Myeloid cell specific deficiency of RORα exacerbates choroidal neovascularization and subretinal inflammation in mice

Abstract

Purpose : Choroidal neovascularization (CNV) leads to visual decline in neovascular AMD, a leading cause of blindness in the elderly. Lipid-cholesterol metabolic pathway has been implicated in AMD pathology, precipitated by chronic non-resolving subretinal inflammation. Retinoic acid receptor-related orphan receptor alpha (RORα), a lipid-cholesterol sensing nuclear receptor has been implicated in neovascular AMD. Previously we found that systemic deficiency of RORα in Rora^sg/sg mice worsened laser-induced CNV, with increased inflammation. Here, we investigated the role of myeloid cell-specific RORα deficiency during aging and in a laser-induced CNV model.

Methods : Aged systemic (Rora^sg/sg) and myeloid specific RORα deficient (LysM^Cre/Rora^fl/fl) mice were examined for fundus imaging with their respective wild type and flox controls. Immunohistochemistry was carried out in RPE/choroid flat mounts and cross sections to visualize microglia/macrophages. Young adult LysM^Cre/Rora^fl/fl and flox control (Rora^fl/fl) mice were characterized for laser-induced CNV. RPE/choroid flat mounts were stained with isolectin to examine CNV lesions at 7 days post laser. Mouse monocyte/macrophage cells (RAW 264.7) were treated with synthetic RORα inverse agonists (SR3335 and SR1001) to assess lipid uptake and lipogenic gene expression in vitro. Chromatin immunoprecipitation (ChIP) assay was performed with retinal lysate to identify RORα target genes.

Results : Aged Rora^sg/sg and LysM^Cre/Rora^fl/fl mice both showed significant increase in fundus lesions compared with their controls, with accumulation of lipid-enriched microglia/macrophages in the subretinal space. LysMCre/Rora^fl/fl mice showed significant increase in CNV lesion size compared with flox controls. Moreover, RORα inhibition with synthetic inverse agonists enhanced macrophage lipid uptake of low-density lipoprotein in RAW 264.7 cells and substantially induced protein levels of PPARγ, a lipogenic gene. Direct binding of RORα to the promoter region of Pparg was confirmed with ChIP assay.

Conclusions : Our findings indicate that genetic deficiency of RORα in myeloid cells worsens subretinal inflammation during aging and exacerbates laser-induced CNV, in part by promoting macrophage lipid uptake and lipogenesis via PPARγ. Hence, RORα is a regulator of myeloid-mediated chronic subretinal inflammation and neovascularization in AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.