Sustainment of bone marrow and spleen function by macrophages

驻留巨噬细胞(MO)通过基本未知的机制维持骨髓中专门的小生境中每天数千亿个血细胞。我的目标是了解常驻MOS的稳态生物学以及生理应激(缺氧、炎症、感染或氧化应激)对它们的调节。本文提出的工作在概念上是创新的，因为尚不清楚这些微环境与文献记载的单核细胞来源的微环境有多大的不同，以及周围微环境对其功能的影响程度。 我们将使用最先进的技术，包括体外低氧曲线分析和高分辨率细胞转录组分析，来研究中央MOS如何维持红母细胞岛中红细胞的产生。 通过在小鼠体内注射氯屈膦酸盐脂质体耗尽MOS的实验模型，我们产生了支持关于中枢MOS的两个主要假说的数据： 假设1.MOS通过参与凋亡的红细胞释放鞘氨醇1磷酸(S1P)的机制促进EPO的原位产生，并以这种方式维持稳定的红细胞生成。 假设2.骨髓稳态红细胞生成和脾应激性红细胞生成对巨噬细胞有不同的要求，因此涉及不同的细胞因子和调节因子。 我们的具体短期目标是： 1.通过S1P-EPO-凋亡轴研究骨髓中枢MOS对基础红细胞生成的调节。 2.研究氯膦酸盐脂质体部分MO消融两周后小鼠脾组织应激反应的性质。 3.比较基础和应激条件下BM岛和脾岛分离的MOS的转录组谱，以确定在低氧反应中的功能差异。 用流式细胞术研究正常小鼠和MO缺陷小鼠红系岛细胞的细胞周期和细胞凋亡，以及它们对S1P、低氧和EPO的反应性。应激反应的特点是应激红系细胞缺氧和扩张，骨髓和脾内MOS的转录本将通过批量RNA测序和功能注释分析进行比较。 这项研究将作为概念的证据，将MOS定位为红细胞凋亡的传感器，并通过自分泌-旁分泌EPO反应控制其生存。哺乳动物红系细胞具有相同的成熟过程，对低氧、炎症或氧化应激的反应在小鼠和其他物种中也有相似之处。我们的研究对于理解控制哺乳动物造血、骨髓和骨功能的生理机制是基础的，并可能改变我们普遍认为的缺氧是主要的红细胞生成调节因子的观点。

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.