Role of choroidal macrophages in regulating lipid homeostasis and immunosuppression of the RPE/choroid

项目摘要 年龄相关性黄斑变性（AMD）患者的治疗选择很少，FDA也没有 已批准的药物可用于最常见的亚型干性AMD。纵向成像研究有 将黄斑中心凹附近玻璃疣的累积确定为进展的最大解剖学风险因素 严重的疾病，这表明视网膜色素上皮细胞（RPE）和 脉络膜是AMD的重要发病机制。此外，组织学和分子研究表明， 黄斑RPE/脉络膜的炎症是AMD的早期统一特征。然而，分子和细胞 负责RPE/脉络膜中脂质积累和炎症的机制仍然很差 明白使用来自150名人类供体的公开可用的批量和单细胞RNA测序数据，我们 证明脉络膜巨噬细胞-脉络膜生态系统的关键组成部分， 维持脉络膜毛细血管和视网膜色素上皮的功能-减少和功能障碍，在黄斑 AMD中的RPE/脉络膜。我们详细介绍了新的转录组学观察相关的分子基础， 人类脉络膜巨噬细胞，证明它们是强大的抗炎和表达几个 参与脂质清除的关键模块。值得注意的是髓系细胞上表达的触发受体的表达， 细胞2（TREM 2），一种在一系列组织中介导脂质稳态和免疫抑制的脂质受体 并且在涉及细胞外物质积累的几种疾病中丢失。利用老鼠的脉络膜组织， 我们采用多种技术来验证与脉络膜巨噬细胞相关的关键发现。我们证明了 脉络膜巨噬细胞在慢性脂质代谢期间维持脉络膜中的脂质稳态中是重要的 挑战，因为这些细胞在高脂肪饮食9周后膨胀并含有几乎所有的过量脂质。这些 这些发现使我们提出了我们的核心假设，即TREM 2+脉络膜巨噬细胞调节了视网膜中的脂质稳态。 RPE/脉络膜和抑制炎症和脉络膜新生血管。我们提出了两个目标，使用TREM 2 敲除小鼠以询问TREM 2信号传导在脉络膜巨噬细胞中关于脂质的作用 RPE/脉络膜中的代谢和免疫及新生血管抑制。我们预计， 该提案将扩大我们对脉络膜巨噬细胞在脉络膜生态系统中的作用的理解， 鉴定TREM 2为AMD中新药物靶点。 PI Seth Fortmann的拟议培训计划由其导师Maria Grant MD博士赞助。 培训计划的总体目标是为PI提供成功职业生涯的强大科学基础 作为一个眼科医生培训计划中包括的经验将有助于PI：1） 获得各种技术的能力，包括生物信息学，流式细胞术，原代细胞培养， 人供体眼睛的离体成像; 2）发展假设驱动的研究; 3）在口腔和 书面形式; 4）有效地将研究与临床结合起来; 5）与其他科学家合作; 6） 负责任地进行研究。

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 human donor eyes; 2) develop hypothesis-driven research; 3) present data in an oral and written format; 4) effectively integrate research with the clinic; 5) collaborate with other scientists; and 6) responsibly conduct research.