肌动蛋白解聚因子cofilin在烟曲霉侵染肺上皮细胞及诱导炎症因子释放中的功能研究

Internalization of Aspergillus fumigatus (Af) into lung alveolar epithelial cells is the key process to escape the immune attack and consequently induce the invasive aspergillosis, which is dependent on the actin cytoskeleton rearrangement. Cofilin is one of the most important actin depolymerization factor which is essential involved in the infection process into host cells by several viruses and bacteria, such as HIV and Salmonella with different mechanism. In the previous work we demonstrated that the conidia of Af is able to stimulate phospholipase D activity that is a critical player in cellular signal transduction; meanwhile the internalization of Listeria monocytogenes, a typical intracellular pathogen into Vero epithelial cells is controlled by cofilin/PLD signaling cascade. Thus, in the present study we will study the distribution and alteration of cofilin activity and expression, and its role in Af internalization into lung epithelial cells and the release of inflammatory factor during the interaction. The possible receptors in epithelial cells or the pathogenic factors from Af itself which might mediate the cofilin activity will be also investigated. In the end, the role of cofilin on PLD activation by Af will be analyzed in detail. By this study the interaction between Af internalization and actin cytoskeleton regulation by cofilin might be elucidated for better understanding on the innate immunity of epithelial cells against Af infection.

烟曲霉孢子侵入肺上皮细胞是烟曲霉逃避免疫攻击，引发侵袭性肺曲霉病的必经步骤，这一过程依赖于肺上皮细胞肌动蛋白骨架的重排，但具体调节机制不清楚。肌动蛋白重排的重要调控因子cofilin在HIV、沙门氏菌等多种病毒及细菌感染细胞过程中发挥重要调控作用，但功能机制不尽相同。我们前期工作证实烟曲霉可激活肺上皮细胞中重要信号蛋白磷脂酶D，而胞内感染模式细菌-李斯特菌侵染上皮细胞受cofilin/磷脂酶D信号级联调控；因此本项目将研究烟曲霉侵染肺上皮细胞过程中cofilin表达及活性变化规律、分析cofilin对烟曲霉侵染以及诱导上皮细胞炎症因子释放的调控规律，探讨可能介导cofilin活性变化的受体及烟曲霉毒力因子，进而阐明cofilin/磷脂酶D信号对烟曲霉内化侵入的调控机制。项目的完成对揭示烟曲霉侵染肺上皮细胞的具体调控途径，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。

烟曲霉是一种重要条件致病真菌，广泛分布于自然界中，其分生孢子直径约 2~5μm，易在空气中悬浮而被吸入肺中并在机体免疫低下时引发感染。肺上皮细胞，尤其是 II 型上皮细胞是机体天然免疫的第一道防线，人们对其在应对烟曲霉感染天然免疫反应中的功能及调控机制所知甚少。Cofilin 是调节细胞肌动蛋白骨架重排的一类重要的肌动蛋白骨架解聚因子，其可切断肌动蛋白丝，调控肌动蛋白骨架的组装，参与细胞迁移、粘附、分裂及凋亡等重要生命活动。本项目研究结果显示，烟曲霉孢子显著诱导肺上皮细胞内cofilin mRNA水平升高，以及胞内cofilin磷酸化水平的短暂升高，并不影响胞内cofilin的蛋白表达量；且烟曲霉孢子诱导胞内cofilin磷酸化水平升高与孢子表面暴露的相关成分有关，但与孢子表面的β-1,3-glucan无关，该过程也不受宿主表面受体dectin-1和CR3直接介导。烟曲霉孢子内化侵入肺上皮细胞后，胞内磷酸化cofilin分布改变，聚集在孢子周围。然而我们发现胞内cofilin表达沉默、过表达或干扰磷酸化循环都降低烟曲霉内化侵入率，暗示烟曲霉孢子内化侵入与cofilin磷酸化循环平衡相关。进一步研究表明RhoA-ROCK-LIMK1-cofilin信号通路参与调控烟曲霉膨胀孢子诱导胞内cofilin磷酸化及其内化侵入肺上皮细胞过程。此外，细胞cofilin磷酸化循环改变对烟曲霉诱导的炎症因子释放有显著影响。烟曲霉分泌的重要毒素gliotoxin不仅显著诱导胞内cofilin磷酸化水平升高及分布改变，同时还诱导细胞肌动蛋白骨架重排。然后进一步证明了ROCK-LIMK信号参与调控gliotoxin诱导肺上皮细胞内cofilin磷酸化及肌动蛋白骨架重排过程。综上所述，在前期工作基础上，本项目深入研究了肌动蛋白解聚因子cofilin在烟曲霉感染肺上皮细胞过程中表达及活性规律，探讨了cofilin磷酸化循环与烟曲霉内化侵入肺上皮细胞的的分子机制以及对炎症因子释放的影响，阐明烟曲霉重要分泌毒素gliotoxin诱导肺上皮细胞形态变化的信号通路。这些工作对进一步理解病原真菌的致病机制具有重要意义，为防控和治疗烟曲霉感染提供了可靠的理论依据。