柔嫩梭菌诱导Breg(IL-10)增多在哮喘气道炎症中的作用及机制研究

Asthma is one of the most common chronic inflammatory diseases of the respiratory system, its pathogenesis has not been fully elucidated. Previous studies have shown that the reduction of regulatory B cell (Breg) can lead to the enhancement of the immune inflammatory effect, and promote chronic airway inflammation, but the mechanism of Breg in asthma is not clear. In the clinical study, we found that the expression of Clostridium leptum (CL) was significantly decreased in the patients with low Breg expression, which suggested that the decrease of the number of Breg might be associated with CL. However, it is necessary to study the effect of Clostridium leptum on the differentiation of Breg and its infiltration in lung tissue, which may affect the inflammatory response. On the basis of previous studies, the project plan is as follows: First, we compare the numbers of Clostridium leptum and Breg of asthma patients and health control, and determine the relationship between Clostridium leptum and Breg in asthma. Secondly, we co-culture Breg with CD4+CD25-T cells and Treg cells respectively, establish the IL-10-/- mouse asthmatic model, and transfer sorted Breg cells to mice during the process of establishing the asthmatic model to determine the effect of CL on Breg differentiation and the role of CL in regulation of asthma immune network, and then investigate the possible mechanism of CL reduction airway inflammation and airway hyper responsiveness in asthma. With above studies, we hope to explore a new strategy to intervene in the development of asthma by CL induced Breg.

哮喘是最常见的呼吸道慢性炎症性疾病之一，其致病机制尚未完全阐明。既往研究表明调节性B细胞 (Breg)减少可导致机体免疫炎症效应上扬，促使哮喘气道慢性炎症形成，但Breg在哮喘中的作用机制尚不明确。本课题组在临床研究中进一步发现哮喘患者在Breg低表达的同时，粪便中柔嫩梭菌(CL)数量也显著减少，提示Breg数量减少可能与柔嫩梭菌相关，但肠道柔嫩梭菌是否会通过调控Breg分化及其在肺组织的浸润，进而影响哮喘炎症反应需深入研究。本项目拟在此基础上，进行如下研究：首先对比哮喘患者和正常个体体内Breg和肠道中CL数量，明确二者和哮喘的关系；然后构建小鼠哮喘模型和体外细胞混合培养体系，利用在体基因敲除及Breg回输，调控Breg数量，对比CL添加与否的差异，探讨CL诱导Breg增多、调控哮喘免疫网络，进而减轻哮喘气道炎症的作用及可能机制。探索以CL诱导Breg为切入点干预哮喘发生发展的新策略。

哮喘是最常见的呼吸道慢性炎症性疾病之一，近20年来其发病率在全世界均呈上升趋势，为社会和家庭带来了沉重的精神压力和经济负担。哮喘的主要致病机制是免疫细胞功能紊乱，其中调节性细胞数量减少及功能降低是其中的关键环节之一，但目前缺乏有效的调节性细胞诱导手段。近年研究发现柔嫩梭菌可显著干预免疫细胞功能，进而影响免疫相关性疾病的进程。因此，我们在前期研究的基础上，较为开创性地在临床→动物→细胞水平三个层面进行研究，首先对比哮喘患者和正常个体体内Breg和肠道中CL数量，明确二者和哮喘的关系；然后构建小鼠哮喘模型和体外细胞混合培养体系，利用在体基因敲除及Breg回输，调控Breg数量，对比CL添加与否的差异，探讨CL诱导Breg增多、调控哮喘免疫网络，进而减轻哮喘气道炎症的作用及可能机制。探索以CL诱导Breg为切入点干预哮喘发生发展的新策略。结果显示：①哮喘患者外周血中Breg细胞百分比和肠道内柔嫩梭菌数量均较健康对照显著减少，并且二者呈正相关。②动物模型中发现：HDM哮喘小鼠肺脏中的Breg细胞和Treg细胞均较正常对照显著减少，并且二者呈正相关；Breg数量减少可加重HDM致敏小鼠的气道炎症和气道高反应性，并且这一效应由IL-10介导，而口服柔嫩梭菌可通过增加Breg数量抑制哮喘气道炎症；给予HDM哮喘小鼠回输Breg细胞，可显著抑制哮喘小鼠肺组织中效应性T细胞Th1，Th2，Th9，Th17细胞亚群数量和相关细胞因子IL-4，IL-5，IL-13，IFN-γ，IL-9，IL-17等的表达，同时，可增加抑制性细胞因子TGF-β和IL-35的表达；并且与口服柔嫩梭菌小鼠体内分离获得的Breg细胞与野生型小鼠体内分离获得的Breg细胞相比，具有更强的抑制效应。③细胞水平发现，Breg可抑制共培养体系中CD4+CD25-T细胞向效应性T细胞亚群Th1、Th2、Th9和Th17分化，同时抑制相应的促炎因子产生；并且将Breg与Treg共培养后，Breg可促进CD4+CD25+T细胞中Foxp3mRNA和蛋白的表达，并且这一效应依赖于细胞-细胞间的直接接触。.上述结果共同表明：柔嫩梭菌可通过诱导Breg（IL-10）这一途径减轻哮喘气道炎症，降低气道高反应性。.