Investigating the function and mechanism of interleukin 7 receptor-expressing pro-angiogenic macrophages during experimental choroidal neovascularization

项目总结： 新生血管性老年性黄斑变性(NAMD)是发达国家导致失明的主要原因， 仅通过抑制血管内皮生长因子(VEGF)进行治疗。虽然非常有效，但15%的 尽管接受了最大限度的抗血管内皮生长因子治疗，患者仍然失去了视力。因此，迫切需要新型的非血管内皮生长因子 治疗。补体途径与AMD相关，巨噬细胞表达补体 在手术切除的脉络膜新生血管(CNV)中发现了蛋白质和受体，以及巨噬细胞。 此外，脉络膜巨噬细胞耗竭会导致脉络膜萎缩，而全球巨噬细胞耗竭 抑制经典单核细胞来源的巨噬细胞可减少实验性CNV面积。因此， 巨噬细胞与人类CNV、稳态血管稳态和病理性脉络膜有关 血管生成。然而，众所周知，巨噬细胞是高度异质的细胞，可以执行许多 功能多样。因此，我们对野生型和经典单核细胞进行了单细胞RNA测序。 研究激光诱导CNV过程中巨噬细胞的来源、异质性和功能。我们 白细胞介素7受体(IL7R)表达的巨噬细胞可能来源于经典单核细胞 并显示促进血管生成的转录组。基于这些数据，我们的中心假设是IL7R+ 巨噬细胞是CNV发病过程中重要的促血管生成细胞。为了检验这一假设，我们制定了 以下是具体目标： 1)证明IL7R+巨噬细胞是CNV所必需的。为此，我们将使用Il7rCre x Rosa26GFP 小鼠命运图IL7R+巨噬细胞，Il7rCre x Cx3cr1iDTR小鼠耗尽IL7R+巨噬细胞，以及 Cx3cr1CrER x Il7rflx小鼠敲除IL7R。我们将检测IL7R+巨噬细胞和IL7R的功能 采用激光诱导的CNV模型。 2)确定IL7R+巨噬细胞来源于经典单核细胞，足以刺激CNV。 为此，我们将使用Ccr2Creer x Rosa26GFP小鼠来定位经典单核细胞，并确定经典单核细胞 单核细胞可分化为IL7R+巨噬细胞。此外，我们还将展示IL7R+巨噬细胞 通过从激光损伤后的小鼠眼内分离IL7R+巨噬细胞，足以刺激血管生成。 然后，我们将测试IL7R+与IL7Rneg巨噬细胞拯救CCR2-/-小鼠CNV面积缩小的能力。 这些目的的完成将决定IL7R+巨噬细胞是CNV所必需的，从经典的 单核细胞，并足以刺激血管生成。这些数据将为基于细胞的抗CNV奠定基础 治疗。以IL7R+巨噬细胞为靶点的细胞疗法，有可能抑制多个亲-巨噬细胞。 同时抑制血管生成途径，而不是单独抑制血管内皮生长因子，并改变NAMD的范式 心理治疗。

PROJECT SUMMARY: Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world, and is treated solely by inhibiting vascular endothelial growth factor (VEGF). Although highly effective, 15% of patients still lose vision despite maximal anti-VEGF therapy. Thus, a critical need exists for novel non-VEGF therapies. The complement pathway is genetically associated with AMD, macrophages express complement proteins and receptors, and macrophages are found in surgically excised choroidal neovascularization (CNV). Additionally, choroidal macrophage depletion results in choroidal atrophy, and both global macrophage depletion and inhibition of classical monocyte-derived macrophages reduce experimental CNV area. Therefore, macrophages are implicated in human CNV, steady state vascular homeostasis, and pathological choroidal angiogenesis. However, macrophages are known to be highly heterogeneous cells that can perform many diverse functions. Therefore, we performed single-cell RNA-sequencing on wildtype and classical monocyte- depleted mice to investigate macrophage origin, heterogeneity, and function during laser-induced CNV. We identified that interleukin-7 receptor (IL7R)-expressing macrophages are likely derived from classical monocytes and display a pro-angiogenic transcriptome. Based upon these data, our central hypothesis is that IL7R+ macrophages are critical pro-angiogenic cells during CNV pathogenesis. To test this hypothesis, we formulated the following specific aims: 1) Demonstrate that IL7R+ macrophages are necessary for CNV. In this aim, we will use Il7rCre x Rosa26GFP mice to fate map IL7R+ macrophages, Il7rCre x Cx3cr1iDTR mice to deplete IL7R+ macrophages, and Cx3cr1CrER x Il7rflox mice to knockout the IL7R. We will test the function of IL7R+ macrophages and the IL7R using the laser-induced CNV model. 2) Determine that IL7R+ macrophages are derived from classical monocytes and are sufficient to stimulate CNV. In this aim, we will use Ccr2CreER x Rosa26GFP mice to fate map classical monocytes and determine if classical monocytes can differentiate into IL7R+ macrophages. Furthermore, we will show that IL7R+ macrophages are sufficient to stimulate angiogenesis by isolating IL7R+ macrophages from mouse eyes after laser injury. We will then test the ability of IL7R+ vs IL7Rneg macrophages to rescue reduced CNV area in Ccr2-/- mice. Completion of these aims will determine that IL7R+ macrophages are necessary for CNV, derived from classical monocytes, and sufficient to stimulate angiogenesis. These data will set the stage for cell-based anti-CNV therapies. Cell-based therapies, targeting IL7R+ macrophages, have the potential to inhibit multiple pro- angiogenic pathways simultaneously, rather than inhibiting VEGF alone, and change the paradigm for nAMD therapy.