Grant: Discovery Grants Program - Individual

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.