P2X7R inhibition activates autophagy and impairs the inflammasome response in the RPE

Abstract

Purpose : P2X7R, a pattern recognition receptor for extracellular ATP, is involved in several pathways relevant to the pathogenesis of age-related macular degeneration (AMD) such as impaired lysosomal function or inflammasome activation. The purpose was to determine the extent that P2X7R inhibition activates autophagy and decreases inflammasome activation in retinal pigment epithelial (RPE) cells.

Methods : Human donor eyes with intermediate AMD were obtained for immunohistochemistry or dissection to obtain pure RPE samples for western blot analysis. The pharmacokinetic profile of JNJ-47965567 was assessed after subcutaneous administration in mice to determine the experimental dosing scheme. The impact of a P2X7R inhibitor (JNJ-47965567) on lysosomal function was assessed in cultured RPE from Cryba1 conditional knockout mice and on inflammasome activation in the RPE of apoB100 mice stressed with intravitreal (IVt) cigarette smoke extract (CSE) or ipS derived RPE cells given CSE (0-500 ug/ml) treatment.

Results : Human AMD samples showed increased immunolabeling for P2X7R in the RPE overlying basal deposits compared to morphologically normal RPE from uninvolved eyes. By Western blot, P2X7R was indeed abundant in the RPE from 3 healthy donor eyes as well as in the RPE from Cryba1 cKO and apoB100 mice. P2X7R inhibition restores the acidic lysosomal pH and restored lysosome-mediated autophagy turnover in chloroquine-treated RPE cells in cultured RPE cells from Cryba1 cKO mice. P2X7R antagonist reduced cleaved IL-1β and IL-18 levels 24 hrs after in IVt CSE injection. In human iPS-RPE cells grown for 4 months and given CSE for 24 hrs, P2X7R inhibition increased autophagy flux with increased LC3I/II conversion and p62 after bafilomycin A, and can increase the level of starvation-mediated autophagy. While CSE induced NLRP3, caspase 1, and cleaved caspase 1, P2X7R inhibition does not influence NLRP3 or caspase 1 levels. However, cleaved caspase 1 and secreted cleaved IL-1 β were reduced by P2X7R inhibition.

Conclusions : P2X7R is expressed and induced in human RPE in vivo and by oxidative stress such as CSE. P2X7R inhibition both in iPS-RPE, cultured mouse RPE, and in the IVt CSE model, can activate autophagy and impair the inflammasome response. Given the contributions of impaired autophagy and augmented inflammasome in AMD pathogenesis, P2X7R inhibition should be further evaluated as a treatment strategy for dry AMD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.