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1.
J Immunol ; 207(12): 3098-3106, 2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34799426

RESUMEN

Rodent mast cells are classified into two major subsets, mucosal mast cells (MMCs) and connective tissue mast cells. MMCs arise from mast cell progenitors that are mobilized from the bone marrow to mucosal tissues in response to allergic inflammation or helminth infection. TGF-ß is known as an inducer of MMC differentiation in mucosal tissues, but we have previously found that Notch receptor-mediated signaling also leads to the differentiation. Here, we examined the relationship between Notch and TGF-ß signaling in MMC differentiation using mouse bone marrow-derived mast cells (BMMCs). We found that the coexistence of Notch and TGF-ß signaling markedly upregulates the expression of MMC markers, mouse mast cell protease (mMCP)-1, mMCP-2, and αE integrin/CD103, more than Notch or TGF-ß signaling alone, and that their signals act interdependently to induce these marker expressions. Notch and TGF-ß-mediated transcription of MMC marker genes were both dependent on the TGF-ß signaling transducer SMAD4. In addition, we also found that Notch signaling markedly upregulated mMCP-1 and mMCP-2 expression levels through epigenetic deregulation of the promoter regions of these genes, but did not affect the promoter of the CD103-encoding gene. Moreover, forced expression of the constitutively active Notch2 intracellular domain in BMMCs showed that Notch signaling promotes the nuclear localization of SMADs 3 and 4 and causes SMAD4-dependent gene transcription. These findings indicate that Notch and TGF-ß signaling play interdependent roles in inducing the differentiation and maturation of MMCs. These roles may contribute to the rapid expansion of the number of MMCs during allergic mucosal inflammation.


Asunto(s)
Mastocitos , Factor de Crecimiento Transformador beta , Animales , Expresión Génica , Inflamación/metabolismo , Mastocitos/metabolismo , Ratones , Membrana Mucosa , Factor de Crecimiento Transformador beta/metabolismo
2.
Int J Mol Sci ; 24(6)2023 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-36983066

RESUMEN

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcεRI on BMMCs, but the mRNA levels of FcεRIα, ß, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcεRI on BMMCs was still observed when protein synthesis or protein transporter was inhibited. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NF-E2-related factor 2 (NRF2)-a master transcription factor of antioxidant stress-in BMMCs, the inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we found that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. The kaempferol-induced upregulation of SHIP1 was also observed in peritoneal MCs. The knockdown of SHIP1 by siRNA significantly enhanced IgE-induced degranulation of BMMCs. A Western blotting analysis showed that IgE-induced phosphorylation of PLCγ was suppressed in kaempferol-treated BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcεRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.


Asunto(s)
Mastocitos , Receptores de IgE , Degranulación de la Célula , Inmunoglobulina E/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Quempferoles/farmacología , Quempferoles/metabolismo , Mastocitos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , ARN Mensajero/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo
3.
J Allergy Clin Immunol ; 147(3): 1063-1076.e9, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-32717254

RESUMEN

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 TH2 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.


Asunto(s)
Desensibilización Inmunológica/métodos , Hipersensibilidad a los Alimentos/inmunología , Células Supresoras de Origen Mieloide/inmunología , Receptores Notch/metabolismo , Células Th2/inmunología , Administración Oral , Alérgenos/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Hipersensibilidad a los Alimentos/terapia , Humanos , Tolerancia Inmunológica , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Transducción de Señal
4.
FASEB J ; 33(10): 11481-11491, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31314592

RESUMEN

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.


Asunto(s)
Movimiento Celular/genética , Células Dendríticas/fisiología , Ganglios Linfáticos/fisiología , Tejido Linfoide/fisiología , Proteínas Proto-Oncogénicas/genética , Receptores CCR7/genética , Transactivadores/genética , Animales , Línea Celular , Femenino , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genética
5.
J Immunol ; 199(8): 2958-2967, 2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28893954

RESUMEN

NR4A3/NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, which is activated in a ligand-independent manner. To examine the role of NR4A3 in gene expression of dendritic cells (DCs), we introduced NR4A3 small interfering RNA (siRNA) into bone marrow-derived DCs and determined the expression levels of mRNA and proteins of cytokines, cell surface molecules, NF-κB signaling-related proteins, and transcription factors. The expression level of NR4A3 was markedly upregulated by TLR-mediated stimulation in DCs. NR4A3 knockdown significantly suppressed LPS, CpG, or poly(I:C)-mediated upregulation of CD80, CD86, IL-10, IL-6, and IL-12. Proliferation and IL-2 production levels of T cells cocultured with NR4A3 knocked-down DCs were significantly lower than that of T cells cocultured with control DCs. Furthermore, the expression of IKKß, IRF4, and IRF8 was significantly decreased in NR4A3 siRNA-introduced bone marrow-derived DCs. The knockdown experiments using siRNAs for IKKß, IRF4, and/or IRF8 indicated that LPS-induced upregulation of IL-10 and IL-6 was reduced in IKKß knocked-down cells, and that the upregulation of IL-12 was suppressed by the knockdown of IRF4 and IRF8. Taken together, these results indicate that NR4A3 is involved in TLR-mediated activation and gene expression of DCs.


Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/inmunología , Activación de Linfocitos , Proteínas del Tejido Nervioso/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal , Receptores Toll-Like/inmunología
6.
Int Immunol ; 29(2): 87-94, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28338898

RESUMEN

PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family, which plays an important role in the development of dendritic cells (DCs). CD11c (encoded by Itgax) is well established as a characteristic marker of hematopoietic lineages including DCs. In the present study, we analyzed the role of PU.1 (encoded by Spi-1) in the expression of CD11c. When small interfering RNA (siRNA) for Spi-1 was introduced into bone marrow-derived DCs (BMDCs), the mRNA level and cell surface expression of CD11c were dramatically reduced. Using reporter assays, the TTCC sequence at -56/-53 was identified to be critical for PU.1-mediated activation of the promoter. An EMSA showed that PU.1 directly bound to this region. ChIP assays demonstrated that a significant amount of PU.1 bound to this region on chromosomal DNA in BMDCs, which was decreased in LPS-stimulated BMDCs in accordance with the reduced levels of mRNAs of Itgax and Spi-1, and the histone acetylation degree. Enforced expression of exogenous PU.1 induced the expression of the CD11c protein on the cell surface of mast cells, whereas control transfectants rarely expressed CD11c. Quantitative RT-PCR also showed that the expression of a transcription factor Irf4, which is a partner molecule of PU.1, was reduced in PU.1-knocked down BMDCs. IRF4 transactivated the Itgax gene in a synergistic manner with PU.1. Taken together, these results indicate that PU.1 functions as a positive regulator of CD11c gene expression by directly binding to the Itgax promoter and through transactivation of the Irf4 gene.


Asunto(s)
Antígeno CD11c/metabolismo , Células Dendríticas/fisiología , Hematopoyesis , Factores Reguladores del Interferón/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Acetilación , Animales , Antígeno CD11c/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Hematopoyesis/genética , Histonas/metabolismo , Factores Reguladores del Interferón/genética , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Transactivadores/genética , Activación Transcripcional
7.
J Immunol ; 196(9): 3559-69, 2016 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-27001956

RESUMEN

Protease activity of papain, a plant-derived occupational allergen homologous to mite major allergens, is essential to IgE/IgG1 production and lung eosinophilia induced by intranasal papain administration in mice, and IL-33 contributes to these responses. In this work, we investigate skin and Ab responses induced by s.c. papain administration into ear lobes and responses induced by subsequent airway challenge with papain. Subcutaneous papain injection induced swelling associated with increased epidermal thickness, dermal inflammation, serum IgE/IgG1 responses, and Th2 cytokine production in draining lymph node cells restimulated in vitro. These responses were markedly less upon s.c. administration of protease inhibitor-treated papain. Results obtained by using mast cell-deficient mice and reconstitution of tissue mast cells suggested the contribution of mast cells to papain-specific IgE/IgG1 responses and eosinophil infiltration. The responses were equivalent between wild-type and IL-33(-/-) mice. After the subsequent airway challenge, the s.c. presensitized wild-type mice showed more severe lung eosinophilia than those without the presensitization. The presensitized IL-33(-/-) mice showed modest lung eosinophilia, which was absent without the presensitization, but its severity and IgE boost by the airway challenge were markedly less than the presensitized wild-type mice, in which protease activity of inhaled papain contributed to the responses. The results suggest that mechanisms for the protease-dependent sensitization differ between skin and airway and that cooperation of mast cell-dependent, IL-33-independent initial sensitization via skin and protease-induced, IL-33-mediated mechanism in re-exposure via airway to protease allergens maximizes the magnitude of the transition from skin inflammation to asthma in natural history of progression of allergic diseases.


Asunto(s)
Alérgenos/administración & dosificación , Alérgenos/inmunología , Hipersensibilidad/inmunología , Interleucina-33/inmunología , Mastocitos/inmunología , Absorción Nasal , Péptido Hidrolasas/inmunología , Absorción Subcutánea , Animales , Asma , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/patología , Eosinófilos/inmunología , Hipersensibilidad/patología , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inflamación , Interleucina-33/deficiencia , Pulmón/inmunología , Ratones , Papaína/administración & dosificación , Papaína/inmunología , Péptido Hidrolasas/administración & dosificación , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/patología , Piel/inmunología , Piel/patología , Células Th2/inmunología
8.
J Allergy Clin Immunol ; 139(3): 987-996.e10, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27555456

RESUMEN

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.


Asunto(s)
Hipersensibilidad a los Alimentos/inmunología , Mucosa Intestinal/inmunología , Mastocitos/inmunología , Receptores Notch/antagonistas & inhibidores , Alérgenos/inmunología , Animales , Citocinas/inmunología , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Femenino , Hipersensibilidad a los Alimentos/tratamiento farmacológico , Hipersensibilidad a los Alimentos/patología , Hiperplasia/tratamiento farmacológico , Hiperplasia/inmunología , Hiperplasia/patología , Intestino Delgado/citología , Mastocitos/patología , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Receptores Notch/inmunología , Transducción de Señal/efectos de los fármacos
10.
J Immunol ; 194(9): 4535-44, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25821223

RESUMEN

Th2-type cytokines and TNF-α secreted by activated mast cells upon cross-linking of FcεRI contribute to the development and maintenance of Th2 immunity to parasites and allergens. We have previously shown that cytokine secretion by mouse mast cells is enhanced by signaling through Notch receptors. In this study, we investigated the molecular mechanisms by which Notch signaling enhances mast cell cytokine production induced by FcεRI cross-linking. FcεRI-mediated production of cytokines, particularly IL-4, was significantly enhanced in mouse bone marrow-derived mast cells by priming with Notch ligands. Western blot analysis showed that Notch signaling augmented and prolonged FcεRI-mediated phosphorylation of MAPKs, mainly JNK and p38 MAPK, through suppression of the expression of SHIP-1, a master negative regulator of FcεRI signaling, resulting in the enhanced production of multiple cytokines. The enhancing effect of Notch ligand priming on multiple cytokine production was abolished by knockdown of Notch2, but not Notch1, and FcεRI-mediated production of multiple cytokines was enhanced by retroviral transduction with the intracellular domain of Notch2. However, only IL-4 production was enhanced by both Notch1 and Notch2. The enhancing effect of Notch signaling on IL-4 production was lost in bone marrow-derived mast cells from mice lacking conserved noncoding sequence 2, which is located at the distal 3' element of the Il4 gene locus and contains Notch effector RBP-J binding sites. These results indicate that Notch2 signaling indirectly enhances the FcεRI-mediated production of multiple cytokines, and both Notch1 and Notch2 signaling directly enhances IL-4 production through the noncoding sequence 2 enhancer of the Il4 gene.


Asunto(s)
Citocinas/biosíntesis , Mastocitos/inmunología , Mastocitos/metabolismo , Receptores de IgE/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Línea Celular , Citocinas/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Inositol Polifosfato 5-Fosfatasas , Ligandos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transcripción Genética
11.
J Immunol ; 195(7): 3427-35, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26297757

RESUMEN

FcεRI, which is composed of α, ß, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-ß1 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-ß1 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-ß/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIß, 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, FcεRIß, 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-ß1 was mimicked by forced expression of Ehf, suggesting that TGF-ß1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.


Asunto(s)
Proteínas Proto-Oncogénicas c-kit/biosíntesis , Receptores de IgE/inmunología , Factores de Transcripción/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células de la Médula Ósea , Degranulación de la Célula/inmunología , Células Cultivadas , Citocinas/biosíntesis , Factor de Transcripción GATA1/biosíntesis , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA2/biosíntesis , Factor de Transcripción GATA2/genética , Inmunoglobulina E/inmunología , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-kit/genética , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Receptores de IgE/biosíntesis , Factor de Transcripción STAT5/biosíntesis , Factor de Transcripción STAT5/genética , Transducción de Señal/inmunología , Proteínas Smad/metabolismo , Factores de Transcripción/biosíntesis , Transcripción Genética/genética , Activación Transcripcional
12.
Allergol Int ; 66(3): 472-478, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28259547

RESUMEN

BACKGROUND: Interleukin-33 (IL-33) is an alarmin cytokine that binds to the interleukin 1 receptor-like 1 protein ST2. Clock is a key circadian gene that is essential for endogenous clockworks in mammals. This study investigated whether Clock temporally regulated IL-33-mediated responses in mast cells. METHODS: The kinetics of IL-33-mediated IL-6, IL-13, and TNF-α productions were compared between bone marrow-derived mast cells (BMMCs) from wild-type and Clock-mutated mice (ClockΔ19/Δ19 mice). The kinetics of the neutrophil influx into the peritoneal cavity or expression of IL-13 and Gob-5 in the lung in response to IL-33 were compared between wild-type and ClockΔ19/Δ19 mice. We also examined the kinetics of ST2 expression in mast cells and its association with Clock expression. RESULTS: There was a time-of-day-dependent variation in IL-33-mediated IL-6, IL-13, and TNF-α production in wild-type BMMCs, which was absent in Clock-mutated BMMCs. IL-33-induced neutrophil infiltration into the peritoneal cavity also showed a time-of-day-dependent variation in wild-type mice, which was absent in ClockΔ19/Δ19 mice. Furthermore, IL-33-induced IL-13 and Gob-5 expression in the lung exhibited a time-of-day-dependent variation in wild-type mice. These temporal variations in IL-33-mediated mast cell responses were associated with temporal variations of ST2 expression in mast cells. In addition, CLOCK bound to the promoter region of ST2 and Clock deletion resulted in down-regulation of ST2 expression in mast cells. CONCLUSIONS: CLOCK temporally gates mast cell responses to IL-33 via regulation of ST2 expression. Our findings provide novel insights into IL-33/mast cell-associated physiology and pathologies.


Asunto(s)
Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Animales , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica , Proteína 1 Similar al Receptor de Interleucina-1/genética , Interleucina-33/farmacología , Masculino , Mastocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fotoperiodo
13.
J Immunol ; 192(8): 3936-46, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24639354

RESUMEN

The high-affinity IgE receptor, FcεRI, which is composed of α-, ß-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of ß mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIß). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIß, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIß transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.


Asunto(s)
Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica , Mastocitos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de IgE/genética , Transactivadores/metabolismo , Línea Celular , Membrana Celular/metabolismo , Inmunoprecipitación de Cromatina , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA2/genética , Técnicas de Silenciamiento del Gen , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/genética , Activación Transcripcional
14.
Allergol Int ; 65(1): 44-51, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26666495

RESUMEN

BACKGROUND: Patients with house dust mite (HDM) allergy or Ascariasis produce serum IgE specific to the antigens of HDM or nematode Ascaris, respectively. Although human IgE cross-reactivity has been reported between HDM and Ascaris antigens, it remains unclear whether it contributes to the pathogenesis of allergic diseases. We herein investigated the induction of cross-reactive antibodies and T cells in mice and effects of airway exposure to HDM antigens after preimmunization with Ascaris antigens. METHODS: Mice were intraperitoneally immunized with HDM or Ascaris antigens with Alum, followed by the intranasal administration of HDM antigens. Serum antigen-specific IgE and IgG were measured by ELISA. Cytokine release in splenocytes from Ascaris-immunized mice upon in vitro restimulation with HDM antigens were measured by ELISA. RESULTS: Immunization with Ascaris or HDM antigens induced cross-reactive IgG1. Splenocytes from Ascaris-immunized mice released IL-5 and IL-13 in response to the restimulation with HDM antigens. Subsequent airway exposure to HDM antigens promoted the induction of HDM-specific IgE and upregulation of HDM-specific IgG1 in Ascaris-immunized mice, whereas these responses were not detected or smaller without the Ascaris presensitization. CONCLUSIONS: We demonstrated that the immunization of naïve mice with Ascaris antigens induced production of antibodies and differentiation of Th2 cells, which were cross-reactive to HDM antigens, and accelerated induction of serum HDM-specific IgE upon subsequent airway exposure to HDM antigens in mice. These results suggest that sensitization to HDM towards IgE-mediated allergic diseases is faster in individuals with a previous history of Ascaris infection than in those without presensitization to Ascaris.


Asunto(s)
Antígenos Dermatofagoides/inmunología , Antígenos Helmínticos/inmunología , Ascaris/inmunología , Hipersensibilidad/inmunología , Inmunización , Inmunoglobulina E/inmunología , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Hipersensibilidad/sangre , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Bazo/citología , Bazo/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología
15.
J Immunol ; 190(9): 4489-99, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23547117

RESUMEN

How the innate and adaptive immune systems cooperate in the natural history of allergic diseases has been largely unknown. Plant-derived allergen, papain, and mite allergens, Der f 1 and Der p 1, belong to the same family of cysteine proteases. We examined the role of protease allergens in the induction of Ab production and airway inflammation after repeated intranasal administration without adjuvants and that in basophil/mast cell stimulation in vitro. Papain induced papain-specific IgE/IgG1 and lung eosinophilia. Der f 1 induced Der f 1-specific IgG1 and eosinophilia. Although papain-, Der f 1-, and Der p 1-stimulated basophils expressed allergy-inducing cytokines, including IL-4 in vitro, basophil-depleting Ab and mast cell deficiency did not suppress the papain-induced in vivo responses. Protease inhibitor-treated allergens and a catalytic site mutant did not induce the responses. These results indicate that protease activity is essential to Ab production and eosinophilia in vivo and basophil activation in vitro. IL-33-deficient mice lacked eosinophilia and had reduced papain-specific IgE/IgG1. Coadministration of OVA with papain induced OVA-specific IgE/IgG1, which was reduced in IL-33-deficient mice. We demonstrated IL-33 release, subsequent IL-33-dependent IL-5/IL-13 release, and activation of T1/ST2-expressing lineage(-)CD25(+)CD44(+) innate lymphoid cells in the lung after papain inhalation, suggesting the contribution of the IL-33-type 2 innate lymphoid cell-IL-5/IL-13 axis to the papain-induced airway eosinophilia. Rag2-deficient mice, which lack adaptive immune cells, showed significant, but less severe, eosinophilia. Collectively, these results suggest cooperation of adaptive immune cells and IL-33-responsive innate cells in protease-dependent allergic airway inflammation.


Asunto(s)
Inmunidad Adaptativa/inmunología , Alérgenos/inmunología , Proteasas de Cisteína/inmunología , Hipersensibilidad/inmunología , Inmunidad Innata/inmunología , Interleucinas/inmunología , Pulmón/inmunología , Animales , Formación de Anticuerpos/inmunología , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Basófilos/inmunología , Cisteína Endopeptidasas/inmunología , Femenino , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Inflamación/inmunología , Interleucina-13/inmunología , Interleucina-33 , Interleucina-5/inmunología , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Papaína/inmunología , Eosinofilia Pulmonar/inmunología
16.
J Allergy Clin Immunol ; 133(2): 568-75, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24060274

RESUMEN

BACKGROUND: It remains elusive how allergic symptoms exhibit prominent 24-hour variations. In mammals the circadian clocks present in nearly all cells, including mast cells, drive the daily rhythms of physiology. Recently, we have shown that the circadian clocks drive the daily rhythms in IgE/mast cell-mediated allergic reactions. However, the precise mechanisms, particularly the specific roles of the mast cell-intrinsic clockwork in temporal regulation, remain unclear. OBJECTIVE: We determined whether the mast cell clockwork contributes to the temporal regulation of IgE/mast cell-mediated allergic reaction. METHODS: The kinetics of a time of day-dependent variation in passive cutaneous anaphylactic reactions were compared between mast cell-deficient mice reconstituted with bone marrow-derived cultured mast cells generated from mice with a wild-type allele and a dominant negative type mutation of the key clock gene Clock. We also examined the temporal responses of wild-type and Clock-mutated bone marrow-derived cultured mast cells to IgE stimulation in vitro. Furthermore, factors influencing the mast cell clockwork were determined by using in vivo imaging. RESULTS: The Clock mutation in mast cells resulted in the absence of temporal variations in IgE-mediated degranulation in mast cells both in vivo and in vitro associated with the loss of temporal regulation of FcεRI expression and signaling. Additionally, adrenalectomy abolished the mast cell clockwork in vivo. CONCLUSION: The mast cell-intrinsic clockwork, entrained by humoral factors from the adrenal gland, primarily contributes to the temporal regulation of IgE/mast cell-mediated allergic reactions. Our results reveal a novel regulatory mechanism for IgE-mediated mast cell responses that might underlie the circadian pathophysiology in patients with allergic diseases.


Asunto(s)
Anafilaxia/inmunología , Ritmo Circadiano/inmunología , Mastocitos/inmunología , Glándulas Suprarrenales/inmunología , Anafilaxia/fisiopatología , Animales , Proteínas CLOCK/genética , Inmunoglobulina E/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Circadianas Period/genética , Receptores de IgE/inmunología
17.
Allergol Int ; 64(3): 241-7, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26117255

RESUMEN

BACKGROUND: The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-specific gene expression and a deficiency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufficient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated. METHODS: Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immunoprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1. RESULTS: Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-specific genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-specific genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1. CONCLUSIONS: These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.


Asunto(s)
Factores de Transcripción NFATC/metabolismo , Osteoclastos/patología , Osteoporosis/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos , Factores de Transcripción NFATC/genética , Osteoporosis/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Transactivadores/genética , Activación Transcripcional , Transcriptoma
19.
Asian Pac J Allergy Immunol ; 32(2): 178-84, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25003733

RESUMEN

BACKGROUND: Maternal milk-borne transforming growth factor (TGF-ß plays a potential role in the development of the mucosal immune system in infants. However, it remains unclear what factors determine TGF-ß levels in breast milk. We hypothesized that microbial pressures during pregnancy might affect the expression levels of TGF-ß in colostrum. OBJECTIVE: This study compared TGF-ß2 levels in colostrum of lactating women living in Japan and Nepal with contrasting hygiene statuses. Additionally, we identified environmental and intrinsic factors influencing TGF-ß levels in colostrum. METHODS: Breast milk samples and structured questionnaires were collected from 80 women living in Japan and 208 women living in Nepal. A robust regression model was used to identify factors associated with colostral TGF-ß levels. RESULTS: Analysis using the Mann-Whitney U test showed that TGF-ß levels were significantly higher in Japanese women than in Nepalese women. Japanese women who consumed animal milk daily during pregnancy and had atopic dermatitis expressed lower levels of TGF-ß in colostrum, as compared to Japanese women who did not. Among Nepalese women, large family size and higher birth order were associated with lower TGF-ß levels and women who gave birth to infants with low birth weight had higher expression of TGF-ß levels in milk than women who gave birth to infants with normal birth weight. CONCLUSION: The results suggest that induction of TGF-ß levels in colostrum depends on differences in the ethnicity of lactating women. Consumption of animal protein and parturition characteristics may affect TGF-ß levels in breast milk, and may explain differences in these levels in breast milk between countries.


Asunto(s)
Calostro/metabolismo , Dermatitis Atópica/metabolismo , Lactancia , Complicaciones del Embarazo/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Adulto , Animales , Bovinos , Proteínas en la Dieta/administración & dosificación , Femenino , Humanos , Japón , Leche , Nepal , Embarazo , Encuestas y Cuestionarios
20.
J Biol Chem ; 287(39): 32689-96, 2012 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-22865859

RESUMEN

The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.


Asunto(s)
Basófilos/metabolismo , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica/fisiología , Mastocitos/metabolismo , Receptores de Superficie Celular/biosíntesis , Elementos de Respuesta/fisiología , Transactivadores/metabolismo , Basófilos/citología , Línea Celular Tumoral , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/genética , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Masculino , Mastocitos/citología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/genética , Transactivadores/genética , Transcripción Genética/fisiología
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