In vivo function of macrophage in healthy and diseased erythropoiesis

描述（由申请人提供）： 50 年前，Marcel Bessis 提出了特定骨髓 (BM) 微环境促进谱系定型血液前体细胞分化的观点，他在开创性研究中描述了成红细胞岛，这是一种由中央巨噬细胞 (M) 组成的结构，周围环绕着处于不同分化阶段的成红细胞。尽管使用体外重建分析的研究已明确表明骨髓源性 M¿ 在红细胞生成中发挥作用，但目前没有体内证据表明 M 是出生后红细胞生成所必需的或什至发挥作用。体内知识缺乏的一个主要原因是 BM M 的性质定义不明确，并且直到最近还缺乏特定的遗传模型。我们已经定义了 BM 单核吞噬细胞的子集，并表明 CD169 M¿ 调节造血干细胞生态位。我们的初步研究还表明，BM CD169 M¿ 促进红细胞生成，因为它们的选择性体内消耗会损害 5-氟尿嘧啶或苯肼诱导的贫血后红细胞生成的恢复。此外，在由突变 JAK2V617F 转基因过表达引起的真性红细胞增多症 (PV) 遗传模型中，我们发现 M¿ 消耗使血细胞比容正常化，这表明 PV 中的红细胞生成出乎意料地取决于来自微环境的信号。基于这些初步结果，我们建议进一步评估 BM M¿ 在健康和患病红细胞生成中的分子基础和功能。在具体目标 1 中，我们将定义调节 BM 红细胞生态位（红细胞岛）的体内分子机制。我们将与 M. Narla 博士 (NYBC) 和 J. Chasis (伯克利) 合作表征 CD169 红细胞岛，并通过与 M. Merad 博士 (西奈山) 合作通过流式细胞术和转录谱分析获得对中央 M¿ 的新见解。我们将为先前建议在成红细胞岛中发挥作用的候选 M¿ 受体（Vcam1、Itgav 和 Maea）生成巨噬细胞特异性遗传模型。在具体目标 2 中，我们将检查骨髓 M¿在慢性患病红细胞中的贡献。我们将与 S. Rivella 博士（康奈尔大学）合作分析红细胞生成应激模型，例如镰状细胞病和地中海贫血。然后，我们将与 J.Zhao 博士（俄克拉荷马州）和 Tony Green（剑桥）合作，使用第一个目标中生成的 M¿ 特异性 CD169-DTR 耗竭和 M¿ 特异性遗传模型来评估转基因 JAK2V617F 模型中巨噬细胞的影响。在具体目标 3 中，我们将研究扩大中央巨噬细胞在清髓术后红细胞生成恢复中的潜在治疗益处。我们将使用转基因模型（与爱因斯坦 R. Stanley 博士合作的 Csf1 过表达）和药理学方法（长效 IL-4 复合物）。这些研究将首次明确 BM 巨噬细胞在红细胞生成中的体内作用，并可能导致在以红细胞扩张为特征的疾病中调节红细胞生成的新方法。 （摘要完）

DESCRIPTION (provided by applicant): The notion that a specific bone marrow (BM) microenvironment promotes the differentiation of lineage- committed blood precursors was suggested 50 years ago by Marcel Bessis where he described in seminal studies the erythroblastic island, a structure made of a central macrophage (M) surrounded by erythroblasts at varying stages of differentiation. Although studies using in vitro reconstitution analyses have clearly suggested a role for marrow-derived M¿ in erythropoiesis, there is currently no in vivo evidence that M¿ are required or even play a role in postnatal erythropoiesis. A major reason for the deficit of in vivo knowledge stems from the poorly defined nature of BM M¿ and, until recently, the lack of specific genetic models. We have defined subsets of BM mononuclear phagocytes and showed that CD169+ M¿ regulated the hematopoietic stem cell niche. Our preliminary studies also suggest that BM CD169+ M¿ promote erythropoiesis since their selective in vivo depletion compromised erythropoietic recovery after 5-fluorouracil- or phenylhydrazine-induced anemia. Additionally, in a genetic model of polycythemia vera (PV) from transgenic overexpression of mutated JAK2V617F, we have found that M¿ depletion normalized the hematocrit, suggesting that erythropoiesis in PV, unexpectedly, depends on signals from the microenvironment. Based on these preliminary results, we propose to evaluate further the molecular basis and function of BM M¿ in healthy and diseased erythropoiesis. In Specific Aim 1, we will define in vivo molecular mechanisms regulating the BM erythroid niche (erythroblastic island). We will characterize CD169+ erythroblastic islands in collaboration with Drs M. Narla (NYBC) and J. Chasis (Berkeley) and get new insight on central M¿ by flow cytometry and transcriptional profiling collaboration with Dr. M. Merad (Mt. Sinai). We will generate macrophage-specific genetic models for candidate M¿ receptors previously suggested to play a role in the erythroblastic island (Vcam1, Itgav, and Maea). In Specific Aim 2, we will examine the contribution of bone marrow M¿ in chronically diseased erythron. We will analyze models of erythropoietic stress such as sickle cell disease and thalassemia, the latter in collaboration with Dr. S. Rivella (Cornell). We will then evaluate the impact of macrophage in transgenic JAK2V617F models in collaboration with Dr. J. Zhao (Oklahoma) and Tony Green (Cambridge) using M¿-specific CD169-DTR depletion and M¿-specific genetic models generated in the first aim. In Specific Aim 3, we will examine the potential therapeutic benefit of expanding the central macrophage in the erythropoietic recovery after myeloablation. We will use transgenic models (Csf1 overexpression in collaboration with Dr. R. Stanley, Einstein) and pharmacological approaches (long-acting IL-4 complexes). These studies will define for the first time the in vivo roles of BM macrophages in erythropoiesis and will likely lead to novel ways to regulate erythropoiesis in diseases characterized by erythron expansion. (End of Abstract)