Decline and dysfunction of FOLR2 ⁺ choroidal macrophages in AMD. (A) Frequency of FOLR2⁺ macrophages (MФ) in scRNA-seq data relative to all myeloid cells. Sample sizes represent unique donors by location and disease group. One-way ANOVA with Holm–Sidak correction. (B) FOLR2 normalized gene expression in RPE/choroid bulk RNA-seq data. Sample sizes represent unique donors by location and disease group. One-way ANOVA with Holm–Sidak correction. (C) Plot showing marker genes of FOLR2⁺ macrophages from scRNA-seq data. Marker genes highlighted by gray box are those used for the heatmap in D. X axis shows proportion of raw counts derived from FOLR2⁺ macrophages divided by the total raw counts for that gene. (D) Heatmap of the top 17 marker genes for FOLR2⁺ macrophages from C. (E) Example of ex vivo imaging from an AMD donor eye. Top left = fundus image using epi-illumination. Top middle = fundus image using trans-illumination. Top right = en-face OCT showing location of B scan below. Scale bars = 200 μm. (F) Representative flow cytometry plots from nondiseased (left) and AMD (right) donors showing FOLR2⁺ and FOLR2⁻ macrophages. (G) Frequency of FOLR2⁺ macrophages relative to all macrophages from flow cytometry. Sample sizes are individual eyes. Atrophic AMD (orange) and neovascular AMD (magenta). Student’s t test. (H) Absolute counts of FOLR2⁺ macrophages per mm² of choroidal tissue from flow cytometry. Sample sizes are individual eyes. Atrophic AMD (orange) and neovascular AMD (magenta). Student’s t test. (I) Flow cytometry percent positive staining for cleaved caspase 3 and Ki67 staining in FOLR2⁺ macrophages from nondiseased (n = 9 eyes) and AMD (n = 8) donors. Student’s t test. (J) Representative immunofluorescence of nondiseased and AMD choroids stained with FOLR2, Ki67, and IBA1. Scale bar = 100 μm. (K) Volcano plot of differentially expressed genes (DEGs) in AMD versus nondiseased macular FOLR2⁺ macrophages from scRNA-seq data. Blue = genes upregulated in AMD (499 genes). Yellow = genes upregulated in nondiseased (297 genes). (L) GSEA of DEGs from K showing selected pathways. Dashed lines represent significance cutoffs. (M) Representative immunofluorescence of human choroids incubated with dil-LDL from nondiseased (top) and AMD (bottom) donors. Scale bars = 100 μm. (N) Histograms of dil-LDL staining in FOLR2⁺ macrophages from representative nondiseased and AMD donors. Dashed line is cutoff from negative control. (O) Flow cytometry MFI of dil-LDL staining in FOLR2⁺ macrophages from nondiseased (n = 6 eyes) and AMD (n = 4 eyes) choroids. Student’s t test. (P) Flow cytometry MFI of Nile red, LDLR, CD206, CD36, PLTP, and ABCA1 staining in FOLR2⁺ macrophages from nondiseased (n = 9 eyes) and AMD (n = 8 eyes) donors. Student’s t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. MFI; mean fluorescent intensity.