呼吸道细菌定植促进哮喘气道炎症与免疫失衡的研究

Sustained or recurrent bacterial infection in respiratory tract trigger recurrent attacks in patients with asthma (especially refractory asthma).Bacterial colonization is not only the basis of infection,but also a risk factor for asthma. Toll-like receptors (TLRs)can recognize exogenous microorganisms.TLRs are expressed on CD4+T cells subsets , and these cells can be directly activated by TLR agonists. Therefore, TLRs are found to act as adjuvant receptors that create a bridge between innate and adaptive immunity, and to have important roles in the induction of adaptive immunity. Based on the research literature and the applicant's preliminary studies, we propose a hypothesis that bacterial colonization of the respiratory tract could cause Thl7/Treg cells and their transcription factors Foxp3/RORγt imbalance,which may lead to chronic airway inflammation and airway hyperresponsiveness in asthma through TLRs signaling pathway.The pathogenesis of the Gram-positive and/or Gram-negative bacterial colonization of the respiratory tract on inducing the immune imbalances which leads to chronic airway inflammation, hyperresponsiveness and remodeling is studied in in vitro and in vivo experiments on a model of persistent colonization of respiratory tract in asthma mice by Streptococcus pneumoniae and/or Haemophilus influenzae. Furthermore, using TLR2 and TLR4 gene RNA interference(RNAi) technology and concurrent rescue of RNAi escape strategy construct(RESC), we investigate the the role of TLR2 and TLR4 signal pathway in asthma immune response and the mechanism of lentiviral vector-mediated TLR2 and TLR4 shRNA in the treatment of asthma. There will be profound theoretical significance and practical value of this research findings for the prevention of asthma and its immune targeted therapy.

呼吸道持续或者反复的细菌感染是导致哮喘（尤其是难治性哮喘）患者反复发病的原因。细菌定植既是感染的基础，也是哮喘的危险因素。Toll样受体（TLRs）可识别外源微生物，并在CD4+T细胞亚群上表达，是天然免疫和获得性免疫的桥梁。基于国内外文献及申请者的前期研究，我们提出设想，呼吸道定植菌通过TLRs信号通路，引起Treg／Thl7细胞及相应转录因子Foxp3/RORγt的失衡表达，从而导致哮喘的慢性气道炎症、气道重塑及气道高反应。本课题通过哮喘动物模型的体内外实验，研究肺炎链球菌和/或流感嗜血杆菌在呼吸道定植引起免疫失衡，导致慢性气道炎症及气道重塑的致病机理。结合使用基因干扰技术和RESC救援机制，明确TLR2及TLR4在哮喘免疫反应中的信号传递作用，以及慢病毒载体介导的TLR2及TLR4的shRNA治疗哮喘的机制，研究成果对于哮喘预防及其免疫靶向治疗具有重要的基础理论意义和应用价值。

呼吸道细菌定植是导致哮喘（尤其是难治性哮喘）患者反复发病、病情进行性加重的原因。本研究采用分子生物学、流式细胞仪、免疫组化等手段和在体、离体和细胞水平的小鼠哮喘模型，首次建立呼吸道细菌定植的哮喘动物模型，并发现：（1）呼吸道细菌定植加重了哮喘气道炎症、气道重塑和气道高反应，其机制与炎症介质释放有关；（2）呼吸道细菌定植加重了哮喘的免疫紊乱，使Treg／Th17及相应的转录因子Foxp3/RORγt表达失衡；（3）呼吸道革兰氏阳性菌和阴性菌的定植分别上调哮喘动物CD4+上TLR2和TLR4信号的表达，TLR的表达与Treg、Th17变化相关；（4）TLRs基因敲除将缓解上述病理改变和免疫失衡。上述结果证实了我们提出“呼吸道定植细菌通过TLRs信号通路，引起Treg／Th17细胞及相应转录因子Foxp3/RORγt的失衡表达，从而导致哮喘的慢性气道炎症及气道高反应”的假说，为哮喘的防治提供一个新的靶点。