RESUMO
Allergic rhinitis (AR) is caused by type I hypersensitivity reaction in the nasal tissues. The interaction between CD300f and its ligand ceramide suppresses immunoglobulin E (IgE)-mediated mast cell activation. However, whether CD300f inhibits the development of allergic rhinitis (AR) remains elusive. We aimed to investigate the roles of CD300f in the development of AR and the effectiveness of intranasal administration of ceramide liposomes on AR in murine models. We used ragweed pollen-induced AR models in mice. Notably, CD300f deficiency did not significantly influence the ragweed-specific IgE production, but increased the frequency of mast cell-dependent sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in our models. Similar results were also obtained for MCPT5-exprssing mast cell-specific loss of CD300f. Importantly, intranasal administration of ceramide liposomes reduced the frequency of sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in AR models. Thus, CD300f-ceramide interaction, predominantly in mast cells, alleviates the symptoms and progression of AR. Therefore, intranasal administration of ceramide liposomes may be a promising therapeutic approach against AR by targeting CD300f.
Assuntos
Lipossomos , Rinite Alérgica , Animais , Camundongos , Administração Intranasal , Espirro , Ceramidas , Modelos Animais de Doenças , Rinite Alérgica/tratamento farmacológico , Imunoglobulina E , Mucosa Nasal , Camundongos Endogâmicos BALB C , OvalbuminaRESUMO
Background: Recently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model. Material and methods: Allergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin. Results: Protein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin. Conclusion: Our results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.
Assuntos
Anafilaxia , Oncorhynchus kisutch , Animais , Camundongos , Tropomiosina , Actinas , Salmão , Sulfato de Amônio , Modelos Animais de Doenças , Pyroglyphidae , Alérgenos , Imunoglobulina ERESUMO
Background: Patients with food allergy often suffer from atopic dermatitis, in which Staphylococcus aureus colonization is frequently observed. Staphylococcus aureus δ-toxin activates mast cells and promotes T helper 2 type skin inflammation in the tape-stripped murine skin. However, the physiological effects of δ-toxin present on the steady-state skin remain unknown. We aimed to investigate whether δ-toxin present on the steady-state skin impacts the development of food allergy. Material and methods: The non-tape-stripped skins of wild-type, KitW-sh/W-sh, or ST2-deficient mice were treated with ovalbumin (OVA) with or without δ-toxin before intragastric administration of OVA. The frequency of diarrhea, numbers of jejunum or skin mast cells, and serum levels of OVA-specific IgE were measured. Conventional dendritic cell 2 (cDC2) in skin and lymph nodes (LN) were analyzed. The cytokine levels in the skin tissues or culture supernatants of δ-toxin-stimulated murine keratinocytes were measured. Anti-IL-1α antibody-pretreated mice were analyzed. Results: Stimulation with δ-toxin induced the release of IL-1α, but not IL-33, in murine keratinocytes. Epicutaneous treatment with OVA and δ-toxin induced the local production of IL-1α. This treatment induced the translocation of OVA-loaded cDC2 from skin to draining LN and OVA-specific IgE production, independently of mast cells and ST2. This resulted in OVA-administered food allergic responses. In these models, pretreatment with anti-IL-1α antibody inhibited the cDC2 activation and OVA-specific IgE production, thereby dampening food allergic responses. Conclusion: Even without tape stripping, δ-toxin present on skin enhances epicutaneous sensitization to food allergen in an IL-1α-dependent manner, thereby promoting the development of food allergy.
Assuntos
Dermatite Atópica , Hipersensibilidade Alimentar , Camundongos , Animais , Staphylococcus aureus , Modelos Animais de Doenças , Proteína 1 Semelhante a Receptor de Interleucina-1 , Imunoglobulina E , Ovalbumina , ExotoxinasRESUMO
Oral allergy syndrome (OAS) is an IgE-mediated immediate food allergy that is localized to the oral mucosa. Pollen food allergy syndrome (PFAS), a pollinosis-associated OAS, is caused by cross-reactivity between food and pollen allergens. However, we need to more precisely understand the underlying pathogenesis of OAS/PFAS. In the present study, we developed a method to comprehensively identify cross-reactive allergens by using murine model of OAS and protein microarray technology. We focused on lip angioedema, which is one of the most common symptoms of OAS, and confirmed that mast cells reside in the tissues inside the lower lip of the mice. Interestingly, when the food allergen ovalbumin (OVA) was injected inside the lower lip of mice with high levels of OVA-specific IgE followed by an intravenous injection of the Evans blue dye, we found immediate dye extravasation in the skin of the neck in a mast cell-dependent manner. In addition, the degree of mast cell degranulation in the oral cavity, reflecting the severity of oral allergic responses, can be estimated by measuring the amount of extravasated dye in the skin. Therefore, we used this model of OAS to examine IgE cross-reactive allergens in vivo. Protein microarray analysis showed that serum IgE from mice intraperitoneally sensitized with ragweed pollen, one of the major pollens causing pollinosis, bound highly to protein extracts from several edible plants including black peppercorn and fennel. We confirmed that the levels of black pepper-specific IgE and fennel-specific IgE were significantly higher in the serum from ragweed pollen-sensitized mice than in the serum from non-sensitized control mice. Importantly, analysis of murine model of OAS showed that the injection of black pepper or fennel extract induced apparent oral allergic responses in ragweed pollen-sensitized mice. These results indicate IgE cross-reactivity of ragweed pollen with black pepper and fennel. In conclusion, we developed mouse model of OAS to identify IgE cross-reactive pollen and food allergens, which will help understand the pathogenesis of OAS/PFAS.
Assuntos
Fluorocarbonos , Foeniculum , Hipersensibilidade Alimentar , Piper nigrum , Rinite Alérgica Sazonal , Alérgenos/análise , Animais , Antígenos de Plantas , Modelos Animais de Doenças , Hipersensibilidade Alimentar/etiologia , Imunoglobulina E , Camundongos , Extratos Vegetais , PólenAssuntos
Interleucina-18 , Cirrose Hepática , Transplante de Fígado/métodos , Fígado , Proteínas NLR/genética , Doença de Papillon-Lefevre , Adolescente , Autoimunidade/imunologia , Feminino , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-18/análise , Interleucina-18/imunologia , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Cirrose Hepática/cirurgia , Mutação , Doença de Papillon-Lefevre/genética , Doença de Papillon-Lefevre/imunologia , Doença de Papillon-Lefevre/fisiopatologia , Doença de Papillon-Lefevre/terapia , Resultado do TratamentoAssuntos
Ceramidas/metabolismo , Hipersensibilidade Alimentar/metabolismo , Receptores Imunológicos/metabolismo , Animais , Anticorpos/imunologia , Ceramidas/imunologia , Modelos Animais de Doenças , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Mucosa Intestinal/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Knockout , Receptores Imunológicos/genética , Células Th2/efeitos dos fármacos , Células Th2/imunologiaAssuntos
Domínios de Imunoglobulina/genética , Anafilaxia Cutânea Passiva/imunologia , Receptores Imunológicos/genética , Substituição de Aminoácidos/imunologia , Animais , Linhagem Celular , Ceramidas/imunologia , Ceramidas/metabolismo , Humanos , Lisina/genética , Mastócitos , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Esfingomielinas/imunologia , Esfingomielinas/metabolismoRESUMO
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 CD300b-/- 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.
Assuntos
Neutrófilos/metabolismo , Óxido Nítrico/metabolismo , Receptores Imunológicos/metabolismo , Esfingosina/análogos & derivados , Zimosan/farmacologia , Animais , Artrite/genética , Artrite/metabolismo , Células Dendríticas/metabolismo , Inflamação/genética , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores Imunológicos/genética , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo , Esfingosina/farmacologia , Receptor 4 Toll-Like/metabolismo , Zimosan/metabolismoAssuntos
Hipersensibilidade/imunologia , Mastócitos/imunologia , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores Imunológicos/imunologia , Receptores de Neuropeptídeos/imunologia , Animais , Linhagem Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Receptores Imunológicos/genética , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
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.
Assuntos
Biomarcadores/metabolismo , Medula Óssea/imunologia , Células Dendríticas/fisiologia , Linfonodos/imunologia , Receptores Imunológicos/metabolismo , Baço/imunologia , Animais , Citometria de Fluxo , Células HEK293 , Humanos , Tolerância Imunológica , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores Imunológicos/genética , Transdução de SinaisRESUMO
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 CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mastócitos/metabolismo , Monócitos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de IgG/metabolismo , Receptores Imunológicos/agonistas , Esfingomielinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligantes , Mastócitos/citologia , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/imunologia , Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de IgG/química , Receptores de IgG/genética , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Ceramidas/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Peritonite/etiologia , Peritonite/metabolismo , Receptores Imunológicos/metabolismo , Sepse/etiologia , Sepse/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Biópsia , Ceramidas/antagonistas & inibidores , Fatores Quimiotáticos/biossíntese , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Peritonite/mortalidade , Peritonite/patologia , Receptores Imunológicos/antagonistas & inibidores , Sepse/mortalidade , Sepse/patologiaRESUMO
LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo 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 CD300f-/- 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 CD300f-/- 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 in vivo.
Assuntos
Ceramidas/metabolismo , Dermatite/prevenção & controle , Lipopolissacarídeos/toxicidade , Receptores Imunológicos/metabolismo , Animais , Quimiotaxia de Leucócito , Dermatite/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Receptores Imunológicos/genéticaRESUMO
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.
Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Ceramidas/fisiologia , Colite/prevenção & controle , Mastócitos/fisiologia , Receptores Imunológicos/fisiologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Fase G1/genética , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Fase de Repouso do Ciclo Celular/genética , Células 3T3 , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p57/biossíntese , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , MicroRNAs/biossíntese , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteína do Retinoblastoma/biossíntese , Proteínas de Ligação a Selênio/biossíntese , Proteínas de Ligação a Selênio/genética , Células-Tronco/citologia , Proteína Supressora de Tumor p53/biossínteseRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Crise Blástica/metabolismo , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Leucemia Mieloide Aguda/mortalidade , Neoplasias Experimentais/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Substituição de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Benzamidas/farmacologia , Crise Blástica/genética , Crise Blástica/patologia , Feminino , Proteínas de Homeodomínio/genética , Humanos , Mesilato de Imatinib , Interleucina-3/biossíntese , Interleucina-3/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas de Fusão Oncogênica/genética , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Pirimidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição HES-1 , Fatores de Poliadenilação e Clivagem de mRNA/genéticaAssuntos
Ceramidas/farmacologia , Mastócitos/imunologia , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Receptores Imunológicos/agonistas , Esfingomielinas/farmacologia , Animais , Degranulação Celular , Linhagem Celular , Retroalimentação Fisiológica , Humanos , Ligantes , Camundongos , Camundongos Mutantes , Anafilaxia Cutânea Passiva/imunologia , Engenharia de Proteínas , Receptores de IgE/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transgenes/genéticaRESUMO
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-terminaltruncating 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 2mediated (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-MTinduced 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.
Assuntos
Proteínas Mutantes/genética , Mutação , Síndromes Mielodisplásicas/genética , Proteínas Repressoras/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Lectinas Tipo C/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Mielopoese/genética , Fragmentos de Peptídeos/genética , Receptores de Superfície Celular/genéticaRESUMO
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.