University of Kiel, University Medical Center, Department of Ophthalmology, Arnold-Heller-Str. 3, Haus B2, 24105, Kiel, Germany.
University of Kiel, University Medical Center, Department of Ophthalmology, Arnold-Heller-Str. 3, Haus B2, 24105, Kiel, Germany.
Exp Eye Res. 2020 Nov;200:108214. doi: 10.1016/j.exer.2020.108214. Epub 2020 Sep 6.
Degenerative ocular disorders like age-related macular degeneration (AMD) are associated with long-term pro-inflammatory signals on retinal pigment epithelial (RPE) cells. In this study, we investigated the effect of long term treatment of RPE cells with agonists of toll-like receptor (TLR) -3 (Polyinosinic:polycytidylic acid, Poly I:C), TLR-4 (lipopolysaccharide, LPS) and the pro-inflammatory cytokine TNFα.
All tests were conducted with primary porcine RPE. Cells were stimulated with Poly I:C (1, 10, 100 μg/ml), LPS (0.1, 1, 10 μg/ml) or TNFα (12.5, 25 or 50 ng/ml) for 1 day, 7 days or 4 weeks. Cell viability tests (MTT) were additionally tested in ARPE-19 cells. Cytokine secretion (IL-6, IL-1β, IL-8, TNFα, TGF-β) was tested in ELISA, phagocytosis in a microscopic assay, and expression of RPE65 in Western blot. Barrier function was tested in transwell-cultured cells by measuring transepithelial resistance for up to 3 days.
LPS and TNFα significantly reduce cell viability after 1 day and 7 days, Poly I:C after 7 days and 4 weeks. LPS, Poly I:C and TNFα significantly induce the secretion of IL-6 and IL-8 at all tested time points. IL-1β is increased by LPS and Poly I:C after 1 day, but not by TNFα. TNFα secretion is increased by Poly I:C and LPS after 1 day but not at later time points. TGF-β secretion is not influenced by any stimulus. Concerning RPE function, LPS decreased phagocytosis after 7 days, while Poly I:C and TNFα showed no effect. RPE65 expression was strongly reduced by TNFα and LPS after 4 weeks. Wound healing capacity was reduced by Poly I:C but induced by LPS after 7 d and 4 w. Barrier function was not affected by Poly I:C or LPS, while TNFα reduced barrier function after 1 h, 4 h and 3 days.
Long term pro-inflammatory stimuli reduce RPE viability, barrier properties and cellular function and induce pro-inflammatory cytokines and therefore may contribute directly to atrophic changes in AMD.
与年龄相关性黄斑变性（AMD）等退行性眼部疾病相关的是视网膜色素上皮（RPE）细胞长期存在促炎信号。在这项研究中，我们研究了用 Toll 样受体（TLR）-3（聚肌苷酸：聚胞苷酸，Poly I:C）、TLR-4（脂多糖，LPS）和促炎细胞因子 TNFα 长期刺激 RPE 细胞的效果。
所有测试均使用原代猪 RPE 进行。用 Poly I:C（1、10、100μg/ml）、LPS（0.1、1、10μg/ml）或 TNFα（12.5、25 或 50ng/ml）刺激细胞 1 天、7 天或 4 周。MTT 细胞活力测试还在 ARPE-19 细胞中进行。ELISA 检测细胞因子分泌（IL-6、IL-1β、IL-8、TNFα、TGF-β），显微镜检测吞噬作用，Western blot 检测 RPE65 表达。通过测量跨上皮电阻，在 Transwell 培养的细胞中测试屏障功能，持续 3 天。
LPS 和 TNFα 在 1 天和 7 天后显著降低细胞活力，Poly I:C 在 7 天和 4 周后降低细胞活力。LPS、Poly I:C 和 TNFα 在所有测试时间点均显著诱导 IL-6 和 IL-8 的分泌。LPS 和 Poly I:C 在 1 天后增加 IL-1β 的分泌，但 TNFα 则没有。TNFα 分泌在 1 天后由 Poly I:C 和 LPS 增加，但在随后的时间点没有增加。TGF-β 分泌不受任何刺激的影响。关于 RPE 功能，LPS 在 7 天后降低吞噬作用，而 Poly I:C 和 TNFα 则没有影响。RPE65 表达在 4 周后被 TNFα 和 LPS 强烈降低。Poly I:C 降低了伤口愈合能力，但 LPS 在 7 天后和 4 周后诱导了该能力。Poly I:C 或 LPS 不影响屏障功能，而 TNFα 在 1 小时、4 小时和 3 天后降低了屏障功能。
长期的促炎刺激物降低了 RPE 的活力、屏障特性和细胞功能，并诱导了促炎细胞因子，因此可能直接导致 AMD 的萎缩性变化。
