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. Author manuscript; available in PMC: 2018 May 27.
Published in final edited form as: Nat Med. 2017 Nov 27;24(1):50–61. doi: 10.1038/nm.4450

Figure 3. Non-canonical inflammasome activation and RPE degeneration induced by Alu RNA is mediated by interferon signaling.

Figure 3

(a) Top, fundus photographs of eyes of WT (n = 7 eyes) and Ifnar−/− (n = 14 eyes) mice subretinally injected with Alu expression plasmid (pAlu) or empty vector (pNull). Bottom, immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the above eyes showing RPE cell boundaries. (b) Immunoblot of pro-caspase-11 (pro-Casp1) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ifnar−/− mouse RPE cells mock transfected or transfected with Alu RNA. (c) Immunoblot of pro-caspase-4 in IFN-β-treated human RPE cells. (d) IFN-β secretion by human RPE cells transfected with Alu expression plasmid (pAlu) or empty vector (pNull). Data presented are mean ± SD; n = 3 independent experiments; *P = 0.0012, two-tailed t test. (e) Immunoblot of phosphorylated STAT2 (pSTAT2) and total STAT2 in human RPE cells transfected with Alu expression plasmid (pAlu) or empty vector (pNull) or DICER1 or control (Ctr) anti-sense oligonucleotides (AS). (f) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT mice subject to Alu RNA co-administration with IFN-β neutralizing antibody (n = 6 eyes) or Isotype IgG (n = 4 eyes). (g) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of WT (n = 6 eyes) or Irf3−/− (n = 6 eyes) or Stat2−/− (n = 7 eyes) mice subretinally injected with Alu expression plasmid (pAlu) or empty vector (pNull). (h) Immunolocalization of IFN-β in the RPE of human geographic atrophy eyes and age-matched unaffected controls. Representative image from control and Dry AMD eyes are presented, n = 4 eyes. (i) Abundance of IFN-β mRNA in the RPE of the human geographic atrophy eyes compared to age-matched healthy controls, (Data presented are mean ± SEM; n = 4 eyes; *P = 0.018, two-tailed t test). 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 a, f and g, 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.