TLR4信号强度决定新生鼠和成年鼠再次感染RSV后转归的机制研究

Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract diseases (bronchiolitis and pneumonia) during infancy and early childhood. RSV-F protein is one of the ligand of TLR4. Two polymorphic point mutations in the human TLR4 gene, Asp299Gly and Thr399Ile, have been associated with severe RSV disease in infants and young children. These mutations were linked with LPS hyporesponsiveness in vitro and in vivo. The applicant found previously that the initial infection in neonatal mice and adult mice resulted in Th2/Th1-biased immune responses, respectively. Dendritic cells play an important role in these age-dependent responses. The crosstalk between RSV and airway epithelial cells (AEC) regulates DC migration and maturation through epithelial cell-derived cytokines, such as IL-33. It has been shown that level of inhaled LPS signaling through TLR4 and the distinct TLR4-expressing cell compartment determines the type of immune responses and airway inflammation. Based on these observations, the applicant hypothesize that “the level of TLR4 signaling determines the sequel of age-related RSV infection.” The weak TLR4 signaling during neonatal RSV infection induces AEC to release IL-33, which in turn promotes Th2 polarization. Upon re-infection these mice will develop asthma-like responses. To test this hypothesis, the role of TLR4 in RSV re-infection, and by which mechanisms it exert its functions were investigated by using TLR4 knock out mice, DC adopted transfer experiments, cytokine and antibody treatment experiments. Our investigation will elucidate for the first time the effects of TLR4 signaling on the development of age-dependent airway inflammation and hyperresponsiveness.

呼吸道合胞病毒（RSV）F蛋白是TLR4的配体。研究显示：携带TLR4突变基因型（LPS低反应性）的儿童感染RSV易致严重下呼吸道感染。申请者前期工作发现新生鼠/成年鼠初次感染RSV后分别出现Th2/Th1应答偏移，且与肺脏DC密切相关。RSV与呼吸道上皮细胞（AEC）的对话，可通过释放细胞因子调控DC向肺组织的趋化和成熟。哮喘研究发现，TLR4既可引起Th1应答和炎症反应，又是Th2应答的必要条件，取决于TLR4信号强弱及其所在的细胞类型。据此提出假说：“TLR4信号的强弱决定不同鼠龄动物再次感染RSV后的转归”。新生鼠感染RSV产生的TLR4信号较弱，导致AEC分泌IL-33调节DC功能，促进Th2应答，再次感染后出现哮喘样反应。本项目拟利用基因敲除小鼠、过继转移实验、细胞因子和抗体干预手段，以再次感染后呼吸道反应作为主要检测指标，阐明TLR4参与不同鼠龄动物再次感染后转归的作用机制。

新生期/婴儿期是呼吸道病毒感染以及过敏性疾病发病的“窗口期”, 针对这一时期肺部免疫的年龄特点，建立合适年龄的动物模型，阐明该阶段病毒/过敏原进入机体后，与呼吸道上皮细胞、固有免疫细胞及适应性免疫细胞的相互作用，是研究肺部免疫相关疾病的当务之急。绝大多数儿童哮喘属于过敏性哮喘。源自呼吸道和免疫系统对环境中过敏原的高敏感性并产生过度反应，导致气道炎症和狭窄。“农场效应”是指农场长大的孩子不易患过敏性哮喘，“卫生假说”对此的解释是农场环境增加了接触各种微生物及其产物（如LPS）的机会，但机制尚未阐明。本研究首先发现致敏期LPS处理具有年龄依赖性差异效应：即在削弱OVA诱导的Th2型呼吸道过敏性炎症的同时，仅成年鼠诱导Th17偏移，而新生鼠LPS处理抑制Th2应答，但未诱发Th17偏移。目前认为肺部Th17细胞和中性粒细胞与更为严重的激素抵抗型哮喘有关，可能并不是有益的，这表明在“时间窗”内，致敏期LPS处理将使患者受益。继而，我们成功建立了模拟农场效应的新生期LPS暴露模型，首先证实了BALB/c小鼠新生期LPS暴露可抑制后续OVA诱导的呼吸道过敏性炎症。进而通过RNA测序技术，发现新生期LPS暴露可引起肺组织IRG1表达升高，并可持续至成年期。IRG1是线粒体相关酶，负责催化三羧酸循环中间产物顺乌头酸脱羧产生衣康酸。衣康酸是兼具免疫调节功能的代谢物，在巨噬细胞发挥多重抗炎作用。随后发现激发期给予衣康酸类似物（DMI）可抑制过敏性哮喘反应，由此将IRG1/衣康酸轴与“农场效应”联系在一起。另外，我们发现新生鼠肺脏pDC缺如是导致新生鼠哮喘反应加重的重要因素，肺脏pDC可通过分泌IFN-抑制上皮来源细胞因子的分泌，以发挥其负调控作用；最后，通过体外培养BMDC，我们发现品系和年龄可影响BMDC的表型、功能以及对LPS刺激的反应强度。本项目的顺利完成一方面深化我们对肺组织呼吸道上皮细胞、ILC2、DC以及免疫效应细胞（Th2）之间作用网络的认识，发现LPS与呼吸道哮喘样反应之间的年龄依赖性作用机制，另一方面发现IRG1/衣康酸轴可能为“农场效应”的机制之一，衣康酸也可作为治疗过敏性哮喘的潜在药物进行转化。