Mitochondrial DNA acts as pro-inflammatory in AMD

Abstract

Purpose: Both mitochondrial damage and inflammation have been shown to be associated with age-related macular degeneration (AMD) and neurodegenerative diseases. Interestingly, mitochondrial DNA (mtDNA) has been recently reported to be pro-inflammatory, acting as a damage-associated molecular pattern (DAMP) in inflammatory cells, as well as neural and cardiac cells. However, whether mtDNA can do so in retinal pigment epithelial (RPE) cells remains unclear. The purpose of our study is to explore whether mtDNA is pro-inflammatory in RPE cells, potentially contributing to the pathogenesis of AMD.

Methods: APRE-19 cells were transfected with mtDNA by X-tremeGene (Roche) and the inflammatory cytokines IL6, IL8, and pro-IL1beta were analyzed by western blot at 48h. Furthermore, NLRP3 inflammasome components such as mature IL1b and caspase-1 were assessed by ELISA (R&D) and FLICA probe (Immunochemistry) respectively. The effect of mtDNA size, sequence and oxidation status was investigated, and an inhibitor of NF-κB (Bay 11-7082, Sigma) was used to determine the pathway of cytokine expression.

Results: mtDNA induced secretion of IL6 (p<0.01) and IL8 (p<0.05) from the RPE cell. The induction was dependent on the size of mtDNA but not on the specific mtDNA sequence. Interestingly, mtDNA induced an even stronger response when oxidized (p<0.05). Furthermore, the induction was mediated by activation of the NF-kb pathway, as NFkb inhibition abrograted the response. In addition, mtDNA primed the NLRP3 inflammasome as evidenced by pro-IL1b and pro-caspase-1 induction (p<0.05), and it was also able to activate it as indicated by caspase-1 activation and mature IL-1b secretion (p<0.01). The priming effect was also size-dependent and stronger with oxidized mtDNA (p<0.05).

Conclusions: mtDNA can induce inflammatory cytokine expression as well as NLRP3 inflammasome priming and activation in human RPE cells. This study contributes to our understanding of the potential pro-inflammatory role of mtDNA in the pathogenesis of AMD.