973项目发现炎症小体识别新生隐球菌感染新机制

　　

　　11月15日，国际免疫学期刊Journal of Immunology 在线发表了中国科学院上海巴斯德研究所孟广勋课题组的最新研究成果Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes (《内吞的新生隐球菌激活经典的caspase-1和非经典的caspase-8炎症小体》)。该研究发现了NLRP3炎症小体识别胞内新生隐球菌感染后介导炎性细胞因子IL-1b成熟和细胞死亡的新机制。

　　新生隐球菌（Cryptococcus neoformans）是一种常见的机会性致病真菌，可在正常人群和免疫缺陷者中感染引起隐球菌病，每年可造成60多万艾滋病（AIDS）患者死亡。新生隐球菌的细胞壁外有一层荚膜，能够抵抗宿主免疫细胞的吞噬，但目前对于新生隐球菌感染的致病机制和宿主免疫反应机制的研究仍较为缺乏。NLRP3炎症小体是以NLRP3、ASC、Caspase-1为核心蛋白组成的大分子复合体，主要功能是识别外源感染和内部损伤等危险信号，诱导细胞因子IL-1b和IL-18的分泌。NLRP3炎症小体广泛参与识别多种病原体，前期研究已发现，生物膜和荚膜缺陷形式的新生隐球菌能够激活炎症小体（Cell Research, 2013; Microbes Infect. 2014）。

　　上海巴斯德所天然免疫课题组研究生陈明宽等在研究员孟广勋指导下，发现新生隐球菌野生株在抗体或补体调理作用下能够被宿主免疫细胞吞噬，激活经典的NLRP3-ASC-caspase-1炎症小体。在caspase-1缺失的条件下，新生隐球菌激活了非典型的NLRP3-ASC-caspase-8炎症小体，介导IL-1β的成熟和宿主细胞的死亡。进一步研究发现，新生隐球菌感染激活经典和非经典的炎症小体都依赖于吞噬溶酶体膜的渗漏和钾离子外流。此外，酵母聚糖在caspase-1缺失条件下，也能激活非经典的NLRP3-ASC-caspase-8炎症小体。研究结果揭示了宿主天然免疫系统识别新生隐球菌感染的新通路，也提示调控NLRP3信号可能帮助宿主控制隐球菌病。

　　该研究得到了国家自然科学基金委面上项目、科技部“973”项目和农业部重大专项等经费支持。

　　图1. A-B，来源于野生型小鼠的BMDC与新生隐球菌H99菌株共培养，并加入荚膜成分GXM抗体调理，免疫荧光法检测ASC聚合点的形成（A），或者检测TUNEL和ASC聚合体的形成（B)。C-D，来源于Nlrp3, Asc, Caspase-1等基因缺陷的小鼠和野生型小鼠的BMDC与新生隐球菌共培养6小时，检测上清中IL-1β水平（C），细胞裂解后用caspase-8抗体进行蛋白免疫印迹检测（D）。

图2. 真菌病原激活经典和非经典NLRP3炎症小体模式图

原文摘要：

Internalized Cryptococcus neoformans Activates the Canonical Caspase-1 and the Noncanonical Caspase-8 Inflammasomes

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococccosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also activated the canonical NLRP3–apoptosis-associated speck-like protein containing a CARD (ASC)–caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3–ASC–caspase-8 inflammasome as well, which resulted in robust IL-1β secretion and cell death from caspase-1–deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans–induced activation of either the canonical NLRP3–ASC–caspase-1 inflammasome or the noncanonical NLRP3–ASC–caspase-8 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3–ASC–caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge.