呼吸道合胞病毒感染支气管上皮细胞介导IL-8致Th17/Treg失衡

Th17/Treg imbalance mediated by IL-8 in RSV-infected bronchial epithelial cells

目的:探讨呼吸道合胞病毒(respiratory syncytial virus,RSV)感染导致哮喘易感性增加的机制,观察人支气管上皮细胞感染RSV后IL-8的表达,以及IL-8对Th17/调节性T淋巴细胞(regulatory T cells,Treg)分化的调节作用。方法:将人支气管上皮细胞(human bronchial epithelial cells,HBECs)分为对照组和RSV感染组,构建并验证RSV持续感染HBECs模型,real-time PCR检测对照组和RSV感染组中IL-8 m RNA的表达;ELISA检测感染细胞上清液中IL-8的浓度。提取健康人外周血淋巴细胞并将其分为空白对照组和IL-8作用组,参照ELISA检测到的浓度将IL-8作用于淋巴细胞24 h,采用流式细胞仪检测淋巴细胞中Th17和Treg亚群的分布情况。结果:本实验成功构建RSV持续感染HBECs模型,感染后的细胞仍能够继续分裂传代,并可检测到RSV持续存在的证据。免疫荧光显示细胞内有RSV致病蛋白的荧光表达;电镜下观察到感染细胞的线粒体水肿和内质网扩张,出现核周裂隙以及合胞现象,细胞核、胞浆内有病毒颗粒分布。Real-time PCR和ELISA分别检测到RSV感染组细胞内IL-8 m RNA表达和上清液中IL-8的分泌水平均高于对照组(均P<0.05)。进一步将IL-8作用于淋巴细胞,流式细胞学结果表明IL-8作用组淋巴细胞中Th17亚群比率增高(P<0.05),但Treg亚群比率与对照组比较,差异无统计学意义(P>0.05)。结论:RSV感染气道上皮细胞后可通过过度分泌IL-8而引起Th 17/Treg亚群分化异常。

Objective：To explore the mechanisms for an increase in susceptibility of asthma induced by respiratory syncytial virus（RSV）,to observe the expression of interleukin-8（IL-8）in human bronchial epithelial cells（HBECs） after RSV infection and to invesigate the regulatory effect of IL-8 on Thl7/Treg differentiation.Methods：HBECs were divided into a control group and a RSV infected group.The RSVE-infected model of HBECs was established and examined.The expression of IL-8 mRNA was detected by real-time PCR,and the levels of IL-8 were measured by ELISA.Peripheral blood lymphocytes in healthy people were extracted and divided into a control group and an IL-8 treatment group.Based on concentration of IL-8 in RSV-infected HBECs,lymphocytes were treated by a matched concentration of human recombinant IL-8 for 24 h.The distribution of Thl7 and Treg subsets in lymphocytes were examined by flow cytometry.Results：The RSV-infected HBECs model was successfully established.The infected HBECs were still able to split and passage.The RSV could be detected in every passage in the infected cells.Virus particles indicated by bright yellow green fluorescence were seen under fluorescence microscope.Edema of mitochondrias,expansion of endoplasmic reticulum,fissure around nucleus and intracellular virus particles were all observed under electron microscope.The expression IL-8 mRNA were significantly enhanced in the RSV-infected group,and the level of IL-8 in the RSV-infected group was higher than that in the control group（P〈0.05）.After IL-8 treatment for 24 h,the ratio of Thl7 subsets in lymphocytes were dramatically increased compared to the control group（P〈0.05）,but there was no difference in the ratio of Treg subsets between the 2 groups（P〉0.05）.Conclusion：Over-secretion of IL-8 by the RSV-infected HBECs may promote the differentiation of Thl7 subsets and maintain the Thl7/Tred imbalance.