School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
HUSLAB, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
FASEB J. 2020 May;34(5):6437-6448. doi: 10.1096/fj.201902355RR. Epub 2020 Mar 19.
DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1β mRNAs were detected with qRT-PCR and secreted amounts of IL-1β, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1β was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1β but NLRP3 contributed only to the secretion of IL-1β. At the mechanistic level, the release of IL-1β was regulated by K efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1β.
DNA 损伤在衰老的有丝分裂后视网膜色素上皮 (RPE) 细胞中积累，这是与年龄相关性黄斑变性发展相关的现象。在这项研究中，我们通过紫外线 B (UVB) 照射在白细胞介素-1α (IL-1α) 引发的 ARPE-19 细胞中诱导 DNA 损伤，并检查了炎症小体介导的信号。为了揭示炎症小体激活的机制，在 UVB 照射之前，细胞还被暴露于高浓度的细胞外氯化钾、N-乙酰半胱氨酸或线粒体靶向抗氧化剂 MitoTEMPO。用 qRT-PCR 检测白细胞介素-18 (IL-18) 和白细胞介素-1β mRNA 的水平，用 ELISA 测量 IL-1β、IL-18 和半胱天冬酶-1 的分泌量。通过使用 NLRP3 特异性 siRNA 验证核苷酸结合域和富含亮氨酸重复吡喃含蛋白 3 (NLRP3) 在 UVB 诱导的炎症小体激活中的作用。用细胞通透性 2',7'-二氯二氢荧光素二乙酸酯 (H DCFDA) 指示剂在 UVB 暴露后立即测量活性氧 (ROS) 水平，用基于细胞的 ELISA 测定环丁烷嘧啶二聚体的水平，并用商业发光测定法测定细胞外三磷酸腺苷 (ATP) 的水平。我们发现 ARPE-19 细胞中组成性表达 pro-IL-18，而 pro-IL-1β 的表达可通过 IL-1α 引发诱导。UVB 诱导成熟的 IL-18 和 IL-1β 的释放，但 NLRP3 仅有助于 IL-1β 的分泌。在机制水平上，IL-1β 的释放受 K+外流调节，而 IL-18 的分泌依赖于 ROS 的产生。除了 K+外流外，细胞在 UVB 照射后释放 ATP。总的来说，我们的数据表明，UVB 明显刺激成熟的 IL-18 作为 ROS 诱导的结果释放，并且这种反应与 DNA 损伤有关。此外，在人 RPE 细胞中，K+外流介导 UVB 激活的 NLRP3 炎症小体信号，导致 IL-1β 的加工。
