A central role for P2X7 receptors in human microglia
A central role for P2X7 receptors in human microglia
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DOI：
10.1186/s12974-018-1353-8
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发表时间：
2018-11-21
影响因子：
9.3
通讯作者：
Egan, Terrance M.
中科院分区：
文献类型：
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作者：
Janks, Laura;Sharma, Cristian V. R.;Egan, Terrance M.
背景资料：ATP门控离子型P2 X7受体（P2 X7 R）具有不寻常的功能，作为一个小的阳离子通道和质膜透化的触发器。在鼠小胶质细胞中，P2 X7 R介导的透化是小胶质细胞活化、增殖和IL-1 β释放的基础。然而，P2 X7 R在初级成人人类小胶质细胞中的作用知之甚少。方法：我们使用膜片钳电生理学记录培养的初级人类小胶质细胞中的ATP门控电流;共聚焦显微镜测量膜起泡;荧光显微镜证明膜透化，半胱天冬酶-1活化，磷脂酰丝氨酸易位和吞噬作用;和基于试剂盒的测定来测量细胞因子水平。我们发现，ATP门控内向电流促进与ATP的重复应用预期的电流通过P2 X7 R和P2 X7 R拮抗剂抑制这些电流。P2 X7 R拮抗剂也阻止了ATP诱导的大阳离子荧光染料的摄取，而靶向泛连接蛋白-1通道的药物则没有效果。相反，ATP不诱导吸收阴离子染料。阳离子染料的摄取被靶向Cl-通道的药物阻断。最后，我们发现，ATP激活caspase-1和抑制吞噬作用，这些影响都被阻断P2 X7 R和Cl-通道拮抗剂。结论：我们的研究结果表明，在文化中的主要人类小胶质细胞表达功能P2 X7 Rs，刺激ATP门控阳离子电流和摄取大分子量阳离子染料。重要的是，我们的数据表明，从小鼠免疫细胞的工作中得出的假设准确地预测了P2 X7 Rs在许多人类先天免疫功能，如吞噬作用和半胱天冬酶-1激活中的重要作用。因此，P2 X7 R代表了人类神经炎性疾病治疗干预的有吸引力的靶标。
Background: The ATP-gated ionotropic P2X7 receptor (P2X7R) has the unusual ability to function as a small cation channel and a trigger for permeabilization of plasmalemmal membranes. In murine microglia, P2X7R-mediated permeabilization is fundamental to microglial activation, proliferation, and IL-1 beta release. However, the role of the P2X7R in primary adult human microglia is poorly understood.Methods: We used patch-clamp electrophysiology to record ATP-gated current in cultured primary human microglia; confocal microscopy to measure membrane blebbing; fluorescence microscopy to demonstrate membrane permeabilization, caspase-1 activation, phosphatidylserine translocation, and phagocytosis; and kit-based assays to measure cytokine levels.Results: We found that ATP-gated inward currents facilitated with repetitive applications of ATP as expected for current through P2X7Rs and that P2X7R antagonists inhibited these currents. P2X7R antagonists also prevented the ATP-induced uptake of large cationic fluorescent dyes whereas drugs that target pannexin-1 channels had no effect. In contrast, ATP did not induce uptake of anionic dyes. The uptake of cationic dyes was blocked by drugs that target Cl- channels. Finally, we found that ATP activates caspase-1 and inhibits phagocytosis, and these effects are blocked by both P2X7R and Cl- channel antagonists.Conclusions: Our results demonstrate that primary human microglia in culture express functional P2X7Rs that stimulate both ATP-gated cationic currents and uptake of large molecular weight cationic dyes. Importantly, our data demonstrate that hypotheses drawn from work on murine immune cells accurately predict the essential role of P2X7Rs in a number of human innate immune functions such as phagocytosis and caspase-1 activation. Therefore, the P2X7R represents an attractive target for therapeutic intervention in human neuroinflammatory disorders.