University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
University of Kiel, Anatomical Institute, Kiel, Germany.
Exp Eye Res. 2020 Oct;199:108167. doi: 10.1016/j.exer.2020.108167. Epub 2020 Jul 28.
In age-related macular degeneration, inflammatory events are presumed to contribute to disease development. A primary suspect of this contribution is the microglia, the innate immune cell of the retina. In addition, retinal pigment epithelium (RPE) cells can be inflammatorily activated. In this study, we investigate the effect of activated RPE cells on retinal microglia and on neuronal cells. RPE cells and microglia were harvested from porcine eyes. In addition, a neuronal cell line (SHSY-5Y) of human origin was used. For inflammatory activation, agonists of toll-like receptors in different concentrations were used: Pam2CSK4 (Pam; TLR-2), Polyinosinic:polycytidylic acid (Poly I:C; TLR-3) and lipopolysaccharid (LPS; TLR-4). Cell viability was investigated with an MTT assay. The secretion of cytokines was assessed in an ELISA and their expression in real-time PCR. There was no effect of the agonists on cell viability in RPE cells. All agonists induced the secretion of IL-6 and IL-8 in RPE cells with the strongest effect induced by LPS. In microglia, pro-inflammatory stimulation increased the metabolic activity. All agonists induced the secretion of IL-1ß, IL-8, and TNFα in microglia cells while in real-time PCR, LPS and Pam induced the expression of IL-6, IL-1ß and iNOS. Direct stimulation of SHSY-5Y with the agonists induced only minor alterations of viability. Stimulated RPE cell supernatant reduced the secretion of TNFα and IL-8 irrespective of the inducing agent in microglia cells. Additionally a slight induction of IL-1ß was found in microglia treated with supernatant of RPE cells treated with Pam. In real time PCR, the supernatant of RPE cells stimulated with LPS significantly reduced the expression of iNOS and IL-6, but not of IL-1ß. Of note, the expression of iNOS was also reduced by naive RPE cells. The treatment of the SHSY-5Y with supernatant of microglia previously treated with RPE conditioned medium significantly decreased SHSY-5Y viability with and without pro-inflammatory treatment. In conclusion, inflammatory activated RPE cells have a regulatory effect on the pro-inflammatory activation of microglia, stressing the importance of the interaction between these two retinal cell types. Microglia treated with RPE supernatant reduced viability of a neuronal cell line, indicating a neurotoxic effect.
在年龄相关性黄斑变性中，炎症事件被认为是导致疾病发展的原因之一。对此有主要的怀疑对象，即小胶质细胞，它是视网膜的先天免疫细胞。此外，视网膜色素上皮细胞（RPE）也可能被炎症激活。在这项研究中，我们研究了激活的 RPE 细胞对视网膜小胶质细胞和神经元细胞的影响。我们从猪眼中提取了 RPE 细胞和小胶质细胞。此外，还使用了源自人类的神经元细胞系（SHSY-5Y）。为了进行炎症激活，我们使用了不同浓度的 Toll 样受体激动剂：Pam2CSK4（Pam；TLR-2）、多聚肌苷酸：多聚胞苷酸（Poly I:C；TLR-3）和脂多糖（LPS；TLR-4）。用 MTT 测定法检测细胞活力。在 ELISA 中评估细胞因子的分泌，并在实时 PCR 中检测其表达。在 RPE 细胞中，激动剂对细胞活力没有影响。所有激动剂均诱导 RPE 细胞分泌 IL-6 和 IL-8，其中 LPS 诱导作用最强。在小胶质细胞中，促炎刺激增加了代谢活性。所有激动剂均诱导小胶质细胞分泌 IL-1β、IL-8 和 TNFα，而在实时 PCR 中，LPS 和 Pam 诱导了 IL-6、IL-1β和 iNOS 的表达。激动剂直接刺激 SHSY-5Y 仅引起细胞活力的微小改变。刺激的 RPE 细胞上清液可降低小胶质细胞中 TNFα和 IL-8 的分泌，而与诱导剂无关。此外，在用 Pam 处理的 RPE 细胞上清液处理的小胶质细胞中还发现了 IL-1β的轻度诱导。在实时 PCR 中，LPS 刺激的 RPE 细胞上清液显著降低了 iNOS 和 IL-6 的表达，但不降低 IL-1β的表达。值得注意的是，未受刺激的 RPE 细胞也降低了 iNOS 的表达。用先前用 RPE 条件培养基处理的小胶质细胞上清液处理 SHSY-5Y 可显著降低 SHSY-5Y 的活力，无论是否进行促炎处理。总之，炎症激活的 RPE 细胞对小胶质细胞的促炎激活具有调节作用，强调了这两种视网膜细胞类型之间相互作用的重要性。用 RPE 上清液处理的小胶质细胞可降低神经元细胞系的活力，表明其具有神经毒性作用。
