Publications by authors named "Junichi Kikuta"

93 Publications

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

Bone 2021 Nov 1;152:116095. Epub 2021 Jul 1.

Department of Immunology and Cell Biology, Graduate School of Medicine & Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; WPI-Immunology Frontier Research Center, Osaka University, Osaka, Japan; Laboratory of Bioimaging and Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bone.2021.116095DOI Listing
November 2021

Arthritis-associated osteoclastogenic macrophages (AtoMs) participate in pathological bone erosion in rheumatoid arthritis.

Immunol Med 2021 Jun 30:1-5. Epub 2021 Jun 30.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Japan.

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.
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http://dx.doi.org/10.1080/25785826.2021.1944547DOI Listing
June 2021

SLPI is a critical mediator that controls PTH-induced bone formation.

Nat Commun 2021 04 9;12(1):2136. Epub 2021 Apr 9.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.

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.
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http://dx.doi.org/10.1038/s41467-021-22402-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035405PMC
April 2021

Small extracellular vesicles derived from interferon-γ pre-conditioned mesenchymal stromal cells effectively treat liver fibrosis.

NPJ Regen Med 2021 Mar 30;6(1):19. Epub 2021 Mar 30.

Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.

Mesenchymal stromal cells (MSCs) are used for ameliorating liver fibrosis and aiding liver regeneration after cirrhosis; Here, we analyzed the therapeutic potential of small extracellular vesicles (sEVs) derived from interferon-γ (IFN-γ) pre-conditioned MSCs (γ-sEVs). γ-sEVs effectively induced anti-inflammatory macrophages with high motility and phagocytic abilities in vitro, while not preventing hepatic stellate cell (HSC; the major source of collagen fiber) activation in vitro. The proteome analysis of MSC-derived sEVs revealed anti-inflammatory macrophage inducible proteins (e.g., annexin-A1, lactotransferrin, and aminopeptidase N) upon IFN-γ stimulation. Furthermore, by enabling CXCR1+ macrophage accumulation in the damaged area, γ-sEVs ameliorated inflammation and fibrosis in the cirrhosis mouse model more effectively than sEVs. Single cell RNA-Seq analysis revealed diverse effects, such as induction of anti-inflammatory macrophages and regulatory T cells, in the cirrhotic liver after γ-sEV administration. Overall, IFN-γ pre-conditioning altered sEVs resulted in efficient tissue repair indicating a new therapeutic strategy.
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http://dx.doi.org/10.1038/s41536-021-00132-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010072PMC
March 2021

Group 2 innate lymphoid cells support hematopoietic recovery under stress conditions.

J Exp Med 2021 May;218(5)

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.

The cell-cycle status of hematopoietic stem and progenitor cells (HSPCs) becomes activated following chemotherapy-induced stress, promoting bone marrow (BM) regeneration; however, the underlying molecular mechanism remains elusive. Here we show that BM-resident group 2 innate lymphoid cells (ILC2s) support the recovery of HSPCs from 5-fluorouracil (5-FU)-induced stress by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF). Mechanistically, IL-33 released from chemo-sensitive B cell progenitors activates MyD88-mediated secretion of GM-CSF in ILC2, suggesting the existence of a B cell-ILC2 axis for maintaining hematopoietic homeostasis. GM-CSF knockout mice treated with 5-FU showed severe loss of myeloid lineage cells, causing lethality, which was rescued by transferring BM ILC2s from wild-type mice. Further, the adoptive transfer of ILC2s to 5-FU-treated mice accelerates hematopoietic recovery, while the reduction of ILC2s results in the opposite effect. Thus, ILC2s may function by "sensing" the damaged BM spaces and subsequently support hematopoietic recovery under stress conditions.
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http://dx.doi.org/10.1084/jem.20200817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941180PMC
May 2021

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

Cell Rep 2020 12;33(12):108542

Research Division, Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., 200, Kajiwara, Kamakura, Kanagawa, 247-8530, Japan.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.
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http://dx.doi.org/10.1016/j.celrep.2020.108542DOI Listing
December 2020

Intravital imaging of orthotopic and ectopic bone.

Inflamm Regen 2020 Nov 2;40(1):26. Epub 2020 Nov 2.

Department of Immunology and Cell Biology, Graduate School of Medicine & Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1186/s41232-020-00135-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604953PMC
November 2020

Expression of leukotriene B receptor 1 defines functionally distinct DCs that control allergic skin inflammation.

Cell Mol Immunol 2021 Jun 9;18(6):1437-1449. Epub 2020 Oct 9.

Department of Biochemistry, Juntendo University Graduate School of Medicine, Tokyo, 113-8421, Japan.

Leukotriene B (LTB) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1 and BLT1 DCs. We also found that BLT1 and BLT1 DCs differentially migrated toward LTB and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB-producing enzyme LTAH knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1 DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1 DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1 DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.
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http://dx.doi.org/10.1038/s41423-020-00559-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167169PMC
June 2021

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

Sci Rep 2020 08 10;10(1):13480. Epub 2020 Aug 10.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan.

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 CXCR1 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.
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http://dx.doi.org/10.1038/s41598-020-70488-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417741PMC
August 2020

An Acid-Activatable Fluorescence Probe for Imaging Osteocytic Bone Resorption Activity in Deep Bone Cavities.

Angew Chem Int Ed Engl 2020 11 8;59(47):20996-21000. Epub 2020 Sep 8.

Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.

A rationally designed pH-activatable fluorescent probe (pHocas-RIS) has been used to measure localised pH levels in osteocytic lacunae in bone tissue. Conjugation of the moderate bone-binding drug risedronate to a pH-activatable BODIPY fluorophore enables the probe to penetrate osteocytic lacunae cavities that are embedded deep within the bone matrix. After injection of pHocas-RIS, any osteocytic lacunae caused by bone-resorbing osteocytes cause the probe to fluoresce in vivo, thus allowing imaging by intravital two-photon excitation microscopy. This pH responsive probe enabled the visualization of the bone mineralizing activities of acid producing osteocytes in real time, thus allowing the study of their central role in remodeling the bone-matrix in healthy and disease states.
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http://dx.doi.org/10.1002/anie.202006388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692916PMC
November 2020

Nonlinear Optics with Near-Infrared Excitation Enable Real-Time Quantitative Diagnosis of Human Cervical Cancers.

Cancer Res 2020 09 23;80(17):3745-3754. Epub 2020 Jul 23.

Department of Immunology and Cell Biology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Histopathologic analysis through biopsy has been one of the most useful methods for the assessment of malignant neoplasms. However, some aspects of the analysis such as invasiveness, evaluation range, and turnaround time from biopsy to report could be improved. Here, we report a novel method for visualizing human cervical tissue three-dimensionally, without biopsy, fixation, or staining, and with sufficient quality for histologic diagnosis. Near-infrared excitation and nonlinear optics were employed to visualize unstained human epithelial tissues of the cervix uteri by constructing images with third-harmonic generation (THG) and second-harmonic generation (SHG). THG images enabled evaluation of nuclear morphology in a quantitative manner with six parameters after image analysis using deep learning. It was also possible to quantitatively assess intraepithelial fibrotic changes based on SHG images and another deep learning analysis. Using each analytical procedure alone, normal and cancerous tissue were classified quantitatively with an AUC ≥0.92. Moreover, a combinatory analysis of THG and SHG images with a machine learning algorithm allowed accurate classification of three-dimensional image files of normal tissue, intraepithelial neoplasia, and invasive carcinoma with a weighted kappa coefficient of 0.86. Our method enables real-time noninvasive diagnosis of cervical lesions, thus constituting a potential tool to dramatically change early detection. SIGNIFICANCE: This study proposes a novel method for diagnosing cancer using nonlinear optics, which enables visualization of histologic features of living tissues without the need for any biopsy or staining dye.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0348DOI Listing
September 2020

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

Inflamm Regen 2020 6;40:15. Epub 2020 Jul 6.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.

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.
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http://dx.doi.org/10.1186/s41232-020-00127-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336645PMC
July 2020

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

Int Immunol 2020 10;32(11):727-736

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Yamada-oka, Suita, Osaka, Japan.

The sympathetic nervous system plays critical roles in the differentiation, maturation and recruitment of immune cells under homeostatic conditions, and in responses to environmental stimuli, although its role in the migratory control of immune cells during acute inflammation remains unclear. In this study, using an advanced intravital bone imaging system established in our laboratory, we demonstrated that the sympathetic nervous system locally regulates neutrophil egress from the bone marrow for mobilization to inflammatory foci. We found that sympathetic neurons were located close to blood vessels in the bone marrow cavity; moreover, upon lipopolysaccharide (LPS) administration, local sympathectomy delayed neutrophil egress from the bone marrow and increased the proportion of neutrophils that remained in place. We also showed that vascular endothelial cells produced C-X-C motif chemokine ligand 1 (CXCL1), which is responsible for neutrophil egress out of the bone marrow. Its expression was up-regulated during acute inflammation, and was suppressed by β-adrenergic receptor blockade, which was accompanied with inhibition of neutrophil egress into the systemic circulation. Furthermore, systemic β-adrenergic signaling blockade decreased the recruitment of neutrophils in the lung under conditions of acute systemic inflammation. Taken together, the results of this study first suggested a new regulatory system, wherein local sympathetic nervous activation promoted neutrophil egress by enhancing Cxcl1 expression in bone marrow endothelial cells in a β-adrenergic signaling-dependent manner, contributing to the recruitment of neutrophils at the onset of inflammation in vivo.
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http://dx.doi.org/10.1093/intimm/dxaa025DOI Listing
October 2020

In vivo dynamic analysis of BMP-2-induced ectopic bone formation.

Sci Rep 2020 03 16;10(1):4751. Epub 2020 Mar 16.

Department of Immunology and Cell Biology, Graduate School of Medicine & Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1038/s41598-020-61825-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076033PMC
March 2020

Thrombomodulin induces anti-inflammatory effects by inhibiting the rolling adhesion of leukocytes in vivo.

J Pharmacol Sci 2020 May 14;143(1):17-22. Epub 2020 Jan 14.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka 565-0871, Japan. Electronic address:

Thrombomodulin (TM) is an integral membrane protein expressed on the surface of vascular endothelial cells that suppresses blood coagulation. Recent studies have shown that TM exhibits anti-inflammatory effects by inhibiting leukocyte recruitment. However, the actual modes of action of TM in vivo remain unclear. Here, we describe the pharmacological effects of recombinant human soluble TM (TM alfa) on leukocyte dynamics in living mice using intravital imaging techniques. Under control conditions, neutrophils exhibited three distinct types of adhesion behavior in vessels: 1) "non-adhesion", in which cells flowed without vessel adhesion; 2) "rolling adhesion", in which cells transiently interacted with the endothelium; and 3) "tight binding", in which cells bound strongly to the endothelial cells. Compared to control conditions, local lipopolysaccharide stimulation resulted in an increased frequency of rolling adhesion that was not homogeneously distributed on vessel walls but occurred at specific endothelial sites. Under inflammatory conditions, TM alfa, particularly the D1 domain which is a lectin-like region of TM, significantly decreased the frequency of rolling adhesion, but did not influence the number of tight bindings. This was the first study to demonstrate that TM alfa exerts anti-inflammatory effects by inhibiting rolling adhesion of neutrophils to vascular endothelial cells in living mice.
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http://dx.doi.org/10.1016/j.jphs.2020.01.001DOI Listing
May 2020

Identification of a novel arthritis-associated osteoclast precursor macrophage regulated by FoxM1.

Nat Immunol 2019 12 18;20(12):1631-1643. Epub 2019 Nov 18.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.

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 CXCR1Ly6CF4/80I-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 CXCR1HLA-DRCD11cCD80CD86 cells that corresponded to mouse AtoMs, and human osteoclastogenesis was inhibited by the FoxM1 inhibitor thiostrepton, constituting a potential target for rheumatoid arthritis treatment.
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http://dx.doi.org/10.1038/s41590-019-0526-7DOI Listing
December 2019

Intravital multiphoton microscopy as a novel tool in the field of immunopharmacology.

Pharmacol Ther 2020 02 2;206:107429. Epub 2019 Nov 2.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan; WPI-Immunology Frontier Research Center, Osaka University, Osaka, Japan. Electronic address:

Intravital microscopy with multiphoton excitation is a recently developed optical imaging technique for deep tissue imaging without fixation or sectioning, which permits examination of fundamental concepts regarding the dynamic nature of cells under physiological and pathological conditions in living animals. This novel technique also offers exciting opportunities for pharmacological research by providing new platforms for the study of cellular dynamics in response to drugs in vivo. Moreover, fluorescent chemical probes for functional or molecular analysis in single cells in vivo play important roles in pharmacology. For example, we have recently revealed the pharmacodynamic actions of different biological agents for the treatment of rheumatoid arthritis (RA) in vivo by directly visualizing drug-induced cellular behaviors and functions of osteoclasts on bone surfaces. This review focuses on the principles and advantages of intravital imaging for the dissection of pharmacological mechanisms, and discusses how such imaging can contribute to the drug development process, introducing recent trials that evaluated the in vivo pharmacological effects of various agents.
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http://dx.doi.org/10.1016/j.pharmthera.2019.107429DOI Listing
February 2020

Mesenchymal stem cells cultured under hypoxic conditions had a greater therapeutic effect on mice with liver cirrhosis compared to those cultured under normal oxygen conditions.

Regen Ther 2019 Dec 20;11:269-281. Epub 2019 Sep 20.

Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.

Background: Mesenchymal stem cells (MSCs) can be easily expanded. They can be acquired from medical waste such as adipose and umbilical cord tissues, are influenced by culturing conditions, and exert anti-inflammatory, antioxidant, anti-fibrotic, and angiogenic effects. We analyzed the multi-directional effects of MSCs cultured under hypoxic conditions and their underlying mechanisms in the treatment of liver cirrhosis in a mouse model.

Methods: Human bone marrow-derived MSCs cultured under hypoxic (5% O; hypoMSCs) and normoxic (21% O; norMSCs) conditions were compared by cap analysis of gene expression (CAGE) with or without serum from liver cirrhosis patients. The therapeutic effects of MSCs, including serum liver enzyme induction, fibrosis regression, and hepatic oxidative stress, were evaluated by injecting 1 × 10, 2 × 10, or 4 × 10 MSCs/mouse into the tail veins of mice with carbon tetrachloride (CCl)-induced liver cirrhosis. Intravital imaging was performed with a two-photon excitation microscope to confirm the various MSC migration paths to the liver.

Results: CAGE analysis revealed that the RNA expression levels of prostaglandin E synthase () and miR210 were significantly higher in hypoMSCs than in norMSCs. analysis revealed that both hypoMSCs and norMSCs reduced serum alanine aminotransferase, oxidative stress, and fibrosis compared to that in control mice in a dose-dependent manner. However, hypoMSCs had stronger therapeutic effects than norMSCs. We confirmed this observation by an study in which hypoMSCs changed macrophage polarity to an anti-inflammatory phenotype via prostaglandin E2 (PGE2) stimulation. In addition, miR210 reduced the rate of hepatocyte apoptosis. Intravital imaging after MSC administration showed that both cell types were primarily trapped in the lungs. Relatively a few hypoMSCs and norMSCs migrated to the liver. There were no significant differences in their distributions.

Conclusion: The therapeutic effect of hypoMSCs was mediated by PGE2 and miR210 production and was greater than that of norMSCs. Therefore, MSCs can be manipulated to improve their therapeutic efficacy in the treatment of liver cirrhosis and could potentially serve in effective cell therapy. MSCs produce several factors with multidirectional effects and function as "conducting cells" in liver cirrhosis.
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http://dx.doi.org/10.1016/j.reth.2019.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813562PMC
December 2019

Multicolor Imaging with Fluorescent Probes Revealed the Dynamics and Function of Osteoclast Proton Pumps.

ACS Cent Sci 2019 Jun 1;5(6):1059-1066. Epub 2019 May 1.

Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

two-photon fluorescence imaging is a powerful modality to monitor cell dynamics in biomedical studies. To detect protein functions in living animals in real-time, fluorescent probes must show a quick response to the target function in specific tissues. Here, we developed a rhodamine-based small-molecule fluorescent probe called Red-pHocas (red pH-activatable fluorescent probe for osteoclast activity sensing) to reversibly detect the acidic environments for the spatiotemporal analysis of the function of osteoclast proton pumps. The introduction of electron-withdrawing -alkyl substituents in the rhodamine spirolactam fluorophore remarkably increased the kinetics of the fluorescence response to acidic pHs, which allowed the rapid and reversible monitoring of acidic compartments and the analysis of the dynamics of osteoclast proton pumps during osteoclastic bone resorption. multicolor two-photon imaging using Red-pHocas in fluorescent reporter mice revealed that bone acidification occurred synchronously with the accumulation of proton pumps onto the bone surfaces. To our knowledge, this is the first study to demonstrate the direct involvement of osteoclast proton pumps in bone acidification under intravital conditions by means of an imaging probe.
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http://dx.doi.org/10.1021/acscentsci.9b00220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598158PMC
June 2019

Imaging the Bone-Immune Cell Interaction in Bone Destruction.

Front Immunol 2019 26;10:596. Epub 2019 Mar 26.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.

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 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 , 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 and would also be useful for evaluating novel therapies in animal models of bone-destroying diseases.
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http://dx.doi.org/10.3389/fimmu.2019.00596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443987PMC
July 2020

GPR31-dependent dendrite protrusion of intestinal CX3CR1 cells by bacterial metabolites.

Nature 2019 02 23;566(7742):110-114. Epub 2019 Jan 23.

Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Suita, Japan.

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1 cells) regulate immune responses. CX3CR1 cells take up luminal antigens by protruding their dendrites into the lumen. However, it remains unclear how dendrite protrusion by CX3CR1 cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1 cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1 cells, showed defective dendrite protrusions of CX3CR1 cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1 cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1 cells of wild-type mice, but not of Gpr31b mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1 cells in the small intestine of wild-type mice, but not in that of Gpr31b mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1 cells.
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http://dx.doi.org/10.1038/s41586-019-0884-1DOI Listing
February 2019

Dynamic Analyses of the Short-Term Effects of Different Bisphosphonates Using Intravital Two-Photon Microscopy.

JBMR Plus 2018 Nov 22;2(6):362-366. Epub 2018 Jun 22.

Department of Immunology and Cell Biology Graduate School of Medicine and Frontier Biosciences Osaka University 2-2 Yamada-oka Suita Osaka 565-0871 Japan.

Bisphosphonates are commonly used for the treatment of bone disorders such as osteoporosis; however, the mechanism by which they affect the dynamics of living mature osteoclasts in vivo remains unknown. Here, we describe the short-term effects of different bisphosphonates on controlling the bone resorptive activity of mature osteoclasts in living bone tissues of mice using intravital two-photon microscopy with a pH-sensing chemical fluorescent probe. Three types of nitrogen-containing bisphosphonates, risedronate, alendronate, and minodronate, inhibited osteoclastic acidification during osteoporotic conditions just 12 hours after i.v. injection. Among the three types of drugs, risedronate was the most effective at increasing osteoclast motility and changing the localization of proton pumps, which led to an inhibition of bone resorption. Together, these results demonstrate that the intravital imaging system is a useful tool for evaluating the similarities and differences in currently used antibone resorptive drugs. © 2018 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237210PMC
November 2018

Mesenchymal Stem Cells and Induced Bone Marrow-Derived Macrophages Synergistically Improve Liver Fibrosis in Mice.

Stem Cells Transl Med 2019 03 5;8(3):271-284. Epub 2018 Nov 5.

Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.

We describe a novel therapeutic approach for cirrhosis using mesenchymal stem cells (MSCs) and colony-stimulating factor-1-induced bone marrow-derived macrophages (id-BMMs) and analyze the mechanisms underlying fibrosis improvement and regeneration. Mouse MSCs and id-BMMs were cultured from mouse bone marrow and their interactions analyzed in vitro. MSCs, id-BMMs, and a combination therapy using MSCs and id-BMMs were administered to mice with CCl -induced cirrhosis. Fibrosis regression, liver regeneration, and liver-migrating host cells were evaluated. Administered cell behavior was also tracked by intravital imaging. In coculture, MSCs induced switching of id-BMMs toward the M2 phenotype with high phagocytic activity. In vivo, the combination therapy reduced liver fibrosis (associated with increased matrix metalloproteinases expression), increased hepatocyte proliferation (associated with increased hepatocyte growth factor, vascular endothelial growth factor, and oncostatin M in the liver), and reduced blood levels of liver enzymes, more effectively than MSCs or id-BMMs monotherapy. Intravital imaging showed that after combination cell administration, a large number of id-BMMs, which phagocytosed hepatocyte debris and were retained in the liver for more than 7 days, along with a few MSCs, the majority of which were trapped in the lung, migrated to the fibrotic area in the liver. Host macrophages and neutrophils infiltrated after combination therapy and contributed to liver fibrosis regression and promoted regeneration along with administered cells. Indirect effector MSCs and direct effector id-BMMs synergistically improved cirrhosis along with host cells in mice. These studies pave the way for new treatments for cirrhosis. Stem Cells Translational Medicine 2019;8:271&284.
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http://dx.doi.org/10.1002/sctm.18-0105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392382PMC
March 2019

Intravital imaging with two-photon microscopy reveals cellular dynamics in the ischeamia-reperfused rat heart.

Sci Rep 2018 10 30;8(1):15991. Epub 2018 Oct 30.

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

Recent advances in intravital microscopy have provided insight into dynamic biological events at the cellular level in both healthy and pathological tissue. However, real-time in vivo cellular imaging of the beating heart has not been fully established, mainly due to the difficulty of obtaining clear images through cycles of cardiac and respiratory motion. Here we report the successful recording of clear in vivo moving images of the beating rat heart by two-photon microscopy facilitated by cardiothoracic surgery and a novel cardiac stabiliser. Subcellular dynamics of the major cardiac components including the myocardium and its subcellular structures (i.e., nuclei and myofibrils) and mitochondrial distribution in cardiac myocytes were visualised for 4-5 h in green fluorescent protein-expressing transgenic Lewis rats at 15 frames/s. We also observed ischaemia/reperfusion (I/R) injury-induced suppression of the contraction/relaxation cycle and the consequent increase in cell permeability and leukocyte accumulation in cardiac tissue. I/R injury was induced in other transgenic mouse lines to further clarify the biological events in cardiac tissue. This imaging system can serve as an alternative modality for real time monitoring in animal models and cardiological drug screening, and can contribute to the development of more effective treatments for cardiac diseases.
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http://dx.doi.org/10.1038/s41598-018-34295-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207786PMC
October 2018

Histone methylation regulator PTIP is required to maintain normal and leukemic bone marrow niches.

Proc Natl Acad Sci U S A 2018 10 8;115(43):E10137-E10146. Epub 2018 Oct 8.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX 77030;

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 (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.
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http://dx.doi.org/10.1073/pnas.1806019115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205459PMC
October 2018

Chiral cell sliding drives left-right asymmetric organ twisting.

Elife 2018 06 12;7. Epub 2018 Jun 12.

Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe, Japan.

Polarized epithelial morphogenesis is an essential process in animal development. While this process is mostly attributed to directional cell intercalation, it can also be induced by other mechanisms. Using live-imaging analysis and a three-dimensional vertex model, we identified 'cell sliding,' a novel mechanism driving epithelial morphogenesis, in which cells directionally change their position relative to their subjacent (posterior) neighbors by sliding in one direction. In embryonic hindgut, an initial left-right (LR) asymmetry of the cell shape (cell chirality in three dimensions), which occurs intrinsically before tissue deformation, is converted through LR asymmetric cell sliding into a directional axial twisting of the epithelial tube. In a inversion mutant showing inverted cell chirality and hindgut rotation, cell sliding occurs in the opposite direction to that in wild-type. Unlike directional cell intercalation, cell sliding does not require junctional remodeling. Cell sliding may also be involved in other cases of LR-polarized epithelial morphogenesis.
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http://dx.doi.org/10.7554/eLife.32506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997448PMC
June 2018

visualisation of different modes of action of biological DMARDs inhibiting osteoclastic bone resorption.

Ann Rheum Dis 2018 08 28;77(8):1219-1225. Epub 2018 Apr 28.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.

Objectives: Osteoclasts play critical roles in inflammatory bone destruction. Precursor cell migration, cell differentiation, and functional cell activation are all in play. Biological disease-modifying antirheumatic drugs (DMARDs) have been shown to significantly inhibit both bone erosion as well as synovitis, although how such agents reduce osteoclastic bone destruction has not been fully explained. Here, we used an intravital time-lapse imaging technique to directly visualise mature osteoclasts and their precursors, and explored how different biological DMARDs acted .

Methods: Lipopolysaccharide (LPS) was injected into the calvarial periosteum of fluorescent reporter mice to induce inflammatory bone destruction. Time-lapse imaging was performed via intravital multiphoton microscopy 5 days after LPS injection. Biological DMARDs, including monoclonal antibodies (mAbs) against the interleukin (IL) 6 receptor (IL-6R) and tumour necrosis factor α (TNFα), or cytotoxic T-lymphocyte-associated protein 4 (CTLA4)-Ig, were intraperitoneally administered at the time of LPS injection. We determined CD80/86 expression levels in mature osteoclasts and their precursors by flow cytometry, quantitative PCR and immunohistochemistry.

Results: Of the biologicals tested, anti-IL-6R and anti-TNFα mAbs affected mature osteoclasts and switched bone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had no action on mature osteoclasts but mobilised osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4-Ig) were prominently expressed only in osteoclast precursor cells, being suppressed during osteoclast maturation.

Conclusions: Intravital imaging revealed that various biological DMARDs acted at specific therapeutic time points during osteoclastic bone destruction, with different efficacies. These results enable us to grasp the real modes of action of drugs, optimising the usage of drug regimens.
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http://dx.doi.org/10.1136/annrheumdis-2017-212880DOI Listing
August 2018

[Homeostasis and Disorder of Musculoskeletal System.Cellular dynamics in musculoskeletal system visualized by intravital imaging techniques.]

Clin Calcium 2018;28(3):367-371

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Japan.

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. We originally established an advanced imaging system to visualize living bone tissues using intravital two-photon microscopy. By means of this system, we revealed the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in bone tissues. This approach facilitates investigation of cellular dynamics in the pathogenesis of musculoskeletal disorders, and would thus be useful for evaluating the efficacy of novel therapeutic agents.
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http://dx.doi.org/CliCa1803367371DOI Listing
February 2019

In vivo imaging of T cell lymphoma infiltration process at the colon.

Sci Rep 2018 03 5;8(1):3978. Epub 2018 Mar 5.

Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902, Japan.

The infiltration and proliferation of cancer cells in the secondary organs are of great interest, since they contribute to cancer metastasis. However, cancer cell dynamics in the secondary organs have not been elucidated at single-cell resolution. In the present study, we established an in vivo model using two-photon microscopy to observe how infiltrating cancer cells form assemblages from single T-cell lymphomas, EL4 cells, in the secondary organs. Using this model, after inoculation of EL4 cells in mice, we discovered that single EL4 cells infiltrated into the colon. In the early stage, sporadic elongated EL4 cells became lodged in small blood vessels. Real-time imaging revealed that, whereas more than 70% of EL4 cells did not move during a 1-hour observation, other EL4 cells irregularly moved even in small vessels and dynamically changed shape upon interacting with other cells. In the late stages, EL4 cells formed small nodules composed of several EL4 cells in blood vessels as well as crypts, suggesting the existence of diverse mechanisms of nodule formation. The present in vivo imaging system is instrumental to dissect cancer cell dynamics during metastasis in other organs at the single-cell level.
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http://dx.doi.org/10.1038/s41598-018-22399-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838227PMC
March 2018

Intravital Imaging of the Heart at the Cellular Level Using Two-Photon Microscopy.

Methods Mol Biol 2018 ;1763:145-151

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.

Recent molecular approaches have provided deeper insight on heart failure. However, real-time in vivo cellular dynamics have not been satisfactorily visualized. Here, we present a detailed protocol for in vivo cellular imaging for visualization of the rat heart using two-photon microscopy.
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http://dx.doi.org/10.1007/978-1-4939-7762-8_14DOI Listing
February 2019
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