胶霉毒素gliotoxin促进烟曲霉内化侵入肺上皮细胞的机制研究

Internalization of Aspergillus fumigatus into lung epithelial cells with obvious cellular morphological change is one of the key steps of lung infection induced by A.fumigatus. This process may be regulated by both cell wall components of A. fumigatus and pattern recognition receptors of lung epithelial cells. But the function of toxins secreted by A. fumigatus in the process is still poorly understood. Gliotoxin (GT) is an important virulence factor of A. fumigatus, and has several immunosuppressive roles including inhibition on phagocytosis of macrophage and neutrophil, T cell proliferation and activation. However, our previous study confirmed that GT significantly promote A. fumigatus internalization into lung epithelial cells and induce the cellular actin cytoskeleton rearrangement accompanying with significant PLD activation. This phenomenon suggested that there maybe significant difference between lung epithelia cells and professional phagocytic cells in response to GT. On the basis of these findings, we propose to further investigate the special signal mechanism how GT promotes A. fumigatus internalization into lung epithelial cells and regulates the release of inflammatory cytokines in vivo and in vitro, respectively. Meanwhile, the signaling pathway from GT to PLD and the role of PLD signal on GT-induced internalization and cytokine-release will be explored as well. This proposal will help to extend the recognition on the role of GT in A. fumigatus infection and immune regulation, and will be of great importance in further clarifying the important role of lung epithelial cell in response to A. fumigatus infection and the development of potential drug targets.

烟曲霉诱发细胞形态改变进而内化侵入肺上皮细胞是其引发肺部感染的关键步骤之一，该过程可能受烟曲霉细胞壁成分和肺上皮细胞的模式识别受体共同调节，但烟曲霉外分泌毒素在该过程中的功能尚不清楚。胶霉毒素（gliotoxin，GT）是烟曲霉分泌的主要毒素因子，具有抑制中性粒细胞和巨噬细胞的吞噬及T细胞增殖和活化等功能。而我们前期研究发现，GT可显著促进烟曲霉孢子内化侵入肺上皮细胞，并诱发细胞肌动蛋白骨架重排和胞内磷脂酶D（PLD）蛋白激活。该现象提示肺上皮细胞对GT的应答机制可能与专性吞噬细胞明显不同。本研究拟此基础上，从细胞和动物水平延续探讨GT促进烟曲霉内化侵入肺上皮细胞及调控细胞炎症因子释放的具体信号机制，并解析PLD对GT信号机制的参与调控规律。项目的完成将有力拓展人们对GT在烟曲霉感染与免疫调节中功能的认识，对进一步阐明肺上皮细胞在应对烟曲霉感染中的重要作用及寻找可能的药物靶点具有重要意义。

胶霉毒素（gliotoxin，GT）是烟曲霉分泌的主要毒素因子，具有抑制中性粒细胞和巨噬细胞的吞噬及T细胞增殖和活化等功能。我们前期研究发现，GT可显著促进烟曲霉孢子内化侵入肺上皮细胞，并诱发细胞形态改变，但是其调控机制仍不清楚。本项目基于前期的研究基础，延续深入探讨了gliotoxin 促进烟曲霉内化侵入的可能调控机制：对细胞肌动蛋白骨架重排和炎症因子释放的影响。研究结果如下：首先确定了gliotoxin诱导A549细胞形态改变主要是由细胞肌动蛋白骨架重排引起的。cofilin是一类肌动蛋白解聚因子，其活性变化在一系列以细胞肌动蛋白骨架重排为基础的重要生命活动过程中发挥重要调控作用。我们的检测结果表明gliotoxin显著诱导A549细胞内磷酸化cofilin（p-cofilin）水平短暂升高和亚细胞分布改变，p-cofilin在细胞边缘处与F-actin共定位；进而我们通过改变cofilin表达和磷酸化状态证明了cofilin参与gliotoxin诱导的肌动蛋白骨架重排和烟曲霉孢子内化侵入肺上皮细胞过程。并进一步证明了cofilin上游信号Cdc42/RhoA-LIMK1信号和cAMP/PKA-LIMK1信号均参与该过程，且Cdc42信号在gliotoxin诱导的烟曲霉孢子内化侵入肺上皮细胞过程中发挥主要调控作用。在动物水平发现，在醋酸氢化可的松诱导免疫缺陷小鼠中，外源 gliotoxin 有助于烟曲霉侵袭肺组织。肺上皮屏障与肌动蛋白骨架重排之间有着紧密的关联，我们初步发现了gliotoxin可诱导肺上皮细胞内紧密连接蛋白的表达和分布的改变。还发现肺上皮细胞应对烟曲霉感染过程中，不同的上皮细胞释放炎症因子规律不同。烟曲霉通过激活NF-kB信号显著诱导支气管上皮细胞Beas2B释放多种炎症因子，而除IFN-γ外gliotoxin不参与其他炎症因子释放过程，但是烟曲霉却显著抑制肺泡上皮细胞A549释放多种炎症因子，同时gliotoxin也抑制A549细胞释放这些炎症因子，推测这种差异可能是由于两种细胞在肺组织中分布不同而导致其功能不同引起的。项目的完成将拓展人们对gliotoxin诱导烟曲霉内化侵入肺上皮细胞机制以及诱导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提供一定理论基础，具有重要意义。