PROJECT SUMMARY Few therapeutic options exist for patients with age-related macular degeneration (AMD), and no FDA approved drugs are available for the most common subtype, dry AMD. Longitudinal imaging studies have identified the accumulation of drusen near the fovea as the single greatest anatomic risk factor for progression to severe disease, suggesting that loss of lipid homeostasis in the retinal pigmented epithelium (RPE) and choroid is a critical disease mechanism underlying AMD. Moreover, histologic and molecular studies have shown that inflammation of the macular RPE/choroid is an early, unifying feature of AMD. Yet, the molecular and cellular mechanisms responsible for the accumulation of lipid and inflammation in the RPE/choroid remain poorly understood. Using publicly available bulk and single-cell RNA-sequencing data from 150 human donors, we demonstrate that choroidal macrophages—a critical component of the choroidal ecosystem required for maintenance of the choriocapillaris and RPE function—are decreased and dysfunctional in the macular RPE/choroid in AMD. We detail novel transcriptomic observations related to the molecular underpinnings of choroidal macrophages in humans, demonstrating that they are robustly anti-inflammatory and express several key modules involved in lipid clearance. Of note is the expression of triggering receptor expressed on myeloid cells 2 (TREM2), a liporeceptor that mediates lipid homeostasis and immunosuppression in a range of tissues and is lost in several diseases involving accumulation of extracellular material. Using choroidal tissue from mice, we employ a variety of techniques to validate key findings related to choroidal macrophages. We show that choroidal macrophages are important in maintaining lipid homeostasis in the choroid during times of chronic lipid challenge, as these cells expand and contain nearly all of the excess lipid after 9 weeks of high-fat diet. These findings led us to our core hypothesis that TREM2+ choroidal macrophages modulate lipid homeostasis in the RPE/choroid and suppress inflammation and choroidal neovascularization. We propose two Aims using TREM2 knock-out mice to interrogate the role of TREM2 signaling in choroidal macrophages with respect to lipid metabolism and immune and neovascular suppression in the RPE/choroid. We anticipate the findings from this proposal will expand our understanding of the role of choroidal macrophages in the choroidal ecosystem and will identify TREM2 as a novel drug target in AMD. The proposed training plan for the PI, Seth Fortmann, is sponsored by his mentor, Dr. Maria Grant MD. The overall goal of the training plan is to provide the PI with a strong scientific foundation for a successful career as a physician-scientist ophthalmologist. Included in the training plan are experiences that will help the PI: 1) gain competence in a variety of techniques including bioinformatics, flow cytometry, primary cell culture, and ex vivo imaging of h...