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Claire H Mitchell, Farraj Albalawi, Jason Lim, Wennan Lu; Priming of the NLRP3 inflammasome in optic nerve head astrocytes by mechanical strain and stimulation of the P2X7 receptor. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3009.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma pathogenesis involves multiple inflammatory responses including contributions from the NLRP3 inflammasome. This is a multiprotein complex leading to maturation and release of IL-1β. There are two steps in inflammasome involvement; in the priming stage relevant genes and proteins are upregulated, while in the activation stage these proteins are assembled and then mediate steps in IL-1β release. While stimulation of the P2X7 receptor by extracellular ATP can activate the inflammasome, its ability to prime the inflammasome is unclear. Optic nerve head astrocytes receive much of the strain in glaucoma, and this strain leads to the release of ATP and autostimulation of the P2X7 receptor. In the present study we ask whether this mechanical strain and P2X7R stimulation can also prime the inflammasome components.
For the in vitro experiments, RNA was extracted from isolated rat optic nerve head astrocytes after a 4 hrs swelling by a 30% hypotonic solution. For in vivo experiments, intraocular pressure (IOP) was elevated to 50-60 mm Hg for 4 hours in 3 month old rats, and RNA was extracted 24 hrs later. qPCR was used to measure the relative expression of inflammasome-related genes.
Swelling rat optic nerve head astrocytes for 4 hours upregulated mRNA for NLRP3 and IL-1β but not caspase 1. The NFκB inhibitor Bay11-7082 prevented upregulation of both genes. Inhibiting the P2X7R with Brilliant blue G (BBG), A839977, or A740003 prevented the swelling-induced upregulation of IL-1β but not NLRP3. In vivo, the expression of IL-1β, NLRP3 and caspase 1 were all increased at the mRNA level in optic nerve head of rat eyes subjected to a moderate elevation of IOP. Intravitreal injection of BBG prevented the rise in IL-1β but not NLRP3, consistent with the effect of P2X7R antagonists found in vitro.
Mechanical strain of the optic nerve head astrocytes primed the inflammasome genes IL-1β and NLRP3 in vitro and in vivo. While the upregulation of both genes required NFκB, only the rise in IL-1β was dependent on P2X7R activity. This implies that the mechanosensitive release of ATP from optic nerve head astrocytes may itself contribute to the priming of at least one inflammasome component, and may thus increase the inflammatory state of the optic nerve head in glaucoma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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