 677-88(2006).
[4] Lundy S.K. et al. Cells of the synovium in rheumatoid arthritis. T lymphocytes. Arthritis Res Ther. 9, 202(2007).
[5] Sutton C, et al. A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J Exp Med. 203, 1685-91(2006).
[6] Lubberts E. IL-17/TH17 targeting: on the road to prevent chronic destructive arthritis? Cytokine. 41, 84-91(2007).
[7] Zaba L. C. et al. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Thl7 responses. J Exp Med. 204, 3183-94(2007)
[8] Kuligowska M. et al. Role of interleukin-17 in cartilage and bone destruction in rheumatoid arthritis. Ortop Traumatol Rehabil. 6, 235-41(2006).