RESUMEN
Maoto, a traditional Japanese medicine (Kampo), is widely used to treat upper respiratory tract infections, including influenza virus infection. Although maoto is known to inhibit pro-inflammatory responses in a rodent model of acute inflammation, its underlying mechanism remains to be determined. In this study, we investigated the involvement of immune responses and noradrenergic function in the inhibitory action of maoto. In a mouse model of polyI:C-induced acute inflammation, maoto was administered orally in conjunction with intraperitoneal injection of PolyI:C (6 mg/kg), and blood was collected after 2 h for measurement of plasma cytokines by ELISA. Maoto significantly decreased PolyI:C-induced TNF-α levels and increased IL-10 production. Neither pretreatment with IL-10 neutralizing antibodies nor T-cell deficiency using nude mice modified the inhibitory effect of maoto, indicating that the anti-inflammatory effects of maoto are independent of IL-10 and T cells. Furthermore, the inhibitory effects of maoto on PolyI:C-induced TNF-α production were not observed in ex vivo splenocytes, suggesting that maoto does not act directly on inflammatory cells. Lastly, pretreatment with a ß-adrenergic receptor antagonist partially cancelled the anti-inflammatory effects of maoto. Collectively, these results suggest that maoto mediates its anti-inflammatory effects via ß-adrenergic receptors in vivo.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Antiinflamatorios/farmacología , Inflamación/prevención & control , Interleucina-10/genética , Extractos Vegetales/farmacología , Receptores Adrenérgicos beta/genética , Administración Oral , Animales , Modelos Animales de Enfermedad , Efedrina/farmacología , Regulación de la Expresión Génica , Inyecciones Intraperitoneales , Interleucina-10/agonistas , Interleucina-10/inmunología , Japón , Masculino , Medicina Kampo/métodos , Ratones Endogámicos BALB C , Ratones Desnudos , Poli I-C/administración & dosificación , Poli I-C/antagonistas & inhibidores , Receptores Adrenérgicos beta/inmunología , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Airway epithelial barrier dysfunction is increasingly recognized as a key feature of asthma and other lung diseases. Respiratory viruses are responsible for a large fraction of asthma exacerbations, and are particularly potent at disrupting epithelial barrier function through pattern recognition receptor engagement leading to tight junction dysfunction. Although different mechanisms of barrier dysfunction have been described, relatively little is known about whether barrier integrity can be promoted to limit disease. Here, we tested three classes of drugs commonly prescribed to treat asthma for their ability to promote barrier function using a cell culture model of virus-induced airway epithelial barrier disruption. Specifically, we studied the corticosteroid budesonide, the long acting beta-agonist formoterol, and the leukotriene receptor antagonist montelukast for their ability to promote barrier integrity of a monolayer of human bronchial epithelial cells (16HBE) before exposure to the viral mimetic double-stranded RNA. Of the three, only budesonide treatment limited transepithelial electrical resistance and small molecule permeability (4 kDa FITC-dextran flux). Next, we used a mouse model of acute dsRNA challenge that induces transient epithelial barrier disruption in vivo, and studied the effects budesonide when administered prophylactically or therapeutically. We found that budesonide similarly protected against dsRNA-induced airway barrier disruption in the lung, independently of its effects on airway inflammation. Taken together, these data suggest that an under-appreciated effect of inhaled budesonide is to maintain or promote airway epithelial barrier integrity during respiratory viral infections.
Asunto(s)
Asma/tratamiento farmacológico , Bronquios/efectos de los fármacos , Broncodilatadores/farmacología , Budesonida/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Poli I-C/antagonistas & inhibidores , Acetatos/farmacología , Administración por Inhalación , Animales , Asma/inducido químicamente , Asma/metabolismo , Asma/patología , Bronquios/metabolismo , Bronquios/patología , Línea Celular , Ciclopropanos/farmacología , Dextranos/metabolismo , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fumarato de Formoterol/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Imitación Molecular , Poli I-C/farmacología , Quinolinas/farmacología , ARN Bicatenario/antagonistas & inhibidores , ARN Bicatenario/farmacología , ARN Viral/antagonistas & inhibidores , ARN Viral/farmacología , Sulfuros/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
Vitamin C is well documented to have antiviral functions; however, there is limited information about its effect on airway epithelial cells-the first cells to encounter infections. Here, we examined the effect of vitamin C on human bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells, and observed that sodium-dependent vitamin C transporter 2 (SVCT2) was the primary vitamin C transporter. Transcriptomic analysis revealed that treating BEAS-2B cells with vitamin C led to a significant upregulation of several metabolic pathways and interferon-stimulated genes (ISGs) along with a downregulation of pathways involved in lung injury and inflammation. Remarkably, vitamin C also enhanced the expression of the viral-sensing receptors retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), which was confirmed at the protein and functional levels. In addition, the lungs of l-gulono-γ-lactone oxidase knockout (GULO-KO) mice also displayed a marked decrease in these genes compared to wild-type controls. Collectively, our findings indicate that vitamin C acts at multiple levels to exert its antiviral and protective functions in the lungs.
Asunto(s)
Antivirales/farmacología , Ácido Ascórbico/farmacología , Células Epiteliales/efectos de los fármacos , Helicasa Inducida por Interferón IFIH1/genética , Receptores de Ácido Retinoico/genética , Transportadores de Sodio Acoplados a la Vitamina C/genética , Animales , Transporte Biológico , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular Transformada , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferón-alfa/antagonistas & inhibidores , Interferón-alfa/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , L-Gulonolactona Oxidasa/deficiencia , L-Gulonolactona Oxidasa/genética , Ratones , Ratones Noqueados , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Ácido Retinoico/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , TranscriptomaRESUMEN
Ze339, an herbal extract from Petasites hybridus leaves is effective in treatment of allergic rhinitis by inhibition of a local production of IL-8 and eicosanoid LTB4 in allergen-challenged patients. However, the mechanism of action and anti-inflammatory potential in virally induced exacerbation of the upper airways is unknown. This study investigates the anti-inflammatory mechanisms of Ze339 on primary human nasal epithelial cells (HNECs) upon viral, bacterial and pro-inflammatory triggers. To investigate the influence of viral and bacterial infections on the airways, HNECs were stimulated with viral mimics, bacterial toll-like-receptor (TLR)-ligands or cytokines, in presence or absence of Ze339. The study uncovers Ze339 modulated changes in pro-inflammatory mediators and decreased neutrophil chemotaxis as well as a reduction of the nuclear translocation and phosphorylation of STAT molecules. Taken together, this study suggests that phyto drug Ze339 specifically targets STAT-signalling pathways in HNECs and has high potential as a broad anti-inflammatory drug that exceeds current indication. © 2016 BioFactors, 43(3):388-399, 2017.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Petasites/química , Extractos Vegetales/farmacología , Factores de Transcripción STAT/antagonistas & inhibidores , Sesquiterpenos/farmacología , Movimiento Celular/efectos de los fármacos , Quimiocinas/antagonistas & inhibidores , Quimiocinas/biosíntesis , Células Epiteliales/citología , Células Epiteliales/metabolismo , Flagelina/antagonistas & inhibidores , Flagelina/farmacología , Regulación de la Expresión Génica , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/farmacología , Interleucina-4/antagonistas & inhibidores , Interleucina-4/farmacología , Lipopéptidos/antagonistas & inhibidores , Lipopéptidos/farmacología , Cavidad Nasal/citología , Cavidad Nasal/efectos de los fármacos , Cavidad Nasal/metabolismo , Neutrófilos/efectos de los fármacos , Hojas de la Planta/química , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , Cultivo Primario de Células , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de SeñalRESUMEN
In the central nervous system, viral infection can induce inflammation by up-regulating pro-inflammatory mediators that contribute to enhanced infiltration of immune cells into the central nervous areas. Celastrol is known to exert various regulatory functions, including anti-microbial activities. In this study, we investigated the regulatory effects and the mechanisms of action of celastrol against astrocytes activated with polyinosinic-polycytidylic acid (poly(I:C)), a synthetic dsRNA, as a model of pro-inflammatory mediated responses. Celastrol significantly inhibited poly(I:C)-induced expression of adhesion molecules, such as ICAM-1/VCAM-1, and chemokines, such as CCL2, CXCL8, and CXCL10, in CRT-MG human astroglioma cells. In addition, celastrol significantly suppressed poly(I:C)-induced activation of JNK MAPK and STAT1 signaling pathways. Furthermore, celastrol significantly suppressed poly(I:C)-induced activation of the NF-κB signaling pathway. These results suggest that celastrol may exert its regulatory activity by inhibiting poly(I:C)-induced expression of pro-inflammatory mediators by suppressing activation of JNK MAPK-STAT1/NF-κB in astrocytes. [BMB Reports 2017; 50(1): 25-30].
Asunto(s)
Astrocitos/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Poli I-C/farmacología , Triterpenos/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Astrocitos/metabolismo , Línea Celular , Quimiocinas/biosíntesis , Quimiocinas/metabolismo , Humanos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Triterpenos Pentacíclicos , Poli I-C/antagonistas & inhibidores , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/metabolismoRESUMEN
AIMS: Keratinocytes are the predominant cells in the epidermis, exerting their primary role of physical barrier through sophisticated differentiation process. In addition, keratinocytes contribute to the activation of innate immunity, providing the surveillant role against external pathogens. It has been known that chronic skin inflammatory disease such as psoriasis can be provoked by viral pathogens including double-stranded RNA. In this study, we demonstrated that rosmarinic acid (RA) has an inhibitory potential on inflammatory reaction induced by double-stranded RNA mimic poly(I:C) in epidermal keratinocytes. MAIN METHODS: We cultured human epidermal keratinocytes and induced inflammatory reaction by poly(I:C) treatment. The effect of RA on inflammatory reaction of keratinocytes was determined by RT-PCR and Western blot. KEY FINDINGS: RA significantly inhibited poly(I:C)-induced expression of inflammatory cytokines including IL-1ß, IL-6, IL-8, CCL20, and TNF-α, and downregulated NF-κB signaling pathway in human keratinocytes. In addition, RA significantly inhibited poly(I:C)-induced inflammasome activation, in terms of secretion of active form of IL-1ß and caspase-1. Furthermore, RA markedly inhibited poly(I:C)-induced NLRP3 and ASC expression. SIGNIFICANCE: These results indicate that RA can inhibit poly(I:C)-induced inflammatory reaction of keratinocytes, and suggest that it may be a potential candidate for the treatment of psoriasis.
Asunto(s)
Cinamatos/farmacología , Depsidos/farmacología , Inflamación/prevención & control , Queratinocitos/efectos de los fármacos , Poli I-C/farmacología , Células Cultivadas , Citocinas/metabolismo , Humanos , Queratinocitos/metabolismo , Poli I-C/antagonistas & inhibidores , Ácido RosmarínicoRESUMEN
Tyrosine phosphorylation of insulin receptor beta (IRß) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRß and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRß and anti-phosphotyrosine antibodies, recombinant IRß and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRß and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRß, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRß, reduces PKR association with the receptor, IRß in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells.
Asunto(s)
Insulina/farmacología , Poli I-C/antagonistas & inhibidores , Biosíntesis de Proteínas , Treonina/metabolismo , Tirosina/metabolismo , eIF-2 Quinasa/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Lipopolisacáridos/farmacología , Fosforilación , Poli I-C/farmacología , ARN Bicatenario/farmacología , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transducción de Señal , Tirfostinos/farmacología , eIF-2 Quinasa/genéticaRESUMEN
Scavenger receptor A (SRA) has been implicated in the processes of tumor invasion and acts as an immunosuppressor during therapeutic cancer vaccination. Pharmacological inhibition of SRA function thus holds a great potential to improve treatment outcome of cancer therapy. Macromolecular natural product sennoside B was recently shown to block SRA function. Here we report the identification and characterization of a small molecule SRA inhibitor rhein. Rhein, a deconstructed analog of sennoside B, reversed the suppressive activity of SRA in dendritic cell-primed T cell activation, indicated by transcription activation of il2 gene and production of IL-2. Rhein also inhibited SRA ligand polyinosinic:polycytidylic acid (poly(I:C)) induced activation of transcriptional factors, including interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1). Additionally, this newly identified lead compound was docked into the homology models of the SRA cysteine rich domain to gain insights into its interaction with the receptor. It was then found that rhein can favorably interact with SRA cysteine rich domain. Collectively, rhein, being the first identified small molecule inhibitors for SRA, warrants further structure-activity relationship studies, which may lead to development of novel pharmacological intervention for cancer therapy.
Asunto(s)
Antraquinonas/síntesis química , Antraquinonas/farmacología , Receptores Depuradores de Clase A/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Células Dendríticas/efectos de los fármacos , Diseño de Fármacos , Humanos , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Poli I-C/antagonistas & inhibidores , Extracto de Senna/química , Extracto de Senna/farmacología , Senósidos , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Receptores Toll-Like/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , beta-Galactosidasa/antagonistas & inhibidoresRESUMEN
Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte-endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-ß, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.
Asunto(s)
Encéfalo/citología , Chalconas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Encéfalo/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/metabolismo , Lipopéptidos/antagonistas & inhibidores , Lipopéptidos/farmacología , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacologíaRESUMEN
Prenatal maternal infection is an environmental risk factor for neurodevelopmental psychiatric illness and disease-associated cognitive impairments. Modeling this epidemiological link in animals shows that prenatal immune challenge is capable of inducing long-lasting deficits in numerous cognitive domains. Here, we combined a neonatal cross-fostering design with a mouse model of prenatal immune challenge induced by maternal gestational treatment with the viral mimetic poly(I:C) to dissect the relative contribution of prenatal and postnatal maternal effects on the offspring. We show that offspring prenatally exposed to poly(I:C) display significant impairments in spatial matching-to-position working memory and spatial novelty presence regardless of whether they are raised by gestationally immune-challenged or non-challenged control surrogate mothers. Likewise, prenatally immune challenged offspring exhibit reduced glutamic acid decarboxylase 65-kDa (GAD65) and 67-kDa (GAD67) gene expression in the adult medial prefrontal cortex and dorsal hippocampus largely independently of the postnatal rearing conditions. In addition, we confirm that being raised by a gestationally immune-challenged surrogate mother is sufficient to increase the offspring's locomotor response to systemic amphetamine treatment. Our data thus suggest that prenatal infection-induced deficits in spatial short-term memory are mediated by prenatal maternal effects on the offspring. At the same time, our study adds further weight to the notion that being reared by a surrogate mother that experienced immune activation during pregnancy may constitute a risk factor for specific dopaminergic abnormalities.
Asunto(s)
Glutamato Descarboxilasa/deficiencia , Exposición Materna , Animales , Animales Recién Nacidos/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Glutamato Descarboxilasa/genética , Hipocampo/enzimología , Hipocampo/inmunología , Hipocampo/patología , Masculino , Exposición Materna/efectos adversos , Trastornos de la Memoria/enzimología , Trastornos de la Memoria/genética , Trastornos de la Memoria/inmunología , Ratones , Ratones Endogámicos C57BL , Poli I-C/antagonistas & inhibidores , Poli I-C/toxicidad , Atención Posnatal , Corteza Prefrontal/enzimología , Corteza Prefrontal/inmunología , Corteza Prefrontal/patología , Embarazo , Diagnóstico Prenatal , Distribución Aleatoria , Ácido gamma-Aminobutírico/genéticaRESUMEN
BACKGROUND: Propolis is a bee product with various biological properties, including an antiviral activity when taken orally. However, its mechanisms at the cellular and molecular level are not well understood. RESULTS: We investigated the effect of propolis on antiviral signaling in A549 cells transfected with double-stranded RNA (dsRNA), a model for viral infection. Pretreatment of the cells with propolis inhibited poly I:C (synthetic dsRNA)-induced interferon (IFN)-ß expression. Propolis had no effect on the dsRNA-induced expression of RIG-I-like receptors (RLRs), which are known as intracellular viral RNA sensors. As to the effect on antiviral executor genes, propolis enhanced myxovirus resistance 1 (MX1) expression, whereas interferon-inducible gene 6-16 (G1P3) and 2'-5'-oligoadenylate synthetase (OAS) were unaffected. All of these genes belong to the IFN-inducible genes, suggesting that the effect of propolis on antiviral signaling is not necessarily mediated by the autocrine regulation by IFN-ß. Propolis pretreatment inhibited dsRNA-induced interleukin-8 (IL8) and CCL5 expression, and consequently lowered polymorphonuclear leukocyte (PMN) chemotactic activity in the cell-conditioned medium. CONCLUSION: Taken together, these results suggest that propolis may suppress excess inflammatory responses without affecting the innate immunity during viral infection.
Asunto(s)
Antivirales/farmacología , Interferón beta/genética , Neutrófilos/efectos de los fármacos , Própolis/farmacología , ARN Bicatenario/antagonistas & inhibidores , Adenocarcinoma , Brasil , Línea Celular Tumoral , Quimiotaxis de Leucocito/efectos de los fármacos , Medios de Cultivo Condicionados , Expresión Génica/efectos de los fármacos , Humanos , Interferón beta/antagonistas & inhibidores , Neoplasias Pulmonares , Neutrófilos/fisiología , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , ARN Bicatenario/fisiología , TransfecciónRESUMEN
RNA receptors such as TLR3 and retinoid acid-inducible gene I/melanoma differentiation-associated gene 5 play essential roles in innate immunity to RNA viruses. However, how innate immunity to RNAs is controlled at the molecular level is not well understood. We describe in this study a new regulatory pathway of anti-RNA immunity that is composed of PI3K, its target GTPase Rac, and the newly described immune regulator TNF-α-induced protein 8 like-2 (TIPE2, or TNFAIP8L2). Polyinosinic-polycytidylic acid [Poly (I:C)], a dsRNA receptor ligand, activates Rac via its guanine nucleotide exchange factor Tiam; this leads to the activation of cytokine genes and, paradoxically, downregulation of the Tipe2 gene. TIPE2 is a negative regulator of immunity; its deficiency leads to hyperactivation of the PI3K-Rac pathway as exemplified by enhanced AKT, Rac, P21-activated kinase, and IFN regulatory factor 3 activities. As a consequence, TIPE2 knockout myeloid cells are hyperreactive to Poly (I:C) stimulation, and TIPE2 knockout mice are hypersensitive to Poly (I:C)-induced lethality. These results indicate that TIPE2 controls innate immunity to RNA by targeting the PI3K-Rac pathway. Therefore, manipulating TIPE2 or Rac functions can be effective for controlling RNA viral infections.
Asunto(s)
Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/fisiología , Fosfatidilinositol 3-Quinasa/fisiología , Transducción de Señal/inmunología , Proteínas de Unión al GTP rac/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Células L , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de las Quinasa Fosfoinosítidos-3 , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , Transducción de Señal/genética , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Proteínas de Unión al GTP rac/fisiologíaAsunto(s)
Quimiocina CCL2/genética , Conjuntiva/efectos de los fármacos , Dinoprostona/farmacología , Regulación hacia Abajo/fisiología , Limbo de la Córnea/efectos de los fármacos , Quimiocina CCL2/metabolismo , Conjuntiva/citología , Conjuntiva/metabolismo , Dinoprostona/análogos & derivados , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E/metabolismoRESUMEN
Excessive inflammation during bacterial and viral infections is destructive to the host and involves elevated production of proinflammatory cytokines. It is especially deleterious in organs with space constraints such as lung and the CNS. Indeed, a number of viruses that infect lungs, such as avian influenza virus, SARS-associated coronavirus, and respiratory syncytial virus, elicit a very high level of proinflammatory cytokines; however, it is unclear what triggers their production. In this study, we show that IL-17 commonly produced during viral infection specifically augments a proinflammatory response by directly synergizing with antiviral signaling. Costimulation of primary human fibroblasts with IL-17 greatly enhanced respiratory syncytial virus-induced or synthetic dsRNA-based viral mimic polyinosinic:polycytidylic acid-induced expression of proinflammatory genes without affecting expression of IFN-ß-stimulated or IFN-stimulated genes. Knockdown of expression of known mediators of the antiviral signaling pathway revealed that the IL-17-poly(I:C) synergy depends on the presence of the transcriptional factors RelA and IFN regulatory factor 3 and IκB kinases. Moreover, this synergy was blocked by an IκB kinase inhibitor, BAY 11-7082. These findings shed light on the molecular mechanisms behind IL-17-dependent immunopathology observed in viral infections.
Asunto(s)
Antivirales/farmacología , Fibroblastos/inmunología , Fibroblastos/patología , Mediadores de Inflamación/fisiología , Interleucina-17/fisiología , Virus Sincitiales Respiratorios/inmunología , Regulación hacia Arriba/inmunología , Antivirales/metabolismo , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Citocinas/fisiología , Fibroblastos/virología , Regulación de la Expresión Génica/inmunología , Humanos , Quinasa I-kappa B/biosíntesis , Quinasa I-kappa B/genética , Quinasa I-kappa B/fisiología , Mediadores de Inflamación/metabolismo , Factor 3 Regulador del Interferón/biosíntesis , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/fisiología , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , Transducción de Señal/genética , Transducción de Señal/inmunología , Piel/inmunología , Piel/patología , Piel/virología , Factor de Transcripción ReIA/biosíntesis , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/fisiologíaRESUMEN
Tolerogenic dendritic cells (TDC) offer a promising therapeutic potential to ameliorate autoimmune diseases. Reported to inhibit adaptive immune responses, little is known about their innate immunity receptor repertoire. In this study, we compared three types of human TDC (IL-10-DC, dexamethasone (DX)-DC, and 1,25(OH)(2)D(3)-DC) by their TLR expression and response to a set of TLR ligands. TDC are endowed with the same TLR set as standard monocyte-derived dendritic cells but respond differentially to the TLR stimuli Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and flagellin. TDC expressed low or no IL-12-related cytokines and remarkably elevated IL-10 levels. Interestingly, only TDC up-regulated the expression of TLR2 upon stimulation. This boosted the tolerogenic potential of these cells, because IL-10 production was up-regulated in TLR2-stimulated, LPS-primed DX-DC, whereas IL-12 and TNF-alpha secretion remained low. When comparing the TDC subsets, DX-DC and 1,25(OH)(2)D(3)-DC up-regulated TLR2 irrespective of the TLR triggered, whereas in IL-10-DC this effect was only mediated by LPS. Likewise, DX-DC and 1,25(OH)(2)D(3)-DC exhibited impaired ability to mature, reduced allostimulatory properties, and hampered capacity to induce Th1 differentiation. Therefore, both DX-DC and 1,25(OH)(2)D(3)-DC display the strongest tolerogenic and anti-inflammatory features and might be most suitable tools for the treatment of autoimmune diseases.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Tolerancia Inmunológica , Mediadores de Inflamación/antagonistas & inhibidores , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/sangre , Regulación hacia Arriba/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/clasificación , Regulación hacia Abajo/inmunología , Retroalimentación Fisiológica/inmunología , Flagelina/antagonistas & inhibidores , Flagelina/metabolismo , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/fisiología , Ligandos , Lipopéptidos/antagonistas & inhibidores , Lipopéptidos/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/metabolismo , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Poli I-C/antagonistas & inhibidores , Poli I-C/metabolismo , Receptor Toll-Like 2/agonistasRESUMEN
Curcumin, a compound found in the Indian spice turmeric, has anti-inflammatory and immunomodulatory properties, though the mechanism remains unclear. Dendritic cells (DCs) are important to generating an immune response and the effect of curcumin on human DCs has not been explored. The role curcumin in the DC response to bacterial and viral infection was investigated in vitro using LPS and Poly I:C as models of infection. CD14(+) monocytes, isolated from human peripheral blood, were cultured in GM-CSF- and IL-4-supplemented medium to generate immature DCs. Cultures were incubated with curcumin, stimulated with LPS or Poly I:C and functional assays were performed. Curcumin prevents DCs from responding to immunostimulants and inducing CD4(+) T cell proliferation by blocking maturation marker, cytokine and chemokine expression and reducing both migration and endocytosis. These data suggest a therapeutic role for curcumin as an immune suppressant.
Asunto(s)
Adyuvantes Inmunológicos/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/farmacología , Curcumina/farmacología , Células Dendríticas/efectos de los fármacos , Inmunosupresores/farmacología , Adyuvantes Inmunológicos/farmacología , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/inmunología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocinas/metabolismo , Células Dendríticas/inmunología , Endocitosis/efectos de los fármacos , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/inmunología , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos , Poli I-C/antagonistas & inhibidores , Poli I-C/inmunologíaRESUMEN
The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in microglial cells and isolating primary microglia from PPAR-alpha-/- mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-alpha. Interestingly, gemfibrozil induced the activation of p85alpha-associated PI3K (p110beta but not p110alpha) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid beta (Abeta)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-gamma-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Abeta-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-kappaB activation in LPS-, Abeta-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-gamma-, induced microglial expression of iNOS and stimulation of IkappaBalpha expression and inhibition of NF-kappaB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-kappaB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IkappaBalpha.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Gemfibrozilo/farmacología , Proteínas I-kappa B/biosíntesis , Microglía/efectos de los fármacos , Microglía/enzimología , Fosfatidilinositol 3-Quinasas/fisiología , Regulación hacia Arriba/efectos de los fármacos , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/fisiología , Androstadienos/farmacología , Animales , Línea Celular , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Activación Enzimática/efectos de los fármacos , Gemfibrozilo/antagonistas & inhibidores , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Microglía/metabolismo , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/biosíntesis , PPAR alfa/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , Priones/antagonistas & inhibidores , Priones/fisiología , ARN Bicatenario/antagonistas & inhibidores , ARN Bicatenario/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/inmunología , WortmaninaRESUMEN
BACKGROUND: The human endometrium is an important site for contact between the host and pathogens ascending the reproductive tract, and thus plays an important role in female reproductive tract immunity. Previous work in our laboratory has suggested that Toll-like receptors (TLRs) are involved in endometrial epithelial recognition of pathogens and that ligation of endometrial TLRs results in the production of cytokines and chemokines important for both immune and reproductive functions of the endometrium. We have also demonstrated cyclic regulation of TLR3 mRNA and protein expression in human endometrium, suggesting that steroid hormones might play a role in the expression and function of TLR3. In this study, the effects of 17beta-estradiol (E2) and progesterone (P) on TLR3 expression and function in endometrial cell lines were investigated. METHODS: Endometrial epithelial cell lines were cultured and examined for the presence of TLR3 and hormone receptors by endpoint RT-PCR. For hormonal studies, cells were pre-treated with ethanol vehicle, 10(-8) M E2, and/or 10(-7) M P. For antagonist assays, cells were treated with the ER antagonist, ICI 182, 780, or the PR antagonist, RU486, for two hours prior to treatment with hormones. Following hormone or hormone/antagonist pre-treatment, cells were stimulated with vehicle, the synthetic TLR3 ligand, polyinosinic-polycytidylic acid (Poly I:C), a negative dsDNA control, or a positive control. Cytokine and chemokine production post-stimulation was measured by ELISA. The effects of E2 and P on TLR3 mRNA and protein expression were measured using Real Time RT-PCR and FACS analysis, respectively. RESULTS: Stimulation of TLR3-expressing cells with the synthetic TLR3 ligand, Poly I:C, resulted in the production of cytokines and chemokines important for endometrial function and regulation. Suppression of Poly I:C-induced cytokine and chemokine production by cells treated with 10(-8) M E2, but not cells treated with 10(-7) M P, was observed in endometrial epithelial cell lines expressing TLR3 and estrogen receptor alpha (ERalpha). The effects of E2 were not observed on cells which did not express ERalpha or in cells pre-treated with the ER antagonist, ICI 182, 780. Treatment with E2 did not affect TLR3 mRNA or protein expression. However, treatment with E2 did suppress cytokine and chemokine production resulting from TLR3 stimulation with Poly I:C, suggesting that E2 modulates TLR3 function. CONCLUSION: The data presented in this study are the first indication that E2 can markedly alter the innate immune response to dsRNA, providing a previously unreported process by which E2 can alter immune responses.
Asunto(s)
Quimiocinas/biosíntesis , Citocinas/biosíntesis , Estradiol/farmacología , Receptor Toll-Like 3/biosíntesis , Línea Celular , Línea Celular Tumoral , Endometrio/citología , Células Epiteliales , Estradiol/análogos & derivados , Femenino , Fulvestrant , Humanos , Mifepristona/farmacología , Poli I-C/antagonistas & inhibidores , Poli I-C/farmacología , Polidesoxirribonucleótidos/farmacología , Progesterona/farmacología , Receptores de Estrógenos/biosíntesis , Receptores de Progesterona/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacología , Receptor Toll-Like 3/efectos de los fármacosRESUMEN
Here, we investigated the influence of cyclosporin A (CsA) on dendritic cell (DC) generation. With this aim, human DC were propagated from monocytes in serum-free medium with granulocyte macrophage-colony stimulating factor and interleukin-4. DC were then exposed to tumor necrosis factor alpha (TNF-alpha) for maturation. Our results show that CsA does not impair commitment of monocytes into DC, as assessed by loss of CD14 and increase of CD40 and CD1a. However, TNF-alpha-induced DC maturation was affected, as CsA-treated DC expressed lower levels of human leukocyte antigen and costimulatory molecules but sustained levels of CD1a, and less DC expressed DC-lysosomal-associated-membrane-protein (LAMP) and CD83. Accordingly, CsA inhibited the allostimulatory and accessory cell functions of DC. Surprisingly, when other maturation stimuli were used, we observed that CsA significantly inhibited maturation induced by lipopolysaccharides but not by polyribocytidylic acid or CD40 ligand, as assessed by DC phenotype and functions. Therefore, our results indicate that CsA may differentially affect DC maturation.
Asunto(s)
Ciclosporina/farmacología , Células Dendríticas/inmunología , Inmunosupresores/farmacología , Presentación de Antígeno/efectos de los fármacos , Antígenos CD/análisis , Ligando de CD40/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Antígenos HLA-DR/análisis , Cinética , Lipopolisacáridos/antagonistas & inhibidores , Prueba de Cultivo Mixto de Linfocitos , Poli I-C/antagonistas & inhibidores , ARN Bicatenario/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Natural killer (NK) cells can be activated in vitro and in vivo by polyinosinic:polycytidylic acid (poly I:C) through induction of type I interferons or other cytokines. Ethanol suppresses in vivo and ex vivo poly I:C activation of NK cell activity in a mouse model for binge drinking, but it is not known whether this effect is mediated by changes in NK cells or in other cell types (e.g., those that produce NK cell-activating cytokines). Splenocytes were obtained from C57BL/6 [NK cell-competent (NKc)] and C57BL/6 perforin knockout [NK cell-incompetent (NKi)] mice 6 h after administration of ethanol (6 g/kg) or vehicle (VH; dH(2)O). Cells were incubated in vitro 18 h with poly I:C (100 micro g/ml), followed by a 4-h 51Cr release assay with the use of YAC-1 target cells. Results of cell-mixing experiments involving all relevant combinations of splenocytes obtained from NKc and NKi mice treated with VH or ethanol strongly supported the suggestion that NK cells, not other cell types, are the primary target of ethanol-induced suppression of NK cell activation. For example, mixing of splenocytes obtained from ethanol-treated NKc and VH-treated NKi mice or from ethanol-treated NKc and ethanol-treated NKi mice yielded similar cytolytic function. However, mixing of splenocytes obtained from ethanol-treated NKc and VH-treated NKi mice yielded significantly less cytolytic activity than that of splenocytes from VH-treated NKc and ethanol-treated NKi mice. In addition, mixing of splenocytes obtained from VH-treated NKc and NKi mice resulted in lower cytolytic activity than when splenocytes from the NKi mice were treated with ethanol instead of with VH, demonstrating that ethanol did not decrease the function of other cell types. A strikingly similar pattern of results was observed when B6C3F1 mice, rendered NK cell deficient by administration of anti-NK 1.1 monoclonal antibody, were used instead of perforin knockout mice. These results indicate that ethanol suppresses activation of NK cells primarily by suppressing the NK cell response to poly I:C, not by acting on another cell type.