CCR4/TARC信号轴调节支气管哮喘气道Treg/Th17细胞浸润进而调控气道炎症的分子机制

In recent years, allergic asthma has considerably increased in prevalence worl dwide. It can not be cured, but it is preventive and can be controled. The disease is characterized by airway hyperresponsiveness(AHR) and chronic mucosal inflammation mediated by CD4-positive Th2 cells. Peoples always consider that the phenotype of asthma depends on the balance between Th1 and Th2 cells. Recently, more and more studies including our previous researches have found that Treg and Th17 cells also involved in the asthmatic airways inflammatory process, but it has no any report can illuminate the molecular mechanisms how Treg and Th17 cells to work in asthmatic airways inflammatory up to day. Our preliminary research had found that the vast majority of pleural CD4-positive CD25-positive T cells expressed CCR4 on their surface, and we also found that one of the ligands for CCR4-the Thymus and Activation-regulated Chemokine (TARC) level were found increase in bronchoalveolar lavage fluid(BALF) of asthmatic subjects. It is report that bronchial epithelial cells can produce TARC, we speculated that CCR4/TARC signal recruits Treg and Th17 cells to asthmatic airways and promotes airways inflammatory. Our project is aimed to explore the molecular mechanism that Treg and Th17 cells promoting asthmatic airway inflammation through clinical trials and asthmatic models in mice.

哮喘的本质是由CD4+细胞介导的慢性气道非特异性炎症。既往研究发现哮喘的表现型取决于Th1及Th2细胞之间的平衡，最近越来越多的研究包括我们的前期研究均发现Treg细胞也参与到哮喘的气道炎症过程，且该过程涉及Treg细胞及Th17细胞的平衡。目前尚未见有报道引起哮喘气道Treg细胞及Th17细胞平衡变化的分子机制，我们的前期研究已发现过敏性哮喘急性发作期患者外周血Treg细胞数目高于过敏性哮喘稳定期及健康对照者，并发现哮喘患者支气管肺泡灌洗液（BALF）中Th17细胞相关细胞因子IL-17A及胸腺激活可调节趋化因子（TARC）升高，TARC对CD4+细胞有趋化作用。有报道支气管气道上皮细胞能产生TARC，我们推测涉及气道上皮细胞的CCR4/TARC信号轴参与调节Treg细胞及Th17细胞向气道浸润，本项目将通过临床试验及哮喘动物模型验证CCR4/TARC信号调控气道炎症的分子机制.

本项目临床研究部分通过采集支气管哮喘稳定期、急性发作期和正常健对照者BALF及外周血，分离淋巴细胞，完成流式细胞术检测CD4+CD25+Treg细胞及Th17细胞表达，并完成细胞表面CCR4受体检测，完成ELISA法检测BALF上清液及血清趋化因子TARC及细胞因子IL-17A浓度，发现哮喘患者外周血中TH17细胞及Treg细胞表面CCR4受体表达均高于健康对照者，且哮喘患者BALF中TH17细胞及Treg细胞表面CCR4受体表达均高于患者自身外周血。进一步通过制备哮喘动物模型探讨CCR4/TARC信号轴调控CD4+ CD25+Treg细胞/Th17细胞向气道浸润并进一步影响气道炎症，同时完成小鼠气道上皮细胞分离并检测TARC蛋白及mRNA表达，探讨气道上皮细胞在CCR4/TARC信号轴中的作用。结果提示外周血中哮喘组CD4+CD25+Foxp3+Treg占CD4+T淋巴细胞的比例是(3.73%±0.58%)低于对照组(10.05%±2.43%)，差异有统计学意义(p﹤0.01)；BALF中哮喘组CD4+IL-17A+T细胞占CD4+T淋巴细胞的比例(2.12%±0.69%)高于对照组(0.93%±0.41%)，差异有统计学意义(p﹤0.01）。同时Treg/Th17细胞的比值哮喘组(1.95±1.34)低于对照组(13.43±7.65) ，差异有统计学意义(p﹤0.01)。在BALF中，TARC的浓度较哮喘组高于对照组，分别为：哮喘组108.27±62.16 pg/ml，对照组41.07±18.95 pg/ml，差异有统计学意义(p＜0.01)。用RT-PCR检测小鼠气道上皮细胞TARC mRNA表达：OVA致敏后小鼠气道上皮TARC mRNA表达上调。研究结果提示哮喘小鼠肺组织Treg/Th17细胞比例失调参与哮喘肺内炎症发病机制；阐明CD4+CD25+Treg细胞与Th17细胞在支气管哮喘气道炎症发生中的作用以及CCR4/TARC轴参与调控哮喘气道CD4+CD25+Treg细胞与Th17细胞平衡及哮喘气道炎症，为临床治疗支气管哮喘寻找新的途径提供理论基础。