Schepens Eye Research Institute/Massachusetts Eye and Ear, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 2013 Jan 7;54(1):110-20. doi: 10.1167/iovs.12-10655.
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).
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.
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.
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."
评估溶酶体不稳定对 RPE 细胞中 NLRP3 炎性体激活的影响，并探讨炎性体激活如何导致年龄相关性黄斑变性（AMD）的发病机制。
对患有地理萎缩或新生血管性 AMD 的患者的眼部组织切片进行 NLRP3 染色，并与年龄匹配的对照组进行比较。用白细胞介素-1α（IL-1α）处理 ARPE-19 细胞，诱导白细胞介素-1β前体（pro-IL-1β）的表达。通过免疫印迹法评估 NLRP3 炎性体成分（NLRP3、ASC 和 procaspase-1）和 ARPE-19 细胞中 pro-IL-1β的表达。使用溶酶体亲脂性药物 L-亮氨酰-L-亮氨酸甲酯（Leu-Leu-OMe）破坏溶酶体。使用荧光标记的 caspase 抑制剂（FLICA）特异性针对 caspase-1 的 FAM-YVAD-FMK 检测活性 caspase-1。通过免疫印迹法和 ELISA 检测 IL-1β，通过 LDH 定量评估细胞毒性。
受地理萎缩或新生血管性 AMD 影响的眼睛的 RPE 在病变部位出现 NLRP3 染色。发现 ARPE-19 细胞表达 NLRP3、ASC 和 procaspase-1。IL-1α 剂量依赖性地诱导 ARPE-19 细胞中 pro-IL-1β 的表达。Leu-Leu-OMe 诱导的溶酶体不稳定引发 caspase-1 激活、IL-1β 分泌和 ARPE-19 细胞死亡。用化合物 Gly-Phe-CHN(2) 阻断 Leu-Leu-OMe 诱导的溶酶体破坏或用 Z-YVAD-FMK 抑制 caspase-1 可阻断 IL-1β 的释放和 ARPE-19 的细胞毒性。
在 AMD 的进展过程中，NLRP3 在 RPE 中的表达上调，无论是在地理萎缩还是新生血管性 AMD 中均如此。RPE 溶酶体的不稳定诱导 NLRP3 炎性体激活，通过释放促炎细胞因子 IL-1β 和 caspase-1 介导的细胞死亡（称为“pyroptosis”），可能导致 AMD 发病机制。
