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Osteoimmunology highlights the reciprocal interactions between the skeletal and immune systems. Over the past two decades, many molecules that link the two have been identified, including cytokines, receptors and transcription factors, leading to successful translation of research into therapeutic approaches to autoimmune diseases such as rheumatoid arthritis. The development of an intravital imaging system using two-photon microscopy, combined with a variety of fluorescent probes and reporter mouse strains, has provided valuable insights into the real-time dynamics of osteoclasts and immune cells in the bone marrow. This technique is now applied to the synovial tissue of arthritic mice to investigate the pathogenesis of osteoimmune diseases and enables direct observation of complex biological phenomena in vivo. In addition, rapid progress in the next-generation sequencing technologies has provided important insights into the field of osteoimmunology through characterizing individual cells in the synovial microenvironment. Single-cell RNA sequencing (scRNA-seq) dissects cellular heterogeneity within a biological system and enables the identification of specific cells differentiating into mature osteoclasts within the previously defined ‘osteoclast precursor-containing population’. In this review, we will explain the cellular interactions and cytokine milieu involved in inflammatory bone destruction and update how the novel technologies, such as scRNA-seq and intravital imaging, have contributed to better understand the pathogenesis of bone destruction in arthritis.

Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast–osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism., The mechanism by which parathyroid hormone mediates the switch from bone resorption to bone formation is unclear. Here, the authors show that SLPI regulates the communication between osteoblasts and osteoclasts to promote the anabolic effect of parathyroid hormone.

Bone morphogenetic protein (BMP)-2 plays a central role in bone-tissue engineering because of its potent bone-induction ability. However, the process of BMP-induced bone formation in vivo remains poorly elucidated. Here, we aimed to establish a method for intravital imaging of the entire process of BMP-2-induced ectopic bone formation. Using multicolor intravital imaging in transgenic mice, we visualized the spatiotemporal process of bone induction, including appearance and motility of osteoblasts and osteoclasts, angiogenesis, collagen-fiber formation, and bone-mineral deposition. Furthermore, we investigated how PTH1-34 affects BMP-2-induced bone formation, which revealed that PTH1-34 administration accelerated differentiation and increased the motility of osteoblasts, whereas it decreased morphological changes in osteoclasts. This is the first report on visualization of the entire process of BMP-2-induced bone formation using intravital imaging techniques, which, we believe, will contribute to our understanding of ectopic bone formation and provide new parameters for evaluating bone-forming activity.

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two‐photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia‐inducible factor activity. We observe that hypoxia decreases ten‐eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen‐dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation., Within the physiological range of oxygen tension, ten‐eleven translocation (TET) enzymes act as functional oxygen sensors involved in osteoclastogenesis.

Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells that play different roles in different organs by sensing surrounding environmental factors. Initially, it was thought that ILC2s in bone marrow (BM) are progenitors for systemic ILC2s, which migrate to other organs and acquire effector functions. However, accumulating evidence that ILC2s differentiate in peripheral tissues suggests that BM ILC2s may play a specific role in the BM as a unique effector per se. Here, we demonstrate that BM ILC2s highly express the receptor activator of nuclear factor κB ligand (RANKL), a robust cytokine for osteoclast differentiation and activation, and RANKL expression on ILC2s is up-regulated by interleukin (IL)-2, IL-7 and all-trans retinoic acid (ATRA). BM ILC2s co-cultured with BM-derived monocyte/macrophage lineage cells (BMMs) in the presence of IL-7 induce the differentiation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in a RANKL-dependent manner. In contrast, BM ILC2s stimulated with IL-33 down-regulate RANKL expression and convert BMMs differentiation into M2 macrophage-like cells rather than osteoclasts by granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-13 production. Intravital imaging using two-photon microscopy revealed that a depletion of ILC2s prominently impaired in vivo osteoclast activity in an IL-7 plus ATRA-induced bone loss mouse model. These results suggest that ILC2s regulate osteoclast activation and contribute to bone homeostasis in both steady state and IL-33-induced inflammation.

Rheumatoid arthritis is a chronic form of arthritis that causes bone destruction in joints such as the knees and fingers. Over the past two decades, the clinical outcomes of rheumatoid arthritis have improved substantially with the development of biological agents and Janus kinase inhibitors. Osteoclasts are myeloid lineage cells with a unique bone-destroying ability that can lead to joint destruction. On the other hand, osteoclasts play an important role in skeletal homeostasis by supporting bone remodeling together with osteoblasts in the bone marrow under steady-state conditions. However, the same osteoclasts are considered to participate in physiological bone remodeling and joint destruction. We found that pathological osteoclasts have different differentiation pathways and regulatory transcription factors compared to physiological osteoclasts. We also identified arthritis-associated osteoclastogenic macrophages (AtoMs), which are common progenitors of pathological osteoclasts in mice and humans that develop specifically in inflamed synovial tissue. This review presents details of the newly identified AtoMs and the original intravital imaging systems that can visualize synovial tissue and pathological osteoclasts at the pannus-bone interface.

Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.

Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor β. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo.

Bone is a highly dynamic organ that is continuously being remodeled by the reciprocal interactions between bone and immune cells. We have originally established an advanced imaging system for visualizing the in vivo behavior of osteoclasts and their precursors in the bone marrow cavity using two-photon microscopy. Using this system, we found that the blood-enriched lipid mediator, sphingosine-1-phosphate, controlled the migratory behavior of osteoclast precursors. We also developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo, and identified two distinct functional states of differentiated osteoclasts, “bone-resorptive” and “non-resorptive.” Here, we summarize our studies on the dynamics and functions of bone and immune cells within the bone marrow. We further discuss how our intravital imaging techniques can be applied to evaluate the mechanisms of action of biological agents in inflammatory bone destruction. Our intravital imaging techniques would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would also be useful for evaluating novel therapies in animal models of bone-destroying diseases.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

Osteoclasts have a unique bone-destroying capacity, playing key roles in steady-state bone remodeling and arthritic bone erosion. Whether the osteoclasts in these different tissue settings arise from the same precursor states of monocytoid cells is presently unknown. Here, we show that osteoclasts in pannus originate exclusively from circulating bone marrow-derived cells and not from locally resident macrophages. We identify murine CX3CR1hiLy6CintF4/80+I-A+/I-E+ macrophages (termed here arthritis-associated osteoclastogenic macrophages (AtoMs)) as the osteoclast precursor-containing population in the inflamed synovium, comprising a subset distinct from conventional osteoclast precursors in homeostatic bone remodeling. Tamoxifen-inducible Foxm1 deletion suppressed the capacity of AtoMs to differentiate into osteoclasts in vitro and in vivo. Furthermore, synovial samples from human patients with rheumatoid arthritis contained CX3CR1+HLA-DRhiCD11c+CD80-CD86+ cells that corresponded to mouse AtoMs, and human osteoclastogenesis was inhibited by the FoxM1 inhibitor thiostrepton, constituting a potential target for rheumatoid arthritis treatment.

The bone is essential for locomotion, calcium storage, and harboring the hematopoietic stem cells (HSCs) that supply the body with mature blood cells throughout life. HSCs reside at the interface of the bone and bone marrow (BM), where active bone remodeling takes place. Although the cellular components of the BM niche have been characterized, little is known about its epigenetic regulation. Here we find that the histone methylation regulator PTIP (Pax interaction with transcription-activation domain protein-1) is required to maintain the integrity of the BM niche by promoting osteoclast differentiation. PTIP directly promotes chromatin changes required for the expression of Pparγ (peroxisome proliferator-activated receptor-γ), a transcription factor essential for osteoclastogenesis. PTIP deletion leads to a drastic reduction of HSCs in the BM and induces extramedullary hematopoiesis. Furthermore, exposure of acute myeloid leukemia cells to a PTIP-deficient BM microenvironment leads to a reduction in leukemia-initiating cells and increased survival upon transplantation. Taken together, our data identify PTIP as an epigenetic regulator of osteoclastogenesis that is required for the integrity of the BM niche to sustain both normal hematopoiesis and leukemia.

Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts and osteoblasts, the fundamental process and dynamics have remained elusive. To resolve this, we established an intravital bone imaging system using multiphoton microscopy to visualize mature osteoclasts and osteoblasts in living bone.We herein describe the methodology for visualizing the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in living bone tissues using intravital multiphoton microscopy. This approach facilitates investigation of cellular dynamics in the pathogenesis of bone-destructive disorders, such as osteoporosis and rheumatoid arthritis in vivo, and would thus be useful for evaluating the efficacy of novel anti-bone-resorptive drugs.

Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. We have originally established an advanced imaging system for visualizing the in vivo behavior of mature osteoclasts in living bone tissues with intravital multiphoton microscopy. By means of this system, we could grasp the real time-course of osteoclastic bone resorption, and identified two distinct functional states of differentiated osteoclasts, 'bone-resorptive' and 'non-resorptive'. Intravital imaging also revealed that various biologic drugs acted directly on mature osteoclasts during inflammatory bone destruction. In this review, we show the latest data of intravital imaging of osteoclast dynamics.

In clinical, various kinds of anti-bone-resorptive drugs have been used for the treatment of bone disorders such as osteoporosis. However, it remains elusive how they affect the osteoclast dynamics in living bone tissues. We recently succeeded in visualizing cellular dynamics of bone-resorbing osteoclasts and their precursors with intravital bone imaging techniques. We found that active vitamin D analogs could regulate the migratory behavior of circulating osteoclast precursors, whereas bisphosphonates could affect mature osteoclasts and inhibit bone resorption within a short period. Here we show the latest data of in vivo effects of anti-bone resorptive drugs currently developed in the world.

In bone tissues, there are various kinds of cell types, such as osteoclasts, osteoblasts, monocytes, granulocyte, lymphocytes, mesenchymal cells and hematopoietic stem cells. They form a network with each other, and play critical roles in our life activities. The recent development of intravital two-photon imaging has enabled us to visualize the in vivo behavior of bone marrow cells in living bone tissues. This technique facilitates investigation of cellular dynamics in the physiology and pathogenesis of bone disorders in vivo, and would thus be useful for evaluating the efficacy of novel drugs. In this review, we summarize the basis of intravital bone imaging, and also discuss its further application.

Teriparatide, recombinant human PTH(1-34), is the only anabolic agent widely used for osteoporosis. This drug is thought to promote bone formation by modulating bone remodeling system, although the detailed mechanism remains unclear. Recently, we developed a novel intravital imaging technique to visualize mature osteoclasts and mature osteoblasts simultaneously. By means of this system, we revealed the effect of teriparatide on three-dimensional distribution and cell-to-cell interactions between these cells. Advances in these imaging studies may lead to further understanding of the intercellular network in bone metabolism.

There are as many as 200 cell types in the body, and highly sophisticated and varied life phenomena are carried out by cell migration to appropriate places at appropriate times following the appropriate interactions. Recent advances in optical imaging technology using multi-photon excitation microscopy have enabled visualization inside intact bone tissues in living animals without thin sectioning. Using such advanced techniques, the dynamic behaviors of living bone cells on intact bone tissue structures can be elucidated. Here, we focus on recent findings using intravital multi-photon imaging of dynamic biological systems, e.g., bone homeostasis. This novel approach has proven beneficial for understanding the mechanisms underlying the spatiotemporal nature of bone remodeling systems and for evaluating the specific modes of actions of novel drugs currently in development, which will contribute to a new chapter in bone and mineral research.

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial–temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell–cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo., Communication between osteoblasts and osteoclasts is essential for bone homeostasis, but the mode of interaction is unclear. The authors use intravital two-photon microscopy in mice to show that these cells directly interact, regulating activity of osteoclasts, and that the interaction is modulated by parathyroid hormone administration.

Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. We have originally established an advanced imaging system for visualizing in vivo behavior of osteoclasts and their precursors with intravital two-photon microscopy. By means of the system, we found that sphingosine-1-phosphate, a lipid mediator enriched in blood, controlled the migratory behavior of osteoclast precursors. We also developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo, and identified two distinct functional states of differentiated osteoclasts, 'bone-resorptive' and 'non-resorptive'. In this review, we summarize our recent studies on the dynamics and functions of osteoclasts. Our intravital imaging techniques would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would thus be useful for evaluating novel therapies targeting aspects of osteoclast dynamics in patients with bone-destructive diseases.

Bone is a dynamic organ that is continuously turned over during growth, even in adults. During bone remodeling, homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. However, in pathological conditions such as osteoporosis, osteopetrosis, arthritic joint destruction, and bone metastasis, this equilibrium is disrupted. Since osteoclasts are excessively activated in osteolytic diseases, the inhibition of osteoclast function has been a major therapeutic strategy. It has recently been demonstrated that sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. By means of intravital multiphoton imaging of bone tissues, we have recently revealed that the reciprocal action of S1P controls the migration of osteoclast precursors between bone tissues and blood stream. Imaging technologies have enabled us to visualize the in situ behaviors of different cell types in intact tissues. In this review we also discuss future perspectives on this new method in the field of bone biology and medical sciences in general. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Rapid development of fluorescent imaging techniques enables us to understand cellular dynamics in vivo. We have originally established an advanced imaging system for visualizing living bone tissues with intravital two-photon microscopy. By means of the system, we have recently succeeded in visualization of the in vivo behavior of living mature osteoclasts on the bone surface, and identified different functional subsets of osteoclasts in terms of their motility and function, i.e., 'static - bone resorptive' and 'moving - non resorptive'. Pathological conditions changed the composition of these populations as well as the total number of mature osteoclasts. We also found that RANKL-bearing Th17 cells could control bone resorption of mature osteoclasts, demonstrating novel actions of Th17. Furthermore, we have also developed the imaging system to visualize bone destruction by osteoclasts in arthritic joints using intravital two-photon microscopy. In this review, we summarize the latest data of intravital imaging of osteoclast dynamics, and also discuss its further application.

Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. Upon the stimulation of essential factors such as M-CSF and RANKL, osteoclast precursor monocytes attach to the bone surface ( "migration" ), fuse with each other to form giant cells ( "differentiation" ) and mediate bone resorption ( "function" ). To reveal the regulatory mechanism of these three dynamic steps of osteoclastic activity, we have originally established an advanced imaging system for visualizing living bone tissues with intravital multiphoton microscopy. By means of the system, we have recently succeeded in visualization of osteoclast migration, differentiation, and function in living bone tissues in vivo. In this review we summarize the latest data of intravital imaging of osteoclast dynamics, and discuss novel lines of osteoclast-targeted therapies that will impact future treatment of bone destructive diseases.

The mechanisms guiding cells toward bone surfaces are generally unknown. Here, Nevius et al. show that the Gαi protein–coupled receptor EBI2 is expressed in mouse osteoclast precursors to guide these cells toward bone surfaces. Defective EBI2 signaling increased bone mass in male mice and protected female mice from age- and estrogen deficiency–induced osteoporosis., Bone surfaces attract hematopoietic and nonhematopoietic cells, such as osteoclasts (OCs) and osteoblasts (OBs), and are targeted by bone metastatic cancers. However, the mechanisms guiding cells toward bone surfaces are essentially unknown. Here, we show that the Gαi protein–coupled receptor (GPCR) EBI2 is expressed in mouse monocyte/OC precursors (OCPs) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) is secreted abundantly by OBs. Using in vitro time-lapse microscopy and intravital two-photon microscopy, we show that EBI2 enhances the development of large OCs by promoting OCP motility, thus facilitating cell–cell interactions and fusion in vitro and in vivo. EBI2 is also necessary and sufficient for guiding OCPs toward bone surfaces. Interestingly, OCPs also secrete 7α,25-OHC, which promotes autocrine EBI2 signaling and reduces OCP migration toward bone surfaces in vivo. Defective EBI2 signaling led to increased bone mass in male mice and protected female mice from age- and estrogen deficiency–induced osteoporosis. This study identifies a novel pathway involved in OCP homing to the bone surface that may have significant therapeutic potential.

Bone marrow is a highly vascularized organ and the vessel walls have a large number of fenestrations. Various kinds of hematopoietic cells are continuously entering into and egressing from bone marrow cavity through these unique features. Monocytoid osteoclast precursors also migrate across the capillary endothelium, serving as a dynamic point of control of bone homeostasis. We have recently revealed the cellular dynamics of monocytoid osteoclast precursors in vivo by a novel fluorescent imaging technology. Furthermore we found the regulatory mechanism of vascular permeability in bone marrow in vivo by using intravital multiphoton microscopy. Here we show the latest data and also discuss its further application.

Osteoclasts are giant bone-resorbing polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. They play critical roles not only in normal bone homeostasis (remodeling) but also in the pathogenesis of bone-destructive disorders such as osteoporosis and rheumatoid arthritis. However, how the activity of mature osteoclasts is regulated in vivo remains unclear. To answer this question, we recently developed an advanced imaging system to visualize living bone tissues with intravital multiphoton microscopy. Using this system, we succeeded in visualization of mature osteoclasts in living bones. We herein describe the detailed methodology for visualizing bone resorption of mature osteoclasts in living bone marrow and joints using intravital multiphoton microscopy. This approach would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would thus be useful for evaluating novel anti-bone-resorptive drugs.

Various kinds of cell types, such as osteoclasts, osteoblasts, hematopoietic cells, and mesenchymal cells, have been reported to exist in the bone marrow and communicate with each other. Although there have been many previous studies about bone marrow microenvironment, most of them were analyzed by conventional methods such as histological analysis and flow cytometry. These methods could not observe the dynamic cell movement in living bone marrow. Recently rapid development of fluorescent imaging techniques enables us to understand the cellular dynamics in vivo . That's why we have originally established an advanced imaging system for visualizing living bone tissues with intravital two-photon microscopy. Here we show the latest data and the detailed methodology of intravital imaging of bone marrow microenvironment, and also discuss its further application.

International audience; Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1–6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1−/− mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1−/− mice but not in Lpar2−/− and Lpar3−/− animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1−/− osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP+ osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.

By using conventional methods such as histological analysis, many osteoclasts can be observed in the site of bone destruction. However, how the bone-resorptive functions of mature osteoclasts are controlled in vivo remains unclear. To answer this question, we have originally developed an advanced imaging system for visualizing living bone tissues with intravital multiphoton microscopy. Using this system, we succeeded in visualizing bone resorption of mature osteoclasts in living bone. Here we show the latest data and also discuss the further application of intravital bone imaging. This approach would be quite useful for evaluating novel anti-bone-resorptive drugs in vivo .

The migration and positioning of osteoclast precursor monocytes are controlled by the blood-enriched lipid mediator sphingosine-1-phosphate (S1P) and have recently been shown to be critical points of control in osteoclastogenesis and bone homeostasis. Here, we show that calcitriol, which is the hormonally active form of vitamin D, and its therapeutically used analog, eldecalcitol, inhibit bone resorption by modulating this mechanism. Vitamin D analogs have been used clinically for treating osteoporosis, although the mode of its pharmacologic action remains to be fully elucidated. In this study, we found that active vitamin D reduced the expression of S1PR2, a chemorepulsive receptor for blood S1P, on circulating osteoclast precursor monocytes both in vitro and in vivo. Calcitriol- or eldecalcitol-treated monocytoid RAW264.7 cells, which display osteoclast precursor-like properties, migrated readily to S1P. Concordantly, the mobility of circulating CX 3 CR1 + osteoclast precursor monocytes was significantly increased on systemic administration of active vitamin D. These results show a mechanism for active vitamin D in controlling the migratory behavior of circulating osteoclast precursors, and this action should be conducive to limiting osteoclastic bone resorption in vivo.

Rapid development of fluorescent imaging techniques enables us to understand cellular dynamics in vivo . We have originally established an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy. By means of the system we have recently succeeded in visualization of mature osteoclasts in live bones and revealed that RANKL regulates bone-resorptive functions of mature osteoclasts in vivo . We also developed new image analysis software for tracking morphological changes of mature osteoclasts and generated pH-sensing chemical fluorescent probes for detecting bone resorption at local sites on the bone surface. Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static - bone resorptive (R) to moving - nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.

Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes or from bone tissue–resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation “parabiosis” with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX3CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP+ osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP+ osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP+ osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR+ monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted “red” KikGR+ osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.

RA is a chronic autoimmune disease characterized by joint synovial inflammation and progressive cartilage/bone destruction. Although various kinds of RA drug have been developed worldwide, there are currently no established methods for preventing RA-associated bone destruction, the most severe outcome of this disease. One of the major pathogenic factors in arthritic bone destruction is the enhanced activity of osteoclasts at inflammatory sites. Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage haematopoietic precursors. Upon stimulation by cytokines, such as M-CSF and RANK ligand, osteoclast precursor monocytes migrate and attach onto the bone surface (migration). They then fuse with each other to form giant cells (differentiation) and mediate bone resorption (function). In this review, we summarize the current understanding regarding the mechanisms underlying these three dynamic steps of osteoclastic activity and discuss novel lines of osteoclast-targeted therapies that will impact future treatment of RA.

We describe a method to visualize the migration of osteoclast precursors within intact murine bone -marrow in real time using intravital multiphoton microscopy. Conventionally, cell migration has been evaluated using in vitro systems, such as transmigration assays. Although these methods are convenient for quantification and are highly reproducible, these in vitro assay systems may not accurately reflect in vivo cellular behavior. In addition to in vitro analyses, recent technological progress in two-photon excitation-based laser microscopy has enabled the visualization of dynamic cell behavior deep inside intact living organs. Combining this imaging method with in vitro chemoattraction analyses, we have revealed that sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, bidirectionally controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors (GPCRs).

Osteoclasts are 'bone-resorbing' giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. However, it has remained unclear how osteoclast precursors migrate into the bone surface and what controls their migratory behaviors in vivo . To investigate these questions, we utilized an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have recently established. By means of the system we have recently succeeded in visualization of osteoclast precursors in live bones and revealed that sphingosine-1-phosphate (S1P) , a lipid mediator, dynamically regulates migration and localization of osteoclasts and their precursors in vivo . Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Osteoclasts are giant polykaryons responsible for bone resorption. Because an enhancement or loss of osteoclast function leads to bone diseases such as osteoporosis and osteopetrosis, real-time imaging of osteoclast activity in vivo can be of great help for the evaluation of drugs. Herein, pH-activatable chemical probes BAp-M and BAp-E have been developed for the detection of bone-resorbing osteoclasts in vivo. Their acid dissociation constants (pK(a)) were determined as 4.5 and 6.2 by fluorometry in various pH solutions. These pK(a) values should be appropriate to perform selective imaging of bone-resorbing osteoclasts, because synthesized probes cannot fluoresce intrinsically at physiological pH and the pH in the resorption pit is lowered to about 4.5. Furthermore, BAp-M and BAp-E have a bisphosphonate moiety that enabled the probes to localize on bone tissues. The hydroxyapatite (HA) binding assay in vitro was, therefore, performed to confirm the tight binding of the probes to the bone tissues. Our probes showed intense fluorescence at low pH values but no fluorescence signal under physiological pH conditions on HA. Finally, we applied the probes to in vivo imaging of osteoclasts by using intravital two-photon microscopy. As expected, the fluorescence signals of the probes were locally observed between the osteoclasts and bone tissues, that is, in resorption pits. These results indicate that our pH-activatable probes will prove to be a powerful tool for the selective detection of bone-resorbing osteoclasts in vivo, because this is the first instance where in vivo imaging has been conducted in a low-pH region created by bone-resorbing osteoclasts.

Osteoclasts are 'bone-resorbing' giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. However how the activity of mature osteoclasts is regulated in vivo remains unclear. To answer the question, we utilized an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have recently established. By means of the system we have recently succeeded in visualization of mature osteoclasts in live bones and revealed that RANKL regulates bone-resorptive functions of mature osteoclasts in vivo . Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Osteoclasts play critical roles not only in normal bone homeostasis ('remodeling') , but also in the pathogenesis of bone destructive disorders such as osteoporosis, rheumatoid arthritis, and bone metastasis. However, it has not been known how osteoclast precursor monocytes migrate into the bone surface and what controls their migratory behaviors. To reveal these systems, we have recently established a new system for visualizing intact bone tissues and bone marrow cavities in live animals by using an advanced imaging technique with intravital two-photon microscopy. By means of the system we have revealed that sphingosine-1-phosphate (S1P) , a lipid mediator, dynamically regulates migration and localization of osteoclasts and their precursors in vivo . Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. Through intravital multiphoton imaging of bone tissues, we reveal that the bidirectional function of S1P temporospatially regulates the migration of osteoclast precursors within intact bone tissues. Imaging technologies have enabled in situ visualization of the behaviors of several players in intact tissues. In addition, intravital microscopy has the potential to be more widely applied to functional analysis and intervention.