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.

Multicentric carpal-tarsal osteolysis; multicentric osteolysis, nodulosis, and arthropathy; and Winchester syndromes, skeletal dysplasias characterized by carpal/tarsal and epiphyseal abnormalities, are caused by mutations in v-maf musculoaponeurotic fibrosarcoma oncogene ortholog B (MAFB), matrix metalloproteinase (MMP) 2, and MMP14, respectively; however, the underlying pathophysiology is unclear. Osteoclast-mediated osteolysis has been regarded as the main mechanism, but does not explain the skeletal distribution. We hypothesized that MAFB, MMP-2, and MMP-14 have integral roles in carpal/tarsal and epiphyseal bone development. Normal neonatal mouse forepaws were imaged by micro-computed tomography and examined histologically. Murine forepaw ossification occurred sequentially. Subarticular regions of endochondral ossification showed morphologic and calcification patterns that were distinct from archetypical physeal endochondral ossification. This suggests that two different forms of endochondral ossification occur. The skeletal sites showing the greatest abnormality in the carpal-tarsal osteolysis syndromes are regions of subarticular ossification. Thus, abnormal bone formation in areas of subarticular ossification may explain the site-specific distribution of the carpal-tarsal osteolysis phenotype. MafB, Mmp-2, and Mmp-14 were expressed widely, and tartrate-resistant acid phosphatase staining notably was absent in the subarticular regions of the cartilage anlagen and entheses at a time point most relevant to the human osteolysis syndromes. Thus, abnormal peri-articular skeletal development and modeling, rather than excessive bone resorption, may be the underlying pathophysiology of these skeletal syndromes.

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

no abstract available

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune-competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169+ macrophages using a suicide gene knock-in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer-induced bone changes. In human bone metastasis specimens, CD68+ macrophages were consistently located within the tumour mass and at the tumour/bone marrow margins. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected had varied distributions. In the immune-competent mouse model, CD169+ macrophage ablation significantly inhibited prostate cancer-induced woven bone formation suggesting that CD169+ macrophages within pathological woven bone are integral to tumour-induced bone formation. In contrast pan-phagocytic, but not targeted CD169+ macrophage depletion resulted in increased tumour mass, indicating that CD169neg macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour-induced bone modelling.

Background
Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required.

Methods
The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry.

Results
Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease.

Conclusions
The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.

For decades it has been assumed that lethal irradiation in bone marrow transplantation experiments ablates the entire host hematopoietic system but preserves the host non-hematopoietic bone marrow stroma. This postulate is at the basis of transplantation chimera experiments in mice to test involvement of hematopoietic cells versus non-hematopoietic stroma. As BM-Macs support HSC niche homeostasis, we examined whether host-derived BM-Macs persist lethal irradiation and play a role in HSC engraftment. Recipient MacGreen mice (expressing GFP in myeloid cells under the control of Csf1r promoter) were lethally irradiated (11.5Gy) and transplanted with sorted syngeneic B6.SJL CD45.1+ Lin-Kit+Sca1+ sorted cells. Flow cytometry analyses of BM 2-30 weeks (wk) post-transplant confirmed more than 99% donor chimerism of monocytes and granulocytes validating ablation of recipient HSC. In contrast, GFP+CD11b+F4/80+CD169+VCAM-1+ERHR3+Ly6Gneg recipient BM-Macs were detected throughout the time-course. A 5.9 fold expansion of these recipient BM-Macs occurred between wk 2 and 5 ( from 45,000 to 270,000 cells/femur) post-transplant which coincided with increased number of phenotypic donor HSC (GFP-Lin-Kit+Sca1+CD48-CD150+). Host BM-Mac proliferation was cell autonomous in the absence of host HSC and granulocytes. Recipient Macs in spleen displayed different frequency and longevity kinetics that correlated with transient post-Tx splenic extramedullary haematopoiesis. In situ, GFP+F480+ recipient BM-Macs were enriched in perivascular microenvironments within both central BM and endosteal regions. These GFP+ host-derived macrophages persisted long-term forming 8.4% of BM macrophages 7months post-transplant. To evaluate the importance of these host-derived radiation-resistant macrophages, we selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP+ HSC. Depletion of recipient CD169+ Macs abated engraftment of donor phenotypic HSC by 70% at 5 wk post-transplant and reduced BM reconstitution potential in competitive secondary transplants. In conclusion BM contains a myeloablation-resistant self-repopulating Mac subset that is necessary for efficient and/or sustained HSC BM engraftment following transplantation. Therefore interpretation of transplantation chimera experiments must take into account the persistence of this subset of host macrophages that support donor HSC engraftment.

no abstract available

Better understanding of bone growth and regeneration mechanisms within periosteal tissues will improve understanding of bone physiology and pathology. Macrophage contributions to bone biology and repair have been established but specific investigation of periosteal macrophages has not been undertaken. We used an immunohistochemistry approach to characterise macrophages in growing murine bone and within activated periosteum induced in a mouse model of bone injury. Osteal tissue macrophages (osteomacs) and resident macrophages were distributed throughout resting periosteum. Tissues were collected from 4 week old mice and osteomacs were observed intimately associated with sites of periosteal diaphyseal and metaphyseal bone dynamics associated with normal growth. This included F4/80+Mac-2−/low osteomac association with extended tracks of bone formation (modeling) on diphyseal periosteal surfaces. While this recapitulated endosteal osteomac characteristics, there was subtle variance in the morphology and spatial organization of modelling-associated osteomacs, which likely reflects the greater structural complexity of periosteum. We also demonstrated that osteomacs, resident macrophages and inflammatory macrophages (F4/80+Mac-2hi) were associated with the complex bone dynamics occurring within the periosteum at the metaphyseal corticalization zone. These 3 macrophage subsets were also present within activated native periosteum after bone injury across a 9 day time course that spanned the inflammatory through remodeling bone healing phases. This included osteomac association with foci of endochondral ossification within the activated native periosteum. These observations confirm that osteomacs are key components of both osteal tissues, in spite of salient differences between endosteal and periosteal structure and that multiple macrophage subsets are involved in periosteal bone dynamics.

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.