Dectin-1在curdlan诱导的慢性肉芽肿病高炎症反应中的作用机制

Role of Dectin-1 in Hyperinflammation of Chronic Granulomatous Disease Induced by Curdlan

慢性肉芽肿病(CGD)是一种罕见的遗传免疫缺陷综合症.CGD病人最常见为NADPH氧化酶2(NADPH oxidase 2,NOX2)功能缺失造成吞噬细胞内活性氧生成障碍,不能清除外源的细菌与真菌的感染.凝胶多糖curdlan属于1,3-β-葡聚糖,是白色念珠菌细胞壁的主要成分.目前,作为吞噬细胞中主要的模式识别受体,凝集素dectin-1是否参与curdlan诱导巨噬细胞参与免疫炎症反应以及其分子机制不十分清楚.为了研究这一机制,我们采用luminol和Amplex Red法检测活性氧生成水平,ELISA检测炎症因子IL-1β水平以及免疫荧光法检测NF-κB信号通路的激活情况.结果表明,curdlan浓度依赖性上调巨噬细胞活性氧生成,NOX2缺失显著降低活性氧产生,dectin-1缺失部分降低活性氧的生成和部分阻断NF-κB信号通路的激活.NOX2 KO小鼠炎症因子IL-1β水平显著升高,Dectin-1缺失后NOX2 KO小鼠IL-1β水平降低;Dectin-1 KO小鼠的IL-1β水平与WT小鼠比无显著差异,与Dectin-1/NOX2 KO小鼠比差异显著.上述结果提示,curdlan刺激下机体通过巨噬细胞内的dectin-1受体诱导免疫应答,激活NF-KB信号通路释放炎症因子IL-1β,同时通过dectin-1受体激活NOX2释放活性氧清除病原,促进机体修复.在这一过程中dectin-1不是唯一参与的受体.

Chronic granulomatous disease（CGD） is an inherited disorder of the immune system due to mutations in gp91 phox gene which coding for NOX2 protein. Bacterial and fungal infections are most common infections in CGD patients. β-glucan（curdlan） is the main component of the cell wall of Candida albicans. At present, the molecular mechanism of dectin-1 in macrophage which involved in immune inflammation induced by curdlan is not very clear. In order to study the molecular mechanism, we estimated the level of ROS production by luminal and Ampled Red; estimated levels of IL-1β by ELISA; and estimated NF-κB signaling pathway by immunofluorescence assay. Our results showed that curdlan induced ROS production in macrophage in a dose-dependent way. Dectin-1 deficiency can partially reduce ROS generation and partially block the activation of NF-κB signaling pathway. NOX2 deficient mice displayed high level of IL-1β after curdlan injection and IL-1βlevel decreased in NOX2 KO mice when dectin-1 was knocked out. IL-1β level in Dectin-1 KO mice was as same as in WT mice while significantly different from Dectin-1/NOX2 KO mice. In summary, curdlan can activate NOX2 to generate ROS through recognition of foreign pathogens by receptor dectin-1 which existed in macrophage cell surface. Besides that, immune response induced by curdlan activated NF-κB signaling pathway and increased IL-1β level through dectin-1. Dectin-1 was involved in this process, but it was not the only pattern recognition receptor.