Ludwig-Maximilians-University, Medical Policlinic, Department of Internal Medicine, Klinikum der Universita¨t Mu¨nchen, Campus Innenstadt, Munich, Germany.
Invest Ophthalmol Vis Sci. 2011 Aug 17;52(9):6536-44. doi: 10.1167/iovs.10-6993.
PURPOSE. To elucidate the role of Toll-like receptor 3 (TLR3) in the pathogenesis of age-related macular degeneration (AMD) and to investigate the effect of alkylphosphocholines (APCs) on the TLR3-mediated expression of cytokines and growth factors in human retinal pigment epithelial (RPE) cells. METHODS. Confluent cultures of human RPE cells (ARPE-19) were stimulated with poly (I:C) RNA as a well-established ligand for TLR3. Cytokine profiles were determined by RT-PCR on the activation of TLR3. RPE cells were transfected with siRNA specific for TLR3 and RIG-1 to determine the receptors involved. The effect of preincubation of RPE cells with APCs on the expression level of target genes was assessed. RESULTS. Poly (I:C) RNA stimulation led to a dose-dependent increase in the expression of TLR3 and RIG-I. A significant increase in expression levels of IL-6, TNF-α, IL-8, MCP-1, ICAM-1, and BFGF was observed after poly (I:C) RNA stimulation (P < 0.05). This effect was time and dose dependent. No effect on PEDG or VEGF expression was seen. Transfection of RPE cells with siRNA specific for TLR3 reduced poly (I:C) RNA-induced mRNA expression of the genes (P < 0.05). Preincubation of RPE cells with APCs significantly reduced the poly (I:C) RNA-induced expression of the target genes (P < 0.05). CONCLUSIONS. The authors demonstrate that the expression of proinflammatory cytokines and chemokines in RPE cells depends on the activation of TLR3. The induction of downstream gene expression is blocked by siRNA specific for TLR3 and alkylphosphocholines. Therefore, TLR3 should be considered a novel target in AMD therapy.
目的。阐明 Toll 样受体 3（TLR3）在年龄相关性黄斑变性（AMD）发病机制中的作用，并研究烷基磷酸胆碱（APCs）对人视网膜色素上皮（RPE）细胞 TLR3 介导的细胞因子和生长因子表达的影响。
方法。用多聚（I：C）RNA 作为 TLR3 的有效配体刺激人 RPE 细胞（ARPE-19）的汇合培养物。通过 TLR3 激活的 RT-PCR 确定细胞因子谱。用 TLR3 和 RIG-1 的 siRNA 转染 RPE 细胞，以确定涉及的受体。评估 APCs 预处理对靶基因表达水平的影响。
结果。多聚（I：C）RNA 刺激导致 TLR3 和 RIG-I 的表达呈剂量依赖性增加。在用多聚（I：C）RNA 刺激后观察到 IL-6、TNF-α、IL-8、MCP-1、ICAM-1 和 BFGF 的表达水平显著增加（P <0.05）。这种效应是时间和剂量依赖性的。PEDG 或 VEGF 的表达没有影响。用 TLR3 的特异性 siRNA 转染 RPE 细胞可降低多聚（I：C）RNA 诱导的基因的 mRNA 表达（P <0.05）。APCs 的预孵育可显著降低多聚（I：C）RNA 诱导的靶基因表达（P <0.05）。
结论。作者证明 RPE 细胞中促炎细胞因子和趋化因子的表达取决于 TLR3 的激活。TLR3 特异性 siRNA 和烷基磷酸胆碱可阻断下游基因表达的诱导。因此，TLR3 应被视为 AMD 治疗的新靶点。
