Macrophage subpopulations in the human choroid. (A) Subclustering of scRNA-seq data from myeloid cluster in (Fig. 1 I). UMAP plot is shown. (B) scRNA-seq marker genes of the two macrophage (MФ) clusters from A. Log transformed, size factor normalized gene expression is shown. (C) Representative flow cytometry from a nondiseased donor pre-gated on macrophages showing the two macrophage subpopulations from A. (D) Flow cytometry mean fluorescent intensities (MFIs) from FOLR2⁺ versus FOLR2⁻ macrophages. Four of the marker genes from B are shown. All donors were nondiseased (n = 9 eyes). Student’s t test. (E) Live-cell imaging of FACS-sorted FOLR2⁻ and FOLR2⁺ macrophages from a nondiseased donor. Scale bar = 50 μm. (F) Multiplex immunofluorescence showing FOLR2⁻ and FOLR2⁺ macrophages. Scale bar = 50 μm. (G) Volcano plot of differentially expressed genes (DEGs) between FOLR2⁺ versus FOLR2⁻ macrophages from scRNA-seq data of nondiseased donors. Blue = genes upregulated in FOLR2⁺ macrophages (902 genes). Yellow = genes upregulated in FOLR2⁻ macrophages (3,762 genes). Magenta = AMD risk genes. (H) GSEA of DEGs from G showing selected pathways. Dashed lines represent significance cutoff. (I) Nile red staining in a choroidal whole mount from a nondiseased donor. Scale bar = 50 μm. (J) Representative flow cytometry plot from a nondiseased donor showing FOLR2 and Nile red staining in macrophages. (K) Flow cytometry MFI of Nile red staining in FOLR2⁺ and FOLR2⁻ macrophages. All donors were nondiseased (n = 9 eyes). Student’s t test. (L) Representative immunofluorescence of human choroid incubated with dil-LDL. White box is area shown by magnified images to right. Large image scale bar = 100 μm. Small image scale bars = 50 μm. (M) Representative flow cytometry plot from a nondiseased donor showing FOLR2 and dil-LDL staining in macrophages. (N) Flow cytometry MFI of dil-LDL staining in FOLR2⁺ and FOLR2⁻ macrophages. All donors were nondiseased (n = 6 eyes). Student’s t test. (O) Flow cytometry MFI of LDLR, SCARB1, MSR1, CD206, and ABCA1 staining in FOLR2⁺ and FOLR2⁻ macrophages. All donors were nondiseased (n = 9 eyes). Student’s t test. (P) LC/MS lipidomics from FACS-sorted FOLR2⁺ and FOLR2⁻ macrophages from a nondiseased donor. Lipid abundances are shown for FOLR2⁺ macrophages relativized to FOLR2⁻ macrophages. VLC = very long chain; SM = sphingomyelin; LacCer = lactosylceramide; PC= phosphatidylcholine; Cer = ceramide; DG = diglyceride; ST = sterol; MG = monoglyceride; HexCer = hexosylceramide; PC = phosphatidylcholine; TG = triglyceride; PE = phosphatidylethanolamine. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.