BACKGROUND We have previously reported that ATP treatment of M bovis-BCG infected human macrophages induces P2X7 receptor-dependent killing of intracellular mycobacteria. The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes. In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy. RESULTS We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocyte-derived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP). CONCLUSION We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections.

中文翻译：

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ATP 诱导的自噬与快速杀死人单核细胞/巨噬细胞内的细胞内分枝杆菌有关。

背景我们之前已经报道了牛支原体-BCG 感染的人巨噬细胞的 ATP 处理诱导细胞内分枝杆菌的 P2X7 受体依赖性杀伤。介导这种杀菌作用的机制尚未完全表征，但已知是 Ca2+ 依赖性的，并促进含分枝杆菌吞噬体的成熟和酸化。在这项研究中，我们证明了 ATP/P2X7 介导的分枝杆菌杀菌作用也涉及细胞自噬的诱导。结果 我们报告了 3 mM ATP 在处理后 30 分钟内在 THP1 细胞和单核细胞衍生的巨噬细胞中诱导快速细胞自噬，正如蛋白质印迹分析中 LC3-II 条带的表达所揭示的那样。使用不含 Ca2+ 的培养基和选择性 P2X7 激动剂和拮抗剂，显示 ATP 诱导的细胞自噬是 Ca2+ 和 P2X7 受体依赖性的。ATP 处理的、BCG 感染的 MDM 的电子显微镜检查显示细菌存在于特征性双膜自噬体中。共聚焦分析进一步证实，渥曼青霉素对自噬的药理学抑制或用抗 P2X7 抗体预处理巨噬细胞可阻断 ATP 诱导的吞噬溶酶体融合。用 ATP 诱导细胞自噬在时间上也与细胞内分枝杆菌生存力的下降有关，这种下降通过用渥曼青霉素或选择性 P2X7 拮抗剂氧化 ATP (oATP) 处理而受到抑制。结论 我们提供了第一个证据，证明 ATP/P2X7 介导的细胞内分枝杆菌杀伤涉及细胞自噬的诱导。这些发现支持自噬在控制分枝杆菌感染中起关键作用的假设。