Publications by authors named "Jiro Kitaura"

78 Publications

A possible association between a novel NLRP1 mutation and an autoinflammatory disease involving liver cirrhosis.

Hepatology 2021 Mar 19. Epub 2021 Mar 19.

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

The nucleotide-binding domain leucine-rich repeat (LRR)-containing protein family pyrin domain (PYD)-containing protein 1 (NLRP1) forms an inflammasome complex with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. Inflammasome-activated caspase-1 cleaves pro-IL-1β or pro-IL-18 to produce its mature form and induces a type of cell death called pyroptosis through gasdermin D (GSDMD) activation.
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http://dx.doi.org/10.1002/hep.31818DOI Listing
March 2021

Anti-CD321 antibody immunotherapy protects liver against ischemia and reperfusion-induced injury.

Sci Rep 2021 Mar 18;11(1):6312. Epub 2021 Mar 18.

Juntendo Advanced Research Institute for Health Science, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

The prognosis of the liver transplant patients was frequently deteriorated by ischemia and reperfusion injury (IRI) in the liver. Infiltration of inflammatory cells is reported to play critical roles in the pathogenesis of hepatic IRI. Although T lymphocytes, neutrophils and monocytes infiltrated into the liver underwent IRI, we found that neutrophil depletion significantly attenuated the injury and serum liver enzyme levels in a murine model. Interestingly, the expression of CD321/JAM-A/F11R, one of essential molecules for transmigration of circulating leukocytes into inflammatory tissues, was significantly augmented on hepatic sinusoid endothelium at 1 h after ischemia and maintained until 45 min after reperfusion. The intraportal administration of anti-CD321 monoclonal antibody (90G4) significantly inhibited the leukocytes infiltration after reperfusion and diminished the damage responses by hepatic IRI (serum liver enzymes, inflammatory cytokines and hepatocyte cell death). Taken together, presented results demonstrated that blockade of CD321 by 90G4 antibody significantly attenuated hepatic IRI accompanied with substantial inhibition of leukocytes infiltration, particularly inhibition of neutrophil infiltration in the early phase of reperfusion. Thus, our work offers a potent therapeutic target, CD321, for preventing liver IRI.
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http://dx.doi.org/10.1038/s41598-021-85001-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973783PMC
March 2021

Orally desensitized mast cells form a regulatory network with Treg cells for the control of food allergy.

Mucosal Immunol 2020 Dec 10. Epub 2020 Dec 10.

Department of Mucosal Immunology, The University of Tokyo Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

Oral immunotherapy (OIT) is an effective approach to controlling food allergy. Although the detailed molecular and cellular mechanisms of OIT are unknown currently, they must be understood to advance the treatment of allergic diseases in general. To elucidate the mechanisms of OIT, especially during the immunological transition from desensitization to allergy regulation, we generated a clinical OIT murine model and used it to examine immunological events of OIT. We found that in mice that completed OIT successfully, desensitized mast cells (MCs) showed functionally beneficial alterations, such as increased induction of regulatory cytokines and enhanced expansion of regulatory T cells. Importantly, these regulatory-T-cell-mediated inhibitions of allergic responses were dramatically decreased in mice lacking OIT-induced desensitized MC. Collectively, these findings show that the desensitization process modulates the activation of MCs, leading directly to enhanced induction of regulatory-T-cell expansion and promotion of clinical allergic unresponsiveness. Our results suggest that efficiently inducing regulatory MCs is a novel strategy for the treatment of allergic disease.
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http://dx.doi.org/10.1038/s41385-020-00358-3DOI Listing
December 2020

Notch signaling contributes to the establishment of sustained unresponsiveness to food allergens by oral immunotherapy.

J Allergy Clin Immunol 2021 Mar 24;147(3):1063-1076.e9. Epub 2020 Jul 24.

Department of Pediatrics and Adolescent Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Background: Oral immunotherapy (OIT) aims to establish desensitization and sustained unresponsiveness (SU) in patients with food allergy by ingestion of gradually increasing doses of specific food allergens. However, little is known about the mechanisms by which OIT induces SU to specific allergens.

Objectives: We investigated the role of Notch signaling, which controls cell fate decisions in many types of immune cells in the induction of SU by OIT treatment.

Methods: Two types of mouse models, ovalbumin-induced food allergy and OIT, were generated. To elucidate the role of Notch signaling in OIT-induced SU, mice were intraperitoneally injected with the Notch signaling inhibitor N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenylglycine-1,1-dimethylethyl ester during the OIT treatment period.

Results: Ovalbumin-sensitized mice were desensitized and also had SU induced by OIT treatment, whereas repeated challenges with ovalbumin caused the development of severe allergic reactions in ovalbumin-sensitized mice. Administration of N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenylglycine-1,1-dimethylethyl ester to mice during the OIT treatment period inhibited the establishment of SU to ovalbumin but did not affect the induction of desensitization. OIT induced a systemic expansion of IL-10-producing CD4 T cells, including T2 cells, and myeloid-derived suppressor cells (MDSCs), particularly the monocytic MDSC subpopulation. Inhibition of Notch signaling prevented the OIT-induced expansion of those cells. In vitro cultures of bone marrow cells showed that Notch signaling directly promoted the generation of monocytic MDSCs. In addition, the contribution of MDSCs to OIT-induced SU was confirmed by MDSC depletion with the anti-Gr1 antibody.

Conclusion: Notch signaling contributes to the establishment of SU induced by OIT through systemic expansion of immunosuppressive cells, such as IL-10-producing CD4 T cells and MDSCs.
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http://dx.doi.org/10.1016/j.jaci.2020.07.011DOI Listing
March 2021

CD155-Transducing Signaling through TIGIT Plays an Important Role in Transmission of Tolerant State and Suppression Capacity.

Immunohorizons 2018 11 16;2(10):338-348. Epub 2018 Nov 16.

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan;

The precise mechanism of how the regulatory T cell population elicits and maintains tolerant state in activated T cells is poorly understood. To address this issue, we established an in vitro coculture system using mouse T cells and showed that tolerant state is serially passed from preinduced-tolerant T cells into new TCR-stimulated T cells across generations in a dendritic cell-independent manner. In this successive induction process of tolerant state, TIGIT was found to play an important role: TIGIT expression on induced-tolerant T cells was promoted in stimulated T cells cocultured with the tolerant cells. In addition, these stimulated T cells in the coculture also expressed high B lymphocyte-induced maturation protein 1 accompanied by IL-2 suppression. Because CD155, a partner of TIGIT, is known to transduce signaling inside by -interaction with its ligands, these phenotypical changes in TCR-stimulated naive T cells were reproduced when naive T cells were double cross-linked by CD3 and CD155. These results indicate that TIGIT enhanced on tolerant T cells may function as a ligand of its paired receptor CD155 to transduce signaling into its expressing naive T cells to accelerate new TIGIT expressions as well as IL-2 suppression via B lymphocyte-induced maturation protein 1 enhancement. In consideration of these results, we propose a novel process in which tolerant state in T cell population is maintained by successive generation of new tolerant T cells from naive T cells as one of the regulating mechanisms in immune responses.
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http://dx.doi.org/10.4049/immunohorizons.1800033DOI Listing
November 2018

NUP98-HBO1-fusion generates phenotypically and genetically relevant chronic myelomonocytic leukemia pathogenesis.

Blood Adv 2019 04;3(7):1047-1060

Laboratory of Oncology, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.

Chronic myelomonocytic leukemia (CMML) constitutes a hematopoietic stem cell (HSC) disorder characterized by prominent monocytosis and myelodysplasia. Although genome sequencing has revealed the CMML mutation profile, the mechanism of disease development remains unclear. Here we show that aberrant histone acetylation by nucleoporin-98 (NUP98)-HBO1, a newly identified fusion in a patient with CMML, is sufficient to generate clinically relevant CMML pathogenesis. Overexpression of NUP98-HBO1 in murine HSC/progenitors (HSC/Ps) induced diverse CMML phenotypes, such as severe leukocytosis, increased CD115 Ly6C monocytes (an equivalent subpopulation to human classical CD14 CD16 monocytes), macrocytic anemia, thrombocytopenia, megakaryocyte-lineage dysplasia, splenomegaly, and cachexia. A NUP98-HBO1-mediated transcriptional signature in human CD34 cells was specifically activated in HSC/Ps from a CMML patient cohort. Besides critical determinants of monocytic cell fate choice in HSC/Ps, an oncogenic HOXA9 signature was significantly activated by NUP98-HBO1 fusion through aberrant histone acetylation. Increased gene expression level with disease progression was confirmed in our CMML cohort. Genetic disruption of NUP98-HBO1 histone acetyltransferase activity abrogated its leukemogenic potential and disease development in human cells and a mouse model. Furthermore, treatment of azacytidine was effective in our CMML mice. The recapitulation of CMML clinical phenotypes and gene expression profile by the HBO1 fusion suggests our new model as a useful platform for elucidating the central downstream mediators underlying diverse CMML-related mutations and testing multiple compounds, providing novel therapeutic potential.
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http://dx.doi.org/10.1182/bloodadvances.2018025007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457235PMC
April 2019

Receptor-destroying enzyme (RDE) from modulates IgE activity and reduces the initiation of anaphylaxis.

J Biol Chem 2019 04 4;294(17):6659-6669. Epub 2019 Mar 4.

From the Department of Microbiology and Immunology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195,

IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)-treated IgE cannot access its binding site on bone marrow-derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)-treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to lectin, which recognizes poly--acetylglucosamine and poly--acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies.
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http://dx.doi.org/10.1074/jbc.RA118.006375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497938PMC
April 2019

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide-dependent neutrophil recruitment.

Sci Signal 2019 01 15;12(564). Epub 2019 Jan 15.

Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.
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http://dx.doi.org/10.1126/scisignal.aar5514DOI Listing
January 2019

Mouse LIMR3/CD300f is a negative regulator of the antimicrobial activity of neutrophils.

Sci Rep 2018 11 27;8(1):17406. Epub 2018 Nov 27.

Department of Chemotherapy and Mycoses, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

Leukocyte mono-immunoglobulin-like receptor (LMIR)/CD300 proteins comprise a family of immunoglobulin-like receptors that are widely expressed on the immune cell surface in humans and mice. In general, LMIR3/CD300f suppresses the inflammatory response, but it can occasionally promote it. However, the precise roles of LMIR3 in the function of neutrophils remain to be elucidated. In the present study, we investigated LMIR3 expression in mature and immature neutrophils, and evaluated the effects of LMIR3 deficiency in mouse neutrophils. Our results indicated that bone marrow (BM) neutrophils expressed LMIR3 on their cell surface during cell maturation and that surface LMIR3 expression increased in response to Pseudomonas aeruginosa infection in a TLR4/MyD88-dependent manner. LMIR3-knockout (KO) neutrophils displayed significantly increased hypochlorous acid production, and elastase release, as well as significantly augmented cytotoxic activity against P. aeruginosa and Candida albicans; meanwhile, inhibitors of elastase and myeloperoxidase offset this enhanced antimicrobial activity. Furthermore, LMIR3-KO mice were significantly more resistant to Pseudomonas peritonitis and systemic candidiasis, although this may not be entirely due to the enhanced activity of neutrophils. These results demonstrate that LMIR3/CD300f deficiency augments the antimicrobial activity of mouse neutrophils.
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http://dx.doi.org/10.1038/s41598-018-35699-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258681PMC
November 2018

The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells.

Sci Rep 2018 09 24;8(1):14237. Epub 2018 Sep 24.

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan.

Immunoglobulin E (IgE) plays a central role in the pathogenesis of Type I hypersensitivity through interaction with a high-affinity receptor (FcεRIα). For therapeutic applications, substantial attention has been focused recently on the blockade of the IgE interaction with FcεRIα. While exploring better options for preventing allergic diseases, we found that the Fab fragment of the rat anti-murine IgE antibody (Fab-6HD5) strongly inhibited passive cutaneous anaphylaxis (PCA) in vivo, as well as spleen tyrosine kinase (Syk) activity and β-hexosaminidase release from basophilic leukemia cells in vitro. The in vivo effects of Fab-6HD5 pre-administration were maintained over a long period of time for at least 10 days. Using flow cytometry analysis, we also found that Fab-6HD5 did not recognize the IgE Cε3 domain containing specific binding sites for FcεRIα. Furthermore, deletion-mapping studies revealed that Fab-6HD5 recognized conformational epitopes on the Cε2 domain of IgE. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcRIα, our results suggest that the specific binding of Fab-6HD5 to the Cε2 domain prevents allergic reactions through destabilizing the preformed IgE-FcεRIα complex on rat mast cells. Although the present study was performed using animal models, these findings support the idea that a certain antibody directed against IgE CH domains may contribute to preventing allergic diseases through interacting with IgE-FcεRIα complex.
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http://dx.doi.org/10.1038/s41598-018-32200-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155129PMC
September 2018

Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Sci Rep 2018 05 29;8(1):8259. Epub 2018 May 29.

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.
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http://dx.doi.org/10.1038/s41598-018-25646-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974347PMC
May 2018

IL-31 is crucial for induction of pruritus, but not inflammation, in contact hypersensitivity.

Sci Rep 2018 04 27;8(1):6639. Epub 2018 Apr 27.

Laboratory of Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.

IL-31, which is a member of the IL-6 family of cytokines, is produced mainly by activated CD4 T cells, in particular activated Th2 cells, suggesting a contribution to development of type-2 immune responses. IL-31 was reported to be increased in specimens from patients with atopic dermatitis, and IL-31-transgenic mice develop atopic dermatitis-like skin inflammation, which is involved in the pathogenesis of atopic dermatitis. However, the role of IL-31 in development of contact dermatitis/contact hypersensitivity (CHS), which is mediated by hapten-specific T cells, including Th2 cells, is not fully understood. Therefore, we investigated this using IL-31-deficient (Il31) mice, which we newly generated. We demonstrated that the mice showed normal migration and maturation of skin dendritic cells and induction of hapten-specific T cells in the sensitization phase of FITC-induced CHS, and normal induction of local inflammation in the elicitation phase of FITC- and DNFB-induced CHS. On the other hand, those mice showed reduced scratching frequency and duration during FITC- and/or DNFB-induced CHS. Our findings suggest that IL-31 is responsible for pruritus, but not induction of local skin inflammation, during CHS induced by FITC and DNFB.
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http://dx.doi.org/10.1038/s41598-018-25094-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923199PMC
April 2018

The CD300e molecule in mice is an immune-activating receptor.

J Biol Chem 2018 03 22;293(10):3793-3805. Epub 2018 Jan 22.

From the Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421,

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115Ly-6C peripheral blood monocytes, corresponding to CD14CD16 human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115Ly-6C monocytes. Stimulation with sphingomyelin failed to activate the CD115Ly-6C mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14CD16 human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.
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http://dx.doi.org/10.1074/jbc.RA117.000696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846139PMC
March 2018

Role of the Ceramide-CD300f Interaction in Gram-Negative Bacterial Skin Infections.

J Invest Dermatol 2018 05 5;138(5):1221-1224. Epub 2017 Dec 5.

Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan; Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2017.11.025DOI Listing
May 2018

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sci Rep 2017 06 27;7(1):4298. Epub 2017 Jun 27.

Atopy Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.
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http://dx.doi.org/10.1038/s41598-017-04647-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487349PMC
June 2017

Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation.

J Biol Chem 2017 02 10;292(7):2924-2932. Epub 2017 Jan 10.

From the Atopy Research Center and

LPS triggers inflammatory responses; however, the negative regulation of LPS responses remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling .
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http://dx.doi.org/10.1074/jbc.M116.768366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314187PMC
February 2017

Pharmacologic inhibition of Notch signaling suppresses food antigen-induced mucosal mast cell hyperplasia.

J Allergy Clin Immunol 2017 Mar 16;139(3):987-996.e10. Epub 2016 Jul 16.

Department of Pediatrics and Adolescent Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Background: Mucosal mast cells (MMCs) play a central role in the development of symptoms associated with IgE-mediated food allergy. Recently, Notch2-mediated signaling was shown to be involved in proper MMC distribution in the intestinal tract.

Objective: This study aimed to clarify the mechanism by which Notch signaling regulates MMC distribution in the intestinal mucosa. Furthermore, pharmacologic inhibition of Notch signaling was evaluated as a treatment for symptoms associated with experimental food allergy.

Methods: Bone marrow-derived mast cells generated from mice were cultured with Notch ligands, and then expression of genes associated with MMCs was measured in the cells. In addition, the effect of an inhibitor of Notch signaling on food antigen-induced allergic reactions was examined in a mouse model of food allergy.

Results: Notch signaling induced MMC differentiation through upregulation of expression of genes characteristic of MMCs in the presence of IL-3. Some lamina propria cells isolated from the mouse small intestine expressed Notch ligands and were able to upregulate MMC markers in bone marrow-derived mast cells through Notch signaling. In a mouse model of food allergy, administration of a Notch signaling inhibitor led to suppression of food antigen-induced hyperplasia of intestinal MMCs, resulting in alleviation of allergic diarrhea and systemic anaphylaxis.

Conclusion: Notch signaling contributes to differentiation and accumulation of MMCs in the intestinal mucosa. Thus inhibition of Notch signaling alleviates symptoms associated with experimental food allergy. These results raise the possibility that Notch signaling in mast cells is a novel target for therapy in patients with food allergy.
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http://dx.doi.org/10.1016/j.jaci.2016.05.046DOI Listing
March 2017

Novel working hypothesis for pathogenesis of hematological malignancies: combination of mutations-induced cellular phenotypes determines the disease (cMIP-DD).

J Biochem 2016 Jan 20;159(1):17-25. Epub 2015 Nov 20.

Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Recent progress in high-speed sequencing technology has revealed that tumors harbor novel mutations in a variety of genes including those for molecules involved in epigenetics and splicing, some of which were not categorized to previously thought malignancy-related genes. However, despite thorough identification of mutations in solid tumors and hematological malignancies, how these mutations induce cell transformation still remains elusive. In addition, each tumor usually contains multiple mutations or sometimes consists of multiple clones, which makes functional analysis difficult. Fifteen years ago, it was proposed that combination of two types of mutations induce acute leukemia; Class I mutations induce cell growth or inhibit apoptosis while class II mutations block differentiation, co-operating in inducing acute leukemia. This notion has been proven using a variety of mouse models, however most of recently found mutations are not typical class I/II mutations. Although some novel mutations have been found to functionally work as class I or II mutation in leukemogenesis, the classical class I/II theory seems to be too simple to explain the whole story. We here overview the molecular basis of hematological malignancies based on clinical and experimental results, and propose a new working hypothesis for leukemogenesis.
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http://dx.doi.org/10.1093/jb/mvv114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882650PMC
January 2016

The Phosphatidylserine and Phosphatidylethanolamine Receptor CD300a Binds Dengue Virus and Enhances Infection.

J Virol 2016 01 14;90(1):92-102. Epub 2015 Oct 14.

INSERM U944-CNRS 7212, Laboratoire de Pathologie et Virologie Moléculaire, Hôpital Saint-Louis, Paris, France Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris, France Université Paris Diderot, Sorbonne Paris Cité, Hôpital St. Louis, Paris, France

Unlabelled: Dengue virus (DENV) is the etiological agent of the major human arboviral disease. We previously demonstrated that the TIM and TAM families of phosphatidylserine (PtdSer) receptors involved in the phagocytosis of apoptotic cells mediate DENV entry into target cells. We show here that human CD300a, a recently identified phospholipid receptor, also binds directly DENV particles and enhances viral entry. CD300a facilitates infection of the four DENV serotypes, as well as of other mosquito-borne viruses such as West Nile virus and Chikungunya virus. CD300a acts as an attachment factor that enhances DENV internalization through clathrin-mediated endocytosis. CD300a recognizes predominantly phosphatidylethanolamine (PtdEth) and to a lesser extent PtdSer associated with viral particles. Mutation of residues in the IgV domain critical for phospholipid binding abrogate CD300a-mediated enhancement of DENV infection. Finally, we show that CD300a is expressed at the surface of primary macrophages and anti-CD300a polyclonal antibodies partially inhibited DENV infection of these cells. Overall, these data indicate that CD300a is a novel DENV binding receptor that recognizes PtdEth and PtdSer present on virions and enhance infection.

Importance: Dengue disease, caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease of humans and is a major global health concern. The molecular bases of DENV-host cell interactions during virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. We recently discovered that the TIM and TAM proteins, two receptor families involved in the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, interact with DENV particles-associated PtdSer through a mechanism that mimics the recognition of apoptotic cells and mediate DENV infection. In this study, we show that CD300a, a novel identified phospholipid receptor, mediates DENV infection. CD300a-dependent DENV infection relies on the direct recognition of phosphatidylethanolamine and to a lesser extent PtdSer associated with viral particles. This study provides novel insights into the mechanisms that mediate DENV entry and reinforce the concept that DENV uses an apoptotic mimicry strategy for viral entry.
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http://dx.doi.org/10.1128/JVI.01849-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702537PMC
January 2016

The Transcription Factor Ehf Is Involved in TGF-β-Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells.

J Immunol 2015 Oct 21;195(7):3427-35. Epub 2015 Aug 21.

Department of Pediatrics and Adolescent Medicine, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan; and.

FcεRI, which is composed of α, β, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-β1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-β1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-β/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIβ, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIβ, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-β1 was mimicked by forced expression of Ehf, suggesting that TGF-β1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.
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http://dx.doi.org/10.4049/jimmunol.1402856DOI Listing
October 2015

Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation.

Gut 2016 May 11;65(5):777-87. Epub 2015 Feb 11.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Atopy Research Center, Juntendo University School of Medicine, Tokyo, Japan.

Objective: Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide-LMIR3 interaction in the development of IBD.

Design: The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3(-/-), mast cell-deficient Kit(W-sh/W-sh), Kit(W-sh/W-sh)LMIR3(-/-) or Kit(W-sh/W-sh) mice engrafted with WT or LMIR3(-/-) bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes.

Results: LMIR3 deficiency exacerbated DSS-induced colitis in mice. Kit(W-sh/W-sh) mice harbouring LMIR3(-/-) mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide-LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide-LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes.

Conclusions: LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3(-/-) mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide-LMIR3 binding.
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http://dx.doi.org/10.1136/gutjnl-2014-308900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4853571PMC
May 2016

A C-terminal mutant of CCAAT-enhancer-binding protein α (C/EBPα-Cm) downregulates Csf1r, a potent accelerator in the progression of acute myeloid leukemia with C/EBPα-Cm.

Exp Hematol 2015 Apr 20;43(4):300-8.e1. Epub 2014 Dec 20.

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Electronic address:

Two types of CCAAT-enhancer-binding protein α (C/EBPα) mutants are found in acute myeloid leukemia (AML) patients: N-terminal frame-shift mutants (C/EBPα-N(m)) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-C(m)). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-C(m), but not C/EBPα-T60fsX159 belonging to C/EBPα-N(m), alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-C(m) mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-C(m) tested (C/EBPα-S299_K304dup, K313dup, or N321D) by itself induced AML, albeit with different latencies after BMT; C/EBPα-N321D induced AML with the shortest latency. By analyzing the gene expression profiles of C/EBPα-N321D- and mock-transduced c-kit(+)Sca-1(+)Lin(-) cells, we identified Csf1r as a gene downregulated by C/EBPα-N321D. In addition, leukemic cells expressing C/EBPα-C(m) exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-N(m) did not influence Csf1r expression in c-kit(+)Sca-1(+)Lin(-) cells, implying a unique role for C/EBPα-C(m) in downregulating Csf1r. Importantly, Csf1r overexpression collaborated with C/EBPα-N321D to induce fulminant AML with leukocytosis in mouse BMT models to a greater extent than did C/EBPα-N321D alone. Collectively, these results suggest that C/EBPα-C(m)-mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-C(m), which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation.
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http://dx.doi.org/10.1016/j.exphem.2014.11.011DOI Listing
April 2015

The molecular basis of myeloid malignancies.

Proc Jpn Acad Ser B Phys Biol Sci 2014 ;90(10):389-404

Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo.

Myeloid malignancies consist of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and myeloproliferative neoplasm (MPN). The latter two diseases have preleukemic features and frequently evolve to AML. As with solid tumors, multiple mutations are required for leukemogenesis. A decade ago, these gene alterations were subdivided into two categories: class I mutations stimulating cell growth or inhibiting apoptosis; and class II mutations that hamper differentiation of hematopoietic cells. In mouse models, class I mutations such as the Bcr-Abl fusion kinase induce MPN by themselves and some class II mutations such as Runx1 mutations induce MDS. Combinations of class I and class II mutations induce AML in a variety of mouse models. Thus, it was postulated that hematopoietic cells whose differentiation is blocked by class II mutations would autonomously proliferate with class I mutations leading to the development of leukemia. Recent progress in high-speed sequencing has enabled efficient identification of novel mutations in a variety of molecules including epigenetic factors, splicing factors, signaling molecules and proteins in the cohesin complex; most of these are not categorized as either class I or class II mutations. The functional consequences of these mutations are now being extensively investigated. In this article, we will review the molecular basis of hematological malignancies, focusing on mouse models and the interfaces between these models and clinical findings, and revisit the classical class I/II hypothesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335136PMC
http://dx.doi.org/10.2183/pjab.90.389DOI Listing
August 2015

Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells.

Blood 2014 Jun 13;123(25):3932-42. Epub 2014 May 13.

Division of Cellular Therapy, Advanced Clinical Research Center, Division of Stem Cell Signaling, Center for Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;

High levels of HES1 expression are frequently found in BCR-ABL(+) chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC-like disease; however, the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-κB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, CMPs expressing BCR-ABL and Hes1 secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC-like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells.
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http://dx.doi.org/10.1182/blood-2013-01-476747DOI Listing
June 2014

[An inhibitory receptor LMIR3/CD300f recognizes ceramide].

Seikagaku 2013 Dec;85(12):1071-5

Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

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December 2013

A novel cell-cycle-indicator, mVenus-p27K-, identifies quiescent cells and visualizes G0-G1 transition.

Sci Rep 2014 Feb 6;4:4012. Epub 2014 Feb 6.

1] Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan [2] Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

The quiescent (G0) phase of the cell cycle is the reversible phase from which the cells exit from the cell cycle. Due to the difficulty of defining the G0 phase, quiescent cells have not been well characterized. In this study, a fusion protein consisting of mVenus and a defective mutant of CDK inhibitor, p27 (p27K(-)) was shown to be able to identify and isolate a population of quiescent cells and to effectively visualize the G0 to G1 transition. By comparing the expression profiles of the G0 and G1 cells defined by mVenus-p27K(-), we have identified molecular features of quiescent cells. Quiescence is also an important feature of many types of stem cells, and mVenus-p27K(-)-transgenic mice enabled the detection of the quiescent cells with muscle stem cell markers in muscle in vivo. The mVenus-p27K(-) probe could be useful in investigating stem cells as well as quiescent cells.
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http://dx.doi.org/10.1038/srep04012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915272PMC
February 2014