磷脂酸在肺上皮细胞应对烟曲霉感染过程中的功能研究

Invasive pulmonary aspergillosis is characterized with multifocal tissue necrosis, difficult curement and high mortality. Aspergillus fumigatus (A. fumigatus) is the major pathogen of this disease and its conidia are able to induce the internalization into and strong innate immune response of lung epithelial cells. To date the exact regulation mechanism on this process is poorly understood. As a key precursor of all phospholipid, phosphatidic acid (PA) is an important second messenger in the cell and plays a critical role in many physiological events, such as vesicle-mediated transport, cytoskeleton organization, growth and differentiation; however, the role of PA in the infection of A. fumigatus into lung epithelial cells is still unknown. We have demonstrated that A. fumigatus can stimulate the activity of phospholipase D, which is one of the key producer of PA. Thus, in the project we will further study the alteration of PA concentration and subcellular distribution, and its interaction with the phagosome mature process. The effect of PA modulation on the internalization of A. fumigatus into lung epithelial cells and the release of inflammatory factors during the interaction will be also investigated. At the end, we will analyze in detail the possible effect of PA on the pathogenicity of A. fumigatus in immunosuppressed mice. By this study the role of phosphatidic acid signal during A. fumigatus infection might be elucidated for better understanding on the innate immunity of epithelial cells against A. fumigatus infection and possible candidate as drug target against invasive pulmonary aspergillosis.

侵袭性肺曲霉病肺组织破坏严重，治疗困难，致死率很高。烟曲霉是该病的主要病原菌，其孢子可诱发肺上皮细胞对其"吞噬"及释放炎症因子等天然免疫应答，该过程的具体调控机制远未阐明。人体细胞内磷脂代谢的关键前体-磷脂酸（PA）是重要的第二信使分子，参与调控胞吞、生长及分化等重要生理功能，但其在肺上皮细胞应对烟曲霉感染中的功能尚不清楚。磷脂酶D是细胞内PA生成的重要调控蛋白之一，我们已证实其参与调控烟曲霉内化侵入肺上皮细胞。本项目将在此基础上延续研究PA在烟曲霉侵染肺上皮细胞过程中含量和分布变化规律及其与吞噬体成熟的相互作用关系，分析PA对烟曲霉侵染效率以及上皮细胞炎症因子释放等的调控规律，并利用免疫缺陷小鼠模型探讨PA对烟曲霉致病性的影响。项目的完成对揭示磷脂信号在烟曲霉感染过程中的作用，透彻理解肺上皮细胞应对病原真菌感染的天然免疫机制具有重要意义，也为寻找新的药物靶点提供一定理论基础。

磷脂酸(phosphotidic acid, PA)是细胞内磷脂代谢的关键前体，也是重要的第二信使分子之一，参与调控胞吞、胞吐、囊泡运输、细胞骨架重组、细胞迁移、生长、分裂、分化等多种生理活动，已成为重要的药物研究靶点之一；磷脂酶D（phospholipase D, PLD）是细胞内PA生成的重要调控蛋白之一，前期我们已证实其参与调控烟曲霉内化侵入肺上皮细胞。但人们对其参与天然免疫应答的调控机制的理解仍十分有限。本研究在前期研究基础上，延续深入探讨磷脂酸及磷脂酶D在小鼠侵袭性肺曲霉病模型中对烟曲霉致病性的影响规律。研究结果如下： 首先体外实验表明烟曲霉侵染过程中，宿主细胞的PA及催化其生成的PLD有助于烟曲霉孢子内化侵入肺上皮细胞。外源PA也显著促进烟曲霉孢子侵入肺上皮细胞。在肺上皮细胞应对烟曲霉感染过程中，PLD及PA显著影响炎症因子IL-8、MCP-1及β-防御素的释放。体内实验表明在免疫功能正条件下，pld基因敲除降低巨噬细胞的杀烟曲霉孢子的能力；在醋酸氢化可的松诱导的免疫缺陷条件下，pld基因敲除小鼠比野生小鼠更易感染烟曲霉死亡，但是pld基因敲除并不影响肺组织中巨噬细胞的减少和中性粒细胞的增加，即两种小鼠肺组织中巨噬细胞和中性粒细胞变化趋势基本一致；在环磷酰胺诱导的免疫缺陷条件下，pld基因敲除小鼠比野生小鼠也更易感染烟曲霉死亡，而环磷酰胺诱导免疫缺陷模型是中性粒细胞减少症模型。以上结果表明，在免疫缺陷条件下，pld基因敲除小鼠比野生小鼠更易感染烟曲霉死亡的主要原因不是中性粒细胞的功能缺陷，很可能是巨噬细胞的功能缺陷导致的。另外，肺上皮细胞的天然屏障功能可能在pld基因敲除小鼠抵抗烟曲霉感染过程中发挥主要作用。项目的完成将拓展人们对磷脂信号参与天然免疫应答机制的认识，对进一步解析肺上皮细胞应对烟曲霉感染的天然免疫机制以及阐明病原体与宿主细胞相互作用的复杂方式具有重要意义，也为研究新型抗真菌药物提供一定理论基础。