Toyama Prefectural Institute for Pharmaceutical Research

Toyama-shi, Japan

Toyama Prefectural Institute for Pharmaceutical Research

Toyama-shi, Japan
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Yamamoto S.,University of Toyama | Niida S.,Bio Bank Omics Unit | Azuma E.,University of Toyama | Azuma E.,Astellas Pharma Inc. | And 17 more authors.
Scientific Reports | Year: 2015

Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases. © 2015 Macmillan Publishers Limited.


Watanabe Y.,University of Toyama | Nakamura T.,University of Toyama | Ishikawa S.,University of Toyama | Fujisaka S.,University of Toyama | And 19 more authors.
Diabetes | Year: 2012

Recent accumulating evidence suggests that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. Here, we show that a Toll-like receptor (TLR) protein, radioprotective 105 (RP105)/myeloid differentiation protein (MD)-1 complex, contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance. An HFD dramatically increased RP105 mRNA and protein expression in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. RP105 mRNA expression also was significantly increased in the visceral adipose tissue of obese human subjects relative to nonobese subjects. The RP105/MD-1 complex was expressed by most adipose tissue macrophages (ATMs). An HFD increased RP105/MD-1 expression on the M1 subset of ATMs that accumulate in eWAT. Macrophages also acquired this characteristic in coculture with 3T3-L1 adipocytes. RP105 knockout (KO) and MD-1 KO mice had less HFD-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance compared with wild-type (WT) and TLR4 KO mice. Finally, the saturated fatty acids, palmitic and stearic acids, are endogenous ligands for TLR4, but they did not activate RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling and may represent a novel therapeutic target for obesityassociated metabolic disorders. © 2012 by the American Diabetes Association.


Honda H.,Toyama Prefectural Institute for Pharmaceutical Research | Honda H.,University of Toyama | Nagai Y.,University of Toyama | Matsunaga T.,Toyama Prefectural Institute for Pharmaceutical Research | And 8 more authors.
Journal of Leukocyte Biology | Year: 2012

Recent evidences suggest that the extracts of plant products are able to modulate innate immune responses. A saponin GL and a chalcone ILG are representative components of Glycyrrhiza uralensis, which attenuate inflammatory responses mediated by TLRs. Here, we show that GL and ILG suppress different steps of the LPS sensor TLR4/MD-2 complex signaling at the receptor level. Extract of G. uralensis suppressed IL-6 and TNF-α production induced by lipid A moiety of LPS in RAW264.7 cells. Among various G. uralensis-related components of saponins and flavanones/chalcones, GL and ILG could suppress IL-6 production induced by lipid A in dose-dependent manners in RAW264.7 cells. Furthermore, elevation of plasma TNF-α in LPS-injected mice was attenuated by passive administration of GL or ILG. GL and ILG inhibited lipid A-induced NF-κB activation in Ba/F3 cells expressing TLR4/MD-2 and CD14 and BMMs. These components also inhibited activation of MAPKs, including JNK, p38, and ERK in BMMs. In addition, GL and ILG inhibited NF-κB activation and IL-6 production induced by paclitaxel, a nonbacterial TLR4 ligand. Interestingly, GL attenuated the formation of the LPS-TLR4/MD-2 complexes, resulting in inhibition of homodimerization of TLR4. Although ILG did not affect LPS binding to TLR4/MD-2, it could inhibit LPS-induced TLR4 homodimerization. These results imply that GL and ILG modulate the TLR4/MD-2 complex at the receptor level, leading to suppress LPS-induced activation of signaling cascades and cytokine production, but their effects are exerted at different steps of TLR4/MD-2 signaling. © Society for Leukocyte Biology.


Honda H.,Toyama Prefectural Institute for Pharmaceutical Research | Honda H.,University of Toyama | Nagai Y.,University of Toyama | Nagai Y.,Japan Science and Technology Agency | And 11 more authors.
Journal of Leukocyte Biology | Year: 2014

Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-κB activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3-and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1βproduction compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1βand caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases. © Society for Leukocyte Biology.


Fujisaka S.,University of Toyama | Usui I.,University of Toyama | Ikutani M.,University of Toyama | Aminuddin A.,University of Toyama | And 8 more authors.
Diabetologia | Year: 2013

Aims/hypothesis: As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. Methods: The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. Results: Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1β. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1β and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. Conclusions/interpretation: Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs. © 2013 Springer-Verlag Berlin Heidelberg.


Watanabe Y.,University of Toyama | Nagai Y.,University of Toyama | Takatsu K.,University of Toyama | Takatsu K.,Toyama Prefectural Institute for Pharmaceutical Research
Nutrients | Year: 2013

Obesity-associated chronic tissue inflammation is a key contributing factor to type 2 diabetes mellitus, and a number of studies have clearly demonstrated that the immune system and metabolism are highly integrated. Recent advances in deciphering the various immune cells and signaling networks that link the immune and metabolic systems have contributed to our understanding of the pathogenesis of obesity-associated inflammation. Other recent studies have suggested that pattern recognition receptors in the innate immune system recognize various kinds of endogenous and exogenous ligands, and have a crucial role in initiating or promoting obesity-associated chronic inflammation. Importantly, these mediators act on insulin target cells or on insulin-producing cells impairing insulin sensitivity and its secretion. Here, we discuss how various pattern recognition receptors in the immune system underlie the etiology of obesity-associated inflammation and insulin resistance, with a particular focus on the TLR (Toll-like receptor) family protein Radioprotective 105 (RP105)/myeloid differentiation protein-1 (MD-1). © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Takatsu K.,Toyama Prefectural Institute for Pharmaceutical Research
Frontiers in Immunology | Year: 2014

This is a perspective based on the paper "Cloning of complementary DNA encoding T cell replacing factor and identity with B cell growth factor II", by Kinashi T, Harada N, Severinson E, Tanabe T, Sideras P, Konishi M, Azuma C, Tominaga A, Bergstedt-Lindqvist S, Takahashi M, Matsuda F, Yaoita Y, Takatsu K, and Honjo, T. Nature (1986) 32(6092): 70-3. We have been interested in understanding the molecular basis of T-B cell cooperation for antibody formation. Although many investigators had described a number of different soluble factors that appeared to have biological relevance to T-B cell interactions, molecular basis of such active substances remained unknown for a long period of time. In this perspective, I will briefly summarize the history of the initial discovery of T cell-replacing factor/B cell growth factor II that appeared to be involved in B-cell growth and differentiation, and outline the discovery and characterization of interleukin-5. Studies of interleukin-5 have provided strong evidence that a single cytokine exerts a variety of activities on diverse target cells. © 2014 Takatsu.


Sasaki S.,University of Toyama | Nagai Y.,University of Toyama | Yanagibashi T.,University of Toyama | Watanabe Y.,University of Toyama | And 6 more authors.
Molecular Immunology | Year: 2012

MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) and this complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRLlpr/lpr mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor (TLR) 4. We have now established a flow cytometry-based assay to detect the soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRLlpr/lpr mice had higher levels of sMD-1 than control MRL+/+ mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRLlpr/lpr mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRLlpr/lpr kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model. © 2011 Elsevier Ltd.


Itakura A.,Iwaki Meisei University | Itakura A.,Hamamatsu University School of Medicine | Ikutani M.,University of Toyama | Takatsu K.,University of Toyama | And 2 more authors.
International Archives of Allergy and Immunology | Year: 2013

Background: Elicitation of contact hypersensitivity requires antigen-specific immunoglobulin M (IgM) antibodies that trigger recruitment of effector T cells to the local tissue. These antigen-specific IgM antibodies are produced by B-1-like 'initiator B cells'. In this study, we compared susceptibility to hapten-induced contact hypersensitivity between BALB/c and C57BL/6 mice. Methods: BALB/c and C57BL/6 mice were sensitized by painting oxazolone onto the skin and were challenged on the ears with the same hapten on day 4. Ear thickness and serum hapten-specific IgM levels were measured at 24 h post-challenge. Peritoneal cells were harvested and the numbers of B cell subpopulations were counted. Interleukin (IL)-5 was intraperitoneally injected into BALB/c and C57BL/6 mice, and the change in numbers of B cell subpopulations and serum IgM levels was monitored. Results: Oxazolone induced stronger ear swelling and specific IgM responses in BALB/c mice than in C57BL/6 mice. BALB/c mice had higher numbers of peritoneal B-1 cells than C57BL/6 mice at steady state. IL-5 injection increased the number of peritoneal B-1 cells and serum IgM levels more significantly in BALB/mice than in C57BL/6 mice. Conclusions: BALB/c mice exhibit higher susceptibility to hapten-induced contact hypersensitivity than C57BL/6 mice, most likely because they have a higher number of B-1 cells, leading to massive production of hapten-specific IgM antibodies upon contact sensitization. The differences in the number of B-1 cells and IgM responses between the two strains of mice may be attributed to the difference in responsiveness of B-1 cells to IL-5. © 2013 S. Karger AG, Basel.


Yanagibashi T.,University of Toyama | Yanagibashi T.,Toyama Prefectural Institute for Pharmaceutical Research | Nagai Y.,University of Toyama | Nagai Y.,Japan Science and Technology Agency | And 5 more authors.
Immunology Letters | Year: 2015

LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety of immune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon-β (TRIF) pathways, but relatively few studies have examined these pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses in B cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88-/- B cells were markedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF-/- B cells were also impaired in these responses compared with WT B cells, but showed better responses than MyD88-/- B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88-/- B cells showed similar patterns of CSR to WT B cells. However, TRIF-/- B cells showed the impaired in the CSR. Compared with WT and MyD88-/- B cells, TRIF-/- B cells exhibited reduced cell division, fewer IgG1+ cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expression in response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization was impaired in TRIF-/- mice, while MyD88-/- mice exhibited increased IgG1 production. Thus, MyD88 and TRIF pathways differently regulate TLR4-induced immune responses in B cells. © 2014 Elsevier B.V.

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