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
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摘要
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.
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.
引文
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[11]Gao J,Zhao L,Wan YY,et al.Mechanism of action of IL-7 and its potential applications and limitations in cancer immunotherapy[J].Int J Mol Sci,2015,16(5):10267-10280.
[12]Mackall CL,Fry TJ,Gress RE.Harnessing the biology of IL-7 for therapeutic application[J].Nat Rev Immunol,2011,11(5):330-342.
[13]Plumb AW,Patton DT,Seo JH,et al.Interleukin-7,but not thymic stromal lymphopoietin,plays a key role in the T cell response to influenza A virus[J].PLo S One,2012,7(11):e50199.
[14]Vassena L,Miao H,Cimbro R,et al.Treatment with IL-7 prevents the decline of circulating CD4+T cells during the acute phase of SIV infection in rhesus macaques[J].PLo S Pathog,2012,8(4):e1002636.
[15]冮宏生,彭定凤,胡勇钧,等.白细胞介素-7调控T细胞表面TIM-3的表达在冠状动脉粥样硬化性心脏病中的作用[J].免疫学杂志,2017,33(12):1040-1046.
[2]Ooi MH,Wong SC,Lewthwaite P,et al.Clinical features,diagnosis,and management of enterovirus 71[J].Lancet Neurol,2010,9(11):1097-1105.
[3]Cox JA,Hiscox JA,Solomon T,et al.Immunopathogenesis and virus-host interactions of enterovirus 71 in patients with hand,foot and mouth disease[J].Front Microbiol,2017,8:2249.
[4]Okoye AA,Rohankhedkar M,Konfe AL,et al.Effect of IL-7 therapy on naive and memory T cell homeostasis in aged rhesus macaques[J].J Immunol,2015,195(9):4292-4305.
[5]Pellegrini M,Calzascia T,Toe JG,et al.IL-7 engages multiple mechanisms to overcome chronic viral infection and limit organ pathology[J].Cell,2011,144(4):601-613.
[6]Hou H,Kang Y,Zeng Y,et al.Interleukin-7 augments CD8(+)T cells function and promotes viral clearance in chronic hepatitis C virus infection[J].Cytokine,2018,102:26-33.
[7]张小红,王红民,靳莉,等.白细胞介素7对非小细胞肺癌患者CD8+T细胞抗肿瘤活性的影响[J].中华医学杂志,2018,98(30):2429-2433.
[8]Boegel S,L?wer M,Bukur T,et al.A catalog of HLA type,HLA expression,and neo-epitope candidates in human cancer cell lines[J].Oncoimmunology,2014,3(8):e954893.
[9]Kimura MY,Pobezinsky LA,Guinter TI,et al.IL-7signaling must be intermittent,not continuous,during CD8+T cell homeostasis to promote cell survival instead of cell death[J].Nature Immunol,2013,14(2):143-151.
[10]Kerzerho J,Mc Ilvaine EJ,Anthony P,et al.Impact of hepatitis C virus on the circulating levels of IL-7 in HIV-1coinfected women[J].J Acquir Immune Defic Syndr,2016,71(2):172-180.
[11]Gao J,Zhao L,Wan YY,et al.Mechanism of action of IL-7 and its potential applications and limitations in cancer immunotherapy[J].Int J Mol Sci,2015,16(5):10267-10280.
[12]Mackall CL,Fry TJ,Gress RE.Harnessing the biology of IL-7 for therapeutic application[J].Nat Rev Immunol,2011,11(5):330-342.
[13]Plumb AW,Patton DT,Seo JH,et al.Interleukin-7,but not thymic stromal lymphopoietin,plays a key role in the T cell response to influenza A virus[J].PLo S One,2012,7(11):e50199.
[14]Vassena L,Miao H,Cimbro R,et al.Treatment with IL-7 prevents the decline of circulating CD4+T cells during the acute phase of SIV infection in rhesus macaques[J].PLo S Pathog,2012,8(4):e1002636.
[15]冮宏生,彭定凤,胡勇钧,等.白细胞介素-7调控T细胞表面TIM-3的表达在冠状动脉粥样硬化性心脏病中的作用[J].免疫学杂志,2017,33(12):1040-1046.