Publications by authors named "Kumi Izawa"

37 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

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

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

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

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

LEUKOCYTE MONO-IMMUNOGLOBULIN-LIKE RECEPTOR (LMIR3)-MEDIATED INHIBITION OF ALLERGY AND INFLAMMATION.

Authors:
Kumi Izawa

Arerugi 2017 ;66(1):36-41

Atopy Research Center, Juntendo University School of Medicine.

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http://dx.doi.org/10.15036/arerugi.66.36DOI Listing
May 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

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

Hes1 upregulation contributes to the development of FIP1L1-PDGRA-positive leukemia in blast crisis.

Exp Hematol 2014 May 31;42(5):369-379.e3. Epub 2014 Jan 31.

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:

We have previously shown that elevated expression of Hairy enhancer of split 1 (Hes1) contributes to blast crisis transition in Bcr-Abl-positive chronic myelogenous leukemia. Here we investigate whether Hes1 is involved in the development of other myeloid neoplasms. Notably, Hes1 expression was elevated in only a few cases of 65 samples with different types of myeloid neoplasms. Interestingly, elevated expression of Hes1 was found in two of five samples of Fip1-like1 platelet-derived growth factor receptor-α (FIP1L1-PDGFA)-positive myeloid neoplasms associated with eosinophilia. Whereas FIP1L1-PDGFRα alone induced acute T-cell leukemia or myeloproliferative neoplasms in mouse bone marrow transplantation models, mice transplanted with bone marrow cells expressing both Hes1 and FIP1L1-PDGFRα developed acute leukemia characterized by an expansion of myeloid blasts and leukemic cells without eosinophilic granules. FIP1L1-PDGFRα conferred cytokine-independent growth to Hes1-transduced common myeloid progenitors, interleukin-3-dependent cells. Imatinib inhibited the growth of common myeloid progenitors expressing Hes1 with FIP1L1-PDGFRα, but not with imatinib-resistant FIP1L1-PDGFRα mutants harboring T674I or D842V. In contrast, ponatinib efficiently eradicated leukemic cells expressing Hes1 and the imatinib-resistant FLP1L1-PDGFRΑ mutant in vitro and in vivo. Thus, we have established mouse models of FIP1L1-PDGFRA-positive leukemia in myeloid blast crisis, which will help elucidate the pathogenesis of the disease and develop a new treatment for it.
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http://dx.doi.org/10.1016/j.exphem.2014.01.009DOI Listing
May 2014

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations.

J Clin Invest 2013 Nov;123(11):4627-40

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.
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http://dx.doi.org/10.1172/JCI70739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809801PMC
November 2013

Sphingomyelin and ceramide are physiological ligands for human LMIR3/CD300f, inhibiting FcεRI-mediated mast cell activation.

J Allergy Clin Immunol 2014 Jan 12;133(1):270-3.e1-7. Epub 2013 Sep 12.

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

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http://dx.doi.org/10.1016/j.jaci.2013.08.008DOI Listing
January 2014

Human CD300C delivers an Fc receptor-γ-dependent activating signal in mast cells and monocytes and differs from CD300A in ligand recognition.

J Biol Chem 2013 Mar 31;288(11):7662-7675. Epub 2013 Jan 31.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Electronic address:

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.
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http://dx.doi.org/10.1074/jbc.M112.434746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597807PMC
March 2013

The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding to extracellular ceramide.

Immunity 2012 Nov 1;37(5):827-39. Epub 2012 Nov 1.

Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.
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http://dx.doi.org/10.1016/j.immuni.2012.08.018DOI Listing
November 2012

Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice.

Int J Hematol 2012 Nov 25;96(5):638-48. Epub 2012 Oct 25.

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

Activating mutations of c-Kit are frequently found in acute myeloid leukemia (AML) patients harboring t(8;21) chromosomal translocation generating a fusion protein AML1-ETO. Here we show that an active mutant of c-Kit cooperates with AML1-ETO to induce AML in mouse bone marrow transplantation models. Leukemic cells expressing AML1-ETO with c-Kit(D814V) were serially transplantable. Transplantation experiments indicated that lineage(-)c-Kit(+)Sca-1(+) (KSL) leukemic cells, but not lineage(+) leukemic cells, were enriched for leukemia stem cells (LSCs). Comparison of gene expression profiles between KSL leukemic and normal cells delineated that CD200R1 was highly expressed in KSL leukemic cells as compared with KSL normal cells. Upregulation of CD200R1 was verified in lineage(-) leukemic cells, but not in lineage(+) leukemic cells. CD200R1 expression in the lineage(-) leukemic cells was not correlated with the frequency of LSCs, indicating that CD200R1 is not a useful marker for LSCs in these models. Interestingly, CD200R1 was upregulated in KSL cells transduced with AML1-ETO, but not with other leukemogenic mutants, including c-Kit(D814V), AML1(D171N), and AML1(S291fsX300). Consistently, upregulation of CD200R1 in lineage(-) leukemic cells was observed only in the BM of mice suffering from AML1-ETO-positive leukemia. In conclusion, AML1-ETO upregulated CD200R1 in lineage(-) cells, which was characteristic of AML1-ETO-positive leukemia in mice.
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http://dx.doi.org/10.1007/s12185-012-1207-6DOI Listing
November 2012

PRAT4A-dependent expression of cell surface TLR5 on neutrophils, classical monocytes and dendritic cells.

Int Immunol 2012 Oct 25;24(10):613-23. Epub 2012 Jul 25.

Division of Infectious Genetics, Department of Microbiology and Immunology, University of Tokyo, Minatoku, Tokyo108-8639, Japan.

AbstractToll-like receptor 5 (TLR5), a sensor for bacterial flagellin, mounts innate and adaptive immune responses, and has been implicated in infectious diseases, colitis and metabolic syndromes. Although TLR5 is believed to belong to cell surface TLRs, cell surface expression has never been verified. Moreover, it has remained unclear which types of immune cells express TLR5 and contribute to flagellin-dependent responses. In this study we established an anti-mouse TLR5 monoclonal antibody and studied the cell surface expression of TLR5 on immune cells. The macrophage cell line J774 expressed endogenous TLR5 on the cell surface and produced IL-6 and G-CSF in response to flagellin. Cell surface expression of TLR5 and flagellin-induced responses were completely abolished by silencing a TLR-specific chaperone protein associated with TLR4 A (PRAT4A), demonstrating that TLR5 is another client of PRAT4A. In the in vivo immune cells, cell surface TLR5 was mainly found on neutrophils and CD11b (hi) Ly6C (hi) classical monocytes in the bone marrow, circulation, spleen and inflammatory lesions. Ly6C (hi) classical monocytes, but not neutrophils, produced cytokines in response to flagellin. Splenic CD8 (-) CD4 (+) conventional dendritic cells and CD11c (hi) CD11b (hi) lamina propria DCs, also clearly expressed cell surface TLR5. Collectively, cell surface expression of TLR5 is dependent on PRAT4A and restricted to neutrophils, classical monocytes and specific DC subsets.
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http://dx.doi.org/10.1093/intimm/dxs068DOI Listing
October 2012

A soluble form of LMIR5/CD300b amplifies lipopolysaccharide-induced lethal inflammation in sepsis.

J Immunol 2012 Aug 6;189(4):1773-9. Epub 2012 Jul 6.

Division of Cellular Therapy, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan;

Leukocyte mono-Ig-like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (M) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal M via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal M. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation.
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http://dx.doi.org/10.4049/jimmunol.1201139DOI Listing
August 2012