白细胞介素-7在肠道病毒71感染所致的手足口病合并中枢神经系统损害患儿中的表达及对CD8^+T细胞功能的影响

The expression of IL-7 in enterovirus 71-induced hand, foot, and mouth disease with central nervous system damage and its influence to CD8^+T cells

目的观察白细胞介素-7(IL-7)在肠道病毒71感染(EV71)所致的手足口病(HFMD)合并中枢神经系统损害患儿中的表达,研究IL-7对CD8^+T细胞的调控作用。方法入组EV71感染所致的HFMD患儿48例(轻症HFMD 27例、伴有中枢神经系统损害的重症HFMD 21例),同时入组9例年龄、性别相匹配的健康对照者(HC)。分离血浆,纯化CD8^+T细胞,对重症HFMD患儿行腰椎穿刺术取脑脊液。酶联免疫吸附试验检测血浆和脑脊液中IL-7水平,实时定量PCR法检测CD127 mRNA相对表达量。使用重组人IL-7刺激纯化的CD8^+T细胞,观察分泌细胞增殖、分泌细胞因子和IL-7下游信号分子的表达变化。建立CD8^+T细胞和EV71感染的U-87MG细胞的直接接触和间接接触共培养系统,观察重组人IL-7对CD8^+T细胞功能的影响。结果伴有中枢神经系统损伤的重症HFMD患儿血浆中IL-7的表达水平较轻症HFMD和HC显著下降(P<0.000 1)。脑脊液IL-7水平在影像学正常和异常的重症HFMD之间的差异无统计学意义(P=0.218)。CD8^+T细胞绝对计数及CD127 mRNA在CD8^+T细胞中的相对表达量在HC、轻症HFMD和重症HFMD之间的差异无统计学意义(P>0.05)。重组人IL-7刺激不影响重症HFMD患儿外周血纯化的CD8^+T细胞的增殖,但可显著增加CD8^+T细胞分泌干扰素-γ(IFN-γ)和肿瘤坏死因子(TNF-α)的水平,同时CD8^+T细胞中信号传导及转录激活因子5的磷酸化及细胞因子信号抑制物3的表达均显著升高。重组人IL-7刺激可增强重症HFMD患儿CD8^+T细胞的细胞杀伤功能,主要表现为IL-7刺激后直接接触培养系统中靶细胞的死亡比例以及分泌IFN-γ和TNF-α的水平增加,而间接接触培养细胞中靶细胞死亡比例未见显著变化。结论 IL-7在伴有中枢神经系统损伤的重症HFMD患儿中的表达降低可能影响CD8^+T细胞的杀伤功能。

The immunopathogenesis of enterovirus 71(EV71)-induced hand, foot, and mouth disease(HFMD is not fully elucidated. Interleukin-7(IL-7) plays an important role in T cells development, proliferation, and activation. The aim of current study was to investigate the expression of IL-7 in EV71-induced HFMD with central nervous system(CNS) damage, and to access the regulatory function of recombinant IL-7 to CD8^+T cells. A total of48 children with EV71-induced HFMD(including 27 of mild HFMD and 21 of severe HFMD with central nervous system damage) and 9 healthy control(HC)were enrolled in the present study. Plasma was isolated, and peripheral CD8^+T cells were purified.Cerebrospinal fluid(CSF) was also obtained from severe HFMD children by lumbar puncture. The expression of IL-7 in plasma and CSF was measured by ELISA, while CD127 mRNA relative expression was semi-quantified by realtime PCR. Cellular proliferation, cytokine production, and IL-7 signaling downstream molecules were assessed in CD8^+T cells in response to IL-7 stimulation. Direct and indirect contact co-culture system of CD8^+T cells and EV71-infected U-87 MG cells was set up. CD8^+T cells activity was also investigated in response to IL-7 stimulation. Data showed that plasma IL-7 expression in severe HFMD children with CNS damage was significantly down-regulated in comparison with mild HFMD and HC(P<0.000 1). There was no remarkable difference of IL-7 expression in CSF between normal imaging and abnormal imaging of severe HFMD patients(P=0.218). There was no significant difference of either CD8^+T absolute number or CD127 mRNA relative expression within CD8^+T cells among HC, mild HFMD, and severe HFMD(P>0.05). IL-7 stimulation did not affect the proliferation of CD8^+T cells purified from severe HFMD children, however, notably increased interferon-γ(IFN-γ) and tumor necrosis factor-α(TNF-α) production by CD8^+T cells. IL-7 signaling downstream molecules(phosphorylated signal transducers, activators of transcription 5 and suppressor of cytokine signaling 3) were also significantly elevated in CD8^+T cells in response to IL-7 stimulation. IL-7 stimulation elevated cytolytic activity of CD8^+T cells from severe HFMD children, which presented as increased target cell death and enhanced IFN-γ/TNF-α production in direct contact co-culture system in response to IL-7 stimulation, however, comparable target cell death demonstrated no obvious changes in indirect contact co-culture system. The current data indicates that IL-7 expression reduction in EV71-induced severe HFMD patients with CNS damage might affect cytotoxicity of CD8^+T cells.