TLR2通过microRNA调节Th17细胞对A型链球菌的免疫保护的功能和机制研究

Group A Streptococci (GAS) are important human pathogens that threaten human health and life qualitiy.However, experimental vaccines cannot cover all of the GAS serotypes. Our group first demonstrated that CD4+IL-17+ T cells are the dominant T cells induced in mouse nasal-associated lymphoid tissue (NALT) by intranasal (i.n.) infections, but how the recognition of GAS by innate immune cells dictate the type of T cell responses has not been reported. In preliminary studies using a secondary infection model, we found that Toll-like receptor 2 knockout mice (TLR2-/-) had a defect in clearing GAS and a significant decrease of NALT Th17 cells compared with wildtype littermated (WT).We hypothesize that the induction by GAS of dentric cells (DCs) promote the poliarization of CD4+IL-17+ T cells through regulating TLR2-NF-κB signal pathway.Moreover, microRNAs may function as regulators in this process.In this study，mucosal infection of TLR2-/- and WT C57B/6 mice, and the in vitro co-culture of DCs and CD4+ T cells from both NALT and cervical lympho nodes (CLNs) will be used as modles. The role of TLR2 actication in regulating Th17 cell poliarization were assesd by Flow cytometry, ELISA and Realtime-PCR in vivo and in vitro, respectively.Moreover, the mechanism that microRNAs employs to regulate GAS activated TLR2 signal pathway will also be studies by loss and gain-of function studies in DCs. In conlusion, this study will analzye the effect of TLR2 activation on Th17 cell poliarization, as well as the mechanism of miRNAs in this process, thus providing sight into new targets for the prevention and treatment of GAS-related recurrent diseases.

A型链球菌（GAS）感染导致的疾病严重影响人类健康，目前尚没有对所有血清型GAS感染保护的疫苗。本组首次发现鼻腔黏膜感染GAS诱导Th17免疫应答增强。但天然免疫细胞如何识别GAS并引起T细胞免疫反应还未知。预实验显示，与WT小鼠相比，TLR2-/-小鼠在GAS二次感染时清除细菌能力显著降低，鼻相关淋巴组织中Th17细胞数量相应减少。我们推测GAS感染活化树突细胞表面的TLR2，改变TLR2-NF-κB通路的活性从而促进感染部位CD4+IL-17+细胞的分化,该通路中已知的microRNA可能在其中起到调控作用。本研究拟利用TLR2-/-小鼠和GAS黏膜感染模型，以及树突细胞体外刺激和T细胞共培养实验，采用流式、ELISA和实时定量PCR的方法分别从体内和体外观察树突细胞表面TLR2活化在感染后对Th17细胞分化的中的功能和miRNA对这一过程的调控，为GAS感染及复发的防治提供新靶点。

Toll样受体2（Toll like receptor 2, TLR2）主要识别革兰氏阳性细菌细胞壁的肽聚糖成分，目前还没有报道明确证明TLR2与A型链球菌之间的联系。虽然TLR4被认为主要识别革兰氏阴性细菌，近期的报道和我们都发现A型链球菌感染也可激活该受体。A型链球菌可反复感染人类并引起相关的自身免疫性症状，提示对该菌的免疫反应调控很复杂而TLRs可能参与其中。在三年的研究中我们利用了TLR2和TLR4敲除小鼠，主要探索了天然免疫细胞，主要是树突细胞表面的TLR2和TLR4对A型链球菌的识别所引起的天然免疫反应与适应性免疫反应。我们的研究为鼻腔黏膜A型链球菌的感染机制提供以下信息：1）树突细胞上的TLR2和TLR4均可识别A型链球菌，这两个受体的活化进而促进树突细胞活化和炎性细胞因子的分泌；2）天然免疫反应中，TLR2对于A型链球菌初次感染的清除必不可少，TLR2依赖的中性粒细胞效应是清除细菌的关键；3）TLR2影响A型链球菌诱导的T细胞免疫反应，即A型链球菌反复感染诱导的过量Th17反应在TLR2敲除小鼠中降低约50%；4）TLR2抑制分泌型IgA和系统型IgG的产生，同时降低滤泡T细胞（Tfh）和GC B细胞的数量，并且这种抑制只针对于A型链球菌偶联的抗原。但是TLR2的存在并未增加对二次感染的免疫保护。综上，我们的研究发现TLR2的存在导致过量Th17的产生，从而在不增加二次感染后细菌的清除效率的基础上增加了A型链球菌相关自身免疫性疾病发生的风险。此外，TLR4在清除A型链球菌的初次感染，以及A型链球菌反复感染引起的T细胞反应中没有作用。但是抗A型链球菌IgA的分泌和二次感染GAS的清除需要TLR4的存在。我们的研究阐明了TLR2和TLR4在A型链球菌感染中未被认识的功能，从而可被运用于A型链球菌感染及相关疾病的预防，以及疫苗开发中。