Club细胞源性HMGB1在RSV感染后期Th2类细胞因子应答中的作用及其机制研究

In our previous study, Th2 cytokines increased significantly in broncholveolar lavage fluid induced by RSV at the late stage in Balb/c mice, which were associated with chronic airway inflammation and airway hyperresponsiveness. However, the mechanism of type 2 response is unclear. Given High Mobility Group Proteins (HMGB1) , an important member of endogenous injury associated molecular patterns, may be the key regulators of type 2 response. While our preliminary data showed that Club cell, a kind of epithelium with the function of self-renew, may generate HMGB1. Therefore, HMGB1, TLR4 knockout mice will be used and club cell depleting and promoting them to differentiate to ciliated cells will be done in this study to prove the following issues: 1) whether club cell was involved in type 2 response though HMGB1 production in the late stage of RSV infection; 2) whether HMGB1 could regulate transcription factor GATA3, mediating type 2 response though TLR4 signaling pathways; 3) effect of HMGB1 on severity of bronchiolitis and RSV-induced recurrent wheezing in clinical cases and evaluate the feasibility of HMGB1 being a biomarker. This study will further reveal the mechanism of RSV-induced chronic airway inflammatory disease, and identify a biomarker of RSV-induced recurrent wheezing and potential intervention targets.

课题组前期证实呼吸道合胞病毒（RSV）感染后期小鼠支气管肺泡灌洗液中Th2类细胞因子（简称II类因子）显著增高, 导致慢性气道炎症与气道高反应性（AHR），预实验发现：以Club细胞为代表的上皮细胞可能通过产生内源性损伤相关分子模式家族成员——高迁移率族蛋白(HMGB1)介导II类因子产生。本课题拟采用HMGB1、TLR4基因敲除小鼠，通过特异性去除或促进Club细胞分化、抗体阻断等证明：1.RSV感染后期Club细胞通过产生HMGB1介导II类因子升高导致气道炎症与AHR；2.HMGB1激活TLR4信号通路，增强GATA3表达，促进II类因子产生；3.明确HMGB1作为预测RSV感染毛细支气管炎患儿严重程度及后期发生反复喘息的生物学指标可行性。本研究将进一步揭示RSV感染后发生慢性气道炎症性疾病的机制，为寻找预测RSV感染后发生反复喘息的生物学指标及干预靶点提供重要的理论依据。

呼吸道合胞病毒（Respiratory syncytial virus, RSV）是全球范围内引起婴幼儿急性下呼吸道感染最重要、最常见的病毒病原体，生命早期的RSV感染是远期哮喘发生的独立危险因素。课题组前期研究证实RSV感染后期小鼠支气管肺泡灌洗液中Th2类细胞因子（简称II类因子）显著增高, 导致慢性气道炎症与气道高反应性（Airway Hyperresponsiveness，AHR），RSV感染后II类因子升高的机制需进一步研究。本研究通过建立萘特异性去除Club细胞、HMGB1中和抗体干预、TLR4特异性阻断剂干预的动物模型发现：1.RSV感染BALB/c小鼠第7天Club细胞大量破坏，第14、21天较对照组增多，予NA特异性去除Club细胞成功后，HMGB1水平降低，伴随气道炎症细胞降低，II类因子水平降低，提示Club细胞源性HMGB1参与RSV感染后期II类因子产生。2.RSV感染后期TLR4蛋白水平升高，阻断TLR4后II类因子相关气道炎症、AHR降低，提示TLR4参与RSV感染后期II类因子产生；但予HMGB1中和抗体干预后TLR4蛋白水平无明显变化，提示HMGB1参与的II类因子产生不依赖TLR4。通过对RSV感染毛细支气管炎患儿的临床观察随访发现，HMGB1水平与RSV感染毛细支气管炎患儿疾病严重程度评分呈正相关，但其水平对再喘息无预测作用。本研究揭示了RSV感染后发生慢性气道炎症的机制，为临床上干预RSV感染后持续气道炎症提供新靶点及理论依据。本课题相关结果已发表6篇SCI；培养博士研究生4名；在此基础上获得2项国家自然科学基金青年基金及1项省部级课题；成功举办1次国际学术交流会议及十余次国内交流会议。