Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells.

Abstract

Activation of the NLRP3 inflammasome has been implicated in the pathogenesis of AMD. Lipofuscin phototoxicity activates the inflammasome in RPE cells by inducing lysosomal membrane permeabilization (LMP). We investigated the effects of LMP-induced inflammasome activation on the secretion of inflammation-related cytokines and VEGF by RPE cells. In primary human RPE cells and ARPE-19 cells, the inflammasome was activated by L-leucyl-L-leucine methyl ester (Leu-Leu-OMe)- or lipofuscin phototoxicity-induced LMP. Cytokine secretion was measured by protein dot blot and enzyme-linked immunosorbent assays. The polarization of cytokine secretion was assessed in RPE monolayers on permeable membranes. We analyzed the chemotactic and angiogenic effects of secreted cytokines on murine embryonic stem cell-derived microglia cells and human umbilical vascular endothelial cells, respectively. Inflammasome activation in RPE cells was associated with caspase-1-dependent secretion of IL-1β, IL-6, IL-18, GM-CSF, and GRO (CXCL1/2/3), whereas constitutive secretion of VEGF was reduced. Secretion of IL-1β and IL-18 was highly polarized to the apical cell side. Incubation with conditioned media of inflammasome-activated RPE cells induced directed migration of microglia cells (11.0-fold increase) and diminished vascular endothelial cells proliferation (39.0% reduction) and migration (69.3% reduction) as compared with conditioned media of untreated control RPE cells. Lysosomal membrane permeabilization-induced activation of the NLRP3 inflammasome in RPE cells results in apical secretion of inflammatory cytokines with chemotactic effects on microglia cells and reduced constitutive secretion of VEGF. Via these mechanisms, lipofuscin phototoxicity may contribute to local immune processes in the outer retina as observed in AMD.