FOLR2+ Choroidal Macrophages are a Homeostatic Lipid Handling Subset of Phagocytes that are Depleted in AMD

Abstract

Purpose : At steady-state, the choroid contains an abundance of innate and adaptive immune cells. How the choroidal immune system contributes to the pathogenesis of AMD remains unclear. The current study aims to characterize choroidal macrophage dysfunction in AMD.

Methods : Single-cell (n=31 samples; scRNA-seq) and bulk (n=269 samples) RNA-sequencing data from human RPE/choroid were analyzed. Flow cytometry and wholemount immunofluorescence were performed on RPE/choroid from 15 donors. LC-MS lipidomics was used on FACS sorted human choroidal macrophages and human RPE/choroid was treated ex vivo with dil-LDL. Flow cytometry and confocal immunofluorescence were performed on RPE/choroid/sclera from MS4A3^Cre/TdTomato^flox mice for lineage tracing and LYVE1^Cre/CSF1R^floxDTR mice treated with diphtheria toxin for depletion studies.

Results : scRNA-seq of the RPE/choroid in nondiseased and AMD donors revealed two populations of macrophages, which were distinguished on the basis of FOLR2 expression. In AMD samples, FOLR2+ macrophages were depleted. FOLR2+ cells showed decreased expression of pro-inflammatory mediators and increased expression of lipid handling proteins. FOLR2+ macrophages had robust staining with the neutral lipid dye nile red and strong uptake of fluorescently labeled LDL in ex vivo treated human choroids. LC-MS lipidomics revealed a broad increase of multiple lipid species in FOLR2+ macrophages, especially sphingomyelin. Multiplex immunofluorescence on human choroid wholemounts revealed that FOLR2+ macrophages were intimately associated with peripheral nerve bundles, particularly tyrosine hydroxylase+ sympathetic nerves. FOLR2+ macrophages showed high expression of COMT, an enzyme that inactivates catecholamine neurotransmitters. Lineage tracing using MS4A3-Cre reporter mice revealed that >80% of FOLR2+ macrophages were prenatally derived. Using LYVE1-Cre/CSF1R-DTR transgenic mice to deplete FOLR2+ macrophages, we observed a large influx of LY6C+ monocytes at baseline and mass infiltration of LY6G+ neutrophils with laser-CNV.

Conclusions : Our findings identify a unique population of FOLR2-expressing choroidal macrophages that are implicated in lipid metabolism and peripheral nerve homeostasis and are depleted in AMD. Murine studies support that FOLR2+ choroidal macrophages are prenatally derived and that their depletion results in infiltration of proinflammatory cells.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.