柔嫩梭菌诱导Treg/tDC分化在小鼠哮喘气道炎症中的作用及机制研究

Asthma is an inflammatory disease caused by allergen-specific T helper cell 2 (Th2) type responses. During the past several decades, the incidence of asthma is increasing dramatically in the world. Notably, regulatory T cells (Tregs) can suppress Th2 responses by secreting IL-10 and TGFβ. Previous studies have shown that the number and function of Tregs is impaired or altered in allergic patients compared with that in healthy individuals. Therefore, development of therapeutic strategies to increase the numbers and function of Tregs may be highly promising for the intervention of allergic disease. Interestingly, recent studies have shown that administration of certain organisms can modulate immune responses in the airway. This association is consistent with hygiene hypthesis, which posits that exposure microbes prevents the development of allergic diseases. In the journal of Science, Atarashi and colleagues report that inoculation of indigenous Clostridium species (especially for Clostridium leptum and coccoides) in the early lives of mice can promote anti-inflammatory immune responses by expanding and activating Tregs, and up-regulating Foxp3 expression, a key transcription factor in programming the differentiation of Tregs. The C. leptum is the highest population in the intestinal tract of the total number of intestinal bacteria. Our previously studies found, oral feeding with C. leptum increased the percentage and total number of Tregs in the spleens and mediastinal lymph nodes at 14 days post inoculation, and attenuated allergen-induced airway hyper-responsiveness and inflammation by inhibiting Th2 cytokine production, but enhancing IL-10 and TGFβ production in the lungs. Indicating that oral treatment with C. leptum can attenuate allergic airway inflammation in adult mice. However, the mechanism of oral C. leptum drive systemic Treg responses and modulate allergic airway inflammation has not been explored. More recent abservations indicate that tolerogenic DCs are also crucial to ensure immunological peace, at least in part, through control of Tregs. Here, this study is aimed at investigating the impact of oral administration of C. leptum on systemic Treg/tDC responses and allergic airway inflammation in a mouse model of asthma, and establish a cell model of CD4+ T cells and DCs in the presence/absence of C. leptum, to explore the effect of C.leptum on Treg amplification, and its possible mechanism.

免疫紊乱是导致哮喘发生的重要机制之一。随着"新卫生假说"的提出，机体缺乏微生物暴露，致调节性T细胞（Treg）低表达是免疫网络中导致哮喘气道炎症的始动因素，提示Treg是治疗哮喘的一个关键切入点。然而目前尚没有成熟建立的体内诱导Treg方法。2011年发表在Science的研究发现，增加小鼠肠道内柔嫩梭菌数量，可诱导Treg增殖；进一步研究表明耐受型DC（tDC）可能参与这一诱导机制。本课题组在预实验中也已证实口服柔嫩梭菌可诱导小鼠肺组织中Treg数量增多和功能因子Foxp3表达。因此本研究设想通过细胞培养和构建小鼠哮喘模型，采用流式、ELISA、实时定量PCR等技术深入分析柔嫩梭菌诱导Treg/tDC分化，进而减轻哮喘气道炎症和高反应性的作用及可能机制。为哮喘的防治提供新思路。同时有望以此为平台，进行口服柔嫩梭菌安全性、有效性及治疗哮喘预后评估的研究，探索以免疫学为基础的临床治疗途径。

哮喘是最常见的呼吸道慢性炎症性疾病之一，近20年来，哮喘的发病率在全世界均呈上升趋势，且这些病人大多为中青年和儿童，为社会和家庭均带来了沉重的精神压力和经济负担。尽管哮喘的发病机制仍不完全明了，但宿主免疫紊乱，导致调节性T细胞（regulatory T cells, Treg）数量减少及功能降低是其中的关键环节之一。而Atarashi 等于2011年在science上发表的研究显示，增加肠道内梭状芽孢杆菌数量（主要是柔嫩梭菌和球形梭菌）可诱导小鼠体内Treg反应。与此同时，耐受型树突状细胞（tolerogenic dendritic cell, tDC）在维持外周免疫自稳中的作用也日益受到重视。因此本研究首先在动物模型中确定CL对Treg/tDC分化的诱导作用，以及对哮喘小鼠气道炎症的抑制效应；然后进一步在细胞水平验证CL对Treg/tDC的诱导作用，并揭示CL、Treg和tDC的相互作用机制。.结果显示：OVA致敏小鼠体内存在Treg和tDC细胞数目减少及功能降低，从而促使辅助性T细胞Th1、Th2、Th17相关细胞因子的分泌增多，进而导致小鼠气道炎性细胞浸润减少、气道高反应性减轻；口服柔嫩梭菌可增加OVA致敏小鼠体内的Treg和tDC细胞数量，减少Th1、Th2、Th17相关细胞因子的分泌，减轻小鼠气道炎性细胞浸润和气道高反应性。在细胞水平，首先建立DC-CL共培养体系，结果发现CL可增加共培养上清中IL-12p40、IL-10和TGF-β的表达，但对IL-6的表达无影响；进一步建立DCCL-CD4+CD25-T共培养体系，DCCL可促进共培养体系中的CD4+CD25-T 细胞转化为CD4+ CD25+Foxp3+Tregs，并可促进共培养体系中的Foxp3 mRNA表达，并且，诱导产生的Treg的抑制功能显著强于原始Treg，而且Treg的抑制功能随Treg比例增高而增强；此外，DC细胞可显著增加共培养体系中效应性T细胞亚群Th1、Th2和Th17的百分比和相关细胞因子分泌，与DC+T cell组比较，DCCL+T cell组的T细胞亚群百分比和相关细胞因子分泌均明显减少，但仍然高于T cell对照组。.上述结果共同表明：柔嫩梭菌可通过诱导Treg/tDC这一途径减轻哮喘气道炎症，降低气道高反应性。