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.

National Institute of Health (NIH)
National Eye Institute (NEI)
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U.S. National Eye Institute
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