Neonatal Osteomacs and Bone Marrow Macrophages Differ in Phenotypic Marker Expression and Function

Abstract

Osteomacs (OM) are specialized bone‐resident macrophages that are a component of the hematopoietic niche and support bone formation. Also located in the niche are a second subset of macrophages, namely bone marrow–derived macrophages (BM Mφ). We previously reported that a subpopulation of OM co‐express both CD166 and CSF1R, the receptor for macrophage colony‐stimulating factor (MCSF), and that OM form more bone‐resorbing osteoclasts than BM Mφ. Reported here are single‐cell quantitative RT‐PCR (qRT‐PCR), mass cytometry (CyTOF), and marker‐specific functional studies that further identify differences between OM and BM Mφ from neonatal C57Bl/6 mice. Although OM express higher levels of CSF1R and MCSF, they do not respond to MCSF‐induced proliferation, in contrast to BM Mφ. Moreover, receptor activator of NF‐κB ligand (RANKL), without the addition of MCSF, was sufficient to induce osteoclast formation in OM but not BM Mφ cultures. OM express higher levels of CD166 than BM Mφ, and we found that osteoclast formation by CD166−/− OM was reduced compared with wild‐type (WT) OM, whereas CD166−/− BM Mφ showed enhanced osteoclast formation. CD110/c‐Mpl, the receptor for thrombopoietin (TPO), was also higher in OM, but TPO did not alter OM‐derived osteoclast formation, whereas TPO stimulated BM Mφ osteoclast formation. CyTOF analyses demonstrated OM uniquely co‐express CD86 and CD206, markers of M1 and M2 polarized macrophages, respectively. OM performed equivalent phagocytosis in response to LPS or IL‐4/IL‐10, which induce polarization to M1 and M2 subtypes, respectively, whereas BM Mφ were less competent at phagocytosis when polarized to the M2 subtype. Moreover, in contrast to BM Mφ, LPS treatment of OM led to the upregulation of CD80, an M1 marker, as well as IL‐10 and IL‐6, known anti‐inflammatory cytokines. Overall, these data reveal that OM and BM Mφ are distinct subgroups of macrophages, whose phenotypic and functional differences in proliferation, phagocytosis, and osteoclast formation may contribute physiological specificity during health and disease. © 2021 American Society for Bone and Mineral Research (ASBMR).

Determination of the physiological range of oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy

“…In bone marrow, there are two types of bone resident macrophages; bone marrow macrophage and osteal macrophage. Both types of macrophages express CD169, but CD206 is expressed in osteal macrophage, but not in bone marrow macrophages 17 . We evaluated the CD169 and CD206 expression levels in vitro by culturing BMMs under the oxygen concentration of 5% and 2%.…”

Osteal macrophages support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model

“…An obvious interpretation for this phenomenon is that osteomacs are increased to supply the estrogen-deficiencyinduced elevated demand for osteoclast precursors. Although in vitro studies support this interpretation, (23,66,67) it is challenged by in vivo studies indicating that macrophages do not function efficiently in this capacity. (68,69) Notably, in mice treated with an exogenous supraphysiologic receptor activator of nuclear factor κB ligand (RANKL), osteomacs remained TRAPnegative and continued to be a prominent cell within the endosteal microenvironment despite substantially elevated osteoclastogenesis.…”

Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice

“…The media was then replaced with 100 μl fresh medium containing 20 μl of CellTiter 96 AQueous One Solution (CellTiter 96® AQeous Non-Radioactive Cell Proliferation Assay (MTS) kit, Promega, Madison, WI). Cells were incubated at 37 °C for 3 h and cell viability was estimated by measuring the optical density (OD) at 490 nm ( Mohamad et al, 2021 ). To minimize the contribution of estrogen, phenol-free αMEM media and charcoal-stripped FBS (Biowest) were used in a separate experiment.…”