Nucleoside reverse transcriptase inhibitor (NRTI) protection against iron cytotoxicity

Abstract

Purpose: Iron-induced toxicity is implicated in multiple diseases, including age-related macular degeneration (AMD). This toxicity has been attributed to reactive oxygen species (ROS) generation through Fenton chemistry. Given the role of ROS in NLRP3 inflammasome activation, we hypothesized that the inflammasome was also involved in cytotoxicity.

Methods: Antibody staining of NLRP3 was performed in mice deficient in the iron transporters Ceruloplasmin (Cp) and Hephaestin (Heph). Metal overload was performed by media supplementation or subretinal injection in cell culture or mice, respectively. RPE cell integrity was assessed by zona occludens 1 (ZO-1) staining. Quantitative PCR (qPCR) and western blotting were performed to detect inflammasome priming and activation, respectively. Alu, B1, and B2 RNA accumulation was detected by Northern blotting or in situ hybridization (ISH). Degradation of Alu RNA in cells was detected by Northern blotting following transfection of a synthetic labeled Alu RNA. Poly(C)-binding protein 2 (PCBP2) binding of Alu RNA was assessed by pulldown assay and immunoprecipitation. Recombinant DICER1 and PCBP2 protein were used to assess in vitro cleavage efficiency of Alu RNA. The nucleoside reverse transcriptase inhibitor (NRTI) d4T was administered following iron overload.

Results: Mice deficient in iron transport proteins showed increased NLRP3 retinal staining and B1/B2 RNA staining. Fenton-capable metals all disrupted RPE cell integrity, but only iron toxicity was protected by genetic ablation of Nlrp3 or Casp1/Casp11. Inflammasome activation was specific to iron overload. Iron overload of human RPE cells and mouse eyes induced Alu RNA accumulation and B1 and B2 RNA accumulation, respectively. Iron overload did not affect Alu RNA transcription but impaired Alu RNA degradation. PCBP2 bound to Alu RNA and enhanced DICER1-mediated degradation of Alu RNA, but iron inhibited both. d4T administration protected the RPE against iron overload.

Conclusions: Iron is a novel NLRP3 inflammasome agonist, and metal-induced inflammasome activation is unique to iron. PCBP2 binds Alu RNA and is required for efficient degradation of Alu RNA by DICER1. The NRTI d4T was found to protect against RPE degeneration following iron overload. These results suggest that inflammasome activation may contribute to iron toxicity, and that NRTI therapy may be effective in other diseases of iron metabolism.