Publications by authors named "Allison Pettit"

57 Publications

Macrophages form erythropoietic niches and regulate iron homeostasis to adapt erythropoiesis in response to infections and inflammation.

Exp Hematol 2021 Sep 6. Epub 2021 Sep 6.

Mater Research Institute - The University of Queensland, Woolloongabba, QLD, Australia.

It has recently emerged that tissue resident macrophages are key regulators of several stem cell niches orchestrating tissue formation during development, as well as postnatally where they also organise the repair and regeneration of many tissues including the haemopoietic tissue. The fact that macrophages are also master regulators and effectors of innate immunity and inflammation allows them to co-ordinate haematopoietic response to infections, injuries and inflammation. After recently reviewing the roles of phagocytes and macrophages in regulating normal and pathological haematopoietic stem cell niches, we now focus on the key roles of macrophages in regulating erythropoiesis and iron homeostasis. We review herein the recent advances in understanding how macrophages at the centre of erythroblastic islands form an erythropoietic niche that controls the terminal differentiation and maturation of erythroblasts into reticulocytes, how red pulp macrophages in the spleen control iron recycling and homeostasis, how these macrophages coordinate emergency erythropoiesis in response to blood loss, infections and inflammation and how persistent infections or inflammation can lead to anaemia of inflammation via macrophages. Finally, we discuss the technical challenges associated with the molecular characterisation of erythroid island macrophages and red pulp macrophages.
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http://dx.doi.org/10.1016/j.exphem.2021.08.011DOI Listing
September 2021

Treatment with a long-acting chimeric CSF1 molecule enhances fracture healing of healthy and osteoporotic bones.

Biomaterials 2021 08 3;275:120936. Epub 2021 Jun 3.

Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, 4102, Australia. Electronic address:

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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http://dx.doi.org/10.1016/j.biomaterials.2021.120936DOI Listing
August 2021

Role of macrophages and phagocytes in orchestrating normal and pathologic hematopoietic niches.

Exp Hematol 2021 Aug 21;100:12-31.e1. Epub 2021 Jul 21.

Mater Research Institute, University of Queensland, Woolloongabba, QLD, Australia.

The bone marrow (BM) contains a mosaic of niches specialized in supporting different maturity stages of hematopoietic stem and progenitor cells such as hematopoietic stem cells and myeloid, lymphoid, and erythroid progenitors. Recent advances in BM imaging and conditional gene knockout mice have revealed that niches are a complex network of cells of mesenchymal, endothelial, neuronal, and hematopoietic origins, together with local physicochemical parameters. Within these complex structures, phagocytes, such as neutrophils, macrophages, and dendritic cells, all of which are of hematopoietic origin, have been found to be important in regulating several niches in the BM, including hematopoietic stem cell niches, erythropoietic niches, and niches involved in endosteal bone formation. There is also increasing evidence that these macrophages have an important role in adapting hematopoiesis, erythropoiesis, and bone formation in response to inflammatory stressors and play a key part in maintaining the integrity and function of these. Likewise, there is also accumulating evidence that subsets of monocytes, macrophages, and other phagocytes contribute to the progression and response to treatment of several lymphoid malignancies such as multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma, as well as lymphoblastic leukemia, and may also play a role in myelodysplastic syndrome and myeloproliferative neoplasms associated with Noonan syndrome and aplastic anemia. In this review, the potential functions of macrophages and other phagocytes in normal and pathologic niches are discussed, as are the challenges in studying BM and other tissue-resident macrophages at the molecular level.
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http://dx.doi.org/10.1016/j.exphem.2021.07.001DOI Listing
August 2021

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

J Bone Miner Res 2021 Jul 19. Epub 2021 Jul 19.

Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia.

Osteal macrophages (osteomacs) support osteoblast function and promote bone anabolism, but their contribution to osteoporosis has not been explored. Although mouse ovariectomy (OVX) models have been repeatedly used, variation in strain, experimental design and assessment modalities have contributed to no single model being confirmed as comprehensively replicating the full gamut of osteoporosis pathological manifestations. We validated an OVX model in adult C3H/HeJ mice and demonstrated that it presents with human postmenopausal osteoporosis features with reduced bone volume in axial and appendicular bone and bone loss in both trabecular and cortical bone including increased cortical porosity. Bone loss was associated with increased osteoclasts on trabecular and endocortical bone and decreased osteoblasts on trabecular bone. Importantly, this OVX model was characterized by delayed fracture healing. Using this validated model, we demonstrated that osteomacs are increased post-OVX on both trabecular and endocortical bone. Dual F4/80 (pan-macrophage marker) and tartrate-resistant acid phosphatase (TRAP) staining revealed osteomacs frequently located near TRAP osteoclasts and contained TRAP intracellular vesicles. Using an in vivo inducible macrophage depletion model that does not simultaneously deplete osteoclasts, we observed that osteomac loss was associated with elevated extracellular TRAP in bone marrow interstitium and increased serum TRAP. Using in vitro high-resolution confocal imaging of mixed osteoclast-macrophage cultures on bone substrate, we observed macrophages juxtaposed to osteoclast basolateral functional secretory domains scavenging degraded bone byproducts. These data demonstrate a role for osteomacs in supporting osteoclastic bone resorption through phagocytosis and sequestration of resorption byproducts. Overall, our data expose a novel role for osteomacs in supporting osteoclast function and provide the first evidence of their involvement in osteoporosis pathogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4413DOI Listing
July 2021

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation.

PLoS Genet 2021 Jun 3;17(6):e1009605. Epub 2021 Jun 3.

Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, Brisbane, Qld, Australia.

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, CD43hi 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.
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http://dx.doi.org/10.1371/journal.pgen.1009605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8205168PMC
June 2021

Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL-1β release.

J Exp Med 2021 May;218(5)

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.
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http://dx.doi.org/10.1084/jem.20201452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933984PMC
May 2021

Stable colony-stimulating factor 1 fusion protein treatment increases hematopoietic stem cell pool and enhances their mobilisation in mice.

J Hematol Oncol 2021 01 6;14(1). Epub 2021 Jan 6.

Mater Research Institute-The University of Queensland, Faculty of Medicine, Translational Research Institute, 37 Kent St, Woolloongabba, 4102, Australia.

Background: 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.

Methods: 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.

Results: 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 CD48CD150 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 CD48CD150 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.

Conclusion: These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.
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http://dx.doi.org/10.1186/s13045-020-00997-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786999PMC
January 2021

A Transgenic Line That Reports CSF1R Protein Expression Provides a Definitive Marker for the Mouse Mononuclear Phagocyte System.

J Immunol 2020 12 2;205(11):3154-3166. Epub 2020 Nov 2.

Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia;

The proliferation, differentiation, and survival of cells of the mononuclear phagocyte system (MPS; progenitors, monocytes, macrophages, and classical dendritic cells) are controlled by signals from the M-CSF receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters, but none is both universal and lineage restricted. In this article, we report the development and characterization of a CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells, arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly expressed in marrow monocytes and common myeloid progenitors but significantly lower in granulocyte-macrophage progenitors. In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169 resident macrophages, and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations, including classical dendritic cells. Whole mount imaging of nonlymphoid tissues in mice with combined CSF1R-FRed/-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells, including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.
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http://dx.doi.org/10.4049/jimmunol.2000835DOI Listing
December 2020

Imaging flow cytometry reveals that granulocyte colony-stimulating factor treatment causes loss of erythroblastic islands in the mouse bone marrow.

Exp Hematol 2020 02 8;82:33-42. Epub 2020 Feb 8.

Stem Cell Biology Group, Mater Research-University of Queensland Translational Research Institute, Woolloongabba, QLD, Australia. Electronic address:

The erythroblastic island (EBI) is a multicellular structure forming an erythropoietic niche consisting of a central macrophage surrounded by a rosette of maturing erythroblasts. Since their discovery more than 60 years ago, simultaneous quantification and visualization of EBIs remain difficult. Although flow cytometry enables high-throughput quantification of cell aggregates co-expressing macrophage and erythroblast markers, it cannot visually confirm whether the aggregates are genuine EBIs. While immunofluorescence microscopy allows visualization of EBIs, its low throughput limits its use for quantification. In the current study we employed nine-channel imaging flow cytometry (IFC) to develop a method to directly visualize and quantify EBIs in the mouse bone marrow. We found that EBI central macrophages do express F4/80, VCAM-1, and CD169, but not CD11b or Ly6G, and that CD11bLy6GF4/80 granulocytes are found associated at the periphery of 40%-60% EBIs. Furthermore, we show for the first time using IFC that in vivo treatment with the hematopoietic stem cell-mobilizing cytokine granulocyte colony-stimulating factor (G-CSF) reduced EBI frequency in the bone marrow by more than 100-fold. These results indicate that mobilizing doses of G-CSF cause a collapse of EBIs in the bone marrow.
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http://dx.doi.org/10.1016/j.exphem.2020.02.003DOI Listing
February 2020

Deformation behavior of porous PHBV scaffold in compression: A finite element analysis study.

J Mech Behav Biomed Mater 2019 08 17;96:1-8. Epub 2019 Apr 17.

School of Mechanical and Mining Engineering, The University of Queensland, QLD, 4072, Australia.

Macroscopic mechanical properties of porous PHBV bone TE scaffolds have been well studied. However, their mechanical behavior at microscopic level has yet to be explored. In this study, the micro-mechanical behavior of a PHBV bone scaffold under compression was investigated using a numerical method that combines micro-computed tomography (μ-CT) and finite element analysis (FEA). It was found that the use of a linear-elastic model resulted in an overestimation of the stiffness of the scaffold, whereas a more realistic estimation of the scaffold's deformation behavior was obtained by utilizing a bilinear material model. The onset of plastic deformation occurred in the very early stage of loading resulting in significantly reduced stiffness of the scaffold. The non-uniform and arbitrary microstructure of the scaffold led to a heterogeneous stress distribution within the porous construct, which was subjected to a mixture of compressive and tensile stresses. Nevertheless, the resultant stress contours showed that the scaffold experienced primarily elastic deformation when it was loaded up to 0.003 strain, while localized plastic deformation occurred at sharp corners and necked regions of the micro-struts. The scaffold expanded slightly in the horizontal direction as it was compressed and the change in geometries of pores within the scaffold was insignificant. The proposed method provides a valuable tool to study the localized mechanical behavior of bone scaffolds in micrometer scale with arbitrary porous architecture. This approach could prove highly useful for guiding the fabrication of scaffolds that have anatomy specific mechanical properties and porous architecture.
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http://dx.doi.org/10.1016/j.jmbbm.2019.04.030DOI Listing
August 2019

Inhibition of JAK1/2 Tyrosine Kinases Reduces Neurogenic Heterotopic Ossification After Spinal Cord Injury.

Front Immunol 2019 7;10:377. Epub 2019 Mar 7.

Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia.

Neurogenic heterotopic ossifications (NHO) are very incapacitating complications of traumatic brain and spinal cord injuries (SCI) which manifest as abnormal formation of bone tissue in periarticular muscles. NHO are debilitating as they cause pain, partial or total joint ankylosis and vascular and nerve compression. NHO pathogenesis is unknown and the only effective treatment remains surgical resection, however once resected, NHO can re-occur. To further understand NHO pathogenesis, we developed the first animal model of NHO following SCI in genetically unmodified mice, which mimics most clinical features of NHO in patients. We have previously shown that the combination of (1) a central nervous system lesion (SCI) and (2) muscular damage (via an intramuscular injection of cardiotoxin) is required for NHO development. Furthermore, macrophages within the injured muscle play a critical role in driving NHO pathogenesis. More recently we demonstrated that macrophage-derived oncostatin M (OSM) is a key mediator of both human and mouse NHO. We now report that inflammatory monocytes infiltrate the injured muscles of SCI mice developing NHO at significantly higher levels compared to mice without SCI. Muscle infiltrating monocytes and neutrophils expressed OSM whereas mouse muscle satellite and interstitial cell expressed the OSM receptor (OSMR). recombinant mouse OSM induced tyrosine phosphorylation of the transcription factor STAT3, a downstream target of OSMR:gp130 signaling in muscle progenitor cells. As STAT3 is tyrosine phosphorylated by JAK1/2 tyrosine kinases downstream of OSMR:gp130, we demonstrated that the JAK1/2 tyrosine kinase inhibitor ruxolitinib blocked OSM driven STAT3 tyrosine phosphorylation in mouse muscle progenitor cells. We further demonstrated that STAT3 tyrosine phosphorylation was not only significantly higher but persisted for a longer duration in injured muscles of SCI mice developing NHO compared to mice with muscle injury without SCI. Finally, administration of ruxolitinib for 7 days post-surgery significantly reduced STAT3 phosphorylation in injured muscles as well as NHO volume at all analyzed time-points up to 3 weeks post-surgery. Our results identify the JAK/STAT3 signaling pathway as a potential therapeutic target to reduce NHO development following SCI.
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http://dx.doi.org/10.3389/fimmu.2019.00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417366PMC
July 2020

Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment.

Blood 2018 08 26;132(7):735-749. Epub 2018 Jun 26.

Mater Research Institute-The University of Queensland, Faculty of Medicine, The University of Queensland, Woolloongabba, QLD, Australia.

Distinct subsets of resident tissue macrophages are important in hematopoietic stem cell niche homeostasis and erythropoiesis. We used a myeloid reporter gene (-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.
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http://dx.doi.org/10.1182/blood-2018-01-829663DOI Listing
August 2018

CD169 macrophages are critical for osteoblast maintenance and promote intramembranous and endochondral ossification during bone repair.

Biomaterials 2019 03 22;196:51-66. Epub 2017 Oct 22.

Bones and Immunology Laboratory, Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, 4102, Australia; The University of Queensland, Faculty of Medicine, Herston, Queensland, 4092, Australia. Electronic address:

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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http://dx.doi.org/10.1016/j.biomaterials.2017.10.033DOI Listing
March 2019

Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications.

JCI Insight 2017 11 2;2(21). Epub 2017 Nov 2.

Inserm UMR-S-MD1197, Paris 11 University, Paul Brousse Hospital, Villejuif, France.

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.
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http://dx.doi.org/10.1172/jci.insight.96034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752299PMC
November 2017

Characterization of Normal Murine Carpal Bone Development Prompts Re-Evaluation of Pathologic Osteolysis as the Cause of Human Carpal-Tarsal Osteolysis Disorders.

Am J Pathol 2017 Sep 1;187(9):1923-1934. Epub 2017 Jul 1.

Translational Research Institute, Brisbane, Queensland, Australia; University of Queensland Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia; Mater Research Institute-UQ, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajpath.2017.05.007DOI Listing
September 2017

Osteomacs and Bone Regeneration.

Curr Osteoporos Rep 2017 08;15(4):385-395

Bones and Immunology Laboratory, Cancer Biology and Care Program, Mater Research Institute - The University of Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD, 4102, Australia.

Purpose Of Review: Mounting evidence supporting the critical contribution of macrophages, in particular osteal macrophages, to bone regeneration is reviewed. We specifically examine the potential role of macrophages in the basic multicellular units coordinating lifelong bone regeneration via remodelling and bone regeneration in response to injury. We review and discuss the distinctions between macrophage and osteoclast contributions to bone homeostasis, particularly the dichotomous role of the colony-stimulating factor 1-colony-stimulating factor 1 receptor axis.

Recent Findings: The impact of inflammation associated with aging and other hallmarks of aging, including senescence, on macrophage function is addressed in the context of osteoporosis and delayed fracture repair. Resident macrophages versus recruited macrophage contributions to fracture healing are also discussed. We identify some of the remaining knowledge gaps that will need to be closed in order to maximise benefits from therapeutically modulating or mimicking the function of macrophages to improve bone health and regeneration over a lifetime.
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http://dx.doi.org/10.1007/s11914-017-0384-xDOI Listing
August 2017

Early anti-inflammatory intervention ameliorates axial disease in the proteoglycan-induced spondylitis mouse model of ankylosing spondylitis.

BMC Musculoskelet Disord 2017 05 30;18(1):228. Epub 2017 May 30.

Mater Research Institute-The University of Queensland, Faculty of Medicine, Translational Research Institute, 37 Kent St, Woolloongabba, QLD, 4102, Australia.

Background: Ankylosing spondylitis (AS) is characterised by immune-mediated arthritis and osteoproliferation, ultimately leading to joint ankylosis. Whether inflammation is necessary for osteoproliferation is controversial, fuelled by the unclear efficacy of anti-inflammatory treatments on radiographic progression. In proteoglycan-induced spondylitis (PGISp), a mouse model of AS, inflammation is the prerequisite for osteoproliferation as osteoproliferation was only observed following inflammation-driven intervertebral disc (IVD) destruction. We hypothesised that early intervention with a potent anti-inflammatory therapy would protect IVD integrity and consequently alter disease progression.

Methods: PGISp mice received vehicle or a combination of etanercept (ETN) plus prednisolone (PRD) therapy for 2 or 6 weeks initiated at an early disease stage. Peripheral arthritis was scored longitudinally. Spinal disease was assessed using a semi-quantitative histological scoring regimen including inflammation, joint destruction and excessive tissue formation.

Results: ETN + PRD therapy significantly delayed the onset of peripheral arthritis. IVD integrity was significantly protected when treatment was commenced in early disease. Six-weeks of treatment resulted in trends towards reductions in intervertebral joint damage and excessive tissue formation. IVD score distribution was dichotomized, likely reflecting the extent of axial disease at initiation of therapy. In the sub-group of mice with high IVD destruction scores, ETN + PRD treatment significantly reduced IVD destruction severity, inflammation and bone erosion and reduced cartilage damage and excessive tissue formation.

Conclusions: Early intervention with anti-inflammatory treatment not only improved inflammatory symptoms but also ameliorated structural damage of spine in PGISp mice. This preclinical observation suggests that early anti-inflammatory intervention may slow radiographic progression in AS patients.
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http://dx.doi.org/10.1186/s12891-017-1600-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450150PMC
May 2017

Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells.

Blood 2017 05 11;129(21):2939-2949. Epub 2017 Apr 11.

Division of Oncology, Department of Medicine, and.

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.
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http://dx.doi.org/10.1182/blood-2016-10-746909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445573PMC
May 2017

Resting and injury-induced inflamed periosteum contain multiple macrophage subsets that are located at sites of bone growth and regeneration.

Immunol Cell Biol 2017 01 15;95(1):7-16. Epub 2016 Nov 15.

The University of Queensland, Institute for Molecular Bioscience, St Lucia, Queensland, Australia.

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 characterize 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. In tissues collected from 4-week-old mice, osteomacs were observed intimately associated with sites of periosteal diaphyseal and metaphyseal bone dynamics associated with normal growth. This included F4/80Mac-2 osteomac association with extended tracks of bone formation (modeling) on diphyseal periosteal surfaces. Although this recapitulated endosteal osteomac characteristics, there was subtle variance in the morphology and spatial organization of periosteal modeling-associated osteomacs, which likely reflects the greater structural complexity of periosteum. Osteomacs, resident macrophages and inflammatory macrophages (F4/80Mac-2) were associated with the complex bone dynamics occurring within the periosteum at the metaphyseal corticalization zone. These three 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.
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http://dx.doi.org/10.1038/icb.2016.74DOI Listing
January 2017

Role of bone marrow macrophages in controlling homeostasis and repair in bone and bone marrow niches.

Semin Cell Dev Biol 2017 01 10;61:12-21. Epub 2016 Aug 10.

Blood and Bone Diseases Program, Mater Research Institute - The University of Queensland, Faculty of Medicine and Biomedical Sciences, Translational Research Institute, 37 Kent Street, Woolloongabba, 4102, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.semcdb.2016.08.009DOI Listing
January 2017

CD169(+) macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer.

J Pathol 2016 06 27;239(2):218-30. Epub 2016 Apr 27.

Faculty of Medicine and Biomedical Sciences, Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Australia.

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. 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 cell, but not targeted CD169(+) macrophage depletion resulted in increased tumour mass, indicating that CD169(-) 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. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.4718DOI Listing
June 2016

Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: sequential not parallel processes.

Arthritis Res Ther 2016 Jan 29;18:35. Epub 2016 Jan 29.

The University of Queensland Diamantina Institute, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD, 4102, Australia.

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.
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http://dx.doi.org/10.1186/s13075-015-0805-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734853PMC
January 2016

Osteoclasts control reactivation of dormant myeloma cells by remodelling the endosteal niche.

Nat Commun 2015 Dec 3;6:8983. Epub 2015 Dec 3.

Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia.

Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.
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http://dx.doi.org/10.1038/ncomms9983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686867PMC
December 2015

Macrophages: Their Emerging Roles in Bone.

J Bone Miner Res 2015 Dec;30(12):2140-9

Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, MI, USA.

Macrophages are present in nearly all tissues and are critical for development, homeostasis, and regeneration. Resident tissue macrophages of bone, termed osteal macrophages, are recently classified myeloid cells that are distinct from osteoclasts. Osteal macrophages are located immediately adjacent to osteoblasts, regulate bone formation, and play diverse roles in skeletal homeostasis. Genetic or pharmacological modulation of macrophages in vivo results in significant bone phenotypes, and these phenotypes depend on which macrophage subsets are altered. Macrophages are also key mediators of osseous wound healing and fracture repair, with distinct roles at various stages of the repair process. A central function of macrophages is their phagocytic ability. Each day, billions of cells die in the body and efferocytosis (phagocytosis of apoptotic cells) is a critical process in both clearing dead cells and recruitment of replacement progenitor cells to maintain homeostasis. Recent data suggest a role for efferocytosis in bone biology and these new mechanisms are outlined. Finally, although macrophages have an established role in primary tumors, emerging evidence suggests that macrophages in bone support cancers which preferentially metastasize to the skeleton. Collectively, this developing area of osteoimmunology raises new questions and promises to provide novel insights into pathophysiologic conditions as well as therapeutic and regenerative approaches vital for skeletal health.
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http://dx.doi.org/10.1002/jbmr.2735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876707PMC
December 2015

Intrauterine Bone Marrow Transplantation in Osteogenesis Imperfecta Mice Yields Donor Osteoclasts and Osteomacs but Not Osteoblasts.

Stem Cell Reports 2015 Nov 29;5(5):682-689. Epub 2015 Oct 29.

UQ Centre for Clinical Research, The University of Queensland, Herston, QLD 4029, Australia; Royal Brisbane and Women's Hospital, Centre for Advanced Prenatal Care, Herston, QLD 4029, Australia.

In this article, Millard and colleagues show that intrauterine bone marrow transplantation in the oim/oim mouse model of osteogenesis imperfecta yields hematopoietic microchimerism in the absence of donor osteopoiesis or phenotypic improvement. Bone-associated donor cells were not bone-forming osteoblasts, but osteoclasts (bone resorbing cells of the hematopoietic lineage) and osteal macrophages (bone regulatory cells of the hematopoietic lineage).
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http://dx.doi.org/10.1016/j.stemcr.2015.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649292PMC
November 2015

Tissue engineered humanized bone supports human hematopoiesis in vivo.

Biomaterials 2015 Aug 19;61:103-14. Epub 2015 May 19.

Stem Cell Biology Group and Stem Cells and Cancer Group - Blood and Bone Diseases Program, Mater Research Institute - The University of Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD 4102, Brisbane, Australia; School of Medicine, The University of Queensland, 288 Herston Road, Herston, QLD 4006, Brisbane, Australia. Electronic address:

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.
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http://dx.doi.org/10.1016/j.biomaterials.2015.04.057DOI Listing
August 2015

Neurological heterotopic ossification following spinal cord injury is triggered by macrophage-mediated inflammation in muscle.

J Pathol 2015 Jun 26;236(2):229-40. Epub 2015 Mar 26.

Blood and Bone Diseases Programme, Mater Research Institute, University of Queensland, Woolloongabba, Australia.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.
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http://dx.doi.org/10.1002/path.4519DOI Listing
June 2015

Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification.

Am J Pathol 2014 Dec 5;184(12):3192-204. Epub 2014 Oct 5.

Bone and Immunology Laboratory, Mater Research Institute-UQ, Translational Research Institute, The University of Queensland, Woolloongabba, Queensland, Australia; UQ-Centre for Clinical Research, Faculty of Health Sciences, The University of Queensland, Herston, Queensland, Australia. Electronic address:

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-2(neg)), 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.
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http://dx.doi.org/10.1016/j.ajpath.2014.08.017DOI Listing
December 2014

Deletion of bone-marrow-derived receptor for AGEs (RAGE) improves renal function in an experimental mouse model of diabetes.

Diabetologia 2014 Sep 24;57(9):1977-85. Epub 2014 Jun 24.

Department of Nephrology, Monash Medical Centre, Monash Health, Clayton, Melbourne, VIC, Australia.

Aims/hypothesis: The AGEs and the receptor for AGEs (RAGE) are known contributors to diabetic complications. RAGE also has a physiological role in innate and adaptive immunity and is expressed on immune cells. The aim of this study was to determine whether deletion of RAGE from bone-marrow-derived cells influences the pathogenesis of experimental diabetic nephropathy.

Methods: Groups (n = 8/group) of lethally irradiated 8 week old wild-type (WT) mice were reconstituted with bone marrow from WT (WT → WT) or RAGE-deficient (RG) mice (RG → WT). Diabetes was induced using multiple low doses of streptozotocin after 8 weeks of bone marrow reconstitution and mice were followed for a further 24 weeks.

Results: Compared with diabetic WT mice reconstituted with WT bone marrow, diabetic WT mice reconstituted with RG bone marrow had lower urinary albumin excretion and podocyte loss, more normal creatinine clearance and less tubulo-interstitial injury and fibrosis. However, glomerular collagen IV deposition, glomerulosclerosis and cortical levels of TGF-β were not different among diabetic mouse groups. The renal tubulo-interstitium of diabetic RG → WT mice also contained fewer infiltrating CD68(+) macrophages that were activated. Diabetic RG → WT mice had lower renal cortical concentrations of CC chemokine ligand 2 (CCL2), macrophage inhibitory factor (MIF) and IL-6 than diabetic WT → WT mice. Renal cortical RAGE ligands S100 calgranulin (S100A)8/9 and AGEs, but not high mobility box protein B-1 (HMGB-1) were also decreased in diabetic RG → WT compared with diabetic WT → WT mice. In vitro, bone-marrow-derived macrophages from WT but not RG mice stimulated collagen IV production in cultured proximal tubule cells.

Conclusions/interpretation: These studies suggest that RAGE expression on haemopoietically derived immune cells contributes to the functional changes seen in diabetic nephropathy by promoting macrophage infiltration and renal tubulo-interstitial damage.
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http://dx.doi.org/10.1007/s00125-014-3291-zDOI Listing
September 2014
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