Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored. We performed multi-parameter flow cytometry on wildtype (WT) and Ccr2−/− mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1CreER/+ ; Rosa26zsGFP/+ mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function. We identified 4 macrophage subsets in mice: microglia, MHCII+CD11c−, MHCII+CD11c+, and MHCII−. Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII− macrophages are long lived, tissue resident macrophages, while MHCII+CD11c− and MHCII+CD11c+ macrophages are partially replenished from blood monocytes. After laser injury, MHCII+CD11c− macrophages are entirely derived from classical monocytes, MHCII− macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCII+CD11c+ macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCII+CD11C− and three MHCII+CD11C+ populations. One MHCII+CD11C+ subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCII+CD11C− and two MHCII+CD11C+ subsets, including the disease-associated cluster. The upregulated MHCII+CD11C− pro-angiogenic genes were unique compared to the increased MHCII+CD11C+ angiogenesis genes. Macrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.

中文翻译：

---

稳态和实验性脉络膜新生血管形成过程中的眼部巨噬细胞起源和异质性

新生血管年龄相关性黄斑变性 (nAMD) 通常会因异常血管生成而导致视力丧失，称为脉络膜新生血管 (CNV)。巨噬细胞是实验性 CNV 所必需的异质细胞，存在于人类 CNV 样本中，可以显示多种功能，这取决于它们的起源和组织微环境。尽管存在这些关联，脉络膜巨噬细胞异质性仍未得到探索。我们在激光损伤后对野生型 (WT) 和 Ccr2-/- 小鼠进行了多参数流式细胞术，以识别巨噬细胞亚型，并确定哪些亚群源自经典单核细胞。为了在稳态和激光损伤后绘制组织驻留巨噬细胞的命运图，我们使用了 Cx3cr1CreER/+；Rosa26zsGFP/+ 小鼠模型。我们重新分析了先前发表的来自健康和 nAMD 患者的人类脉络膜样本的单细胞 RNA-seq，以研究人类巨噬细胞的异质性、疾病关联和功能。我们在小鼠中鉴定了 4 个巨噬细胞亚群：小胶质细胞、MHCII+CD11c-、MHCII+CD11c+ 和 MHCII-。小胶质细胞是稳定状态下的组织驻留巨噬细胞，不受激光损伤的影响。在稳定状态下，MHCII- 巨噬细胞是长寿的组织驻留巨噬细胞，而 MHCII+CD11c- 和 MHCII+CD11c+ 巨噬细胞部分由血液单核细胞补充。激光损伤后，MHCII+CD11c-巨噬细胞完全来源于经典单核细胞，MHCII-巨噬细胞来源于经典单核细胞（90%）和组织驻留巨噬细胞的扩增（10%），MHCII+CD11c+巨噬细胞来源于经典单核细胞（ 70%), 非经典单核细胞 (10%) 和组织驻留巨噬细胞的扩增 (20%)。人类脉络膜的单细胞 RNA-seq 分析发现了 5 个巨噬细胞亚群：两个 MHCII+CD11C- 和三个 MHCII+CD11C+ 群体。一个 MHCII+CD11C+ 子集有 78% 来自 nAMD 患者。差异表达分析确定了一个 MHCII+CD11C- 和两个 MHCII+CD11C+ 亚群中促血管生成基因表达的上调，包括疾病相关簇。与增加的 MHCII+CD11C+ 血管生成基因相比，上调的 MHCII+CD11C- 促血管生成基因是独一无二的。巨噬细胞起源影响小鼠稳态和激光损伤后的异质性。小鼠和人类患者都表现出相似的巨噬细胞亚型。人类脉络膜中存在两种离散的促血管生成巨噬细胞群。针对性强，