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Anu Kauppinen, Niina Piippo, Eveliina Korhonen, Maria Hytti, Kati Kinnunen, Kai Kaarniranta; Activation of NLRP3 by intracellular aggregates in RPE cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5742.
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© ARVO (1962-2015); The Authors (2016-present)
NLRP3 inflammasome promotes inflammation by the caspase-1-mediated release of IL-1β and IL-18, and its activation has been associated with the pathogenesis of age-related macular degeneration (AMD). We have previously shown that insufficient degradation of intracellular waste material serves as an activation signal for NLRP3 in human retinal pigment epithelial (RPE) cells. NLRP3 is a pattern recognition receptor (PRR) with several activation mechanisms, and in this study, we have explored the mechanism(s) contributing to the inflammasome activation induced by dysfunctional intracellular cleaning.
NLRP3 was activated in IL-1α-primed human ARPE-19 cells by the proteasome inhibitor MG-132 and the autophagy inhibitor Bafilomycin A. We measured extracellular ATP using a commercial kit, and inhibited ATP-receptors by a P2X7 inhibitor in cell cultures. For preventing potassium efflux, cell cultures were exposed to high extracellular potassium concentration or glyburide. Cathepsin B activity was measured using a commercial assay. In order to study the role of oxidative stress, mitoTEMPO, APDC (ammonium pyrrolidine dithiocarbamate), or NAC (N-acetylcysteine) were added to the cell cultures. Cellular reactive oxygen species (ROS) were detected using the DCFDA (2’,7’-dichlorofluorescin diacetate) assay.
Inhibition of P2X7 receptors did not prevent the secretion of IL-1β although increased levels of extracellular ATP could have been able to initiate the response. Blocking the potassium efflux showed a weak response but cathepsin B seemed not to play any role in the inflammasome activation in our model. The treatment increased intracellular oxidative (P < 0.001), and ROS inhibitors mitoTEMPO (P < 0.05) and APDC (P < 0.001) but not NAC alleviated the release of IL-1β.
According to our results, oxidative stress plays a major role in the activation of NLRP3 inflammasome in human RPE cells with declined functionality of intracellular clearance systems. Especially inhibitors of NADPH oxidase and mitochondria-derived ROS appeared efficient.
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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