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Ivana Apicella, Shinichi Fukuda, Shuichiro Hirahara, Shaobin Wang, Siddharth Narendran, Nagaraj Kerur, Bradley Gelfand, Jayakrishna Ambati; Multifarious evidence for NLRP3 inflammasome expression and activation in retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):765.
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© ARVO (1962-2015); The Authors (2016-present)
Although several studies have investigated the expression and role of NLRP3 in inflammatory cells, data on retinal pigmented epithelium (RPE) cells are still incomplete. Our aim was to directly ascertain NLRP3 expression and activation in RPE cells under basal and inflammatory conditions, as well as of upstream (P2X7) and downstream (ASC, Caspase-1) inflammasome components.
Primary human RPE cells in culture were transfected with Alu RNA. NLRP3 mRNA and protein abundance were quantified by real-time PCR and western blotting respectively. Interaction of NLRP3 with ASC was monitored by proximity ligation assay (PLA). Selective ablation of NLRP3, ASC, Caspase-1, or P2X7 in the RPE was achieved by RPE-specific expression of Cre recombinase in mice “floxed” for these genes. Selective expression of NLRP3 in the RPE was achieved by subretinal injection of NLRP3-encoding plasmid in Nlrp3-deficient mice. The effect of subretinal administration of Alu RNA or control was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of RPE flat mounts.
In human RPE cells, Alu RNA induced 7-fold increase in the abundance of NLRP3 transcripts, including of a novel truncated isoform, and also induced interaction of NLRP3 with ASC. NLRP3 ko mice were protected against RPE degeneration induced by Alu RNA, whereas reconstitution of NLRP3 in the RPE restored degeneration. Alu RNA did not induce degeneration in mice lacking NLRP3, ASC, Caspase-1, or P2X7 selectively in the RPE.
We have demonstrated that human and mouse RPE express functional NLRP3 and that this is necessary and sufficient for Alu RNA-induced RPE degeneration. We have also identified a novel NLRP3 isoform, whose expression and function warrant further study. RPE cells express functional P2X7, NLRP3, ASC, and Caspase-1, each of which is critical for Alu RNA-induced RPE degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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