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Wen A. Tseng, Kati Kinnunen, Thuzar Thein, Meredith Gregory-Ksander, Bruce Ksander, Patricia A. D'Amore; Activation Of The NALP3 Inflammasome In Retinal Pigment Epithelial (RPE) Cells: Implications For AMD. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2299.
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A hallmark of early-stage age-related macular degeneration (AMD) is accumulation of insoluble drusen deposits beneath the retinal pigment epithelium (RPE). Evidence suggests this increased drusen deposition may be a consequence of impaired RPE function secondary to lysosomal dysfunction. Moreover, recent studies have shown that lysosomal destabilization induced by phagocytosis of indigestible material activates the NALP3 inflammasome in some cell types, resulting in IL-1β secretion. This study investigates whether RPE possess the NALP3 inflammasome, and whether lysosomal destabilization can activate the NALP3 inflammasome in RPE cells, leading to IL-1β release.
The human RPE cell line ARPE-19 was used in these studies. To induce expression of pro-IL-1β, ARPE-19 cells were primed for 48 hr with IL-1α. Expression of inflammasome components and pro-IL-1β in ARPE-19 cells was evaluated by western blot and immunohistochemistry (IHC). Lysosomal destabilization was induced by the methyl ester Leu-Leu-OMe. Mature IL-1β was measured by ELISA. Caspase-1 activation was assessed through the use of FAM-YVAD-FMK, a fluorescent probe that binds to active caspase-1, but not to the inactive precursor. The caspase-1 inhibitor Z-YVAD-FMK was used to test whether IL-1β release from ARPE-19 cells is caspase-1-dependent.
ARPE-19 cells were demonstrated by western blot and/or IHC to express the inflammasome components, NALP3, ASC, and procaspase-1. Treatment of IL-1α-primed ARPE-19 cells with the lysosome-destabilizing agent Leu-Leu-OMe induced significant release of mature IL-1β. Lysosomal destabilization induced by Leu-Leu-OMe also triggered activation of caspase-1 in ARPE-19 cells. The caspase-1 inhibitor Z-YVAD-FMK suppressed Leu-Leu-OMe-induced release of IL-1β from ARPE-19 cells, demonstrating that the release of IL-1β was caspase-1-dependent.
The NALP3 inflammasome is triggered in RPE cells in response to lysosomal destabilization, leading to release of IL-1β. These results support the hypothesis that inflammasome activation may serve as an early trigger of inflammation in the pathogenesis of AMD.
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