肺炎支原体促发或加剧支气管哮喘免疫病理新机制：CARDS毒素调控Th细胞分化的作用

Mycoplasma pneumoniae (MP) infection is closely associated with the development of asthma and potential related to community-acquired respiratory distress syndrome (CARDS) toxin secreted by MP. However, the mechanism is unclear. Previous studies have demonstrated that costimulatory molecule B7-H3 is the key factor for Th cell differentiation. Our study shows that CARDS toxin could regulate Th2 and Th17 cell differentiation via expression of B7-H3 in macrophages and CARDS toxin combined with tumor necrosis factor (TNF)-α could induce higher expression of B7-H3. Thus， we presume that CARDS toxin regulate Th2 and Th17 cell differentiation via expression of B7-H3 in macrophages which lead to immunopathogenesis of asthma. In view of this theory, the aims of this study is, based on theNLRP3-/-, TNF-α-/- and B7-H3-/- mice, to explore the function and mechanism of B7-H3 expression in macrophages stimulated by CARDS toxin and /or TNF-α, as well as the effect of intervention of NLRP3, TNF-α or B7-H3 in vivo. The present study will provide new clue for treatment of immunopathogenesis of asthma caused by MP infection.

肺炎支原体（MP）感染与哮喘发生发展密切相关，可能与其分泌的CARDS毒素调控Th细胞分化有关，但机制不明。既往研究证实巨噬细胞表达的B7-H3是Th细胞分化的关键分子。本项目组预初实验发现CARDS毒素诱导巨噬细胞高表达B7-H3并调控Th2/Th17细胞分化，且TNF-α联合CARDS毒素可促进巨噬细胞表达更高的B7-H3。推测CARDS毒素通过介导B7-H3的表达而调控Th细胞分化进而参与哮喘免疫病理。鉴此，本项目拟借助NLRP3、TNF-α、B7-H3敲基因小鼠等手段，通过体内外试验进一步探讨1）CARDS调控巨噬细胞表达B7-H3的作用及机制；2）TNF-α促进巨噬细胞高表达B7-H3的作用及机制；3）体内NLRP3、TNF-α和B7-H3干预的临床治疗效果，为MP促发或加剧哮喘免疫病理的治疗提供新线索。

CARDS毒素是肺炎支原体特有的外分泌毒素，具有空泡变性和核糖基化功能。其在感染或哮喘中的作用还有待进一步阐述。本项目以巨噬细胞、上皮细胞以及树突状细胞为切入点，研究CARDS毒素调控巨噬细胞、上皮细胞以及树突状细胞的作用及在哮喘和感染中的作用机制。.本项目组首先构建了MPN372（CARDS毒素基因）真核生物表达载体，并纯化了CARDS毒素，与HELA细胞共育24h，结果发现，与medium对照组比较，CARDS毒素可以使宿主细胞空泡化，提示所构建的CARDS具有生物学功能。CARDS毒素刺激巨噬细胞高表达共刺激分子B7-H3。同时，CARDS毒素诱导小鼠肺部炎性细胞浸润及肺部高表达B7-H3。CARDS毒素刺激巨噬细胞后再与CD4+CD45RA+初始性T细胞共育，可以使得初始性T细胞GATA3、ROR-γt表达增加。其次，CARDS毒素可以促进Flt3L诱导的DCs调控Th1细胞分化，Th1细胞可以高分泌IFN-γ，而IFN-γ可以诱导巨噬细胞分泌CXCL9，进而CXCL9趋化Th1细胞至肺组织，从而形成一个I型免疫反应回路，提示I型免疫反应在MPP患儿中的重要作用。再次，CARDS毒素以浓度依赖的形式刺激上皮细胞A549高表达MUC5AC和MUC5B，提示CARDS毒素可能参与MPP患儿塑型支气管炎的形成。项目组获得了一株CARDS毒素的单克隆抗体MM03H，其能显著阻断荧光标记的CARDS毒素进入巨噬细胞，且存在浓度依赖性，提示单克隆抗体MM03H具有阻断功能。.综上，CARDS毒素具有较强的诱导炎症反应的能力，可作为重症/难治性支原体感染的生物标志物之一，深入研究其促炎机制为临床疾病干预提供新靶标。