Immune-related mechanisms in the pathogenic processes of retinal degeneration

视网膜变性发病过程中的免疫相关机制

• 批准号: 8737672
• 负责人: Igal Gery
• 金额: $ 21.28万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8737672
• 关键词: 7-ketocholesterol; Biological Products; Caspase; Caspase-1; Cell Culture Techniques; Cell Line; Cells; Cholesterol; Diabetic Retinopathy; Drusen; Enzymes; Exposure to; Eye diseases; Family; Generations; Human; Human Cell Line; Immune; Inflammatory; Interleukin-1 beta; Interleukin-18; Interleukins; Measures; Medicine; Microglia; Mouse Cell Line; Muller' s cell; Multiprotein Complexes; Myeloid Cells; Nature; Pathogenesis; Population; Process; Production; Reporting; Retina; Retinal; Retinal Degeneration; Silicon Dioxide; Stimulus; Testing; Time; Western Blotting; cytokine; fetal; interleukin-1beta-converting enzyme inhibitor; monocyte; neovascular; oxidized low density lipoprotein; secretion process

The study was extended in 2013 to examine the capacity of retinal, retina-related and myeloid cells to generate inflammasome when exposed in culture to drusen components and other stimuli. The tested cells were primary human fetal RPE and cell lines of human RPE (ARPE19), as well as primary cultures of human microglia and cell lines of mouse microglia (BV-2) and of human monocytes (THP). Also tested: a cell line of human Muller cells. The two drusen components tested in this study for induction of inflammasome production are 7-ketocholesterol and oxidized LDL; the latter component has been tested in FY 2013 for the first time. In addition, inflammasome generation was tested in cell cultures stimulated by crystals of silica and of cholesterol. Generation of inflammasomes was determined by increased expression of NLRP3, measured by quantitative PCR, production of caspase-1, determined by Western blotting and, mainly, by measuring levels of IL-1beta and IL-18 in the culture supernatants, using commercial kits. The specific activity of caspase-1 was also confirmed by the suppressive effects of caspase-1 inhibitor. Microglial and myeloid cells were found to be superior to RPE cells in their capacity to generate inflammasomes, as indicated by higher production levels of the inflammasome components and products, mentioned above. Unlike these two families of cells, cultured Muller cells produced just marginal levels of the inflammasome molecules, thus underscoring the capacity of RPE cells to generate inflammasomes. Importantly, a fundamental difference was noted between RPE cells and myeloid cells (microglia and monocytes) in their preferential production of IL-18 (RPE cells) or IL-2beta (myeloid cells). This observation is of significance in view of a recent report (Doyle et al., Nature Medicine, 2012, 18:791), showing the protective activity of IL-18 against the neovascular process.

这项研究于2013年扩展，以检查视网膜，视网膜相关和髓样细胞在培养物中暴露于Drusen成分和其他刺激的能力。测试的细胞是原代人胎儿RPE和人RPE的细胞系（ARPE19），以及人类小胶质细胞和小鼠小胶质细胞（BV-2）和人单核细胞（THP）的细胞系的原代培养物。还测试了人类穆勒细胞的细胞系。 在这项研究中测试的两个drusen成分用于诱导炎性体的产生，为7-酮胆脂酯和氧化的LDL。后一个组件首次在2013财年进行了测试。此外，在二氧化硅和胆固醇晶体刺激的细胞培养物中测试了炎性体的产生。通过使用商业套件在培养上清液中测量IL-1BETA和IL-18的水平，通过定量PCR，CASPASE-1的产生，caspase-1的产生，caspase-1的产生，caspase-1的产生，caspase-1的产生，caspase-1的产生，caspase-1的产生，caspase-1的产生，caspase-1的产生来确定炎症的产生。 caspase-1抑制剂的抑制作用也证实了caspase-1的比活性。 发现小胶质细胞和髓样细胞以其产生炎症的能力优于RPE细胞，如上所述，炎性体成分和产物的产生水平较高，如上所述。与这两个细胞家族不同，培养的Muller细胞仅产生了炎性体分子的边际水平，因此突显了RPE细胞产生炎症体的能力。 重要的是，RPE细胞和髓样细胞（小胶质细胞和单核细胞）在其优先生产IL-18（RPE细胞）或IL-2BETA（髓样细胞）中的基本差异。鉴于最近的一份报告（Doyle等人，自然医学，2012，12，18：791），这一观察结果具有重要意义，显示了IL-18对新生血管过程的保护活性。