Mitochondrial DNA has a pro-inflammatory role in AMD

Abstract

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly of industrialized nations, and there is increasing evidence to support a role for chronic inflammation in its pathogenesis. Mitochondrial DNA (mtDNA) has been recently reported to be pro-inflammatory in various diseases such as Alzheimer's and heart failure. Here, we report that intracellular mtDNA induces ARPE-19 cells to secrete inflammatory cytokines IL-6 and IL-8, which have been consistently associated with AMD onset and progression. The induction was dependent on the size of mtDNA, but not on specific sequence. Oxidative stress plays a major role in the development of AMD, and our findings indicate that mtDNA induces IL-6 and IL-8 more potently when oxidized. Cytokine induction was mediated by STING (Stimulator of Interferon Genes) and NF-κB as evidenced by abrogation of the cytokine response with the use of specific inhibitors (siRNA and BAY 11-7082, respectively). Finally, mtDNA primed the NLRP3 inflammasome. This study contributes to our understanding of the potential pro-inflammatory role of mtDNA in the pathogenesis of AMD.

Keywords: Age-related macular degeneration; Inflammation; Mitochondrial DNA; NLRP3 inflammasome; Retinal pigment epithelium.

Figure 1. mtDNA induces IL-6 and IL-8 secretion from ARPE-19 cells

(A) IL-6 and IL-8 secretion from ARPE-19 as determined by western blot of culture media 48h post-transfection with 1µg/mL mtDNA. Right, quantitation by densitometry. *p < 0.05 versus control and mtDNA groups; #p < 0.001 versus all other groups; §p < 0.01 versus all other groups. (B) ARPE-19 cells 24h after treatment with FAM-tagged mtDNA with or without transfection agent. mtDNA in green, DAPI in blue. (C) ARPE-19 viability determined by MTT 48h post-transfection with 1µg/mL mtDNA.

Figure 2. Oxidized and larger mtDNA fragments induce a stronger cytokine response

(A) IL-6 and IL-8 secretion from ARPE-19 cells 48h post-transfection with 1µg/mL of 2-kb mtDNA fragments of different sequences but similar sizes (schematic). Right, quantitation by densitometry. (B) IL-6 and IL-8 secretion from ARPE-19 cells 48h post-transfection with 1µg/mL of mtDNA fragments of different sizes (schematic). Right, quantitation by densitometry. *p < 0.05 versus all other groups. (C) IL-6 and IL-8 secretion from ARPE-19 cells 48h post-transfection with 1µg/mL of mtDNA or oxidized mtDNA. Right, quantitation by densitometry. *p < 0.01 versus control. (D) Time course of IL-6 and IL-8 induction by 1µg/mL of mtDNA. (E) Effect of mtDNA dose on IL-6 and IL-8 secretion at 48h.

Figure 3. mtDNA induces IL-6 and IL-8 secretion through STING

(A) STING immunoblotting of untreated ARPE-19 and primary human RPE lysates alongside a THP-1 lysate (positive control for STING). 40ug of total protein loaded per lane. Prominent band seen at STING’s predicted molecular weight (≈40kDa). (B) ARPE-19 cells were treated for a total of 96h with 20nM of either STING siRNA or control siRNA and lysates immunoblotted with STING monoclonal antibody. Right, quantitation by densitometry. *p < 0.001 versus control siRNA. (C&D) Along with mtDNA transfection, ARPE-19 cells were pre and co-treated with STING or control siRNA, and culture media were collected at 48h and subjected to western blot for IL-6 (C) and ELISA for IL-8 (D), as western blot did not provide sufficient resolution to study differences in IL-8 levels. *p < 0.05; §p < 0.01 versus all other groups.

Figure 4. mtDNA induces IL-6 and IL-8 secretion via NF-κB

(A) Nuclear fractionation efficiency verified with tata-binding protein (TBP) and b-tubulin. (B) p65 NF-κB ELISA performed on nuclear isolates of ARPE-19 cells 24h post-transfection with mtDNA. *p < 0.01 versus control. (C) A20 expression level 48h post-transfection of mtDNA. Right, quantitation by densitometry. *p < 0.05 versus control. (D) ARPE-19 cells were pre-incubated for 30 minutes with an irreversible NF-κB inhibitor (Bay 11–7082) before mtDNA transfection. After 48h, culture media were collected and immunoblotted for IL-6 and IL-8. Right, quantitation by densitometry. *p < 0.05 versus control and [mtDNA + Bay 5uM] groups.

Figure 5. mtDNA primes the NLRP3 inflammasome through STING and NF-κB

(A) Immunoblots of ARPE-19 lysates 48h after mtDNA transfection. Right, quantitation by densitometry. *p < 0.05 versus control. (B) Along with mtDNA transfection, ARPE-19 cells were pre and co-treated with STING or control siRNA, and lysates were collected at 48h and immunoblotted for pro-IL-1β. Right, quantitation by densitometry. *p < 0.05 versus all other groups. (C) ARPE-19 cells were pre-incubated for 30 minutes with an irreversible NF-κB inhibitor (Bay 11–7082) before mtDNA transfection. After 48h, lysates were collected and immunoblotted for pro-IL-1β. Right, quantitation by densitometry. *p < 0.05 versus all other groups.

Figure 6. mtDNA and the NLRP3 inflammasome in the RPE

(A) IL-1β and IL-18 ELISA on culture media of ARPE-19 cells 48h post-transfection with mtDNA. Results are mean ± SEM, n=4. *p < 0.01 versus control. (B) ARPE-19 caspase-1 activity assessed 24h after mtDNA transfection using fluorescent FLICA probe FAM-YVAD-FMK.