Changes In Osteoclast Numbers Research Articles

Neutrophils Regulate Bone Formation Via IL-1b Secretion

The bone marrow (BM) is surrounded by a shell of cortical bone that protects the hematopoietic stem cells (HSC) from external aggressions. Within the BM, progenitors for bone and blood coexist and, intriguingly, the same mesenchymal progenitors that give rise to bone also form niches that regulate hematopoiesis. Whether, and if so how, the hematopoietic system regulates the formation of its protective osteal shell is poorly understood. Among HSC descendants, innate immune cells have been involved in maintaining homeostasis in multiple organs, and neutrophils in particular have been shown to regulate the BM stroma both at steady-state and during stress, and to contribute to vascular repair. Hence, we examined MRP8cre iDTR mice (iDTR) which allow for inducible neutropenia upon diphtheria toxin injection. We found that acute neutropenia in these mice caused bone mass reduction (48% reduction) as determined by μCT, while no changes in osteoclast numbers. We confirmed this reduction using the calcein double labeling assay, altogether indicating that neutrophils regulate bone formation. We found a similar osteoporotic phenotype in MRP8cre CXCR4fl mice (CXCR4ΔN), in which only the return of circulating neutrophils to the BM is compromised, which suggested that it is peripheral-experienced neutrophils returning to the BM that exert the regulatory effect on the bone-producing mesenchymal cells. Concomitant with bone loss, we found that the number of hematopoietic progenitors were elevated in both in the iDTR and CXCR4ΔN models as assessed by flow cytometry, CFU-C assays and limited dilution reconstitution assays, suggesting that neutrophils counteract natural, age-related alterations in the BM. To identify the mechanisms by which neutrophils regulate bone formation, we searched for factors differentially produced by neutrophils returning to the BM compared with marrow-residing neutrophils. Transcriptomic analyses of neutrophils that migrated to the BM in a parabiotic model identified 8 soluble factors that were not produced by immature neutrophils. Among these factors, we found that interleukin 1 beta (Il1b) was highly expressed in returning neutrophils by qRT-PCR and protein levels, and single-cell RNAseq demonstrated that the production of Il1b was a hallmark of neutrophil maturation and egress into blood. To explore whether this cytokine mediated the osteogenic program of neutrophils in the BM, we generated mice with neutrophil-specific deletion of Il1b ; MRP8cre Il1bfl mice (Il1bΔN). Single-cell RNA-seq analysis of CD45-Ter119-CD31-CXCL12+ stroma cells from control CXCL12GFP mice, iDTR CXCL12GFP and Il1bΔN CXCL12GFP demonstrated marked anomalies in the differentiation of mesenchymal stem cell (MSC) into osteoblast lineage cells (OLC) and, to a lesser extent, adipocytic-lineage cells in the absence of neutrophils or neutrophil-derived Il1b. In vitro cultures confirmed a modulatory effect of Il1b on MSC differentiation. These findings correlated with the close proximity of neutrophils to CXCL12GFP+ MSC over random distribution, and with high expression of interleukin 1 receptor type1 (Il1r1) in OLC, altogether suggesting that neutrophils modulate the osteogenic program of MSC through Il1b secretion. To explore physiological contexts in which neutrophils modulate bone formation, we first examined the circadian transcriptome of total BM cells and found that Il1b was one of the few cytokines whose transcript levels in the BM followed a tight circadian pattern. We found that depletion of neutrophils fully ablated Il1b expression at all time points, raising the possibility that neutrophils enable diurnal differentiation of mesenchymal cells to produce bone. Indeed, the calcein double labeling assay assays revealed marked circadian patterns of bone synthesis that were significantly blunted in neutropenic and Il1bΔN mice. We propose that bone and blood lineages have co-evolved as an interlocked system that ensures both physical protection and cellular output of this organ.

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms.

Activating-mutations in NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) cause neonatal-onset multisystem inflammatory disease. However, the ontogeny of skeletal anomalies in this disorder is poorly understood. Mice globally expressing the D301N mutation in Nlrp3 (D303N in human) model the human phenotype, including systemic inflammation and skeletal deformities. To gain insights into the skeletal manifestations, we generated mice in which the expression of D301N Nlrp3 (Nlrp3( D301N)) is restricted to myeloid cells. These mice exhibit systemic inflammation and severe osteopenia (∼ 60% lower bone mass) similar to mice globally expressing the knock-in mutation, consistent with the paradigm of innate immune-driven cryopyrinopathies. Because systemic inflammation may indirectly affect bone homeostasis, we engineered mice in which Nlrp3( D301N) is expressed specifically in osteoclasts, the cells that resorb bone. These mice also develop ∼ 50% lower bone mass due to increased osteolysis, but there is no systemic inflammation and no change in osteoclast number. Mechanistically, aside from its role in IL-1β maturation, Nlrp3( D301N) expression enhances osteoclast bone resorbing ability through reorganization of actin cytoskeleton while promoting the degradation of poly(ADP-ribose) polymerase 1, an inhibitor of osteoclastogenesis. Thus, NLRP3 inflammasome activation is not restricted to the production of proinflammatory mediators but also leads to cytokine-autonomous responses.

Loss of the PGE2 receptor EP1 enhances bone acquisition, which protects against age and ovariectomy-induced impairments in bone strength

PGE2 exerts anabolic and catabolic effects on bone through the discrete actions of four prostanoid receptors (EP1–4). We have previously demonstrated that loss EP1 accelerates fracture repair by enhancing bone formation. In the present study we defined the role of EP1 in bone maintenance and homeostasis during aging and in response to ovariectomy. The femur and L4 vertebrae of wild type (WT) and EP1−/− mice were examined at 2-months, 6-months, and 1-year of age, and in WT and EP1−/− mice following ovariectomy (OVX) or sham surgery. Bone volume fraction, trabecular architecture and mechanical properties were maintained during aging in EP1−/− mice to a greater degree than age-matched WT mice. Moreover, significant increases in bone formation rate (BFR) (+60%) and mineral apposition rate (MAR) (+50%) were observed in EP1−/−, relative to WT, while no change in osteoclast number and osteoclast surface were observed. Following OVX, loss of EP1 was protective against bone loss in both femur and L4 vertebrae, with increased bone volume/total volume (BV/TV) (+32% in femur) and max load at failure (+10% in femur) relative to WT OVX, likely resulting from the increased bone formation rate that was observed in these mice. Taken together these studies identify inhibition of EP1 as a potential therapeutic approach to suppress bone loss in aged or post-menopausal patients.

Carbon-11 labeled cathepsin K inhibitors: syntheses and preliminary in vivo evaluation.

Cathepsin K is a cysteine peptidase primarily located in osteoclasts, cells involved in normal growth and remodeling of bone but that are also responsible for bone loss in osteolytic diseases such as osteoporosis. In vivo imaging of cathepsin K may provide a method to assess changes in osteoclast numbers in such disease states. To that end, two high-affinity and selective cathepsin K inhibitors were radiolabeled with carbon-11. In vivo microPET imaging studies demonstrated uptake and prolonged retention of radioactivity in actively growing or remodeling bone regions (e.g., distal ulnar, carpal, distal and proximal humeral, distal femur, proximal tibia, tail vertebrae). Uptake into bone could be blocked by pre- or co-injection of unlabeled ligand, supporting a specific and saturable binding mechanism for radiotracer localization. These proof-of-concept studies indicate that radiolabeled cathepsin K inhibitors may have potential as in vivo imaging radiotracers for assessing changes of osteoclast numbers in osteolytic diseases.

Loss of Functional NADPH Oxidase 2 Protects Against Alcohol‐Induced Bone Resorption in Female p47phox−/− Mice

In bone, NADPH oxidase (NOX)-derived reactive oxygen species (ROS) superoxide and/or hydrogen peroxide are an important stimulus for osteoclast differentiation and activity. Previously, we have demonstrated that chronic ethanol (EtOH) consumption generates excess NOX-dependent ROS in osteoblasts, which functions to stimulate nuclear factor kappa-β receptor ligand (RANKL)-RANK signaling, thus increasing osteoclastogenesis and activity. This activity can be blocked by co-administration of EtOH with the pan-NOX inhibitor diphenylene idonium (DPI). To test whether EtOH-induced bone loss is dependent on a functional NOX2 enzyme, 6-week-old female C57BL/6J-Ncf1/p47phox(-/-) (p47phox KO) and wild-type (WT) mice were pair-fed EtOH diets for 40days. Bone loss was assessed by 3-point bending, micro-computed tomography and static histomorphometric analysis. Additionally, ST2 cultured cells were co-treated with EtOH and NOX inhibitors, DPI, gliotoxin, and plumbagin, after which changes in ROS production, and in RANKL and NOX mRNA expression were analyzed. In WT mice, EtOH treatment significantly reduced bone density and mechanical strength, and increased total osteoclast number and activity. In EtOH-treated p47phox KO mice, bone density and mechanical strength were completely preserved. EtOH p47phox KO mice had no changes in osteoclast numbers or activity, and no elevations in serum CTX or RANKL gene expression (p<0.05). In both WT and p47phox KO mice, EtOH feeding reduced biochemical markers of bone formation (p<0.05). In vitro EtOH exposure of ST2 cells increased ROS, which was blocked by pretreating with DPI or the NOX2 inhibitor gliotoxin. EtOH-induced RANKL and NOX2 gene expression were inhibited by the NOX4-specific inhibitor plumbagin. These data suggest that NOX2-derived ROS is necessary for EtOH-induced bone resorption. In osteoblasts, NOX2 and NOX4 appear to work in tandem to increase RANKL expression, whereas EtOH-mediated inhibition of bone formation occurs via a NOX2-independent mechanism.

The Effect of Myeloma Cells on Bone Homeostasis Is Heterogenous and Correlates with Underlying Genetic Lesions and Bone Disease In Vivo

AbstractAbstract 4052Osteolytic bone disease is a major clinical burden in multiple myeloma. Uncoupling of normal homestatic bone remodelling by an increase in osteoclast numbers and suppression of osteoblasts is the main causative mechanism. Subgroups of patients, identified by recurrent IgH translocation partners and D-type cyclin expression, have different clinical features, including prevalence of bone disease. Molecular groups characterised by t(4:14) or t(14:16) translocations, have a lower incidence of lytic bone lesions when compared with subgroups characterised by t(11:14) and/or cyclin D1 expression. We hypothesize that while osteoclast activation is a common phenomenon, osteolysis depends upon the presence and degree of osteoblast suppression, which in turn is dictated by the underlying genetic lesion. We have recently established a medullary model of myeloma using the human KMS12BM cell line that bears t(11;14). This model exhibits lytic bone disease as evidenced by micro-computed-tomography (microCT) and histomorphometry, accompanied by increased osteoclastogenesis and suppressed osteoblast numbers. These features are typical of the clinical disease bearing t(11;14). In addition, we have established a second medullary model of an alternative molecular disease group using the MM1.s human myeloma cell line, bearing t(14;16). Non-irradiated β2M NOD/SCID mice intravenously injected with either the KMS12BM or MM1s cells develop tumours confined to the bone marrow with little extramedullary disease. Histomorphometric analysis of femora from diseased animals revealed several striking differences in bone biology between these two models. Early disease progression in the KMS12BM model is characterised by an increase in osteoblast numbers (p < 0.05) with little change in osteoclast numbers at trabecular surfaces. As homeostatic bone remodelling processes become uncoupled in advanced disease there is a loss of osteoblasts and an increase in osteoclast numbers. In contrast, in the MM1.s model, there are no significant changes in osteoblast or osteoclast numbers at the trabecular surfaces. The distinct impact of t(11:14) and t(14:16) modelled disease on bone is further illustrated by microCT analysis of femora of diseased animals. Femora of KMS12BM mice show a reduction in trabecular number and thickness (p < 0.05) with bone loss further highlighted by an increased structure model index (SMI, p < 0.05). MM1.s disease results in increased bone volume (p < 0.01) with a concordant increase in trabecular thickness (p < 0.05) and number (p < 0.01), and little change in SMI. Importantly, while bone disease in the KMS12BM model is typified by osteolytic lesions, MM1.s animals do not appear to develop osteolytic lesions, despite perturbation of bone biology. These models, representing distinct genetic subtypes of myeloma, produce different clinico-pathological effects on bone remodelling, perhaps by differential effects on osteoblast biology. They will facilitate the identification of critical molecular pathways involved in osteoblast suppression leading to bone loss. Disclosures:No relevant conflicts of interest to declare.

Complex Dynamics of Osteoclast Formation and Death in Long-Term Cultures

BackgroundOsteoclasts, cells responsible for bone resorption, contribute to the development of degenerative, metabolic and neoplastic bone diseases, which are often characterized by persistent changes in bone microenvironment. We aimed to investigate the dynamics of osteoclast formation and death in cultures that considerably exceeded the length of standard protocol and to design a mathematical model describing osteoclastogenesis.Methodology/Principal FindingsRAW 264.7 monocytic cells fuse to form multinucleated osteoclasts upon treatment with pro-resorptive cytokine RANKL. We have found that in long-term experiments (15–26 days), the dynamics of changes in osteoclast numbers was remarkably complex and qualitatively variable in different experiments. Whereas 19 of 46 experiments exhibited single peak of osteoclast formation, in 27 experiments we observed development of successive waves of osteoclast formation and death. Periodic changes in osteoclast numbers were confirmed in long-term cultures of mouse bone marrow cells treated with M-CSF and RANKL. Because the dynamics of changes in osteoclast numbers was found to be largely independent of monocytes, a two-species model of ordinary differential equations describing the changes in osteoclasts and monocytes was ineffective in recapitulating the oscillations in osteoclast numbers. Following experimental observation that medium collected from mature osteoclasts inhibited osteoclastogenesis in fresh cultures, we introduced a third variable, factor f, to describe osteoclast-derived inhibitor. This model allowed us to simulate the oscillatory changes in osteoclasts, which were coupled to oscillatory changes in the factor f, whereas monocytes changed exponentially. Importantly, to achieve the experimentally observed oscillations with increasing amplitude, we also had to assume that osteoclast presence stimulates osteoclast formation.Conclusions/SignificanceThis study identifies the critical role for osteoclast autocrine regulation in controlling long-term dynamic of osteoclast formation and death and describes the complementary roles for negative and positive feedback mediators in determining the sharp dynamics of activation and inactivation of osteoclasts.

A Histomorphologic Study of Scoliosis in Pinealectomized Chickens

Pinealectomy was used to induce scoliosis in Broiler chickens, and a histologic investigation of the pinealectomized chickens was performed. To investigate the incidence of scoliosis in pinealectomized Broiler chickens and to determine histologic changes in vertebrae in the pinealectomized chickens before the occurrence of a secondary vertebral wedging deformity due to scoliosis. White Leghorn chickens have previously been used as an experimental model of scoliosis, but the occurrence of scoliosis in these chickens following pinealectomy has varied in different reports and is not always high. It is accepted that the development of scoliosis has a close association with growth, but the etiology and mechanism of the disease remain unknown. Histologic findings in vertebrae after the occurrence of scoliosis include those caused by secondary changes due to mechanical compression, and no histologic data for the period before the occurrence of scoliosis are available. Study 1. A total of 100 female "Broiler" chickens were pinealectomized, and the frequency with which scoliosis occurred was investigated. Study 2. Sixty female Broiler chickens were divided into three groups: the control group (Group C, n = 20), the sham operation group (Group S, n = 20), and the pinealectomy group (Group P, n = 20). Each group was then subdivided into two groups according to the time of death: 3 days after the operation (Group 3-C, 3-S, 3-P, n = 10), and 6 days after the operation (Group 6-C, 6-S, 6-P, n = 10). Decalcified thin sagittal sections were made using a tartrate-resistant acid phosphatase (TRAP) stain. Histologic examinations of the growth plate, trabecular structure, and osteoclast number were performed. The incidence of scoliosis in the pinealectomized Broiler chickens was 93.6%. Nine of the 10 chickens in Group 6-P showed scoliosis deformity, while the presence of scoliosis was unclear in chickens in Group 3-P. The osteoclast number increased significantly in Group 3-P, compared with Groups 3-C and 3-S, and the trabecular thickness was greater in Group 3-P than in Groups 3-C and 3-S. There was no significant change in the growth plate or in other aspects of the trabecular structure, except for trabecular thickness, in any of the groups. The results show that the incidence of scoliosis using pinealectomized Broiler chickens is sufficient to study histologic changes of the vertebral body before onset of scoliosis. We found that the osteoclast number and trabecular thickness increased in pinealectomized chickens after 3 days after surgery, just before scoliosis began to develop, and that no change in the growth plate occurred. This suggests that there is no association between changes in the growth plate and the development of scoliosis. However, the change in osteoclast number may have an association with the development of scoliosis, through changes in bone modeling.

Characterization of bone structure in leptin receptor-deficient Zucker (fa/fa) rats.

To investigate the role of leptin in bone formation, the skeleton of the obese female leptin receptor-deficient Zucker rat was examined using pQCT, microCT, and histomorphometry. A trend toward decreasing structural and bone formation parameters in these rats as they age suggest that leptin has a small positive effect on bone. Evidence in the literature has suggested the possible role of leptin in bone formation. Leptin deficiency or leptin receptor deficiency results in higher bone mass. In an attempt to further investigate leptin's role in bone formation, we examined the skeleton of obese leptin receptor-deficient Zucker rats. Female leptin receptor-deficient Zucker (fa/fa) rats and their homozygous (Fa/Fa) and heterozygous (Fa/fa) lean controls were used at 9 and 15 weeks of age (n = 5). Bone mineral density of the proximal tibia was measured by peripheral quantitative computed tomography (pQCT). Microcomputed tomography (microCT) was used for the analysis of trabecular architecture in the proximal tibia metaphysis and cortical bone at the tibia-fibula junction. Static and dynamic parameters of bone resorption and formation were quantitated by histomorphometry. Statistical analysis was performed by Dunnett's one-way ANOVA. Analysis of the proximal tibia by pQCT show no significant differences in the bone mineral density of obese rats compared with their corresponding lean controls in either age group. Trabecular architecture measured by microCT indicate a trends toward decreasing bone volume (BV/TV) in the obese animals, evident by a decrease in trabecular number and thickness with an increase in trabecular separation. Histomorphometric evaluation further shows significant increases in osteoclast surface in the obese rats at both 9 and 15 weeks without a change in osteoclast number. Osteoid surface in the obese animals was also found to be decreased by 15 weeks of age. Fluorescent-based measurements of bone formation were not significantly different. Differences in the cortical compartment were not observed at either age. Based on the observed skeletal phenotype of the Zucker (fa/fa) rat, it is suggested that leptin exerts a positive effect on bone.

Bioactive parathyroid hormone in pregnant rats and fetuses

Parathyroid function at the end of gestation (day 21) was investigated by measuring plasma calcium (PCa), immunoreactive parathyroid hormone (iPTH), bioactive parathyroid hormone (bioPTH; cytochemical bioassay), and bone histology in intact and thyroparathyroidectomized (TPTX; day 12, ether anesthesia) rats and their fetuses. In pregnant intact rats, PCa was significantly lower, and iPTH, bioPTH, and osteoclast number were higher than in nonpregnant rats. In fetuses, PCa was higher than maternal PCa and correlated with fetal bioPTH. TPTX suppressed maternal bioPTH and decreased iPTH and osteoclast number, whereas fetal iPTH and bioPTH were decreased with no change in osteoclast number. Fetal PCa was near normal and was correlated with maternal PCa but not with fetal bioPTH. The fetomaternal calcium gradient was maintained and even increased. This study shows that there is maternal physiological hyperparathyroidism and functional fetal parathyroid glands at the end of gestation in the rat. Parathyroid hormone does not seem to be responsible for maintaining the high fetomaternal calcium gradient in TPTX animals.