Sustainment of bone marrow and spleen function by macrophages

每天有数千亿个血细胞在骨髓中的专门小生境中，由常驻巨噬细胞（MO）通过基本上未知的机制维持。我的目标是了解居民MO的稳态生物学及其生理应激（缺氧，炎症，感染或氧化应激）的调制。本文提出的工作在概念上是创新的，因为尚不清楚这些MO与有据可查的单核细胞来源的MO有多大区别，以及周围微环境在多大程度上影响它们的功能。 我们将使用最先进的技术，包括缺氧概况的离体分析和高分辨率细胞转录组分析，以研究中央MO如何维持成红细胞岛中红细胞的产生。 通过在小鼠中注射氯膦酸盐脂质体以消耗MO的体内实验模型，我们生成了支持关于中枢MO的两个主要假设的数据： 假设1. MO通过涉及由凋亡成红细胞释放鞘氨醇1磷酸（S1 P）的机制促进EPO的原位产生，并以这种方式保持稳态红细胞生成。 假设2.骨髓稳态红细胞生成和脾应激红细胞生成对MO具有相反的要求，因此涉及不同的细胞参与者和调节因子。 我们的具体短期目标是： 1.探讨骨髓中心MO通过S1 P-EPO-凋亡轴对基础红细胞生成的调节作用。 2.表征氯膦酸盐脂质体部分MO消融后两周脾脏中产生的应激反应的性质。 3.比较基础和应激条件下从BM和脾岛分离的MO中的转录组谱，以鉴定对缺氧反应的功能差异。 将在对照小鼠和MO缺陷小鼠中通过流式细胞术研究成红细胞岛细胞的细胞周期和凋亡，以及它们对S1 P、缺氧和EPO的反应性。通过MO消融在脾脏中产生的应激反应将在缺氧和应激红系细胞扩增方面进行表征，并将通过批量RNA测序和功能注释分析来分析和比较骨髓和脾脏驻留MO的转录组。 这项调查将作为概念的证明，定位MO作为成红细胞凋亡的传感器和控制器，通过自分泌-旁分泌EPO反应的生存。哺乳动物红系细胞具有相同的成熟过程，并且在小鼠和其他物种中预期对缺氧、炎症或氧化应激的反应之间具有相似性。我们的研究是了解控制哺乳动物造血，骨髓和骨功能的生理机制的基础，并可能改变我们普遍认为缺氧是主要的红细胞生成调节剂。

Hundreds of billion blood cells every day in specialized niches in the bone marrow sustained by resident macrophages (MOs) through largely unknown mechanisms. My goal is to understand the steady state biology of resident MOs and their modulation by physiological stresses (hypoxia, inflammatory, infection or oxidative stress). The work herein proposed is conceptually innovative as it is unknown how distinct these MOs are from well-documented monocyte-derived MOs, and to what extent the surrounding microenvironment influences their function. We will use state-of-the-art technologies, including ex-vivo analysis of hypoxia profiles and high-resolution cell transcriptome analysis to study how central MOs sustain production of red blood cells in erythroblastic islands. By means of an experimental in vivo model of clodronate liposome injection in mice to deplete MOs, we have generated data supporting two major hypotheses concerning central MOs: Hypothesis 1. MOs contribute to in situ production of EPO through a mechanism involving sphingosine 1 phosphate (S1P) release by apoptotic erythroblasts, and in this manner preserve steady state erythropoiesis. Hypothesis 2. Bone marrow steady state erythropoiesis and splenic stress erythropoiesis have contrasting requirements for MOs, and as such involve distinct cellular players and regulatory factors. Our specific short-term objectives are to: 1. Investigate the regulation of basal erythropoiesis by bone marrow central MOs through the S1P-EPO-apoptosis axis. 2. Characterize the nature of the stress response generated in the spleen two weeks after partial MO ablation with clodronate liposomes. 3. Compare the transcriptome profiles in MOs isolated from BM and spleen islands under basal and stress conditions to identify functional differences in the responses to hypoxia. The cell cycle and apoptosis of cells from erythroblastic islands will be studied in control mice, and in MO-deficient mice by flow cytometry, as well as their responsiveness to S1P, hypoxia and EPO. The stress response generated in the spleen by MO ablation will be characterized in terms of hypoxia and expansion of stress erythroid cells, and transcriptomes of bone marrow and splenic resident MOs will be analyzed and compared by bulk RNA sequencing and functional annotation analysis. This investigation will serve as proof of concept to position MOs as sensors of erythroblast apoptosis and controllers of their survival through autocrine-paracrine EPO responses. Mammalian erythroid cells share identical maturation processes, and similarities among the responses to hypoxic, inflammatory or oxidant stresses are expected in mice and other species. Our research is fundamental for understanding the physiological mechanisms controlling mammalian hematopoiesis, bone marrow and bone function and may change our pervasive view of hypoxia as the major erythropoiesis regulator.