Publications by authors named "Takahiro Adachi"

54 Publications

Recognition of acrolein-specific epitopes by B cell receptors triggers an innate immune response.

J Biol Chem 2021 Apr 8:100648. Epub 2021 Apr 8.

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657, Japan; Japan Agency for Medical Research and Development, CREST, Tokyo, Japan. Electronic address:

Natural antibodies, predominantly immunoglobulin M (IgM), play an important role in the defense against pathogens and in maintaining homeostasis against oxidized molecules known as oxidation-specific epitopes, such as those contained in oxidized low-density lipoproteins. However, due to the complexity of the oxidized products, very few individual epitopes have been characterized in detail. In the present study, to identify endogenous sources of oxidation-specific epitopes, we stimulated mouse spleen and peritoneal cavity (PerC) cells in vitro with bovine serum albumin (BSA) modified with a variety of lipid peroxidation-related carbonyl compounds, and identified the acrolein-modified BSA (acrBSA) as the most efficient trigger studied for the production of IgM in PerC cells. The acrolein-specific epitopes accelerated the differentiation of B-1a cells, a fetal-derived B cell lineage, to plasma cells. In addition, acrBSA was specifically bound to B-1a cells, suggesting the presence of an acrolein-specific IgM-B cell receptor (BCR). A hybridoma, RE-G25, producing an acrolein-specific IgM, was established from the PerC cells and was indeed identified as a population of B cells expressing a specific IgM-BCR. In addition, we analyzed the BCR repertoire of acrolein-specific B cells and identified the most frequent IgM heavy chain gene segments of the B cells. These data established the presence of innate B cells expressing the acrolein-specific BCR, and suggested that in addition to our understanding of acrolein as a toxic aldehyde, acrolein may play a role as a trigger of the innate immune response.
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http://dx.doi.org/10.1016/j.jbc.2021.100648DOI Listing
April 2021

Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method.

Biochem Biophys Res Commun 2021 Mar 30;545:14-19. Epub 2021 Jan 30.

Innate Immunity Laboratory, Department of Cell Biological Science, Faculty of Advanced Life Science, Hokkaido University, Kita-21, Nishi-11, Kita-ku, Sapporo, Hokkaido, 001-0021, Japan; Innate Immunity Laboratory, Graduate School of Life Science, Hokkaido University, Kita-21, Nishi-11, Kita-ku, Sapporo, Hokkaido, 001-0021, Japan. Electronic address:

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.
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http://dx.doi.org/10.1016/j.bbrc.2021.01.050DOI Listing
March 2021

CEACAM1 specifically suppresses B cell receptor signaling-mediated activation.

Biochem Biophys Res Commun 2021 Jan 21;535:99-105. Epub 2020 Dec 21.

Department of Advanced Therapeutics for GI Diseases, Graduate School of Medical Science, TMDU, Tokyo, Japan. Electronic address:

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressed in T cells may regulate immune responses in the gut. In addition to T cells, B cells are also an important population in the gut-associated lymphoid tissues that orchestrate mucosal homeostasis. However, the role of CEACAM1 in B cells has not been elucidated. We herein analyzed mature B cells to determine the functions of CEACAM1. Flow cytometry revealed high expression of CEACAM1 on B cells in secondary lymphoid tissues. Cytokine production induced by activation of B cell receptor (BCR) signaling was suppressed by CEACAM1 signaling in contrast to that associated with either Toll-like receptor 4 or CD40 signaling. Confocal microscopy revealed co-localization of CEACAM1 and BCR when activated with anti-Igμ F(ab') fragment. Overexpression of CEACAM1 in a murine B cell line, A20, resulted in reduced expressions of activation surface markers with decreased Ca influx after BCR signal activation. Overexpression of CEACAM1 suppressed BCR signal cascade in A20 cells in association with decreased spontaneous proliferation. Our results suggest that CEACAM1 can regulate BCR-mediated mature B cell activation in lymphoid tissues. Therefore, further studies of this molecule may lead to greater insights into the mechanisms of immune responses within peripheral tissues and the potential treatment of inflammatory diseases.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.126DOI Listing
January 2021

Visualization of mechanical stress-mediated Ca signaling in the gut using intravital imaging.

Biosci Microbiota Food Health 2020 11;39(4):209-218. Epub 2020 Jun 11.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

Mechanosensory systems have been implicated in the maintenance of gut homeostasis, but details on the related mechanisms are scarce. Recently, we generated a conditional Ca biosensor yellow cameleon 3.60 (YC3.60)-expressing transgenic mouse model and established a five-dimensional (5D; x, y, z, time, and Ca) intravital imaging system for investigating lymphoid tissues and enteric epithelial cell responses. To validate this gut-sensing system, we visualized responses of enteric nervous system (ENS) cells in Nestin-Cre/YC3.60 mice with specific YC3.60 expression. The ENS, including the myenteric (Auerbach's) and submucous (Meissner's) plexuses, could be visualized without staining in this mouse line, indicating that the probe produced sufficient fluorescent intensity. Furthermore, the myenteric plexus exhibited Ca signaling during peristalsis without stimulation. Nerve endings on the surface of enteric epithelia also exhibited Ca signaling without stimulation. Mechanical stress induced transient salient Ca flux in the myenteric plexus and in enteric epithelial cells in the Nestin-Cre/YC3.60 and the CAG-Cre/YC3.60 lines, respectively. Furthermore, the potential TRPM7 inhibitors were shown to attenuate mechanical stress-mediated Ca signaling. These data indicate that the present intravital imaging system can be used to visualize mechanosensory Ca signaling in ENS cells and enteric epithelial cells.
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http://dx.doi.org/10.12938/bmfh.2019-054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573108PMC
June 2020

and β-Glucan Paramylon Induce Ca Signaling in Intestinal Tract Epithelial, Immune, and Neural Cells.

Nutrients 2020 Jul 30;12(8). Epub 2020 Jul 30.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.

The intestinal tract contains over half of all immune cells and peripheral nerves and manages the beneficial interactions between food compounds and the host. Paramylon is a β-1,3-glucan storage polysaccharide from () that exerts immunostimulatory activities by affecting cytokine production. This study investigated the signaling mechanisms that regulate the beneficial interactions between food compounds and the intestinal tract using cell type-specific calcium (Ca) imaging in vivo and in vitro. We successfully visualized - and paramylon-mediated Ca signaling in vivo in intestinal epithelial cells from mice ubiquitously expressing the Yellow Cameleon 3.60 (YC3.60) Ca biosensor. Moreover, in vivo Ca imaging demonstrated that the intraperitoneal injection of both and paramylon stimulated dendritic cells (DCs) in Peyer's patches, indicating that paramylon is an active component of that affects the immune system. In addition, in vitro Ca imaging in dorsal root ganglia indicated that , but not paramylon, triggers Ca signaling in the sensory nervous system innervating the intestine. Thus, this study is the first to successfully visualize the direct effect of β-1,3-glucan on DCs in vivo and will help elucidate the mechanisms via which and paramylon exert various effects in the intestinal tract.
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http://dx.doi.org/10.3390/nu12082293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468862PMC
July 2020

Isolation of food-derived bacteria inducing interleukin-22 in B cells.

Biosci Microbiota Food Health 2020 21;39(1):1-9. Epub 2019 Sep 21.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

Recently, we found a novel function of the lactic acid bacterium derived from miso, a fermented soy paste, that induces interleukin (IL)-22 production in B cells preferentially. IL-22 plays a critical role in barrier functions in the gut and skin. We further screened other bacteria species, namely, , , , , , , and , in addition to and found that some of them possessed robust IL-22-inducible function in B cells . This process resulted in the augmented expression of activation markers CD86 and CD69 on B and T cells, respectively. However, these observations were not correlated with IL-22 production. We isolated sc-09 from miso and determined it to be the best strain to induce robust IL-22 production in B cells. Furthermore, feeding sc-09 to mice augmented the barrier function of the skin regardless of gut microbiota.
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http://dx.doi.org/10.12938/bmfh.19-012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971416PMC
September 2019

Dual real-time in vivo monitoring system of the brain-gut axis.

Biochem Biophys Res Commun 2020 04 26;524(2):340-345. Epub 2020 Jan 26.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan. Electronic address:

The brain-gut axis which is an interaction between recognition and emotion and the gut sensory system for food and microbiota is important for health. However, there is no real-time monitoring system of the brain and the gut simultaneously so far. We attempted to establish a dual real-time monitoring system for the brain-gut axis by a combination of intravital Ca imaging of the gut and electroencephalogram. Using a conditional Yellow Cameleon 3.60 expression mouse line, we performed intravital imaging of the gut, electrophysiological recordings of the vagus nerve, and electroencephalogram recordings of the various cortical regions simultaneously upon capsaicin stimuli as a positive control. Upon capsaicin administration into the small intestinal lumen, a simultaneous response of Ca signal in the enteric nervous system and cortical local field potentials (LFPs) was successfully observed. Both of them responded immediately upon capsaicin stimuli. Capsaicin triggered a significant increase in the frequency of vagus nerve spikes and a significant decrease in the slow-wave power of cortical LFPs. Furthermore, capsaicin induced delayed and sustained Ca signal in intestinal epithelial cells and then suppressed intestinal motility. The dual real-time monitoring system of the brain and the gut enables to dissect the interaction between the brain and the gut over time with precision.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.090DOI Listing
April 2020

Commensal-bacteria-derived butyrate promotes the T-cell-independent IgA response in the colon.

Int Immunol 2020 04;32(4):243-258

Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Science, Keio University, Tokyo, Japan.

Secretory immunoglobulin A (SIgA), the most abundant antibody isotype in the body, maintains a mutual relationship with commensal bacteria and acts as a primary barrier at the mucosal surface. Colonization by commensal bacteria induces an IgA response, at least partly through a T-cell-independent process. However, the mechanism underlying the commensal-bacteria-induced T-cell-independent IgA response has yet to be fully clarified. Here, we show that commensal-bacteria-derived butyrate promotes T-cell-independent IgA class switching recombination (CSR) in the mouse colon. Notably, the butyrate concentration in human stools correlated positively with the amount of IgA. Butyrate up-regulated the production of transforming growth factor β1 and all-trans retinoic acid by CD103+CD11b+ dendritic cells, both of which are critical for T-cell-independent IgA CSR. This effect was mediated by G-protein-coupled receptor 41 (GPR41/FFA3) and GPR109a/HCA2, and the inhibition of histone deacetylase. The butyrate-induced IgA response reinforced the colonic barrier function, preventing systemic bacterial dissemination under inflammatory conditions. These observations demonstrate that commensal-bacteria-derived butyrate contributes to the maintenance of the gut immune homeostasis by facilitating the T-cell-independent IgA response in the colon.
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http://dx.doi.org/10.1093/intimm/dxz078DOI Listing
April 2020

Propolis induces Ca signaling in immune cells.

Biosci Microbiota Food Health 2019 24;38(4):141-149. Epub 2019 Aug 24.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

Propolis possesses several immunological functions. We recently generated a conditional Ca biosensor yellow cameleon (YC3.60) transgenic mouse line and established a five-dimensional (5D) (x, y, z, time, and Ca signaling) system for intravital imaging of lymphoid tissues, including Peyer's patches (PPs). To assess the effects of propolis on immune cells, we analyzed Ca signaling and using CD11c-Cre/YC3.60 transgenic mice, in which CD11c dendritic cells (DCs) specifically express YC3.60. We found that propolis induced Ca signaling in DCs in the PPs. Intravital imaging of PPs also showed that an intraperitoneal injection of propolis augmented Ca signaling in CD11c cells, suggesting that propolis possesses immune-stimulating activity. Furthermore, CD11c cells in PPs in mice administrated propolis indicated an increase in Ca signaling. Our results indicate that propolis induces immunogenicity under physiological conditions.
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http://dx.doi.org/10.12938/bmfh.19-011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856514PMC
August 2019

Corticotropin-releasing hormone is significantly upregulated in the mouse paraventricular nucleus following a single oral dose of cinnamtannin A2 as an (-)-epicatechin tetramer.

J Clin Biochem Nutr 2019 Jul 7;65(1):29-33. Epub 2019 Jun 7.

Department of Bioscience and Engineering, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan.

Cinnamtannin A2, an (-)-epicatechin tetramer, was reported to have potent physiological activity. Cinnamtannin A2 is rarely absorbed from the gastrointestinal tract into the blood and the mechanisms of its beneficial activities are unknown. Cinnamtannin A2 reported to increase sympathetic nervous activity, which was induced by various stressors. In present study, we examined the stress response in the mouse paraventricular nucleus following a single oral dose of cinnamtannin A2 by monitoring mRNA expression of corticotropin-releasing hormone (CRH) and c-fos using hybridization. Corticotropin-releasing hormone mRNA showed a tendency to increase at 15 min and significantly increased at 60 min following a single oral administration of 100 µg/kg cinnamtannin A2. After a single dose of 10 µg/kg cinnamtannin A2, there was significant upregulation of CRH mRNA at 60 min. These results suggested that cinnamtannin A2 was recognized as a stressor in central nervous system and this may lead to its beneficial effects on circulation and metabolism.
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http://dx.doi.org/10.3164/jcbn.19-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667379PMC
July 2019

Pivotal role of STIM2, but not STIM1, in IL-4 production by IL-3-stimulated murine basophils.

Sci Signal 2019 04 9;12(576). Epub 2019 Apr 9.

Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.

Basophils have nonredundant roles in various immune responses that require Ca influx. Here, we examined the role of two Ca sensors, stromal interaction molecule 1 and 2 (STIM1 and STIM2), in basophil activation. We found that loss of STIM1, but not STIM2, impaired basophil IL-4 production after stimulation with immunoglobulin E (IgE)-containing immune complexes. In contrast, when basophils were stimulated with IL-3, loss of STIM2, but not STIM1, reduced basophil IL-4 production. This difference in STIM proteins was associated with distinct time courses of Ca influx and transcription of the gene that were elicited by each stimulus. Similarly, basophil-specific STIM1 expression was required for IgE-driven chronic allergic inflammation in vivo, whereas STIM2 was required for IL-4 production after combined IL-3 and IL-33 treatment in mice. These data indicate that STIM1 and STIM2 have differential roles in the production of IL-4, which are stimulus dependent. Furthermore, these results illustrate the vital role of STIM2 in basophils, which is often considered to be less important than STIM1.
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http://dx.doi.org/10.1126/scisignal.aav2060DOI Listing
April 2019

Isolation of immune-regulatory Tetragenococcus halophilus from miso.

PLoS One 2018 26;13(12):e0208821. Epub 2018 Dec 26.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Tetragenococcus halophilus is a halophilic lactic acid bacterium that exists in the traditional Japanese seasoning miso-a fermented soy paste. Considering the popularity of miso as a component of healthy diet, we attempted to evaluate the immunoregulatory functions of T. halophilus spices isolated from miso. We screened 56 strains that facilitated the upregulation of activation markers such as CD86 and CD69 on B cells and T cells in vitro. Of these, 7 strains (Nos. 1, 3, 13, 15, 19, 30, and 31) were found to preferentially induce the CD86 expression on B cells. Furthermore, DNA microarray analysis revealed that T. halophilus strain No. 1 significantly augmented the gene expressions of CD86, CD70, IL-10, INF-γ, and IL-22 in B cells. We confirmed these results at the protein level by flow cytometry. Mice feeding diet containing 1% T. halophilus No. 1 exhibited significantly greater IgA production in the serum. Furthermore, a diet containing 1% T. halophilus No. 1 augmented ovoalbumin (OVA)-specific IgG titer in mice upon OVA/alum immunization. Thus, we demonstrated that T. halophilus No. 1 is a strong immunomodulatory strain with potential as a probiotic.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208821PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306251PMC
May 2019

Intravital Two-photon Imaging of Ca signaling in Secretory Organs of Yellow Cameleon Transgenic Mice.

Sci Rep 2018 10 26;8(1):15880. Epub 2018 Oct 26.

Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Intracellular calcium ([Ca]i) signaling regulates physiological functions in most cells. In secretory organs, such as the pancreas, salivary gland, and lacrimal gland (LG), [Ca]i elevation in acinar cells triggers fluid secretion, which plays vital roles in the maintenance of functional health across the life-course. It is important to understand the secretory mechanism of secretory organs, but lack of analytic systems available for living animals limits the scope of research to gain deeper insights into the precise mechanism of secretion. We established an intravital imaging system for specific cell types of secretory organs to monitor the [Ca]i changes using mouse line expressing Yellow Cameleon 3.60, a genetically encoded Ca indicator. Elevation of [Ca]i in specific cell types of secretory organs could be monitored after cholinergic stimulation ex vivo and intravitally. We found that a marked attenuation of LG [Ca]i response to cholinergic stimulation was induced under pathological conditions by postganglionic denervation. Intravital Ca imaging in secretory organs will broaden our understanding of the cellular mechanisms in animal models of secretory diseases.
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http://dx.doi.org/10.1038/s41598-018-34347-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203801PMC
October 2018

Comparison of the sympathetic stimulatory abilities of B-type procyanidins based on induction of uncoupling protein-1 in brown adipose tissue (BAT) and increased plasma catecholamine (CA) in mice.

PLoS One 2018 30;13(7):e0201203. Epub 2018 Jul 30.

Department of Bio-science and Engineering, Shibaura Institute of Technology, Munumaku, Saitama, Japan.

Objectives: We previously found that elevated energy expenditure following a single oral dose of flavan 3-ols (FL), a mixture of catechins and B type procyanidins, is caused by sympathetic nerve activation. In the present study, we compared the activity of the FL components (-)-epicatechin (EC; monomer), procyanidin B2 (B2; dimer), procyanidin C1 (C1; trimer), cinnamtannin A2 (A2; tetramer), and more than pentamer fraction (P5).

Methods: Male ICR mice were treated with a single oral dose of FL, EC, B2, C1, A2, or P5. The animals were sacrificed and blood and brown adipose tissue (BAT) sampled. The plasma catecholamine (CA) levels and BAT uncoupling protein (UCP)-1 mRNA expression were determined.

Results: A single dose of 10 mg/kg FL significantly increased plasma CA and UCP-1 mRNA levels. B2, C1, and A2, but not EC and P5 (all at 1 mg/kg), significantly increased plasma adrenaline levels. Plasma noradrenaline was significantly elevated by B2 and A2, but not by EC, C1, or P5. UCP-1 mRNA levels were significantly increased by C1 and P5. In the dose response study of A2, 10-3 mg/kg A2 increased UCP-1 mRNA levels significantly, but not 10-2 and 10-1 mg/kg A2. In addition, combination treatment with 10-1 mg/kg A2 and yohimbine, an α2 adrenalin blocker, remarkably increased UCP-1 mRNA levels.

Conclusion: These results suggest that FL and its components, except EC, increase UCP-1 mRNA and plasma CA with varying efficacy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201203PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066223PMC
January 2019

Histamine Released From Skin-Infiltrating Basophils but Not Mast Cells Is Crucial for Acquired Tick Resistance in Mice.

Front Immunol 2018 3;9:1540. Epub 2018 Jul 3.

Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

Ticks are blood-feeding arthropods that can transmit pathogens to humans and animals, leading to serious infectious diseases such as Lyme disease. After single or multiple tick infestation, some animal species develop resistance to tick feeding, leading to reduced risk of pathogen transmission. In mice infested with larval ticks, both mast cells and basophils reportedly play key roles in the manifestation of acquired tick resistance (ATR), but it remains ill-defined how they contribute to it. Here, we investigated their products responsible for ATR. Treatment of mice with antihistamine abolished the ATR while histamine or histamine H1 receptor agonist reduced tick-feeding even in the first infestation. In accordance with these, mice deficient for histamine production showed little or no ATR, indicating the crucial role for histamine in the expression of ATR. Adoptive transfer of mast cells and basophils derived from histamine-sufficient or deficient mice to recipient mice lacking mast cells and basophils, respectively, revealed that histamine produced by basophils but not mast cells is essential for the manifestation of ATR, in contrast to the case of local and systemic anaphylaxis where mast cell-derived histamine is the major player. During the second but not first tick infestation, basophils accumulated and made a cluster, surrounding a tick mouthpart, in the epidermis whereas mast cells were scattered and localized mainly in the dermis, more distantly from a tick mouthpart. This appears to explain why basophil-derived histamine is much more effective than mast cell-derived one. Histamine-sufficient, but not -deficient mice showed the thickened epidermis at the second tick-feeding site. Taken together, histamine released from skin-infiltrating basophils rather than skin-resident mast cells plays a crucial role in the manifestation of ATR, perhaps through promotion of epidermal hyperplasia that may inhibit tick feeding.
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http://dx.doi.org/10.3389/fimmu.2018.01540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6043789PMC
July 2018

LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

Immunity 2018 07 10;49(1):120-133.e9. Epub 2018 Jul 10.

Deutsches Rheuma-Forschungszentrum, a Leibniz Institute, Charitéplatz 1, 10117 Berlin, Germany; Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France; AP-HP, Hôpital Necker Enfants Malades, Paris, France. Electronic address:

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.
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http://dx.doi.org/10.1016/j.immuni.2018.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057275PMC
July 2018

Single oral administration of flavan 3-ols induces stress responses monitored with stress hormone elevations in the plasma and paraventricular nucleus.

Neurosci Lett 2018 08 11;682:106-111. Epub 2018 Jun 11.

Department of Bioscience and Engineering, Shibaura Institute of Technology, 307 Fukasaku, Munumaku, Saitama, 337-8570, Japan. Electronic address:

We previously confirmed that postprandial alterations in the circulation and metabolism after a single oral dose of flavan 3-ols (mixture of catechin and catechin oligomers) were involved in an increase in sympathetic nervous activity. However, it is well known that, in response to various stresses, activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs together with sympathetic nerve activity, which is associated with activation of the sympathetic-adrenal-medullary (SAM) axis. In this study, we examined whether the HPA axis was activated after a single dose of flavan 3-ols. We administered an oral dose of 10 or 50 mg/kg flavan 3-ols to male ICR mice, removed the brains, and fixed them in paraformaldehyde-phosphate buffer. Other animals that were treated similarly were decapitated, and blood was collected. In the paraventricular nucleus (PVN), c-fos mRNA expression increased significantly at 15 min after administration of either 10 or 50 mg/kg flavan 3-ols. Corticotropin-releasing hormone (CRH) mRNA expression levels significantly increased at 240 min after administration of 10 mg/kg flavan 3-ols, and at 60 min after administration of 50 mg/kg flavan 3-ols. Plasma corticosterone levels were also significantly increased at 240 min after ingestion of 50 mg/kg flavan 3-ols. In this experiment, we confirmed that the ingestion of flavan 3-ols acted as a stressor in mammals with activation both the SAM and HPA axes.
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http://dx.doi.org/10.1016/j.neulet.2018.06.015DOI Listing
August 2018

B cell activation in the cecal patches during the development of an experimental colitis model.

Biochem Biophys Res Commun 2018 02 10;496(2):367-373. Epub 2018 Jan 10.

Department of Gastroenterology, Graduate School of Medical Science, Tokyo Medical and Dental University (TMDU), Tokyo, Japan. Electronic address:

Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca signaling in CP B cells during the early phase of colitis development. These findings suggested that the CP B cells may be involved in the pathogenesis of colitis including inflammatory bowel diseases in humans.
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http://dx.doi.org/10.1016/j.bbrc.2018.01.053DOI Listing
February 2018

Visualization of Probiotic-Mediated Ca Signaling in Intestinal Epithelial Cells .

Front Immunol 2016 16;7:601. Epub 2016 Dec 16.

Biomedical Research Institute, National Institute for Advanced Industrial Science and Technology (AIST) , Tsukuba , Japan.

Probiotics, such as lactic acid bacteria (LAB) and var. , have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (, time, and Ca) intravital imaging systems of lymphoid tissues including those in Peyer's patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed , a probiotic TTCC012 strain, is shown to directly induce Ca signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.
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http://dx.doi.org/10.3389/fimmu.2016.00601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159486PMC
December 2016

CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP.

J Exp Med 2016 11 24;213(12):2691-2706. Epub 2016 Oct 24.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo, Tokyo 113-8510, Japan

Toll-like receptor 7 (TLR7) plays an essential role in development of systemic lupus erythematosus by co-stimulating B cells reactive to the endogenous TLR7 ligand Sm/ribonucleoprotein (RNP), a crucial lupus self-antigen. However, how the TLR7-mediated autoimmune response is regulated is not yet known. In this study, we demonstrate that CD72, an inhibitory B cell co-receptor known to prevent development of lupus, recognizes Sm/RNP at the extracellular C-type lectin-like domain (CTLD) and specifically inhibits B cell response to Sm/RNP. Moreover, the CTLD of CD72, a lupus-susceptible allele, binds to Sm/RNP less strongly than that of lupus-resistant CD72 Reduced binding of CD72 is supported by x-ray crystallographic analysis that reveals a considerable alteration in charge at the putative ligand-binding site. Thus, CD72 appears to specifically inhibit B cell response to the endogenous TLR7 ligand Sm/RNP through CTLD-mediated recognition of Sm/RNP, thereby preventing production of anti-Sm/RNP antibody crucial for development of lupus.
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http://dx.doi.org/10.1084/jem.20160560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110020PMC
November 2016

Intravital imaging of Ca(2+) signals in lymphocytes of Ca(2+) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset.

Sci Rep 2016 Jan 6;6:18738. Epub 2016 Jan 6.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, 113-8510, Japan.

Calcium ion (Ca(2+)) signaling is a typical phenomenon mediated through immune receptors, such as the B-cell antigen receptor (BCR), and it is important for their biological activities. To analyze the signaling of immune receptors together with their in vivo dynamics, we generated stable transgenic mice with the Föster/fluorescence resonance energy transfer (FRET)-based Ca(2+) indicator yellow cameleon 3.60 (YC3.60), based on the Cre/loxP system (YC3.60(flox)). We successfully obtained mice with specific YC3.60 expression in immune or nerve cells as well as mice with ubiquitous expression of this indicator. We established five-dimensional (5D) (x, y, z, time, and Ca(2+)) intravital imaging of lymphoid tissues, including the bone marrow. Furthermore, in autoimmune-prone models, the CD22(-/-) and C57BL/6- lymphoproliferation (lpr)/lpr mouse, Ca(2+) fluxes were augmented, although they did not induce autoimmune disease. Intravital imaging of Ca(2+) signals in lymphocytes may improve assessment of the risk of autoimmune diseases in model animals.
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http://dx.doi.org/10.1038/srep18738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702216PMC
January 2016

Cd72(c) is a modifier gene that regulates Fas(lpr)-induced autoimmune disease.

J Immunol 2013 Jun 24;190(11):5436-45. Epub 2013 Apr 24.

Laboratory of Immunology, Graduate School of Biomedical Sciences, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.

Although modifier genes are extensively studied in various diseases, little is known about modifier genes that regulate autoimmune diseases. Autoimmune disease caused by the Fas(lpr) mutation depends on the genetic background of mouse strains, suggesting a crucial role of modifier genes. MRL/MpJ-Fas(lpr) (MRL/lpr) and AKR/lpr mice develop severe and mild lupus-like autoimmune disease, respectively, whereas this mutation does not cause disease on C57BL/6 (B6) or C3H background. Both MRL and AKR carry the same haplotype of the Cd72 gene encoding an inhibitory BCR coreceptor (CD72(c)), and CD72(c) contains several amino acid substitutions and a deletion in the extracellular region compared with CD72(a) and CD72(b). To address the role of Cd72(c) locus in the regulation of Fas(lpr)-induced autoimmune disease, we generated B6.CD72(c)/lpr and MRL.CD72(b)/lpr congenic mice. Introduction of the chromosomal interval containing Cd72(c) did not cause disease in B6 mice by itself, but caused development of lupus-like disease in the presence of Fas(lpr) on B6 background, clearly demonstrating that this interval contains the modifier gene that regulates Fas(lpr)-induced autoimmune disease. Conversely, MRL.CD72(b)/lpr congenic mice showed milder disease compared with MRL/lpr mice. We further demonstrated that Cd72(c) is a hypofunctional allele in BCR signal inhibition and that CD72 deficiency induces severe autoimmune disease in the presence of Fas(lpr). These results strongly suggest that the Cd72(c) is a crucial modifier gene that regulates Fas(lpr)-induced autoimmune disease due to its reduced activity of B cell signal regulation.
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http://dx.doi.org/10.4049/jimmunol.1203576DOI Listing
June 2013

A system for reconstructing B cell antigen receptor signaling in the mouse myeloma J558L cell line.

Arch Biochem Biophys 2013 May 27;533(1-2):18-24. Epub 2013 Feb 27.

Department of Cell signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca(2+) indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lμv11 cells) permitted monitoring of Ca(2+) mobilization. Reconstituting CD19 in J558Lμv11 cells resulted in detectable BCR-induced Ca(2+) mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lμv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling.
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http://dx.doi.org/10.1016/j.abb.2013.02.008DOI Listing
May 2013

Human CD72 splicing isoform responsible for resistance to systemic lupus erythematosus regulates serum immunoglobulin level and is localized in endoplasmic reticulum.

BMC Immunol 2012 Dec 26;13:72. Epub 2012 Dec 26.

Laboratory of Immunology, School of Biomedical Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

Background: CD72 is an inhibitory co-receptor expressed on B cells. We previously demonstrated significant association of the polymorphism of the CD72 gene with susceptibility to human systemic lupus erythematosus (SLE) in individuals carrying a SLE-susceptible FCGR2B genotype (FCGR2B-232Thr/Thr). The human CD72 locus generates a splicing isoform that lacks exon 8 (CD72Δex8) as well as full-length CD72 (CD72fl), and the CD72 polymorphism regulates exon 8 skipping.

Results: Here we demonstrated that individuals carrying the disease-protective CD72 genotype exhibit significantly lower serum immunoglobulin levels than do individuals carrying other CD72 genotypes (P < 0.05). Although expression level of CD72fl in the peripheral blood B cells was similar regardless of CD72 genotype, the protein level of CD72Δex8 was increased in individuals carrying the disease-protective CD72 genotype, suggesting a crucial role of CD72Δex8 in regulation of antibody production. By expressing these human CD72 isoforms in mouse cell lines, we further demonstrated that CD72Δex8 is accumulated in endoplasmic reticulum (ER) and fails to regulate BCR signaling whereas human CD72fl is efficiently transported to the cell surface and inhibits signaling through the B cell antigen receptor (BCR), as is the case for mouse CD72.

Conclusion: Human CD72 polymorphism appears to regulate antibody production as well as susceptibility to SLE by regulating expression of ER-localizing CD72Δex8.
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http://dx.doi.org/10.1186/1471-2172-13-72DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3565990PMC
December 2012

CD22 serves as a receptor for soluble IgM.

Eur J Immunol 2012 Jan 10;42(1):241-7. Epub 2011 Nov 10.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan.

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB).
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http://dx.doi.org/10.1002/eji.201141899DOI Listing
January 2012

Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering.

Biotechnol Bioeng 2011 Oct 31;108(10):2468-76. Epub 2011 May 31.

Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 Japan.

The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering.
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http://dx.doi.org/10.1002/bit.23192DOI Listing
October 2011

A cell-free assay to estimate the neutralizing capacity of granulocyte-macrophage colony-stimulating factor autoantibodies.

J Immunol Methods 2010 Aug 16;360(1-2):141-8. Epub 2010 Jul 16.

Niigata University Medical and Dental Hospital, Niigata, Japan.

The aim of the project is to develop a novel method estimating granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing capacity with high-throughput and good reproducibility. For that purpose, we designed a cell-free receptor binding assay consisting of a solid-phase recombinant soluble GM-CSF receptor alpha (GMRalpha) and a biotinylated GM-CSF (bGM-CSF). Using this system, competitive inhibition of bGM-CSF binding to soluble GM-CSF receptor alpha (sGMRalpha) by GM-CSF autoantibody or IgG fractions from the sera of patients with pulmonary alveolar proteinosis was examined, resulting in excellent reproducibility. Binding inhibition was correlated with growth inhibition of TF-1 cells, a GM-CSF dependent cell line. These results suggest that our cell-free system can be applied to estimate the neutralizing capacity of GM-CSF autoantibodies ex vivo.
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http://dx.doi.org/10.1016/j.jim.2010.07.001DOI Listing
August 2010

Production of a non-triple helical collagen alpha chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture.

Biotechnol Bioeng 2010 Aug;106(6):860-70

Neosilk Co., Ltd., 3-13-26 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.

We generated transgenic silkworms that synthesized human type I collagen alpha1 chain [alpha1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant alpha1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans-activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the alpha1(I) chain content to 8.0% (4.24 mg per cocoon). The alpha1(I) chain was extracted and purified from the cocoons using a very simple method. The alpha1(I) chain contained no hydroxyprolines due to the absence of prolyl-hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the alpha1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the alpha1(I) chain-coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the alpha1(I) chain-coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human alpha1(I) chain is a promising candidate biomaterial as a high-quality and safe gelatin substitute for cell culture.
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http://dx.doi.org/10.1002/bit.22752DOI Listing
August 2010

Differential phosphorylation of functional tyrosines in CD19 modulates B-lymphocyte activation.

Eur J Immunol 2010 Apr;40(4):1192-204

Department of Dermatology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

CD19 is a B-cell transmembrane molecule that is critical for B-cell activation. CD19 serves as a scaffold protein for key signal transduction molecules including Lyn, PI3K, and Vav, by providing docking sites for these molecules via phosphorylation of CD19-Y(513), CD19-Y(482), and CD19-Y(391). We investigated the process of CD19 tyrosine phophorylation during B-cell activation using Ab specific for each of these phosphorylated tyrosines. BCR engagement induced differential tyrosine phosphorylation, as CD19-Y(513) phophorylation occurred first, and CD19-Y(482) phosphorylation was delayed and transient. Different BCR isotypes exhibited distinct patterns of CD19 phosphorylation: IgG-BCR ligation resulted in faster phosphorylation of CD19-Y(513) and more intense phosphorylation of CD19-Y(391) than IgM-BCR ligation. This affected CD19-mediated downstream pathways involving Vav, PI3K, and Akt. Additionally, the phosphorylation profile of CD19 differed distinctly according to its plasma membrane location. CD19 phosphorylated at Y(513) was almost exclusively located within lipid rafts, whereas phosphorylated Y(482) and Y(391) were found both inside and outside of the rafts. Furthermore, the phosphorylation of all three tyrosines was remarkably enhanced and prolonged following the simultaneous stimulation of BCR and CD40. Thus, variations in phosphorylation patterns may contribute to the complexity of CD19-regulated signal transduction.
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http://dx.doi.org/10.1002/eji.200939848DOI Listing
April 2010