Reactive Oxygen Species and P2X7 receptors are critical for Alu RNA induced RPE degeneration caused by NLRP3 inflammasome

Abstract

 

Purpose:

 

Accumulation of Alu RNA transcripts due to Dicer1 deficiency in RPE is responsible for Geographic atrophy. A Recent study by our group has shown that NLRP3 inflammasome activation is responsible for the Alu RNA cytotoxicity. The goal in this study was to decipher the pathways critical to NLRP3 inflammasome activation in mediating the RPE degeneration

 

Methods:

 

Human RPE cells were transfected with plasmid encoding Alu RNA (pAlu). Production of reactive oxygen species (ROS) was assessed using the ROS-specific probe 2’7’-dichlorofluorescin diacetate. The NLRP3 and IL-18 mRNA priming was of analyzed by real-time qPCR in human RPE cells pre-incubated with ROS inhibitor (APDC). For in vivo models, pAlu and inhibitors (Glipizide, Glyburide and P2X7 receptor antagonist A438079) were delivered by subretinal and intra-vitreous injections respectively. RPE degeneration was assessed by fundus photography and zona occludens 1 (ZO-1) staining of RPE flat mounts.

 

Results:

 

Exposure to Alu RNA induced robust ROS production in human RPE cells. Further, priming of NLRP3 and IL-18 mRNA levels was dependent on ROS generation. Consistent with this, ROS inhibitor APDC prevented RPE degeneration in mice. P2rX₇^-/- mice as well as P2X7 inhibitor treated mice were resistant to Alu RNA induced RPE degeneration suggesting a critical role for P2X7 receptor in activation NLRP3 inflammasome. In alignment with this, NLRP3 inflammasome inhibitor glyburide, which acts downstream of P2X7 also suppressed the Alu RNA induced RPE degeneration.

 

Conclusions:

 

Alu induced RPE degeneration via NLRP3 inflammasome activation was dependent upon NLRP3 and IL-18 mRNA priming by ROS. Additionally purinergic receptor P2X7 was critical for mediating NLRP3 inflammasome activation. Thus our findings suggest that, ROS production and P2X7 receptors are the two critical steps involved the RPE degeneration caused by Alu RNA induced NLRP3 activation.

 

Keywords: age-related macular degeneration • retinal pigment epithelium • inflammation