细胞自噬在肺上皮细胞应对烟曲霉感染及释放炎症因子过程中的功能研究

Aspergillus fumigatus, an important pathogenic fungus of invasive pulmonary aspergilosis is able to induce innate immunological response in pulmonary epithelial cells with internalization and even survival in the cells，which might be a major mechanism for the dissemination of its invasive infection. The precise regulation on this process is still unclear. Autophagy (usually macroautophagy) is an essential degradation pathway in eukaryotic cells to maintain cell homeostasis. It has been recently shown to play a critical role in regulation of host innate immunity in response to many bacterial and viral pathogens, especially in pathogen phagocytosis and intracellular elimination, however, its role and function may variable in different cells against different pathogen. We found that the internalization of A. fumigatus into pulmonary epithelial cells could induce phospholipase D (PLD) activation mediated by dectin-1 receptor in pulmonary epithelial cells. Interestingly, PLD has been shown to interact closely with autophagy signal pathway. Further, preliminary results showed A. fumigatus is indeed able to induce autophagy during its infection into A549 lung epithelial cells. Thus, we will investigate further the formation, translocation and distribution of autophagosome during A. fumigatus internalization into pulmonary epithelial cells and the possible modulators on autophagy (fungal factors and host receptor signal); elucidate the function of autophagy regulating invasion and release of inflammation factors, and analyze the relationship of autophagy with PLD signal. This proposal would help clarify the mechanism of innate immunity response of pulmonary epithelial cells against A. fumigatus internalization, especially the involvement of host autophagy, which might greatly potentiate the development of antifungal drugs.

引发侵袭性肺曲霉病的主要病原真菌烟曲霉可内化侵入肺上皮细胞并在其中存活较长时间，同时诱导细胞炎症因子释放，这是其形成侵袭性感染的重要机制之一，但该过程的具体调控机制仍不清楚。细胞自噬（通常指巨自噬）是真核细胞内维持稳态的重要降解通路，在清除病原体、释放炎症因子等宿主抗感染天然免疫应答中发挥重要作用，但机制不尽相同。我们前期研究发现，在烟曲霉感染过程中肺上皮细胞发生明显自噬，且细胞重要信号蛋白磷脂酶D可显著调控烟曲霉的内化侵入。而磷脂酶D活性可能与细胞自噬密切相关。在此基础上，本项目拟阐明烟曲霉感染过程中肺上皮细胞自噬发生及变化规律，以及自噬对烟曲霉内化侵入及诱导炎症因子释放的影响机制，寻找可能介导自噬的烟曲霉毒力因子及上皮细胞膜受体，揭示肺上皮细胞自噬与磷脂酶D的相互作用关系。这对深入解析肺上皮细胞应对烟曲霉感染的天然免疫应答机制具有重要意义，也为寻找可能的抗真菌药物靶点提供重要科学依据。

细胞自噬（通常指巨自噬）是真核细胞内维持稳态的重要降解通路，在清除病原体、释放炎症因子等宿主抗感染天然免疫应答中发挥重要作用，但机制不尽相同。我们前期研究发现，引发侵袭性肺曲霉病的主要病原真菌烟曲霉在感染肺上皮细胞过程中胞内发生明显自噬现象，且烟曲霉孢子可内化侵入肺上皮细胞并在其中存活较长时间，同时诱导细胞释放炎症因子，这是其形成侵袭性感染的重要机制之一，但该过程的具体调控机制仍不清楚。在此基础上，本项目拟阐明烟曲霉感染过程中肺上皮细胞自噬发生及变化规律，以及自噬对烟曲霉内化侵入及诱导炎症因子释放的影响机制，寻找可能介导自噬的烟曲霉毒力因子及胞内信号传导调控机制。结果显示，烟曲霉感染肺上皮细胞过程中，随着感染时间的延长，胞内自噬标志蛋白LC3-II水平逐渐显著升高，然而我们发现烟曲霉感染肺上皮细胞诱导的LC3-II水平升高效应既不同于传统自噬现象也不同于LAP（LC3-associated phagocytosis ）现象。因为肺上皮细胞内包裹烟曲霉孢子的膜结构均为单层膜结构，而自噬体为双层膜，而自噬起始蛋白ULK1表达沉默显著抑制烟曲霉诱导的LC3-II水平升高，LAP发生的必须蛋白Rubicon和PI3K表达沉默对烟曲霉诱导的LC3-II水平升高无明显影响。同时烟曲霉感染肺上皮细胞过程中，胞内NF-κB信号通路和MAPK信号通路均被激活，调控多种炎症因子的表达和释放。尽管ULK1、Rubicon和PI3K表达沉默均显著降低烟曲霉孢子额侵染能力，但只有ULK1表达沉默显著降低肺上皮细胞杀死烟曲霉孢子的能力，Rubicon表达沉默显著降低IL-8的释放，PI3K表达沉默显著降低细胞释放炎症因子IL-8和MCP-1的能力。此外发现烟曲霉表面的难溶大分子多糖可能是烟曲霉诱导支气管上皮胞内LC3-Ⅱ水平升高的关键刺激因子，表面疏水蛋白和黑色素可能参与LC3-ΙΙ表达过程。而烟曲霉自身分泌的毒力因子gliotoxin对LC3-ΙΙ表达和炎症因子表达调控无显著影响。PLD参与巨噬细胞吞噬和杀灭烟曲霉孢子过程，且发挥重要功能，但是pld基因的敲除并不影响烟曲霉诱导胞内LC3-Ⅱ水平的升高。