Campagno Keith E, Lu Wennan, Sripinun Puttipong, Albalawi Farraj, Cenaj Aurora, Mitchell Claire H
Department of Basic and Translational Science, University of Pennsylvania, Philadelphia, PA, 19104, United States.
Department of Orthodontics, University of Pennsylvania, Philadelphia, PA, 19104, United States.
Exp Eye Res. 2025 Mar;252:110246. doi: 10.1016/j.exer.2025.110246. Epub 2025 Jan 21.
The P2X7 receptor (P2X7R) for extracellular ATP is implicated in several forms of retinal degeneration, including diabetic retinopathy, age-related macular degeneration, and glaucoma. P2X7R stimulation can trigger release of master cytokine IL-1β from microglia in the brain and from macrophages, but evidence of release from retinal microglia is indirect. Isolated mouse and rat retinal microglia, and wholemounts from Cx3CR1 mice, were examined to determine if ATP induced IL-1β release directly from retinal microglial cells and if it also primed expression of IL-1β on an mRNA and protein level. Isolated retinal microglia were ramified and expressed low levels of polarization markers unless provoked. Over 90% of isolated microglial cells expressed P2X7R, with cytoplasmic Ca elevation following receptor stimulation. ATP induced a dose-dependent release of IL-1β from primed microglial cells that was blocked by P2X7R antagonist A839977 and emulated by agonist BzATP. P2X7R stimulation also primed Il1b mRNA in isolated microglia cells. BzATP increased IL-1β immunostaining and GFP fluorescence throughout lamina of retinal wholemounts from CX3CR1 mice. Some of the IL-1β and GFP signals colocalized, particularly in the outer retina, and in projections extending distally through photoreceptor layers. The inner retina had more microglia without IL-1β, and more IL-1β staining without microglia. Substantial IL-1β release was also detected from rat retinal microglial cells, but not optic nerve head astrocytes. In summary, this study implicates microglial cells as a key source of released IL-1β when levels of extracellular ATP are increased following retinal damage, and suggest a greater participation in the outer retina.
细胞外ATP的P2X7受体（P2X7R）与多种形式的视网膜变性有关，包括糖尿病性视网膜病变、年龄相关性黄斑变性和青光眼。P2X7R刺激可触发大脑中的小胶质细胞和巨噬细胞释放主要细胞因子白细胞介素-1β（IL-1β），但视网膜小胶质细胞释放IL-1β的证据是间接的。研究人员对分离的小鼠和大鼠视网膜小胶质细胞以及Cx3CR1小鼠的视网膜全层进行了检测，以确定ATP是否直接诱导视网膜小胶质细胞释放IL-1β，以及它是否还在mRNA和蛋白质水平上启动IL-1β的表达。分离的视网膜小胶质细胞呈分支状，除非受到刺激，否则极化标记物表达水平较低。超过90%的分离小胶质细胞表达P2X7R，受体刺激后细胞质钙升高。ATP诱导经预处理的小胶质细胞剂量依赖性释放IL-1β，这被P2X7R拮抗剂A839977阻断，激动剂BzATP可模拟该过程。P2X7R刺激还可启动分离的小胶质细胞中Il1b mRNA的表达。BzATP增加了CX3CR1小鼠视网膜全层板层中IL-1β免疫染色和绿色荧光蛋白（GFP）荧光。一些IL-1β和GFP信号共定位，特别是在外层视网膜以及通过光感受器层向远端延伸的突起中。内层视网膜有更多没有IL-1β的小胶质细胞，以及更多没有小胶质细胞的IL-1β染色。在大鼠视网膜小胶质细胞中也检测到大量IL-1β释放，但在视神经乳头星形胶质细胞中未检测到。总之，本研究表明，当视网膜损伤后细胞外ATP水平升高时，小胶质细胞是释放IL-1β的关键来源，并提示其在外层视网膜中的参与度更高。
