RESUMEN
Various gut bacteria, including Lactobacillus plantarum, possess several enzymes that produce hydroxy fatty acids (FAs), oxo FAs, conjugated FAs, and partially saturated FAs from polyunsaturated FAs as secondary metabolites. Among these derivatives, we identified 10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC), a γ-linolenic acid (GLA)-derived enon FA, as the most effective immunomodulator, which inhibited the antigen-induced immunoactivation and LPS-induced production of inflammatory cytokines. The treatment with γKetoC significantly suppressed proliferation of CD4+ T cells, LPS-induced activation of bone marrow-derived dendritic cells (BMDCs), and LPS-induced IL-6 release from peritoneal cells, splenocytes, and CD11c+ cells isolated from the spleen. γKetoC also inhibited the release of inflammatory cytokines from BMDCs stimulated with poly-I:C, R-848, or CpG. Further in vitro experiments using an agonist of GPR40/120 suggested the involvement of these GPCRs in the effects of γKetoC on DCs. We also found that γKetoC stimulated the NRF2 pathway in DCs, and the suppressive effects of γKetoC and agonist of GPR40/120 on the release of IL-6 and IL-12 were reduced in Nrf2-/- BMDCs. We evaluated the role of NRF2 in the anti-inflammatory effects of γKetoC in a dextran sodium sulfate-induced colitis model. The oral administration of γKetoC significantly reduced body weight loss, improved stool scores, and attenuated atrophy of the colon, in wild-type C57BL/6 and Nrf2+/- mice with colitis. In contrast, the pathology of colitis was deteriorated in Nrf2-/- mice even with the administration of γKetoC. Collectively, the present results demonstrated the involvement of the NRF2 pathway and GPCRs in γKetoC-mediated anti-inflammatory responses.
Asunto(s)
Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Factor 2 Relacionado con NF-E2 , Receptores Acoplados a Proteínas G , Transducción de Señal , Animales , Masculino , Ratones , Colitis/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/efectos de los fármacos , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/inmunología , Lactobacillus plantarum , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Ácidos Oléicos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.
Asunto(s)
Antiinflamatorios , Fármacos Antiobesidad , Receptores X del Hígado , Obesidad , Animales , Ratones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Receptores X del Hígado/metabolismo , Receptores X del Hígado/agonistas , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Fármacos Antiobesidad/farmacología , Fármacos Antiobesidad/uso terapéutico , Adipogénesis/efectos de los fármacos , Ésteres/química , Colitis/tratamiento farmacológico , Colitis/inducido químicamente , Colitis/metabolismo , Humanos , Mentol/farmacología , Ratones Endogámicos C57BL , Lipopolisacáridos , Factor de Necrosis Tumoral alfa/metabolismo , Células 3T3-L1 , Sulfato de Dextran , Adipocitos/metabolismo , Adipocitos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Canales Catiónicos TRPM/metabolismoRESUMEN
Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs in vitro and in vivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs in vivo and in vitro involving GPR109A, PGE2, and epigenetic regulation.
Asunto(s)
Anafilaxia , Niacina , Ratones , Animales , Anafilaxia/tratamiento farmacológico , Anafilaxia/metabolismo , Niacina/farmacología , Niacina/metabolismo , Dinoprostona/metabolismo , Butiratos/farmacología , Butiratos/metabolismo , Valeratos/metabolismo , Mastocitos/metabolismo , Epigénesis Genética , Inmunoglobulina E/metabolismo , Degranulación de la CélulaRESUMEN
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/metabolismoRESUMEN
Dendritic cells (DCs), which are typical antigen-presenting cells, localize to various sites in the body, particularly the front line of infection as sentinels, and are involved in innate and adaptive immune responses. Although the functions of DCs, such as pathogen-induced cytokine production and antigen-specific T cell activation, are important for host defenses against infection and tumorigenesis, the hyper- and/or extended activation of DCs leads to inflammatory and autoimmune diseases. In the present study, ß-damascone, a major ingredient of rose fragrance, was selected from an aroma library as a candidate compound that suppresses antigen-induced immune responses. ß-Damascone inhibited the functions of DCs, including the antigen-dependent proliferation of T cells, DC-induced Th1 development, and the TLR ligand-induced production of inflammatory cytokines by DCs. The ß-damascone treatment also increased the protein level of the transcription factor NF-E2-related factor 2 (NRF2), which plays key roles in antioxidant responses, and the transcription of Hmox1 and Nqo1, target genes of NRF2, in DCs. Nrf2 -/ - DCs induced Th1-development and produced large amount of IL-12p40 even in the presence of ß-damascone, whereas these functions by Nrf2 +/- DCs were inhibited by ß-damascone under the same conditions. The intake of ß-damascone suppressed ear swelling in contact hypersensitivity (CHS) model mice, but not in CHS-induced Nrf2 -/ - mice. Collectively, the present results indicate the potential of the rose aroma compound ß-damascone, which suppresses DC-mediated immune responses by activating the NRF2 pathway in DCs, for the prevention and/or attenuation of immune-mediated diseases.
RESUMEN
Group 2 innate lymphoid cells (ILC2s) are tissue-resident innate lymphoid cells that express the transcription factor GATA3 as a master regulator, which leads to the production of large amounts of type 2 cytokines, such as IL-5 and IL-13. ILC2s are activated by epithelial cell-derived cytokines, including IL-33 and IL-25, and play a key role in parasite expulsion, allergic responses, tissue repair, and metabolism. In the first five years after the discovery of ILC2s, research mainly focused on their function through cytokine receptors. However, in recent years, their regulatory mechanisms through not only cytokine receptors but also lipids, neuropeptides, and hormones have become a hot topic. For ILC2s that do not recognize foreign antigens, receptor expression of such endogenous factors is important, and the diverse expression patterns create the individuality of ILC2s in each organ. By considering the mechanisms of differentiation and regulation of ILC2s and their role in disease while taking into account spatio-temporal information, it is expected that new therapeutic strategies targeting ILC2s will be developed. Herein, we summarize the current understanding of ILC2s in lung homeostasis and pathology and provide valuable insights that will help to guide the future development of therapeutic methods for ILC2-mediated lung diseases.
Asunto(s)
Inmunidad Innata , Investigación Biomédica Traslacional , Humanos , Linfocitos , Citocinas/metabolismo , Receptores de Citocinas/metabolismoRESUMEN
Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.
Asunto(s)
Betacianinas , Mirabilis , Animales , Ratones , Betacianinas/análisis , Betacianinas/metabolismo , Verduras/metabolismo , Ingeniería Metabólica , Mirabilis/metabolismo , Lipopolisacáridos , Betalaínas/análisis , Betalaínas/metabolismo , Extractos VegetalesRESUMEN
Two new phenylpropanoids (1 and 2) and one new isoflavone glycoside (3), along with nine known compounds (4 - 12), were isolated from the pod of Ceratonia siliqua L. Their chemical structures were elucidated based on extensive spectroscopic analyses (1 D and 2 D NMR, UV, IR, and HRESIMS) and compared with the literature data. In addition, all isolated compounds were evaluated in vitro for inhibitory activity against acetylcholinesterase (AChE). Compounds 4, 5, and 12 showed inhibitory activity against acetylcholinesterase (AChE) with IC50 values ranging from 15.0 to 50.2 µM.
RESUMEN
Mast cells (MCs) play key roles in IgE-mediated immunoresponses, including in the protection against parasitic infections and the onset and/or symptoms of allergic diseases. IgE-mediated activation induces MCs to release mediators, including histamine and leukotriene, as an early response, and to produce cytokines as a late phase response. Attempts have been made to identify novel antiallergic compounds from natural materials such as Chinese medicines and food ingredients. We herein screened approximately 60 compounds and identified salicylaldehyde, an aromatic aldehyde isolated from plant essential oils, as an inhibitor of the IgE-mediated activation of MCs. A degranulation assay, flow cytometric analyses, and enzyme-linked immunosorbent assays revealed that salicylaldehyde inhibited the IgE-mediated degranulation and cytokine expression of bone-marrow-derived MCs (BMMCs). The salicylaldehyde treatment reduced the surface expression level of FcεRI, the high affinity receptor for IgE, on BMMCs, and suppressed the IgE-induced phosphorylation of tyrosine residues in intercellular proteins, possibly Lyn, Syk, and Fyn, in BMMCs. We also examined the effects of salicylaldehyde in vivo using passive anaphylaxis mouse models and found that salicylaldehyde administration significantly enhanced the recovery of a reduced body temperature due to systemic anaphylaxis and markedly suppressed ear swelling, footpad swelling, and vascular permeability in cutaneous anaphylaxis.
Asunto(s)
Anafilaxia , Mastocitos , Aldehídos/metabolismo , Anafilaxia/tratamiento farmacológico , Anafilaxia/metabolismo , Animales , Degranulación de la Célula , Citocinas/metabolismo , Inmunoglobulina E/metabolismo , Mastocitos/metabolismo , Ratones , Receptores de IgE/metabolismo , Transducción de SeñalRESUMEN
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/metabolismoRESUMEN
NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1ß and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1ß in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.
Asunto(s)
Inflamasomas/genética , Factores Reguladores del Interferón/metabolismo , Macrófagos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Regulación de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Factores Reguladores del Interferón/genética , Macrófagos/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Especificidad de la Especie , Células THP-1 , Transactivadores/genética , Células U937RESUMEN
Dendritic cells (DCs) and T cells play important roles in immune regulation, and modulating their function is an approach for developing preventive or therapeutic strategies against immune disorders. Herein, the effect of pterostilbene (PSB) (3',5'-dimethoxy-resveratrol)-a resveratrol-related polyphenol found in blueberries-on immune regulation was evaluated. Using an in vitro co-culture system, PSB was found to exert the strongest inhibitory effect among all tested resveratrol derivatives on DC-mediated T cell proliferation; moreover, PSB treatment decreased the Th1 and Th17 populations and increased the regulatory T cell (Treg) population. Upon co-stimulation with anti-CD3 and anti-CD28 antibodies, PSB inhibited CD4+ T cell proliferation and differentiation into Th1 cells. Additionally, PSB acted on DCs to suppress the lipopolysaccharide-induced transactivation of genes encoding antigen presentation-related molecules and inflammatory cytokines by attenuating the DNA-binding ability of the transcription factor PU.1. Furthermore, PSB promoted DC-mediated Foxp3+ Treg differentiation, and PU.1 knockdown increased DC-induced Treg activity. Oral administration of PSB alleviated the symptoms of dextran sulfate sodium-induced colitis and decreased tumor necrosis factor-α expression in mice. Thus, PSB treatment ameliorates colonic inflammation.
Asunto(s)
Antiinflamatorios/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Células Dendríticas/inmunología , Estilbenos/farmacología , Linfocitos T Reguladores/inmunología , Animales , Antiinflamatorios/uso terapéutico , Línea Celular , Proliferación Celular , Células Cultivadas , Colitis Ulcerosa/inmunología , Colon/efectos de los fármacos , Colon/inmunología , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/metabolismo , Estilbenos/uso terapéutico , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/fisiología , Células TH1/inmunología , Células TH1/fisiología , Células Th17/inmunología , Células Th17/fisiología , Transactivadores/metabolismoRESUMEN
We evaluated the effect of gut bacterial metabolites of polyunsaturated fatty acids on inflammation and found that 10-oxo-cis-6,trans-11-octadecadienoic acid (γKetoC) strikingly suppressed LPS-induced IL-6 release from bone marrow-derived macrophages (BMMs), which was accompanied by reduced mRNA expression of Il6, TNF, and Il1b. γKetoC decreased the cAMP concentration in BMMs, suggesting that γKetoC stimulated G protein-coupled receptors. A Gq agonist significantly suppressed LPS-induced IL-6 expression in BMMs, whereas a Gi inhibitor partially abrogated γKetoC-mediated IL-6 suppression. Cytosolic Ca2+ was markedly increased by γKetoC, which was partly but not fully abrogated by an ion channel inhibitor. Taken together, these data suggest that γKetoC suppresses inflammatory cytokine expression in macrophages primarily through Gq and partially through Gi. γKetoC suppressed osteoclast development and IL-6 expression in synovial fibroblasts from rheumatoid arthritis (RA) patients, suggesting the beneficial effect of γKetoC on the prevention or treatment of RA.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Microbioma Gastrointestinal , Lactobacillales/metabolismo , Monocitos/metabolismo , Animales , Artritis Reumatoide/etiología , Artritis Reumatoide/metabolismo , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Inflamación/etiología , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores Protectores , Células RAW 264.7RESUMEN
PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and macrophages. The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of cancer immunology. To reveal the mechanism by which Pdcd1lg2 gene expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell-specific transcription factors PU.1 and IRF4 decreased PD-L2 expression in GM-CSF-induced mouse bone marrow-derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 gene expression, which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow-derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic regulator in DCs.
Asunto(s)
Células Dendríticas/inmunología , Epigénesis Genética/inmunología , Factores Reguladores del Interferón/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/inmunología , Proteínas Proto-Oncogénicas/inmunología , Transactivadores/inmunología , Activación Transcripcional/inmunología , Animales , Células Dendríticas/citología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
C-C chemokine receptor type 7 (CCR7) is expressed on naïve T cells, B cells, and activated dendritic cells (DCs). We previously demonstrated that the transcription factor PU.1/Spi1 positively regulates the expression of CCR7 in DCs. In the present study, we investigated the role of PU.1 in CCR7 expression in T cells. To confirm whether PU.1 is involved in the expression of CCR7, we conducted a ChIP assay in various T cells purified from splenocytes and thymocytes and found that PU.1 binds to the Ccr7 promoter-proximal region in spleen naïve CD4+ T cells, but not in thymocytes. Small interfering RNA-mediated PU.1 knockdown resulted in decreased CCR7 expression in spleen naïve CD4+ T cells. Compared to naïve CD4+ T cells, Spi1 and Ccr7 mRNA levels decreased in Th1 and Th2 cells, in which PU.1 did not bind to the Ccr7 promoter, suggesting that CCR7 expression decreases due to the dissociation of PU.1 from the Ccr7 promoter during the development of effector T cells from naïve T cells. Collectively, we concluded that CCR7 expression level correlates with the binding level of PU.1 to the Ccr7 promoter and PU.1 acts as a transcriptional activator of the Ccr7 gene in naïve CD4+ T cells.
Asunto(s)
Proteínas Proto-Oncogénicas/metabolismo , Receptores CCR7/genética , Células TH1/metabolismo , Células Th2/metabolismo , Transactivadores/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Técnicas de Silenciamiento del Gen , Ratones , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Células TH1/inmunología , Células Th2/inmunología , Transactivadores/genética , Activación Transcripcional/inmunologíaRESUMEN
Mouse mast cell proteases (mMCP)-1 and -2 are specifically expressed in mucosal mast cells (MCs). However, the transcriptional regulation mechanism of the Mcpt1 and Mcpt2 genes induced in mucosal MCs is largely unknown. In the current study, we found that TGF-ß stimulation drastically induced upregulation of Mcpt1 and Mcpt2 mRNA in mouse bone marrow-derived MCs (BMMCs). TGF-ß-induced expression of Mcpt1 and Mcpt2 was markedly suppressed by transfection with small interfering RNA targeting Smad2 or Smad4 and moderately reduced by Smad3 small interfering RNA. We next examined the roles of the hematopoietic cell-specific transcription factors GATA1 and GATA2 in the expression of Mcpt1 and Mcpt2 and demonstrated that knockdown of GATA1 and GATA2 reduced the mRNA levels of Mcpt1 and Mcpt2 in BMMCs. The recruitment of GATA2 and acetylation of histone H4 of the highly conserved GATA-Smad motifs, which were localized in the distal regions of the Mcpt1 and Mcpt2 genes, were markedly increased by TGF-ß stimulation, whereas the level of GATA2 binding to the proximal GATA motif was not affected by TGF-ß. A reporter assay showed that TGF-ß stimulation upregulated GATA2-mediated transactivation activity in a GATA-Smad motif-dependent manner. We also observed that GATA2 and Smad4 interacted in TGF-ß-stimulated BMMCs via immunoprecipitation and Western blotting analysis. Taken together, these results demonstrate that TGF-ß induced mMCP-1 and -2 expression by accelerating the recruitment of GATA2 to the proximal regions of the Mcpt1 and Mcpt2 genes in mucosal MCs.
Asunto(s)
Quimasas/genética , Inmunidad Mucosa/genética , Mastocitos/inmunología , Activación Transcripcional/inmunología , Animales , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mastocitos/metabolismo , Ratones , Membrana Mucosa/citología , Membrana Mucosa/inmunología , Cultivo Primario de Células , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
Mast cells (MCs) play a central role in IgE-dependent immune responses. PPARγ is a nuclear receptor that is essential for adipocyte differentiation and insulin sensitivity. Although PPARγ is expressed in activated MCs, the effect of PPARγ suppression in IgE-mediated activation of MCs is largely unknown. In the current study, we evaluated the effect of PPARγ knockdown on the function of IgE plus antigen (Ag)-stimulated MCs using siRNA-transfected bone marrow-derived MCs (BMMCs). We found that the mRNA expression level of cytokines in IgE/Ag-stimulated BMMCs was significantly increased in PPARγ knockdown BMMCs, and IgE/Ag-mediated degranulation and the protein production level of TNF-α was moderately increased by PPARγ knockdown, whereas the cell surface expression level of FcεRI was not affected by PPARγ knockdown. Oral administration of pioglitazone (PPARγ agonist) significantly suppressed body temperature change of mice in passive systemic anaphylaxis, supporting the inhibitory functions of PPARγ in IgE/Ag-dependent activation of MCs in vivo. IgE-mediated up-regulation of mRNA levels of Ptgs2 (encoding COX-2) was drastically enhanced in PPARγ knockdown BMMCs. Although several prostaglandin (PG) derivatives are known to be ligands for PPARγ, treatment with a COX inhibitor, acetyl salicylic acid, up-regulated the IgE-mediated increase of Il13, Tnf and Ptgs2 mRNA levels in a synergistic manner with PPARγ siRNA. Knockdown of COX-1 and/or COX-2 by siRNA showed that suppression of IgE/Ag-mediated activation was mainly dependent on COX-1. Taken together, these results indicate that PPARγ suppresses IgE/Ag-induced transactivation of cytokine genes and the Ptgs2 gene in MCs in a manner distinguishable from that of PGs.
Asunto(s)
Células de la Médula Ósea/inmunología , Inmunoglobulina E/inmunología , Mastocitos/inmunología , PPAR gamma/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR gamma/agonistas , PPAR gamma/deficiencia , ARN Interferente Pequeño/farmacologíaRESUMEN
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/genéticaRESUMEN
The immunosuppressive activity of myriocin (ISP-1), a lead compound of fingolimod (FTY720), is derived from its 2-amino-1,3-propandiol structure. A non-proteinogenic amino acid, (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH), that contains this structure, was recently identified as a biosynthetic intermediate of a dipeptide secondary metabolite, vazabitide A, in Streptmyces sp. SANK 60404; however its effect on adaptive immunity has not yet been examined. In this study, we examined whether DADH suppresses mixed lymphocyte reaction using mouse bone marrow-derived dendritic cells (BMDCs) and allogeneic splenic T cells. Although T cell proliferation induced by cross-linking CD3 and CD28 were not suppressed by DADH unlike ISP-1, the pre-incubation of BMDCs with DADH but not ISP-1 significantly decreased allogeneic CD8+ T cell expansion. Based on these results, we concluded that DADH suppresses DC-mediated T cell activation by targeting DCs.