Relative contributions of osteal macrophages and osteoclasts to postnatal bone development in CSF1R-deficient rats and phenotype rescue following wild-type bone marrow cell transfer.

Macrophage and osteoclast proliferation, differentiation and survival are regulated by colony-stimulating factor-1 receptor (CSF1R) signaling. Osteopetrosis associated with Csf1 and Csf1r mutations has been attributed to the loss of osteoclasts and deficiency in bone resorption. Here we demonstrate that homozygous Csf1r mutation in rat leads to delayed postnatal skeletal ossification associated with substantial loss of osteal macrophages (osteomacs) in addition to osteoclasts. Osteosclerosis and site-specific skeletal abnormalities were reversed by intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning. Following BMT, IBA1+ macrophages were detected before TRAP+ osteoclasts at sites of ossification restoration. These observations extend evidence that osteomacs independently contribute to bone anabolism and are required for normal postnatal bone growth and morphogenesis.

Reversible expansion of tissue macrophages in response to macrophage colony-stimulating factor (CSF1) transforms systemic lipid and carbohydrate metabolism.

Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome.NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.

Macrophage heterogeneity in the single-cell era: facts and artifacts.

In this spotlight, we review technical issues that compromise single-cell analysis of tissue macrophages, including limited and unrepresentative yields, fragmentation and generation of remnants, and activation during tissue disaggregation. These issues may lead to a misleading definition of subpopulations of macrophages and the expression of macrophage-specific transcripts by unrelated cells. Recognition of the technical limitations of single-cell approaches is required in order to map the full spectrum of tissue-resident macrophage heterogeneity and assess its biological significance.

Spinal cord injury reprograms muscle fibroadipogenic progenitors to form heterotopic bones within muscles.

The cells of origin of neurogenic heterotopic ossifications (NHOs), which develop frequently in the periarticular muscles following spinal cord injuries (SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells (SCs), which are myogenic, and fibroadipogenic progenitors (FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα+ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56+ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.

Therapeutic potential of macrophage colony-stimulating factor in chronic liver disease.

Resident and recruited macrophages control the development and proliferation of the liver. We have previously shown in multiple species that treatment with a macrophage colony stimulating factor (CSF1)-Fc fusion protein initiated hepatocyte proliferation and promoted repair in models of acute hepatic injury in mice. Here, we investigated the impact of CSF1-Fc on resolution of advanced fibrosis and liver regeneration, using a non-resolving toxin-induced model of chronic liver injury and fibrosis in C57BL/6J mice. Co-administration of CSF1-Fc with exposure to thioacetamide (TAA) exacerbated inflammation consistent with monocyte contributions to initiation of pathology. After removal of TAA, either acute or chronic CSF1-Fc treatment promoted liver growth, prevented progression and promoted resolution of fibrosis. Acute CSF1-Fc treatment was also anti-fibrotic and pro-regenerative in a model of partial hepatectomy in mice with established fibrosis. The beneficial impacts of CSF1-Fc treatment were associated with monocyte-macrophage recruitment and increased expression of remodelling enzymes and growth factors. These studies indicate that CSF1-dependent macrophages contribute to both initiation and resolution of fibrotic injury and that CSF1-Fc has therapeutic potential in human liver disease.

Interleukin-1 Is Overexpressed in Injured Muscles Following Spinal Cord Injury and Promotes Neurogenic Heterotopic Ossification.

Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles after severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1ß (IL-1ß) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1ß, we show that IL-1 signaling contributes to NHO development after SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor, or C-C chemokine ligand-2, did not promote NHO development. Finally, using NHO biopsies from SCI and TBI patients, we show that IL-1ß is expressed by CD68+ macrophages. IL-1α and IL-1ß produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether, these data suggest that interleukin-1 promotes NHO development in both humans and mice. © 2021 American Society for Bone and Mineral Research (ASBMR).

Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues.

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.

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

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, when they also organize the repair and regeneration of many tissues including the hemopoietic tissue. The fact that macrophages are also master regulators and effectors of innate immunity and inflammation allows them to coordinate hematopoietic response to infections, injuries, and inflammation. After recently reviewing the roles of phagocytes and macrophages in regulating normal and pathologic hematopoietic 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 center 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 anemia of inflammation via macrophages. Finally, we discuss the technical challenges associated with the molecular characterization of erythroid island macrophages and red pulp macrophages.

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

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.

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

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).

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

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.

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

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.

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

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-1ß 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.

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

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 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. CONCLUSION: These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

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

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/Csf1r-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.

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

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 CD11b+Ly6G+F4/80- 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.

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

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). In vitro 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 in vivo 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 in vivo 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.

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

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.

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

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.