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Mara Supik, Marcia Terluk, Sandra Monetzuma, Deborah A Ferrington; Inflammasome Activation in Primary RPE Cultures from Human Donors with and without Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5248.
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Inflammasome activation is implicated in mediating the degeneration of the retinal pigment epithelium (RPE), which is one of the hallmarks of age-related macular degeneration (AMD). However, how the presence of AMD affects the response of RPE to various stimuli that activate the inflammasome has not been previously investigated. We tested the effect of reagents that promote inflammasome activation in primary RPE cultures from human donors with or without AMD.
The presence of AMD in donor eyes was determined using the Minnesota Grading System (MGS). Primary RPE cell cultures were developed from donors (ages 50 to 80 yrs) without AMD (MGS1, n=5) and with AMD (MGS2 and 3, early and intermediate AMD, n=9). Cells were primed with lipopolysaccharide and interleukin-1α, then treated with reagents that activate inflammasomes (MG132, Rotenone, Bafilomycin A, or ATP). After treatment, the content of inflammasomes (NLRP3, Aim2) and the activation product pro-IL1β were measured by Western Blot. The response to inflammasome activation was compared between cells from MGS1 and AMD donors by t-test analysis.
Basal levels of NLRP3 were detected in all cells. A 3-fold increase in NLRP3 content was observed after activation with ATP in MGS1 cells compared to no increase in AMD cells (p=0.035). MGS1 cells also exhibited a 2-fold increase in NLRP3 after rotenone, but MG132 and Bafilomycin induced only minor effects in all cells. Basal levels of Aim2 were observed in 50% of all cells. In these cells, only rotenone increased Aim2; cells without basal Aim2 were unresponsive. All reagents tested caused a similar increase in expression of pro IL-1β in both MGS1 and AMD cells.
Inflammasome activation was observed in primary RPE cultures from both healthy and AMD donors. However, in vivo exposure to AMD may alter the cell’s response to select inflammasome activators. Our results suggest that primary RPE cultures from human donors provide an excellent model system for mechanistic studies of inflammasome activation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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