Cultured Human Macrophages As a Model for Central Macrophages in Erythroblastic Islands

Erythropoiesis occurs in erythroblastic islands, specific structures in the bone marrow comprising a central macrophage surrounded by erythroid precursors at different stages of terminal differentiation. The central macrophage of the erythroblastic island supports proliferation and differentiation of erythroblasts, as well as phagocytosis of the extruded erythroblast nuclei, the pyrenocytes. Its identity, however, has been poorly characterized. We previously showed that macrophages also enhance in vitro erythropoiesis because they support hematopoietic stem cell (HSC) survival [Heideveld et al. 2015]. Thus, bone marrow macrophages affect all stages of erythropoiesis. The aim of our study is to characterize the relevant human bone marrow macrophages and unravel the mechanism by which they support erythropoiesis with the ultimate goal (i) to optimize erythroblast culture systems that produce erythrocytes for transfusion purposes, and (ii) to target macrophages in vivo to improve erythropoiesis in anemic patients.

Macrophages are a heterogeneous population, that can be divided into pro-inflammatory M1 and anti-inflammatory M2 macrophages. Macrophages that we showed to support stem cell survival, and subsequently enhance the yield of erythroid cell cultures, were characterized as a subclass: M2c-like macrophages. These macrophages were derived from CD14⁺ cells isolated from human peripheral blood mononuclear cells that were cultured in serum-free media supplemented with stem cell factor, erythropoietin and dexamethasone. Within three days these macrophages expressed CD163high, CD169, mannose receptor (MR), CXCR4 and HLA-DR and harbored characteristics of bone marrow resident macrophages. This differentiation process was dependent on glucocorticoid receptor activation. Mass spectrometry of monocytes cultured in presence and absence of dexamethasone showed that expression of CD163 and MR was strictly Dex-dependent, underscoring the role of glucocorticoids in the phenotype of M2c macrophages. Protein ontology analysis revealed dexamethasone-mediated enrichment of lysosome, endocytosis and endothelial development (e.g. STAB1, IL13RA1, CD81, SLC1A3 and FKBP5). We wondered whether these macrophages with increased endosomal and lysosomal capacity not only support stem cell survival and enable erythroid commitment, but also support erythroblastic islands. In mice, it has been shown that clearance of the pyrenocytes by central macrophages occurs presumably via TAM-receptors on the macrophages. Indeed, mRNA expression of cultured M2c-like macrophages showed increased levels of TAM family members MerTK and AXL. Functionally, these macrophages have the capacity to phagocytose zymosan and to bind nuclei. Furthermore, co-culture of the M2c-like macrophages with erythroblasts yielded GPA⁺(erythroid marker)/CD14⁺ cell aggregates that suggested the formation of erythroblastic islands. Interestingly, M2c-like macrophages expressing CD163high, MR and CD169 were also observed in human bone marrow aspirates and human fetal livers resembling macrophages induced in in vitro cultures in presence of dexamethasone.

Currently, we investigate the mechanism by which glucocorticoids induce monocytes to differentiate into macrophages that may be used to model erythroblastic island-mediated erythropoiesis. Knowledge on the function of such a erythroblastic island is lacking by the absence of an in vitro model. Furthermore, targeting this mechanism in vivo may enhance the recovery of erythropoiesis following bone marrow transplantation.

CD14+ cells from peripheral blood positively regulate hematopoietic stem and progenitor cell survival resulting in increased erythroid yield. (2015) Heideveld E, Masiello F, Marra M, Esteghamat F, Yağcı N, von Lindern M, Migliaccio AR, van den Akker E. Haematologica. 100(11):1396-1406