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Macrophages, named for their phagocytic ability, participate in homeostasis, tissue regeneration and inflammatory responses. Bone and adjacent marrow contain multiple functionally ...unique resident tissue macrophage subsets which maintain and regulate anatomically distinct niche environments within these interconnected tissues. Three subsets of bone–bone marrow resident tissue macrophages have been characterised; erythroblastic island macrophages, haematopoietic stem cell niche macrophages and osteal macrophages. The role of these macrophages in controlling homeostasis and repair in bone and bone marrow niches is reviewed in detail.
The skeleton is a common site for cancer metastases with the bone microenvironment providing the appropriate conditions for cancer cell colonization. Once in bone, cancer cells effectively manipulate ...their microenvironment to support their growth and survival. Despite previous efforts to improve treatment modalities, skeletal metastases remain with poor prognoses. This warrants an improved understanding of the mechanisms leading to bone metastasis that will aid development of effective treatments. Macrophages in the tumor microenvironment are termed tumor associated macrophages (TAMs) and their crosstalk with cancer cells is critical in regulating tumorigenicity in multiple cancers. In bone metastases, this crosstalk is also being increasingly implicated but the specific signaling pathways remain incompletely understood. Here, we summarize the reported functions, interactions, and signaling of macrophages with cancer cells during the metastatic cascade to bone. Specifically, we review and discuss how these specific interactions impact macrophages and their profiles to promote tumor development. We also discuss the potential of targeting this crosstalk to inhibit disease progression. Finally, we identify the remaining knowledge gaps that will need to be addressed in order to fully consider therapeutic targeting to improve clinical outcomes in cancer patients.
Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic ...impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43.sup.hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.
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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Apoptosis and efficient efferocytosis are integral to growth, development, and homeostasis. The heterogeneity of these mechanisms in different cells across distinct tissues renders it difficult to ...develop broadly applicable in vivo technologies. Here, we introduced a novel inducible caspase-9 (iCasp9) mouse model which allowed targeted cell apoptosis and further facilitated investigation of concomitant efferocytosis. We generated iCasp9
mice with conditional expression of chemically inducible caspase-9 protein that is triggered in the presence of Cre recombinase. In vitro, bone marrow cells from iCasp9
mice showed expression of the iCasp9 protein when transduced with Cre-expressing adenovirus. Treatment of these cells with the chemical dimerizer (AP20187/AP) resulted in iCasp9 processing and cleaved caspase-3 upregulation, indicating successful apoptosis induction. The in vivo functionality and versatility of this model was demonstrated by crossing iCasp9
mice with CD19-Cre and Osteocalcin (OCN)-Cre mice to target CD19
B cells or OCN
bone-lining osteoblasts. Immunofluorescence and/or immunohistochemical staining in combination with histomorphometric analysis of EGFP, CD19/OCN, and cleaved caspase-3 expression demonstrated that a single dose of AP effectively induced apoptosis in CD19
B cells or OCN
osteoblasts. Examination of the known efferocytes in the target tissues showed that CD19
cell apoptosis was associated with infiltration of dendritic cells into splenic B cell follicles. In the bone, where efferocytosis remains under-explored, the use of iCasp9 provided direct in vivo evidence that macrophages are important mediators of apoptotic osteoblast clearance. Collectively, this study presented the first mouse model of iCasp9 which achieved selective apoptosis, allowing examination of subsequent efferocytosis. Given its unique feature of being controlled by any Cre-expressing mouse lines, the potential applications of this model are extensive and will bring forth more insights into the diversity of mechanisms and cellular effects induced by apoptosis including the physiologically important efferocytic process that follows.
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.
Interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow ...(BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.
•Prolonged inhibition of CXCR4/CXCL12 signaling results in exceptional mobilization along with an expansion of the BM HSPC pool.•Reversible inhibition of the CXCR4/CXCL12 axis may represent a novel strategy to restore damaged BM.
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.
Osteal macrophages (osteomacs) contribute to bone homeostasis and regeneration. To further distinguish their functions from osteoclasts, which share many markers and growth factor requirements, we ...developed a rapid, enzyme-free osteomac enrichment protocol that permitted characterization of minimally manipulated osteomacs by flow cytometry. Osteomacs differ from osteoclasts in expression of Siglec1 (CD169). This distinction was confirmed using the CD169-diphtheria toxin (DT) receptor (DTR) knock-in model. DT treatment of naïve CD169-DTR mice resulted in selective and striking loss of osteomacs, whilst osteoclasts and trabecular bone area were unaffected. Consistent with a previously-reported trophic interaction, osteomac loss was accompanied by a concomitant and proportionately striking reduction in osteoblasts. The impact of CD169+ macrophage depletion was assessed in two models of bone injury that heal via either intramembranous (tibial injury) or endochondral (internally-plated femoral fracture model) ossification. In both models, CD169+ macrophage, including osteomac depletion compromised bone repair. Importantly, DT treatment in CD169-DTR mice did not affect osteoclast frequency in either model. In the femoral fracture model, the magnitude of callus formation correlated with the number of F4/80+ macrophages that persisted within the callus. Overall these observations provide compelling support that CD169+ osteomacs, independent of osteoclasts, provide vital pro-anabolic support to osteoblasts during both bone homeostasis and repair.
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