Mechanisms behind the protein aggregation‐related inflammasome activation in RPE cells

Abstract

PurposeInflammasomes are intracellular protein complexes whose activation results in the caspase‐1‐mediated release of pro‐inflammatory cytokines IL‐1β and IL‐18. We and others have shown that activation of NLRP3 inflammasome is associated with the pathogenesis of age‐related macular degeneration (AMD). Declined proteasomal degradation with concurrent autophagy inhibition is among the risk factors activating the NLRP3 receptor in retinal pigment epithelial (RPE) cells. NLRP3 is known to have various activation mechanisms and in the present study, we have analyzed which of them contribute to the inflammasome activation when protein degradation systems in RPE cells fail.MethodsInflammasome activation was induced in human ARPE‐19 cells by the proteasome inhibitor MG‐132 and the lysosome neutralizer Bafilomycin A. Extracellular ATP was measured, and its receptors were inhibited using a P2X7 inhibitor. Potassium efflux was inhibited using high extracellular ion concentration, and glyburide was added to block the ATP‐mediated potassium efflux. APDC (ammonium pyrrolidine dithiocarbamate) and NAC (N‐acetylcysteine) were used to reduce oxidative stress.ResultsDespite increased extracellular ATP level, the inhibition of P2X7 receptors did not reduce the secretion of IL‐1β. Neither potassium efflux nor cathepsin B contributed to the inflammasome activation in our model, but ROS inhibitors alleviated the IL‐1β production.ConclusionsOur results suggest that oxidative stress plays a role in the inflammasome activation in RPE cells upon declined intracellular clearance.