Figure 6. mtDNA in non-canonical inflammasome activation and RPE degeneration.

(a) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in human RPE cells mock-transfected or transfected with Alu RNA (Data presented are mean ± SEM; n = 3 independent experiments; *P = 0.0018, two-tailed t test). (b) Relative enrichment of mtDNA in cGAS immunoprecipitate in ChIP like pull-down assay. Mock or Alu RNA transfected, indicated mouse embryonic fibroblast (MEF) were analyzed upon HA-cGAS immunoprecipitation with anti-HA antibody or isotype conrol. Data presented are mean ± SEM; n = 3; *P = 0.008, two-tailed t test. (c) Relative abundance of cytosolic mitochondrial DNA (mtDNA) in WT and Ppif^−/− mouse RPE cells mock transfected (n = 4 cell culture replicates) or transfected with Alu RNA (Data presented are mean ± SEM; n = 6 cell culture replicates; *P = 0.004, two-tailed t test). (d) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) and Ppif^−/− (n = 12 eyes) mice subretinally injected with vehicle or Alu RNA. (e) Immunoblot for procaspase-1 (procasp-1) and p20 cleavage product of caspase-1 in WT and Ppif^−/− mouse RPE cells mock transfected or transfected with Alu RNA. (f) Immunoblot for procaspase-11 (procasp-11) and p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ppif^−/− mouse RPE cells mock transfected or transfected with Alu RNA. (g) Immunoblot for procaspase-4 (procasp-4) and p30 cleavage product of caspase-4 (Casp4 p30) in WT and mitochondrial DNA deficient Rho⁰ ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. (h, i) WT and mitochondrial DNA deficient Rho⁰ ARPE19 human RPE cells mock-transfected or transfected with Alu RNA. (h) IL-18 secretion; data presented are mean ± SD; n = 4 independent experiments; *P = 0.0001, two-tailed t test. (i) IFN- β secretion; data presented are mean ± SD; n = 4 independent experiments; *P = 0.004, two-tailed t test. (j) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the Ppif^−/− mice subretinally co-administered Alu RNA with recombinant IFN-β (n = 5 eyes) or vehicle control (n = 5 eyes); or IFN-β expression plasmid (pIFNB; n = 6 eyes) or empty vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel image of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (SEM)) are shown. In d and j, binary (Healthy %) and morphometric (PM, polymegethism (mean (SEM)) quantification of RPE degeneration are shown (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). Loss of regular hexagonal cellular boundaries in ZO-1 stained flat mounts is indicative of degenerated RPE. The degenerated retinal area is outlined by blue arrowheads in the fundus images.