Figure 4.

APOE inhibits subretinal MP clearance

1. Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).
2. Quantifications of CFSE⁺F4/80⁺ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1^GFP/GFP CFSE⁺ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann–Whitney U-test, C57BL/6J versus Cx3cr1^GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317).
3. Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE⁺ macrophages (experiment repeated three times, inset close-up view).
4. Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1^GFP/GFP CFSE⁺ macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1^GFP/GFP macrophages into C57BL/6J (n = 16–20/group; Mann–Whitney U-test, *P = 0.0024).
5. Quantification of subretinal CFSE⁺ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE⁺ magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1^GFP/GFP mice into C57BL/6J mice (n = 8–12/group; Mann–Whitney U-test, *P = 0.0006).
6. Quantification of subretinal CFSE⁺ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE⁺CD11b FACS-sorted brain microglial cells from C57BL/6J and Cx3cr1^GFP/GFP mice into C57BL/6J mice (n = 9–12/group; Mann–Whitney U-test, *P = 0.0087).
7. Quantification of subretinal CFSE⁺F4/80⁺ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE⁺ macrophages from C57BL/6J, Cx3cr1^GFP/GFP, Cx3cr1^GFP/GFPApoE^−/−, and ApoE^−/− mice into C57BL/6J mice (n = 8–12/group; one-way ANOVA/Dunnett test of Cx3cr1^GFP/GFP versus any other group *P ≤ 0.0001; Mann–Whitney U-test, Cx3cr1^GFP/GFP versus Cx3cr1^GFP/GFPApoE^−/− *P = 0.0006).
8. Quantification of subretinal CFSE⁺F4/80⁺ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE⁺ macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations (n = 6–7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg *P = 0.0488; C57BL/6J versus 100 μg ^‡P = 0.006. Mann–Whitney U-test: C57BL/6J versus 10 μg *P = 0.0012; C57BL/6J versus 100 μg ^‡P = 0.0013).

Data information: All primary cells were prepared from male mice; all recipient C57BL/6J mice were male. Mo: monocytes; MC: microglial cells; Mϕ: macrophages; SCC-A: side scatter detector A. Scale bars: 1 mm (A); 50 μm (C).