Physiological bone remodeling is a highly coordinated process responsible for bone resorption and formation and is necessary to repair damaged bone and to maintain mineral homeostasis. In addition to ...the traditional bone cells (osteoclasts, osteoblasts, and osteocytes) that are necessary for bone remodeling, several immune cells have also been implicated in bone disease. This minireview discusses physiological bone remodeling, outlining the traditional bone biology dogma in light of emerging osteoimmunology data. Specifically discussed in detail are the cellular and molecular mechanisms of bone remodeling, including events that orchestrate the five sequential phases of bone remodeling: activation, resorption, reversal, formation, and termination.
The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine ...femoral fracture model internally fixed using a flexible plate (MouseFix) was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+ Mac-2+ ) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+ Mac-2neg ), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window.
Distinct subsets of resident tissue macrophages are important in hematopoietic stem cell niche homeostasis and erythropoiesis. We used a myeloid reporter gene (Csf1r-eGFP) to dissect the persistence ...of bone marrow and splenic macrophage subsets following lethal irradiation and autologous hematopoietic stem cell transplantation in a mouse model. Multiple recipient bone marrow and splenic macrophage subsets survived after autologous hematopoietic stem cell transplantation with organ-specific persistence kinetics. Short-term persistence (5 weeks) of recipient resident macrophages in spleen paralleled the duration of extramedullary hematopoiesis. In bone marrow, radiation-resistant recipient CD169+ resident macrophages and erythroid-island macrophages self-repopulated long-term after transplantation via autonomous cell division. Posttransplant peak expansion of recipient CD169+ resident macrophage number in bone marrow aligned with the persistent engraftment of phenotypic long-term reconstituting hematopoietic stem cells within bone marrow. Selective depletion of recipient CD169+ macrophages significantly compromised the engraftment of phenotypic long-term reconstituting hematopoietic stem cells and consequently impaired hematopoietic reconstitution. Recipient bone marrow resident macrophages are essential for optimal hematopoietic stem cell transplantation outcomes and could be an important consideration in the development of pretransplant conditioning therapies and/or chemoresistance approaches.
•Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation.•Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.
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In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the ...mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.
Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse ...bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.
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•Genuine macrophages are absent from hematopoietic tissue single-cell preparations•Fragmented remnants of macrophages containing cytoplasm adhere to other cells•Macrophage remnants convolute hematopoietic tissue flow cytometry analysis•Remnant-derived macrophage-RNA is misattributed to other cells in RNA-seq data
Millard et al. demonstrate intact macrophages are absent in hematopoietic tissue single-cell suspensions as they are disrupted during preparation. Macrophage remnants containing membrane and intracellular contents remain attached to other cells within the suspensions. This results in misassignment of macrophage identity and misattribution of macrophage-expressed genes.
Parathyroid hormone (PTH) is the major hormone regulating calcium metabolism. It is also the only FDA-approved drug for osteoporosis treatment that stimulates bone formation when injected daily. ...However, continuous infusion of PTH causes severe bone loss in line with its known catabolic effects. Many studies to understand the dual effects of PTH have been carried out, and in recent years a growing number of molecular and cellular mechanisms underlying these effects have emerged. Here, we outline the present knowledge and conclude that the kinetics of administration and subsequent signaling probably account for the divergent actions of the hormone.
Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC ...prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation.
We administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay.
CSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48
CD150
HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48
CD150
HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment.
These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.
Macrophage-targeted therapies, including macrophage colony-stimulating factor 1 (CSF1), have been shown to have pro-repair impacts post-fracture. Preclinical/clinical applications of CSF1 have been ...expedited by development of chimeric CSF1-Fc which has extended circulating half-life. Here, we used mouse models to investigate the bone regenerative potential of CSF1-Fc in healthy and osteoporotic fracture. We also explored whether combination of CSF1-Fc with interleukin (IL)-4 provided additional fracture healing benefit in osteopenic bone. Micro-computed tomography, in situ histomorphometry, and bone mechanical parameters were used to assess systemic impacts of CSF1-Fc therapy in naive mice (male and female young, adult and geriatric). An intermittent CSF1-Fc regimen was optimized to mitigate undesirable impacts on bone resorption and hepatosplenomegaly, irrespective of age or gender. The intermittent CSF1-Fc regimen was tested in a mid-diaphyseal femoral fracture model in healthy bones with treatment initiated 1-day post-fracture. Weekly CSF1-Fc did not impact osteoclasts but increased osteal macrophages and improved fracture strength. Importantly, this treatment regimen also improved fracture union and strength in an ovariectomy-model of delayed fracture repair. Combining CSF1-Fc with IL-4 initiated 1-week post-fracture reduced the efficacy of CSF1-Fc. This study describes a novel strategy to specifically achieve bone regenerative actions of CSF1-Fc that has the potential to alleviate fragility fracture morbidity and mortality.
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