肺结核患者疗程中外周血辅助性和调节性T细胞的动态研究

摘要/Abstract

摘要： 目的探讨辅助性T细胞17（Th17）和CD4⁺CD25⁺CD127^low调节性T细胞与结核病的发病及抗结核治疗转归的关系。方法纳入对象包括32例活动性肺结核患者、25例Mtb潜伏感染者、45例健康对照者。流式细胞术检测外周血Th17细胞分泌细胞因子IL-17和CD4⁺CD25⁺CD127^low调节性T细胞表达强度。结果以x±s表示，所有数据均使用Prism 4.0统计软件进行分析，两组间比较采用非配对t检验，多组间的比较采用ANOVA方差分析，以P<0.05为差异有统计学意义。结果肺结核患者组治疗前IL-17表达为（3.25±1.68）%，明显低于健康对照组［（4.62±1.46）%］（F=6.633，P<0.0001）。比较活动性肺结核患者组IL-17表达在治疗前、治疗3个月［（4.17±2.27）%］、治疗6个月［（5.58±1.66）%］时的检测结果，发现治疗3个月时高于治疗前，但差异无统计学意义（F=12.244，P=0.057）；而治疗6个月时明显高于治疗前（F=12.244，P<0.0001）和治疗3个月时（F=12.244，P=0.004）。健康对照组CD4⁺CD25⁺CD127^low调节性T细胞表达为（4.97±1.60）%，与Mtb潜伏感染组［（5.00±1.08）%］比较，差异无统计学意义（F=11.986，P=0.937）；活动性肺结核患者组治疗前表达为（6.59±1.73）%，显著高于健康对照组及Mtb潜伏感染组(F=11.986，P<0.0001)。比较活动性肺结核患者组治疗前、治疗3个月［（8.28±2.04）%］、治疗6个月［（7.46±1.87）%］时的CD4⁺CD25⁺CD127^low调节性T细胞表达，发现治疗3个月时的表达明显高于治疗前（F=6.458，P=0.001）；治疗6个月时的表达低于治疗3个月时（F=6.458， P=0.085），但差异无统计学意义；治疗6个月与治疗前的表达相比较，差异无统计学意义（F=6.458，P=0.068）；治疗6个月CD4⁺CD25⁺CD127^low调节性T细胞表达明显高于健康对照组（t=6.255，P<0.0001）。结论结核病患者外周血Th17细胞明显减少，经有效抗结核治疗后，Th17细胞逐渐增加，表明Th17细胞在抗结核免疫中起保护作用。结核病患者外周血CD4⁺CD25⁺CD127^low调节性T细胞明显增多，经抗结核治疗后CD4⁺CD25⁺CD127^low调节性T细胞逐渐减少，进一步说明CD4⁺CD25⁺CD127^low调节性T细胞在抗结核免疫中起抑制作用。

关键词: 结核, 肺/免疫学, T淋巴细胞, 调节性, T淋巴细胞, 辅助诱导

Abstract: Objective To study the relationship between the numbers of Th17 cells, CD4⁺CD25⁺CD127^low Tregs and the tuberculosis (TB) and outcome of anti-TB. Methods Intracellular staining and flow cytometry analysis were used to evaluate IL-17 cytokine secreted by Th17 cells and the responses of CD4⁺CD25⁺CD127^low Treg cells in peripheral blood samples collected from 25 individuals with LTBI, 45 healthy donors (HD), 32 patients with active pulmonary TB. The results were showed as x±s，all data were analyzed by software Prism 4.0. Two groups were compared by t-test，the comparison between groups used ANOVA. There is statistical significance if P<0.05. Results The percentage of IL-17 in TB group ［（3.25±1.68）%］was significantly lower than that in HD group ［（4.62±1.46）%］（F=6.633，P<0.0001）before treatment. The dynamic variation of IL-17 percentage was monitored at different time points: before treatment［（3.25±1.68）%］, at 3 months［（4.17±2.27）%］, and at 6 months after treatment［（5.58±1.66）%］. The percentage of Th17 cells from TB group at 3 months after treatment was higher than that before treatment, but there was no significant difference（F=12.244，P=0.057）. The percentage of Th17 cells at 6 months after treatment was significantly higher than those before treatment（F=12.244，P<0.0001） and at 3 months after treatment（F=12.244，P=0.004）. The percentage of CD4⁺CD25⁺CD127^low treg cells in LTBI group ［（5.00±1.08）%］ was higher than that in HD group ［（4.97±1.60）%］, but there was no significant difference （F=11.986，P=0.937）. The percentage of CD4+CD25+CD127low treg cells in TB group ［（6.59±1.73）%］ was significantly higher than that in HD group and LTBI group (F=11.986，P<0.0001). The Treg cells at 3 months after treatment were significantly higher than that before treatment（F=6.458，P=0.001）. But the Treg cells at 6 months after treatment were lower than that at 3 months after treatment（F=6.458，P=0.085）. There was no significant difference between before treatment and at 6 months after treatment（F=6.458，P=0.068）. The Treg cells at 6 months after treatment were significantly higher than HD group（t=6.255，P<0.0001）. Conclusion The Th17 cells in TB group were low, gradually increased after effective anti-TB treatment, which suggested that Th17 cells play a protective role in the anti-TB immunity. The CD4⁺CD25⁺CD127^low treg cells in TB group were higher, and gradually reduced after effective anti-TB treatment, which suggested that CD4⁺CD25⁺CD127^low treg cells play a supressive role in the anti-TB immunity.

Key words: Tuberculosis, pulmonary/immunology, T-lymphocytes, regulatory, T-lymphocytes, helper-inducer

张影陆坚侯志平张明霞陈心春张兴树邱振纲. 肺结核患者疗程中外周血辅助性和调节性T细胞的动态研究[J]. 中国防痨杂志, 2013, 35(6): 427-432.

ZHANG Ying，LU Jian，HOU Zhi-ping，ZHANG Ming-xia，CHEN Xin-chun，ZHANG Xing-shu，QIU Zhen-gang. The dynamic change of Th17 cells and regulatory T cells in PBMC during the anti-tuberculosis treatment of the patients with pulmonary tuberculosis[J]. Chinese Journal of Antituberculosis, 2013, 35(6): 427-432.

参考文献

［1］Sakaguchi S, Sakaguchi N, Asano M,et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.J Immunol,1995,155（3）:1151-1164．
［2］Belkaid Y,Rouse BT.Natural regulatory T cells in infectious disease.Nat Immunol,2005，6（4）：353-360.
［3］Sasada T,Kimura M,Yoshida Y, et al.CD4⁺CD25⁺regulatory T cells in patients with gastrointestinal malignancies：possible involvement of regulatory T cells in disease progression.Cancer,2003,98（5）:1089-1099．
［4］Wolf D,Wolf AM,Rumpold H, et al.The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer.Clin Cancer Ras,2005,11（23）:8326-8331．
［5］Dannull J,Su Z,Rizzieri D, et al.Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells.J Clin Invest,2005,115（12）:3623-3633．
［6］陈心春,周伯平,邓群益,等.CD4⁺CD25⁺调节性T细胞对肺结核患者特异细胞免疫的调节作用.中国防痨杂志,2008,30（3）:165-169.
［7］Dong C.Differentiation and function of pro-inflammatory Th17 cells. Microbes Infect,2009,11（5）:584-588.
［8］Kotake S,Udagawa N,Takahashi N, et al.IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis.J Clin Invest， 1999,103（9）:1345-1352.
［9］Fujimoto M,Serada S,Mihara M, et al.Interleukin-6 blockade suppresses autoimmune arthritis in mice by the inhibition of inflammatory Th17 responses. Arthritis Rheum,2008,58（12）:3710-3719.
［10］Wilson NJ,Boniface K,Chan JR, et al.Development,cytokine profile and function of human interleukin 17-producing helper T cells.Nat Immunol,2007,8（9）:950-957.
［11］Pitta MG,Romano A,Cabantous S, et al.IL-17 and IL-22 are associated with protection against human kala azar caused by Leishmania donovani.J Clin Invest,2009,119（8）:2379-2387.
［12］Acosta-Rodriguez EV,Rivino L,Geginat J, et al.Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol,2007,8（6）:639-646.
［13］Khader SA,Bell GK,Pearl JE, et al.IL-23 and IL-17 in the establishment of protective pulmonary CD4⁺T cell responses after vaccination and during Mycobacterium tuberculosis challenge.Nat Immunol,2007,8（4）:369-377.
［14］Chen X,Zhang M,Zhu X, et al.Engagement of Toll-like receptor 2 on CD4⁺T cells facilitates local immune responses in patients with tuberculous pleurisy. J Infect Dis,2009,200（3）:399-408.
［15］Paidipally P,Periasamy S,Barnes PF, et al.NKG2D-dependent IL-17 production by human T cells in response to an intracellular pathogen.J Immunol，2009,183（3）:1940-1945.
［16］Scriba TJ,Kalsdorf B,Abrahams DA, et al.Distinct,specific IL-17 and IL-22 producing CD4⁺T cell subsets contribute to the human anti-mycobacterial immune response.J Immunol,2008,180（3）:1962-1970.
［17］Chen X,Zhang M,Liao M, et al.Reduced Th17 response in patients with tuberculosis correlates with IL-6R expression on CD4⁺T Cells.Am J Respir Crit Care Med,2010,181(7):734-742.
［18］Wang T,Lv M,Qian Q, et al.Increased frequencies of T helper type 17 cells in tuberculous pleural effusion. Tuberculosis (Edinb),2011, 91（3）:231-237.
［19］Bettelli E,Korn T,Kuchroo VK.Th17:the third member of the effector T cell trilogy.Curr Opin Irnmtmol,2007,19(6):652-657．
［20］Chen X,Zhou B, Li M. CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress 〖WTB2X〗Mycobacterium tuberculosis〖WTBZ〗 immunity in patients with active disease. Clin Immunol,2007,123（1）:50-59.
［21］孙晨鸣,马海霞,刘光伟,等.CD127与T淋巴细胞.中国生物工程杂志,2007,27(5):113-119．
［22］Liu W,Putnam AL, Xu-Yu Z, et al．CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+Treg cells．J Exp Med,2006,203（7）:1701-1711．
［23］Guyot-Revol V,Innes JA,Hackforth S, et al.Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis.Am J Respir Crit Care Med,2006，173(7):803-810.
［24］Ichiyama K,Yoshida H,Wakabayashi Y, et al.Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat．J Biol Chem，2008,283(25):17003-17008．