Interferon-beta signaling in retinal mononuclear phagocytes attenuates pathological neovascularization

Abstract

Age-related macular degeneration (AMD) is a leading cause of vision loss among the elderly. AMD pathogenesis involves chronic activation of the innate immune system including complement factors and microglia/macrophage reactivity in the retina. Here, we show that lack of interferon-β signaling in the retina accelerates mononuclear phagocyte reactivity and promotes choroidal neovascularization (CNV) in the laser model of neovascular AMD Complete deletion of interferon-α/β receptor (Ifnar) using Ifnar1(-/-) mice significantly enhanced early microglia and macrophage activation in lesion areas. This triggered subsequent vascular leakage and CNV at later stages. Similar findings were obtained in laser-treated Cx3cr1(Cre) (ER):Ifnar1(fl/fl) animals that allowed the tamoxifen-induced conditional depletion of Ifnar in resident mononuclear phagocytes only. Conversely, systemic IFN-β therapy of laser-treated wild-type animals effectively attenuated microgliosis and macrophage responses in the early stage of disease and significantly reduced CNV size in the late phase. Our results reveal a protective role of Ifnar signaling in retinal immune homeostasis and highlight a potential use for IFN-β therapy in the eye to limit chronic inflammation and pathological angiogenesis in AMD.

Keywords: age‐related macular degeneration; choroidal neovascularization; interferon‐beta signaling; macrophages; microglia.

Figure 1. Loss of Ifnar1 signaling elicits inflammatory processes and angiogenesis in laser‐induced retinal damage

1. A

   Experimental design. Laser coagulation was performed in C57BL6/J control and Ifnar1 ^−/− mice. Animals were analyzed 3, 7, and 14 days after laser treatment.

2. B, C

   Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in C57BL6/J controls (B) and Ifnar1 ^−/− (C) mice. Scale bar: 20 μm.

3. D

   Quantification of amoeboid‐shaped mononuclear phagocytes in laser spots. Values show mean ± SD (n = 11–12 retinas; unpaired Student's t‐test: ***P = 0.0004).

4. E

   Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD (n = 42–62 cells; unpaired Student's t‐test: ***P < 0.0001).

5. F–K

   Representative fundus fluorescein angiography images of C57BL6/J (F–H) and Ifnar1 ^−/− (I–K) mice 3, 7, and 14 days after laser‐induced damage.

6. L

   Quantification of vascular leakage by analyzing pixel intensities at 3, 7, and 14 days after laser‐induced retinal damage in C57BL6/J controls versus Ifnar1 ^−/− mice. Values show mean ± SD (n = 14–22 eyes; one‐way ANOVA followed by Tukey's post‐test: ***P < 0.0001).

7. M–P

   Representative images of lectin‐stained choroidal flat mounts 7 and 14 days after laser coagulation in C57BL6/J control mice (M, N) and Ifnar1 ^−/− animals (O, P). Dashed lines indicate CNV areas, and the asterisk marks the central optic nerve head. Scale bar: 200 μm.

8. Q

   Quantification of lectin‐stained CNV areas with ImageJ software. Bars show mean ± SD (n = 4–11 RPE/choroidal flat mounts; one‐way ANOVA followed by Tukey's post‐test: *P = 0.0281).

Figure EV1. Loss of Ifnar1 enhances CNV and leads to accumulation of mononuclear phagocytes in the subretinal space

1. A, B

   Representative images of Iba1 (green) and lectin (red) co‐stained retinal cross sections 3 days after laser coagulation in C57BL6/J controls (A) and Ifnar1 ^−/− (B) mice with nuclear staining (DAPI, blue). The white box indicates the spot where the laser hit the retina. Scale bar 50 μm.

2. C, D

   Representative images of Iba1 (green) stained retinal flat mounts and Iba1 and lectin (red) co‐stained RPE/choroidal flat mounts 7 days after laser coagulation in C57BL6/J (C) and Ifnar1 ^−/− (D) mice. Scale bar 50 μm.

Figure 2. IFN‐β ameliorates microgliosis and inhibits choroidal neovascularization

1. A

   Experimental design. Laser coagulation was performed in all C57BL6/J mice that were either untreated or received 10,000 units IFN‐β i.p. every second day until animals were analyzed 3, 7, and 14 days after laser treatment, respectively.

2. B, C

   Representative Iba1 stainings of retinal flat mounts detecting microglia/macrophages in laser spots 3 days after laser coagulation in control mice (B) or IFN‐β‐treated animals (C). Scale bar: 20 μm.

3. D

   Quantification of amoeboid‐shaped mononuclear phagocytes in laser spots. Values show mean ± SD (n = 7–10 retinas; unpaired Student's t‐test: ***P < 0.0001).

4. E

   Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD (n = 41–62 cells; unpaired Student's t‐test: ***P < 0.0001).

5. F–K

   Representative fundus fluorescein angiography images of control mice (F–H) or IFN‐β‐treated animals (I–K) 3, 7, and 14 days after laser‐induced damage.

6. L

   Quantification of vascular leakage by analyzing pixel intensities at 3, 7, and 14 days after laser‐induced retinal damage in control mice and IFN‐β‐treated animals. Values show mean ± SD (n = 15–22 eyes; one‐way ANOVA followed by Tukey's post‐test: ***P = 0.0004).

7. M–P

   Representative images of lectin‐stained choroidal flat mounts 7 and 14 days after laser coagulation in control mice (M, N) and IFN‐β‐treated animals (O, P). Dashed lines indicate CNV areas, and the asterisk marks the central optic nerve head. Scale bar: 200 μm.

8. Q

   Quantification of lectin‐stained CNV areas with ImageJ software. Bars show mean ± SD (n = 7–12 RPE/choroidal flat mounts; one‐way ANOVA followed by Tukey's post‐test: **P = 0.0038).

Figure EV2. Reduced CNV and subretinal mononuclear phagocyte accumulation in IFN‐ß‐treated mice

1. A, B

   Representative images of Iba1 (green) and lectin (red) co‐stained retinal cross sections 3 days after laser coagulation in C57BL6/J controls (A) and IFN‐ß‐treated C57BL6/J (B) mice with nuclear staining (DAPI, blue). The white box indicates the spot where the laser hit the retina. Scale bar 50 μm.

2. C, D

   Representative images of Iba1 (green) stained retinal flat mounts and Iba1 and lectin (red) co‐stained RPE/choroidal flat mounts 7 days after laser coagulation in C57BL6/J controls (C) and IFN‐ß‐treated C57BL6/J (D) mice. Scale bar 50 μm.

Figure 3. Loss of Ifnar1 signaling in mononuclear phagocytes enhances CNV

1. A

   Experimental design. Laser coagulation was performed in tamoxifen‐treated Cx3cr1 ^CreER , Ifnar1 ^fl/fl, and Cx3cr1 ^CreER:Ifnar1 ^fl/fl mice. Animals were analyzed 3, 7, and 14 days after laser treatment.

2. B–D

   Representative Iba1 staining results of retinal flat mounts detecting microglia/macrophages in laser spot 3 days after laser coagulation in Cx3cr1 ^CreER (B), Ifnar1 ^fl/fl (C), and Cx3cr1 ^CreER:Ifnar1 ^fl/fl (D) mice. Scale bar: 20 μm.

3. E

   Quantification of amoeboid‐shaped mononuclear phagocytes in laser spots. Values show mean ± SD (n = 17–29 retinas; unpaired Student's t‐test: ***P = 0.0003, **P = 0.0044).

4. F

   Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD (n = 44–52 cells; unpaired Student's t‐test: ***P < 0.0001).

5. G–O

   Representative fundus fluorescein angiography images of Cx3cr1 ^CreER (G–I), Ifnar1 ^fl/fl (J–L), and Cx3cr1 ^CreER:Ifnar1 ^fl/fl (M–O) mice 3, 7, and 14 days after laser‐induced damage.

6. P

   Quantification of vascular leakage by analyzing pixel intensities at 3, 7, and 14 days after laser‐induced retinal damage in Cx3cr1 ^CreER , Ifnar1 ^fl/fl, and Cx3cr1 ^CreER:Ifnar1 ^fl/fl mice. Values show mean ± SD (n = 5–12 eyes; one‐way ANOVA followed by Tukey's post‐test: **P = 0.0032, *P = 0.0247).

7. Q–V

   Representative images of lectin‐stained choroidal flat mounts 7 and 14 days after laser coagulation in Cx3cr1 ^CreER (Q, R), Ifnar1 ^fl/fl (S, T), and Cx3cr1 ^CreER:Ifnar1 ^fl/fl (U, V) mice. Dashed lines indicate CNV areas, and the asterisk marks the central optic nerve head. Scale bar: 200 μm.

8. W

   Quantification of lectin‐stained CNV areas with ImageJ software. Bars show mean ± SD (n = 4–11 RPE/choroidal flat mounts; one‐way ANOVA followed by Tukey's post‐test: *P = 0.043, ***P = 0.0007).

Figure EV3. Loss of Ifnar1 in microglia/macrophages leads to their subretinal accumulation and enhances CNV

1. A–C

   Representative images of Iba1 (green) and lectin (red) co‐stained retinal cross sections 3 days after laser coagulation in C57BL6/J controls (A) and Ifnar1 ^−/− (B) and Cx3cr1 ^CreER:Ifnar1 ^fl/fl (C) mice with nuclear staining (DAPI, blue). The white box indicates the spot where the laser hit the retina. Scale bar 50 μm.

2. D–F

   Representative images of Iba1 (green) stained retinal flat mounts and Iba1 and lectin (red) co‐stained RPE/choroidal flat mounts 7 days after laser coagulation in C57BL6/J controls (D) and Ifnar1 ^−/− (E) and Cx3cr1 ^CreER:Ifnar1 ^fl/fl (F) mice. Scale bar 50 μm.