The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells
The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells
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DOI：
10.4049/jimmunol.0802577
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发表时间：
2009-02-15
影响因子：
4.4
通讯作者：
Hashimoto, Makoto
中科院分区：
文献类型：
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作者：
Takenouchi, Takato;Nakai, Masaaki;Hashimoto, Makoto
近年来，自噬被认为与TLR信号通路有关，以消除先天免疫系统中的细胞内病原体。然而，在免疫反应中是否有其他途径调节自噬尚不清楚。鉴于嘌呤能P2X7受体（P2X7R）通路在各种免疫功能（即caspase激活和IL-1 β分泌）中的关键作用，本研究的主要目的是确定P2X7R通路是否可能调节免疫细胞的自噬。我们在MG6小鼠小胶质细胞和原代小胶质细胞中观察到，ATP激活P2X7R可以增加微管相关蛋白1轻链3 (LC3)-II的表达，LC3是LC3的自噬体膜相关形式，以细胞外Ca2+依赖的方式表达。与此一致的是，免疫组织化学显示lc3免疫阳性点的广泛形成，电镜显示atp处理的细胞中自噬体和自噬溶酶体的积累。重要的是，P2X7R活化对LC3-II的上调不受自噬抑制剂（如3-甲基腺嘌呤和PI3K抑制剂）的影响。此外，当溶酶体功能因ATP处理而受损时，自噬溶酶体成分被释放到细胞外空间。同样，利用大肠杆菌生物颗粒进行的吞噬实验表明，atp处理的细胞中吞噬体的成熟受到损害，lc3免疫阳性吞噬溶酶体的大量释放被诱导，微管束呈放射状延伸。综上所述，这些数据提示了一种新的机制，即P2X7R信号通路可能通过损害溶酶体功能来负性调节自噬通量，从而刺激自噬溶酶体/吞噬溶酶体释放到细胞外空间。免疫学杂志，2009,32(2):2051-2062。
Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1 beta secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca2+-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation,of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space. The Journal of Immunology, 2009, 182: 2051-2062.