APOE Isoforms Control Pathogenic Subretinal Inflammation in Age-Related Macular Degeneration

Abstract

Contrary to Alzheimer's disease (AD), the APOE2 allele increases and the APOE4 allele reduces the risk to develop age-related macular degeneration (AMD) compared with the most common APOE3 allele. The underlying mechanism for this association with AMD and the reason for the puzzling difference with AD are unknown. We previously demonstrated that pathogenic subretinal mononuclear phagocytes (MPs) accumulate in Cx3cr1-deficient mice due to the overexpression of APOE, interleukin-6, and CC chemokine ligand 2 (CCL2). We here show using targeted replacement mice expressing the human APOE isoforms (TRE2, TRE3, and TRE4) that MPs of TRE2 mice express increased levels of APOE, interleukin-6, and CCL2 and develop subretinal MP accumulation, photoreceptor degeneration, and exaggerated choroidal neovascularization similar to AMD. Pharmacological inhibition of the cytokine induction inhibited the pathogenic subretinal inflammation. In the context of APOE-dependent subretinal inflammation in Cx3cr1(GFP/GFP) mice, the APOE4 allele led to diminished APOE and CCL2 levels and protected Cx3cr1(GFP/GFP) mice against harmful subretinal MP accumulation observed in Cx3cr1(GFP/GFP)TRE3 mice. Our study shows that pathogenic subretinal inflammation is APOE isoform-dependent and provides the rationale for the previously unexplained implication of the APOE2 isoform as a risk factor and the APOE4 isoform as a protective factor in AMD pathogenesis.

Significance statement: The understanding of how genetic predisposing factors, which play a major role in age-related macular degeneration (AMD), participate in its pathogenesis is an important clue to decipher the pathomechanism and develop efficient therapies. In this study, we used transgenic, targeted replacement mice that carry the three human APOE isoform-defining sequences at the mouse APOE chromosomal location and express the human APOE isoforms. Our study is the first to show how APOE2 provokes and APOE4 inhibits the cardinal AMD features, inflammation, degeneration, and exaggerated neovascularization. Our findings reflect the clinical association of the genetic predisposition that was recently confirmed in a major pooled analysis. They emphasize the role of APOE in inflammation and inflammation in AMD.

Keywords: apolipoprotein E; mononuclear phagocyte; neurodegeneration; neuroinflammation.

Figure 1.

The APOE2 allele leads to age- and stress-related subretinal MP accumulation, retinal degeneration, and exacerbated choroidal neovascularization. A, Representative 12-month-old IBA-1-stained RPE flatmounts of TRE3 and TRE2 mice and quantification of subretinal IBA-1⁺ MPs in 2- and 12-month-old mice of the indicated strains (n = 9–20/group). *p < 0.0001, 12 months versus TRE3 control (ANOVA/Dunnett's multiple-comparison test). B, Quantification of subretinal IBA-1⁺ MPs after a 4 d light challenge followed by 10 d of normal light conditions (d14) of 2-month-old mice of the indicated strains (n = 14–16/group). *p < 0.0001, versus TRE3 control at 14 d (ANOVA/Dunnett's multiple-comparison test). C, Micrographs taken 1000 μm from the optic nerve of 12-month-old TRE3 and TRE2 mice. ONL, Outer nuclear layer. C′, Photoreceptor nuclei rows at increasing distances (−3000 μm: inferior pole; 3000 μm: superior pole) from the optic nerve (0 μm) in 12-month-old mice. C″, Quantification of the area under the curve of photoreceptor nuclei row counts of 2- and 12-month-old transgenic mouse strains (n = 4–7). *p = 0.0102, versus TRE3 control at 12 months (ANOVA/Dunnett's multiple-comparison test). Mice were taken from several (≥3) independent cages for the quantifications. D, CD102 (red) and IBA-1 (green) immunohistochemistry and quantification of subretinal IBA-1⁺ MPs on the RPE counted at a distance of 0–500 μm to CD102⁺ CNV 7 d after the laser injury of 2-month-old mice of the indicated strains (n = 8–10/group). *p < 0.0001 (one-way ANOVA/Dunnett's multiple-comparison test). E, CD102 immunohistochemistry and quantification of CD102 area on RPE/choroidal flatmount from 2-month-old transgenic strains, 7 d after laser injury (n = 8–10/group). *p < 0.0001 (one-way ANOVA/Dunnett's multiple-comparison test). TRE2–4, Targeted replacement mice expressing human APOE isoforms Scale bars: A, C–E, 50 μm .

Figure 2.

The APOE2 allele increases APOE levels in the eye and APOE transcription and IIRC activation in MPs. A, APOE ELISA of homogenates of PBS-perfused posterior segments of 12-month-old TRE2, TRE3, and TRE4 mice (n = 5 or 6/group). *p = 0.0027, versus TRE3 control (one-way ANOVA/Dunnett's multiple-comparison test). ‡p = 0.0027 (one-way ANOVA/Dunnett's multiple-comparison test). B, Immunohistochemistry of APOE (red, top) and IBA-1 (green, bottom) of the subretinal side of a retinal flatmount from a 12-month-old TRE2 mouse (representative of three independent experiments; experiments omitting the primary antibody immunostaining served as negative controls). C, Quantitative RT-PCR of ApoE mRNA normalized with S26 mRNA of liver extracts from transgenic replacement mice expressing human APOE isoforms (TRE2, TRE3, and TRE4 mice, n = 3). D, ELISA quantification of APOE plasma concentrations in transgenic replacement mice expressing human APOE isoforms (TRE2, TRE3, and TRE4 mice, n = 3). *p < 0.0001, versus TRE3 control (ANOVA/ Dunnett's multiple-comparison test). E, Quantitative RT-PCR of ApoE mRNA normalized with S26 mRNA of bone marrow-derived monocytes from transgenic replacement mice expressing human APOE isoforms (TRE2, TRE3, and TRE4 mice) cultured for 3 d with or without porcine photoreceptor outer segments to simulate subretinal monocyte to macrophage differentiation (n = 6/group). *p = 0.0033, Mo+POS versus TRE3 control (ANOVA/ Dunnett's multiple-comparison test). F, Quantitative RT-PCR of ApoE mRNA normalized with S26 mRNA of peritoneal Mφs from TRE mice cultured for 24 h (n = 6). *p < 0.0001, versus TRE3 control (ANOVA/Dunnett's multiple-comparison test). G, APOE-ELISA of supernatants of peritoneal Mφs from TRE mice cultured for 24 h (n = 6). *p < 0.0001, versus TRE3 control (ANOVA/Dunnett's multiple-comparison test). H, Human IL-6 ELISA of supernatants from human monocytes incubated for 24 h in control medium, APOE3 (5 μg/ml), heat-denaturated APOE3 (dAPOE3, 5 μg/ml), APOE3 (5 μg/ml), and polymyxin B (25 μg/ml), APOE3 (5 μg/ml), and rat IgG1 isotype, or human IgA2 isotype control, or mouse IgG1 isotype control, or rat anti-CD14 IgG1 antibody, or human anti-TLR4 IgGA2 antibody, or mouse anti-TLR2 IgG1 antibody (all antibodies at 25 μg/ml) (n = 4–6/group). *p < 0.0001, APOE3 versus CTL (one-way ANOVA/Bonferroni multiple-comparison test). ^#p < 0.0001, dAPOE3 versus APOE3 (one-way ANOVA/Bonferroni multiple-comparison test). ^#p < 0.0001, APOE3 IgG versus APOE3 aCD14 Ab (one-way ANOVA/Bonferroni multiple-comparison test). ^#p < 0.0001, APOE3 IgG versus APOE3 aTLR4 Ab (one-way ANOVA/Bonferroni multiple-comparison test). ^#p < 0.0001, APOE3 IgA versus APOE3 aTLR2 Ab (one-way ANOVA/Bonferroni multiple-comparison test). I, Quantitative RT-PCR of Il-6, Ccl-2, and II1β mRNA normalized with S26 mRNA (n = 6) of peritoneal Mϕs from TRE mice cultured for 24 h. *p = 0.0069, versus TRE3 control IL-6 (ANOVA/Dunnett's multiple-comparison test). *p < 0.0001, versus TRE3 control CCL2 (ANOVA/Dunnett's multiple-comparison test). *p = 0.0097, versus TRE3 control IL-1β (ANOVA/Dunnett's multiple-comparison test). TRE2–4, Targeted replacement mice expressing human APOE isoforms. Scale bar: B, 50 μm.

Figure 3.

IIRC inhibition reduces subretinal MP accumulation and choroidal neovascularization in TRE2 mice in vivo. A, B, Seven day laser-injured IBA-1 (green) and CD102 (red) stained RPE flatmounts of control IgG and anti-CD14-treated TRE2 mice. Quantification of subretinal IBA-1⁺ MPs/impact localized on the lesion surrounding RPE (A) and quantification of CD102⁺ CNV area (B) of TRE2 mice treated with control IgG or CD14-blocking antibodies (intraocular antibody concentration 5 μg/ml; n = 12/group). A, *p = 0.0012 (Mann–Whitney t test). B, *p = 0.0009 (Mann–Whitney t test). TRE2–4, Targeted replacement mice expressing human APOE isoforms; Scale bars: A and B, 50 μm.

Figure 4.

The APOE4 allele protects APOE-overexpressing Cx3cr1^GFP/GFP mice from subretinal MP accumulation, retinal degeneration, and exacerbated choroidal neovascularization. A, Representative 12-month-old IBA-1-stained RPE flatmounts of Cx3cr1^GFP/GFPTRE3 mice and Cx3cr1^GFP/GFPTRE4 mice and quantification of subretinal IBA-1⁺ MPs in 2- and 12-month-old mice of the indicated strains (n = 8–13/group). *p = 0.0034 (Mann–Whitney t test at 12 months). B, Quantification of subretinal IBA-1⁺ MPs after a 4 d light challenge followed by 10 d of normal light conditions (d14) of 2-month-old mice of the indicated strains (n = 18/group). *p = 0.0036 (Mann–Whitney t test at d14). C, Micrographs, taken 1000 μm from the optic nerve of 12-month-old Cx3cr1^GFP/GFPTRE3 mice and Cx3cr1^GFP/GFPTRE4 mice. ONL, Outer nuclear layer. C′, Photoreceptor nuclei rows at increasing distances (−3000 μm: inferior pole; 3000 μm: superior pole) from the optic nerve (0 μm) in 12-month-old mice. C″, Quantification of the area under the curve of photoreceptor nuclei row counts of 12-month-old transgenic mouse strains (n = 5–9). *p = 0.0032 (Mann–Whitney t test). Mice were taken from several (≥3) independent cages for the quantifications. D, CD102 (red) and IBA-1 (green) immunohistochemistry and quantification of subretinal IBA-1⁺ MPs on the RPE counted at a distance of 0–500 μm to CD102⁺ CNV 14 d after the laser injury of 2-month-old mice of the indicated strains (n = 7/group). *p = 0.0182 (Mann–Whitney t test). E, CD102 immunohistochemistry and quantification of CD102 area on RPE/choroidal flatmount from 2-month-old transgenic strains, 14 d after laser injury (n = 7/group). *p = 0.0034 (Mann–Whitney t test). Scale bars: A, C–E, 50 μm.

Figure 5.

The APOE4 allele decreases ocular APOE levels in Cx3cr1^GFP/GFP mice and activates the IIRC inefficiently. A, APOE ELISA of homogenates of PBS-perfused posterior segments of 12-month-old Cx3cr1^GFP/GFPTRE3 mice and Cx3cr1^GFP/GFPTRE4 mice (n = 8–11/group). *p = 0.0276 (Mann–Whitney t test). B, Human IL-6 and CCL2 ELISA of supernatants from human monocytes incubated for 24 h in control medium, APOE3 (5 μg/ml), or APOE4 (5 μg/ml) (n = 6). p < 0.0001, IL-6 and CCL2 (one-way ANOVA/Bonferroni multiple-comparison tests). *p < 0.05, different from control (one-way ANOVA/Bonferroni multiple-comparison tests). ‡p < 0.05, different from APOE3 (one-way ANOVA/Bonferroni multiple-comparison tests). C, D, Quantitative RT-PCR of ApoE mRNA normalized with S26 mRNA (C, n = 4) and APOE-ELISA of supernatants (D, n = 4) of peritoneal Mφs from TRE mice cultured for 24 h. E, Quantitative RT-PCR of Il-6 and Ccl2 mRNA normalized with S26 mRNA (n = 5–10) of peritoneal Mφs from TRE mice cultured for 24 h. ‡p = 0.0035, compared with CCL2 from Cx3cr1G/GTRE3 Mφs (Mann–Whitney t test).