PURPOSE: To evaluate the effect of lysosomal destabilization on NLRP3 inflammasome activation in RPE cells and to investigate the mechanisms by which inflammasome activation may contribute to the pathogenesis of age-related macular degeneration (AMD). METHODS: Human ocular tissue sections from patients with geographic atrophy or neovascular AMD were stained for NLRP3 and compared to tissues from age-matched controls. Expression of the IL-1β precursor, pro-IL-1β, was induced in ARPE-19 cells by IL-1α treatment. Immunoblotting was performed to assess expression of NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1β in ARPE-19 cells. Lysosomes were destabilized using the lysosomotropic agent L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). Active caspase-1 was detected using FAM-YVAD-FMK, a fluorescent-labeled inhibitor of caspases (FLICA) specific for caspase-1. IL-1β was detected by immunoblotting and ELISA, and cytotoxicity was evaluated by LDH quantification. RESULTS: RPE of eyes affected by geographic atrophy or neovascular AMD exhibited NLRP3 staining at lesion sites. ARPE-19 cells were found to express NLRP3, ASC, and procaspase-1. IL-1α dose-dependently induced pro-IL-1β expression in ARPE-19 cells. Lysosomal destabilization induced by Leu-Leu-OMe triggered caspase-1 activation, IL-1β secretion, and ARPE-19 cell death. Blocking Leu-Leu-OMe-induced lysosomal disruption with the compound Gly-Phe-CHN(2) or inhibiting caspase-1 with Z-YVAD-FMK abrogated IL-1β release and ARPE-19 cytotoxicity. CONCLUSIONS: NLRP3 upregulation occurs in the RPE during the pathogenesis of advanced AMD, in both geographic atrophy and neovascular AMD. Destabilization of RPE lysosomes induces NLRP3 inflammasome activation, which may contribute to AMD pathology through the release of the proinflammatory cytokine IL-1β and through caspase-1-mediated cell death, known as "pyroptosis."
PURPOSE: To evaluate the effect of lysosomal destabilization on NLRP3 inflammasome activation in RPE cells and to investigate the mechanisms by which inflammasome activation may contribute to the pathogenesis of age-related macular degeneration (AMD). METHODS:Human ocular tissue sections from patients with geographic atrophy or neovascular AMD were stained for NLRP3 and compared to tissues from age-matched controls. Expression of the IL-1β precursor, pro-IL-1β, was induced in ARPE-19 cells by IL-1α treatment. Immunoblotting was performed to assess expression of NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1β in ARPE-19 cells. Lysosomes were destabilized using the lysosomotropic agent L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). Active caspase-1 was detected using FAM-YVAD-FMK, a fluorescent-labeled inhibitor of caspases (FLICA) specific for caspase-1. IL-1β was detected by immunoblotting and ELISA, and cytotoxicity was evaluated by LDH quantification. RESULTS: RPE of eyes affected by geographic atrophy or neovascular AMD exhibited NLRP3 staining at lesion sites. ARPE-19 cells were found to express NLRP3, ASC, and procaspase-1. IL-1α dose-dependently induced pro-IL-1β expression in ARPE-19 cells. Lysosomal destabilization induced by Leu-Leu-OMe triggered caspase-1 activation, IL-1β secretion, and ARPE-19 cell death. Blocking Leu-Leu-OMe-induced lysosomal disruption with the compound Gly-Phe-CHN(2) or inhibiting caspase-1 with Z-YVAD-FMK abrogated IL-1β release and ARPE-19 cytotoxicity. CONCLUSIONS:NLRP3 upregulation occurs in the RPE during the pathogenesis of advanced AMD, in both geographic atrophy and neovascular AMD. Destabilization of RPE lysosomes induces NLRP3 inflammasome activation, which may contribute to AMD pathology through the release of the proinflammatory cytokine IL-1β and through caspase-1-mediated cell death, known as "pyroptosis."
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