Imaging the bone-immune cell interaction in bone destruction

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.Hasegawa T., Kikuta J., Ishii M.. Imaging the bone-immune cell interaction in bone destruction. Frontiers in Immunology 10, 596 (2019); https://doi.org/10.3389/fimmu.2019.00596

The effects of vitamin D on immune system and inflammatory diseases

Immune cells, including dendritic cells, macrophages, and T and B cells, express the vitamin D receptor and 1α-hydroxylase. In vitro studies have shown that 1,25-dihydroxyvitamin D, the active form of vitamin D, has an anti-inflammatory effect. Recent epidemiological evidence has indicated a significant association between vitamin D deficiency and an increased incidence, or aggravation, of infectious diseases and inflammatory autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. However, the impact of vitamin D on treatment and prevention, particularly in infectious diseases such as the 2019 coronavirus disease (COVID-19), remains controversial. Here, we review recent evidence associated with the relationship between vitamin D and inflammatory diseases and describe the underlying immunomodulatory effect of vitamin D.Ao T., Kikuta J., Ishii M.. The effects of vitamin D on immune system and inflammatory diseases. Biomolecules 11, 1624 (2021); https://doi.org/10.3390/biom11111624

Imaging the Bone-Immune Cell Interaction in Bone Destruction

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 of orthotopic and ectopic bone

Bone homeostasis is dynamically regulated by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. Visualizing and evaluating the dynamics of bone cells in vivo remain difficult using conventional technologies, including histomorphometry and imaging analysis. Over the past two decades, multiphoton microscopy, which can penetrate thick specimens, has been utilized in the field of biological imaging. Using this innovative technique, the in vivo dynamic motion of bone metabolism-related cells and their interactions has been revealed. In this review, we summarize previous approaches used for bone imaging and provide an overview of current bone tissue imaging methods using multiphoton excitation microscopy.Hashimoto K., Kaito T., Kikuta J., et al. Intravital imaging of orthotopic and ectopic bone. Inflammation and Regeneration 40, 26 (2020); https://doi.org/10.1186/s41232-020-00135-6

Local sympathetic neurons promote neutrophil egress from the bone marrow at the onset of acute inflammation

This is a pre-copyedited, author-produced version of an article accepted for publication in International Immunology following peer review. The version of record Tomoka Ao, Junichi Kikuta, Takao Sudo, Yutaka Uchida, Kenta Kobayashi, Masaru Ishii, Local sympathetic neurons promote neutrophil egress from the bone marrow at the onset of acute inflammation, International Immunology, Volume 32, Issue 11, November 2020, Pages 727–736. is available online at: https://doi.org/10.1093/intimm/dxaa025

A case of newly demonstrated coronary spasm 4 months after paclitaxel-eluting stent implantation for in-stent restenosis

SummaryA 56-year-old woman with hypertension and hypercholesterolemia was admitted to our hospital with acute inferior myocardial infarction. The patient had total occlusion of the right coronary artery (RCA) segment 2, and bare-metal stents were placed. Four months later, plain old balloon angioplasty was performed for in-stent restenosis. Follow-up coronary angiography (CAG) 6 months later showed in-stent total occlusion, so a stent-in-stent procedure was performed using paclitaxel-eluting stents (PESs). Four months later, the patient began complaining of early morning chest pain at rest. CAG showed no in-stent restenosis, so coronary spastic angina was suspected. Intracoronary infusion of ergonovine to the right and left coronary arteries revealed spasm of the RCA with total occlusion just proximal to the PES in segment 1. Her chest pain was reproduced with ST-elevation in leads II, III, and aVF, so the diagnosis of coronary spastic angina was made. Treatment with a Ca-channel blocker and nitrates relieved the symptoms. The PES was the probable cause of the coronary spasm

Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo

There have been many attempts to visualize the inflamed joints using multiphoton microscopy. However, due to the hypervascular and multilayered structure of the inflamed synovium, intravital imaging of the deep synovial tissue has been difficult. Here, we established original intravital imaging systems to visualize synovial tissue and pathological osteoclasts at the pannus–bone interface using multiphoton microscopy. Combined with fluorescence-labeling of CTLA-4 Ig, a biological agent used for the treatment of rheumatoid arthritis, we identified that CTLA-4 Ig was distributed predominantly within the inflamed synovium and bound to CX3CR1+ macrophages and CD140a+ fibroblasts 6 h after injection, but not to mature osteoclasts. Intravital imaging of blood and lymphatic vessels in the inflamed synovium further showed that extravasated CTLA-4 Ig was immediately drained through lymphatic vessels under acute arthritic conditions, but the drainage activity was retarded under chronic conditions. These results indicate that this intravital synovial imaging system can serve as a platform for exploring the dynamics of immune cells, osteoclasts, and biological agents within the synovial microenvironment in vivo.Hasegawa T., Kikuta J., Sudo T., et al. Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo. Scientific Reports 10, 13480 (2020); https://doi.org/10.1038/s41598-020-70488-y

Migration arrest of chemoresistant leukemia cells mediated by MRTF-SRF pathway

Background: Dormant chemotherapy-resistant leukemia cells can survive for an extended period before relapse. Nevertheless, the mechanisms underlying the development of chemoresistance in vivo remain unclear. Methods: Using intravital bone imaging, we characterized the behavior of murine acute myeloid leukemia (AML) cells (C1498) in the bone marrow before and after chemotherapy with cytarabine. Results: Proliferative C1498 cells exhibited high motility in the bone marrow. Cytarabine treatment impaired the motility of residual C1498 cells. However, C1498 cells regained their migration potential after relapse. RNA sequencing revealed that cytarabine treatment promoted MRTF-SRF pathway activation. MRTF inhibition using CCG-203971 augmented the anti-tumor effects of chemotherapy in our AML mouse model, as well as suppressed the migration of chemoresistant C1498 cells. Conclusions: These results provide novel insight into the role of cell migration arrest on the development of chemoresistance in AML, as well as provide a strong rationale for the modulation of cellular motility as a therapeutic target for refractory AML.Morimatsu M., Yamashita E., Seno S., et al. Migration arrest of chemoresistant leukemia cells mediated by MRTF-SRF pathway. Inflammation and Regeneration 40, 15 (2020); https://doi.org/10.1186/s41232-020-00127-6

JAK inhibition ameliorates bone destruction by simultaneously targeting mature osteoclasts and their precursors

Background: Rheumatoid arthritis (RA) is characterized by chronic inflammation and resultant cartilage/bone destruction because of aberrantly activated osteoclasts. Recently, novel treatments with several Janus kinase (JAK) inhibitors have been shown to successfully ameliorate arthritis-related inflammation and bone erosion, although their mechanisms of action for limiting bone destruction remain unclear. Here, we examined the effects of a JAK inhibitor on mature osteoclasts and their precursors by intravital multiphoton imaging. Methods: Inflammatory bone destruction was induced by local injection of lipopolysaccharides into transgenic mice carrying reporters for mature osteoclasts or their precursors. Mice were treated with the JAK inhibitor, ABT-317, which selectively inhibits the activation of JAK1, and then subjected to intravital imaging with multiphoton microscopy. We also used RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanism underlying the effects of the JAK inhibitor on osteoclasts. Results: The JAK inhibitor, ABT-317, suppressed bone resorption by blocking the function of mature osteoclasts and by targeting the migratory behaviors of osteoclast precursors to the bone surface. Further exhaustive RNA-Seq analysis demonstrated that Ccr1 expression on osteoclast precursors was suppressed in the JAK inhibitor-treated mice; the CCR1 antagonist, J-113863, altered the migratory behaviors of osteoclast precursors, which led to the inhibition of bone destruction under inflammatory conditions. Conclusions: This is the first study to determine the pharmacological actions by which a JAK inhibitor blocks bone destruction under inflammatory conditions; this inhibition is beneficial because of its dual effects on both mature osteoclasts and immature osteoclast precursors.Yari S., Kikuta J., Shigyo H., et al. JAK inhibition ameliorates bone destruction by simultaneously targeting mature osteoclasts and their precursors. Inflammation and Regeneration 43, 18 (2023); https://doi.org/10.1186/s41232-023-00268-4

Anti-Siglec-15 antibody suppresses bone resorption by inhibiting osteoclast multinucleation without attenuating bone formation

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.Tsukazaki H., Kikuta J., Ao T., et al. Anti-Siglec-15 antibody suppresses bone resorption by inhibiting osteoclast multinucleation without attenuating bone formation. Bone 152, 116095 (2021); https://doi.org/10.1016/j.bone.2021.116095