Tissue-Resident Macrophages in the Bone Marrow Comprise a Diverse Cellular Population Derived from Hematopoietic Stem Cells

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.3910-3910
• Main Authors: Kingsley, Paul D., Rust, Eli T., Kingsley, Alec J., Pinney, Jonathan J., Rivera-Escalera, Fátima, Bennett, Conner P., Vit, Leah A., Livada, Alison C., Morrell, Craig, Elliott, Michael R., McGrath, Kathleen E., Palis, James
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-188067

In the bone marrow, tissue-resident macrophages serve several specific roles including engulfing senescent neutrophils, maintaining hematopoietic stem cells (HSCs), and comprising an important component of the erythroid microenvironment within erythroblastic islands (EBIs). These latter macrophages are thought to “nurture” maturing erythroblasts with iron and cytokines, and to serve as a sink for erythroblast mitochondria, while simultaneously phagocytosing newly formed pyrenocytes. Here we sought to delineate the developmental origin of marrow macrophages, as well as their cellular heterogeneity within the complex cellular milieu of the marrow. Macrophages form an extensive cellular network extending long processes throughout the marrow, which results in cellular injury during dissociation and the presence of macrophage remnants on diverse hematopoietic cell types. We visualized macrophage remnants utilizing imaging flow cytometry to optimize conditions for marrow dissociation and to develop immunophenotyping that removes contaminants to enrich for bona fide macrophages. Importantly, in vitro coculture of marrow macrophages with late-stage erythroid progenitors significantly expanded erythroid output >1.5-fold, consistent with a role of EBI-macrophages in erythroblast maturation. To better define the diversity of macrophages within the marrow we performed single cell transcriptomic analysis using the 10X Genomics Chromium platform of sorted macrophages from mice at steady state and 3 days after induction of anemia by phlebotomy, which resulted in a 50% reduction in hematocrit. Approximately 2/3 of 2,937 steady state and 2,440 anemic marrow cells clustered together in UMAP space with expression consistent with macrophages (Csf1r and genes associated with phagocytosis), while the remaining 1/3 of cells comprised other hematopoietic lineages. Integration of our data with a larger dataset of myeloid cells in the marrow confirmed that our population of macrophages was distinct from other hematopoietic lineages, including monocytes and dendritic cells. Rescaling, dimensionality reduction, and clustering of macrophage cells in UMAP space revealed 8 clusters constituting three “super-clusters”, which we termed A, B, and C. The ‘A’ supercluster was enriched in iron metabolism (ferritin, heme oxygenase, Hfe, ferroportin) and mitochondrial processing (Pink1, Tmem14c) genes. Additionally, the proportion of cells in ‘A’ increased from 25% to 73% of all marrow macropha