Priming and release of cytokine IL-1β in microglial cells from the retina

Abstract

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^+/GFP 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 Ca²⁺ 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^+/GFP 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.

Keywords: Age-dependent neurodegeneration; Astrocyte; Cytokine release; Glaucoma; IL-1beta; NLRP3 inflammasome; Neuroinflammation; P2X7 receptor; Retinal microglia.