马拉色菌感染角质形成细胞与皮脂腺细胞中NLRP1、NLRP3、NLRC4、AIM2炎症小体激活机制

Malassezia yeasts can activate the NLRP3 inflammasome in the dendritic cells, but it is unreported whether they stimulate the inflammasomes in the skin. In our preliminary observation, the immunoreactivity of NLRP1, NLRP3, NLRC4, AIM2, and IL-1β was increased in the skin lesions of Malassezia folliculitis, and the overproduction of NLRP3, Caspase-1 and IL-1β was present in Malassezia-treated HaCaT and SZ95 cells. These results suggest that Malassezia infection might mediate the release of IL-1β via activating NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in the keratinocytes and sebocytes, but their activation mechanism is unknown. This project intends to treat HaCaT and SZ95 cells with 2 Malassezia species, and then to determine the roles of K+, Ca2+, reactive oxygen species, lysosome rupture, and mitochondrial damage, as well as TLR2/4, Dectin-1/2 and NF-κB pathways in the activation of these inflammasomes using different techniques such as RNA interference, flow cytometry, confocal microscopy, transmission electron microscopy, etc. Meanwhile, the Malassezia susceptibility and inflammatory response will be compared among the cutaneous Malassezia infection models in Nlrp1b-/-, Nlrp3-/-, Nlrc4-/-, and Aim2-/- mice. Our results may not only be helpful to elucidate the pathogenesis of Malassezia-associated dermatosis, but also provide a theoretical foundation for exploring new therapeutic targets.

马拉色菌激活树突状细胞中NLRP3炎症小体，但是否激活皮肤中炎症小体未见报道。我们发现马拉色菌毛囊炎皮损中NLRP1、NLRP3、NLRC4、AIM2、IL-1β表达增加，马拉色菌处理HaCaT和SZ95细胞上调NLRP3、Caspase-1、IL-1β表达，提示马拉色菌感染可能激活角质形成细胞和皮脂腺细胞中NLRP1、NLRP3、NLRC4、AIM2炎症小体介导IL-1β释放，但激活机制不明。本项目拟用2种马拉色菌感染HaCaT和SZ95细胞，采用RNA干扰、流式细胞术、激光共焦显微镜、透射电镜等方法，探讨K+、Ca2+、活性氧、溶酶体破裂、线粒体病变及TLR2/4、Dectin-1/2、NF-κB通路在这些炎症小体激活中作用；构建4种炎症小体基因敲除小鼠皮肤马拉色菌感染模型，比较马拉色菌易感性和炎症反应差异。研究结果有助于阐明马拉色菌相关皮肤病发病机制，为探索新的治疗靶点提供理论依据。

免疫组化发现MF皮损中表皮及真皮炎症细胞NLRP3、NLRC4、AIM2、Casp-1、IL-1β、TLR2、TLR4、Dectin-1、Dectin-2、NFκB表达高于正常皮肤（P<0.01-0.05），而NLRP1表达无明显变化（P>0.05）。qRT-PCR显示皮损中NLRP3、Casp-1、IL-1β mRNA水平明显高于正常皮肤（P<0.01-0.05），而NLRP1、NLRC4、AIM2 mRNA水平仅有轻微增加（P>0.05）。结果提示NLRP3、NLRC4、AIM2炎症小体同时激活可能在MF发病中起重要作用。.392 株分离马拉色菌，进行基因鉴定、药物敏感性和胞外酶活性测定。发现本地区以糠秕马拉色菌和球形马拉色菌为主；糠秕马拉色菌多见于花斑糠疹和健康志愿者，而球形马拉色菌在MF中常见。马拉色菌对伊曲康唑、泊沙康唑、伏立康唑、酮康唑较敏感；伊曲康唑与特比萘芬、他克莫司与抗真菌药联合用药可能无临床应用价值。马拉色菌主要分泌磷酸酶、脂酶和蛋白酶；过度产生脂酶可能促进糠秕马拉色菌皮肤定植。.THP-1细胞用佛波酯诱导分化为巨噬细胞，再用不同感染复数（MOI）马拉色菌感染6 ~72 h。发现球形、糠秕马拉色菌感染激活THP-1细胞NLRP4、NLRP10和4个经典炎症小体（NLRP3、NLRC4、AIM2、pyrin）。球形马拉色菌感染可能优先激活NLRP3、NLRC4炎症小体，主要引起IL-18释放；糠秕马拉色菌感染可能主要激活AIM2、pyrin，主要引起IL-1β释放。.我们发现30 MOI球形马拉色菌激活HaCaT细胞中NLRP3炎症小体涉及第一信号分子（TLR2/4、Dectin-1/2）、第二信号分子（K+外流、Ca2+内流、溶酶体损伤、线粒体膜电位下降、ROS积聚）及Syk、NF-κB、Myd88信号通路活化。30 MOI球形马拉色菌激活SZ95细胞中NLRP3炎症小体涉及分泌蛋白酶、细胞吞噬、胞外高浓度K+、K+外流、活性氧产生及TLR2、Dectin-1活化。.此外，我们已构建Nlrp3-/-、Aim2-/-小鼠皮肤马拉色菌感染模型，发现Nlrp3-/-、Aim2-/-小鼠皮损愈合缓慢，皮损IL-1β、IL-18表达明显减少；提示Nlrp3、Aim2基因敲除增加小鼠皮肤马拉色菌感染易感性，减轻皮肤炎症反应。