RESUMEN
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) of the immune system and are specialized to activate T as well as B cell-dependent immune responses. Mature DC are characterized by expression of CD83, a surface molecule that has been postulated to be required for efficient DC activity. Here we show that Leptomycin B (LMB), a highly specific inhibitor of the nuclear export receptor CRM1, abrogates the ability of DC to stimulate T cells in an allogeneic mixed lymphocyte reaction. Interestingly, this effect correlates with down-regulation of CD83, CD80 and CD86 surface expression during DC maturation, whereas other investigated DC surface molecules, such as MHC class I and II molecules are not significantly affected. Analysis of RNA distribution reveals that particularly the stimulated expression of CD83 depended on a functional CRM1 export receptor. Taken together, the presented data show a critical involvement of the CRM1 transport receptor in DC maturation, most likely by enabling efficient nucleo-cytoplasmic translocation of specific mRNAs. Thus, interference with this pathway may provide new strategies to modulate DC function and, subsequently, DC-mediated immune responses.
Asunto(s)
Células Dendríticas/inmunología , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Antígenos CD/biosíntesis , Diferenciación Celular , Células Dendríticas/efectos de los fármacos , Ácidos Grasos Insaturados/farmacología , Humanos , Inmunoglobulinas/biosíntesis , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/biosíntesis , Neutrófilos/inmunología , Proteína Exportina 1 , Antígeno CD83RESUMEN
Dendritic cells are the sentinels of the immune system and as such represent the first-line of defense against incoming pathogens. Upon encounter with harmful antigens, these antigen-presenting cells start to mature and migrate towards the draining lymph nodes to display the antigen to T-lymphocytes, thereby eliciting the immune response of the host. Viruses, including human herpesvirus type I (HSV-1), seek to avoid such immune reactions. Therefore, they developed an arsenal of immune evasion strategies, some of which have been described earlier by our group and others. The secretion of tumor necrosis factor (TNF) represents a typical defense line of the host and it has been shown that this cytokine contributes to the inhibition of viral replication and augments the proliferation of cytotoxic T-lymphocytes. Here we report, that upon infection of mature dendritic cells, HSV-1 very strongly induces the expression of the AU-rich elements (ARE)-binding protein tristetraprolin (TTP), an mRNA-destabilizing protein. One of the best described targets of TTP is the TNF mRNA. This induction is dependent on the phosphorylation of both signal transducer and activator of transcription (STAT1) and p38 in a collaborative manner. By repressing this phosphorylation with specific inhibitors, we were able to reduce TTP mRNA levels. At the same time TNF mRNA levels were increased, suggesting that TNF mRNA is indeed a target of TTP in this setting. In summary, these data underline that HSV-1 induces TTP transcription in order to reduce TNF levels generated by infected mature dendritic cell.
Asunto(s)
Células Dendríticas/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , ARN Mensajero/metabolismo , Factor de Transcripción STAT1/metabolismo , Tristetraprolina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/virología , Humanos , Fosforilación , Tristetraprolina/genética , Regulación hacia ArribaRESUMEN
Selective gene silencing by RNA interference (RNAi) has been shown to be an efficient method for the targeted manipulation of cellular functions. In this study we describe for the first time electroporation as a suitable and efficient method for the delivery of small interfering RNA (siRNA) into murine bone marrow-derived dendritic cells (BM-DC). Using a fluorescein-labeled non-silencing siRNA duplex, we established an electroporation protocol yielding routinely >90% positive cells. We investigated the effects of siRNA electroporation on BM-DC viability, phenotype and ability to induce allogeneic T cell proliferation. Finally, using siRNAs directed against MAPK1 and the transcription factor HIF-1alpha we were able to demonstrate an efficient knock down of cellular mRNA- and protein level in electroporated BM-DC. Furthermore, knocking down the transcription factor HIF-1alpha impeded hypoxic induction of HIF-1alpha target genes. We therefore propose siRNA electroporation into murine BM-DC as an efficient method to manipulate BM-DC function without the use of chemical transfection reagents. This new approach is superior to lipofection regarding detrimental effects of lipid-based transfection agents on BM-DC immunobiology.
Asunto(s)
Células de la Médula Ósea/metabolismo , Células Dendríticas/metabolismo , Electroporación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transfección/métodos , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Hipoxia de la Célula , Supervivencia Celular , Células Cultivadas , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lípidos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fenotipo , Linfocitos T/inmunologíaRESUMEN
Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1alpha on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1alpha protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1alpha significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1alpha and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lipopolisacáridos/farmacología , Animales , Diferenciación Celular , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , ARN Interferente Pequeño/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) of the immune system. Their migration to secondary lymphoid tissues is a crucial step for the priming of T cells and ultimately for the initiation of adaptive immune responses. Therefore, DCs are potential targets for immune evasion strategies of pathogens. The migration of DCs to the T cell areas of lymph nodes is guided by a gradient of chemokines, CCL19 and CCL21, which are constitutively expressed there. CCR7, the receptor for these chemokines, is expressed on activated DCs, enabling their homing to the lymph nodes. However, CCR7 expression alone is not sufficient for efficient migration. Prostaglandin E(2) (PGE(2)) is a mandatory factor for CCR7-mediated migration of DCs and exerts its effects via prostaglandin E(2) receptor 2 (EP2) and prostaglandin E(2) receptor 4 (EP4). In this study, we investigated the effect of herpes simplex virus type 1 (HSV-1) infection of mature monocyte-derived dendritic cells (MODCs) on the EP2 and EP4 receptor expression. Affymetrix analyses and real-time polymerase chain reaction (PCR) demonstrated a dramatic down-regulation of the expression of those receptors. Additional real-time PCR and migration assays with a Deltavhs mutant virus lacking the virion host shutoff (vhs) gene implicate a vhs independent mechanism. Therefore, our results suggest a novel immune evasion strategy for HSV-1.
Asunto(s)
Quimiocinas CC/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virología , Herpesvirus Humano 1/fisiología , Receptores CCR7/metabolismo , Receptores de Prostaglandina E/metabolismo , Quimiotaxis , Humanos , Prostaglandinas E/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina ERESUMEN
Here we report for the first time that MCS-18, a novel natural product isolated from Helleborus purpurascens, is able to inhibit the expression of typical molecules of mature dendritic cells (DC) such as CD80, CD86, and especially of CD83 subsequently leading to a clear and dose-dependent inhibition of the DC-mediated T-cell stimulation. Furthermore, MCS-18 impeded the formation of the typical DC/T-cell clusters, which are essential to induce potent immune responses. Interestingly, MCS-18 also inhibited CCR7 expression on DC which subsequently lead to a dose-dependent block of the CCL19-mediated DC migration. MCS-18 not only inhibited the DC-mediated T-cell stimulation but also the anti-CD3/anti-CD28-mediated T-cell stimulation. Strikingly, MCS-18 also strongly reduced the paralysis associated with the experimental autoimmune encephalomyelitis (EAE), which is a murine model for human multiple sclerosis, in a prophylactic as well as in a "real" therapeutic setting. Even when the EAE was induced for a second time, the MCS-18-treated animals were still protected, suggesting that MCS-18 induces a long-lasting suppressive effect. In addition, and very important for the potential practical application in humans, MCS-18 was also active when administered orally. MCS-18 treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In conclusion, using in vitro as well in vivo assays we were able to show that MCS-18 exerts a strong immunosuppressive activity with remarkable potential for the therapy of diseases characterized by a pathologically over-activated immune system.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Helleborus/química , Inmunosupresores/farmacología , Extractos Vegetales/farmacología , Animales , Autoinmunidad/inmunología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/metabolismo , Inmunosupresores/uso terapéutico , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Fitoterapia , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Mature human dendritic cells (mDCs) are the most powerful APCs known today, having the unique ability to induce primary immune responses. One of the best known surface markers for mDCs is the glycoprotein CD83, which is strongly up-regulated during maturation, together with costimulatory molecules such as CD80 and CD86. When CD83 surface expression was inhibited by interference with the messenger RNA export or by infection with certain viruses, DCs showed a dramatically reduced capability to induce T cell proliferation. However, in these cases side effects on other cellular functions cannot be excluded completely. In this study we present an efficient method to specifically influence CD83 surface expression by the use of RNA interference. We used small-interfering RNA targeted against CD83 and carefully evaluated an electroporation protocol for the delivery of the duplex into the cells. Furthermore, we identified freshly prepared immature DCs as the best target for the application of a CD83 knockdown and we were also able to achieve a long lasting silencing effect for this molecule. Finally, we were able to confirm that CD83 functions as an enhancer during the stimulation of T cells, significantly increases DC-mediated T cell proliferation, and goes hand in hand with clear changes in cytokine expression during T cell priming. These results were obtained for the first time without the use of agents that might cause unwanted side effects, such as low m.w. inhibitors or viruses. Therefore, this method presents a suitable way to influence DC biology.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Activación de Linfocitos , Glicoproteínas de Membrana/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Linfocitos T/inmunología , Antígenos CD/genética , Células Dendríticas/inmunología , Electroporación , Humanos , Inmunoglobulinas/genética , Activación de Linfocitos/genética , Glicoproteínas de Membrana/genética , Monocitos/inmunología , Antígeno CD83RESUMEN
Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism.
Asunto(s)
Antígenos CD/biosíntesis , Células Dendríticas/citología , Herpesvirus Humano 1/metabolismo , Inmunoglobulinas/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular , Separación Celular , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Herpes Simple/inmunología , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Cinética , Leucocitos Mononucleares/virología , Transfección , Ubiquitina-Proteína Ligasas/metabolismo , Antígeno CD83RESUMEN
Selective gene silencing by small interfering RNAs (siRNAs) has been shown to be an efficient method for the targeted manipulation of cellular functions. Chemical transfection reagents represent the current standard technique in siRNA duplex delivery into mammalian cells. However, when trying to manipulate cells isolated from patients in clinical approaches, chemical agents might cause unwanted side effects, such as allergic reactions, or interfere with other cellular functions. In this study we describe electroporation as a suitable and efficient method for the delivery of siRNA into monocyte-derived dendritic cells (moDCs). Using a fluorescein-labeled non-silencing siRNA duplex as a model system, we carefully investigated the effects of siRNA electroporation on moDCs' viability, phenotype, migratory capacity, and ability to induce T-cell mediated immune responses. Finally, by using a standard duplex directed against the nuclear Lamins A and C we were able to demonstrate an efficient knockdown of a cellular messenger RNA in electroporated moDCs. We therefore propose siRNA electroporation into moDCs as an efficient method to manipulate DC function at large cell numbers without the use of chemical transfection reagents. This new approach represents an advantage especially in the light of clinical trials.
Asunto(s)
Células Dendríticas/fisiología , Electroporación/métodos , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Movimiento Celular , Células Dendríticas/inmunología , Silenciador del Gen , Humanos , Inmunofenotipificación , Lamina Tipo A/inmunología , Prueba de Cultivo Mixto de Linfocitos , ARN/química , ARN/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Herpes simplex virus type 1 (HSV-1) is able to establish latency in infected individuals. In order to characterize potential new immune-escape mechanisms, mature dendritic cells (DCs) were infected with HSV-1 and total cellular RNA was isolated from infected and mock-infected populations at different time points. RNA profiling on Affymetrix Human Genome U133A arrays demonstrated a dramatic downregulation of the migration-mediating surface molecules CCR7 and CXCR4, an observation that was further confirmed by RT-PCR and fluorescence-activated cell sorting analyses. Furthermore, migration assays revealed that, upon infection of mature DCs, CCR7- and CXCR4-mediated migration towards the corresponding CCL19 and CXCL12 chemokine gradients was strongly reduced. It is noteworthy that the infection of immature DCs with HSV-1 prior to maturation led to a failure of CCR7 and CXCR4 upregulation during DC maturation and, as a consequence, also induced a block in their migratory capacity. Additional migration assays with a Deltavhs mutant virus lacking the virion host shutoff (vhs) gene, which is known to degrade cellular mRNAs, suggested a vhs-independent mechanism. These results indicate that HSV-1-infected mature DCs are limited in their capacity to migrate to secondary lymphoid organs, the areas of antigen presentation and T-cell stimulation, thus inhibiting an antiviral immune response. This represents a novel, previously unrecognized mechanism for HSV-1 to escape the human immune system.
Asunto(s)
Células Dendríticas/inmunología , Herpesvirus Humano 1/inmunología , Diferenciación Celular , Movimiento Celular , Células Dendríticas/citología , Regulación hacia Abajo , Herpes Simple/inmunología , Humanos , Mutación , ARN Mensajero/análisis , Receptores CCR7 , Receptores CXCR4/análisis , Receptores CXCR4/genética , Receptores de Quimiocina/análisis , Receptores de Quimiocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas , Factores de Tiempo , Proteínas Virales/genéticaRESUMEN
The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Glicoproteínas/fisiología , Lectinas Tipo C/fisiología , Marburgvirus/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Proteínas del Envoltorio Viral/fisiología , Glicoproteína de la Espiga del CoronavirusRESUMEN
The tetravalent guanylhydrazone CNI-1493 (CNI-1493) has been shown to inhibit macrophage activation, reduce systemic inflammation as well as proinflammatory cytokine production. Here we report for the first time that CNI-1493 also influences the biology of dendritic cells (DC). In order to become potent T cell stimulators of DC have to mature. Interestingly, when CNI-1493 was added to the maturation stimulus the expression of a typical DC-maturation marker i.e. CD83 was reduced. Subsequent functional in vitro analyses showed that DC-mediated T-cell stimulation was clearly reduced in CNI-1493-treated DC, underlining the functional impact that CNI-1493 on DC biology. Furthermore, the effect of CNI-1493 was analyzed in vivo using the experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice. Interestingly, in a prophylactic treatment regimen CNI-1493 prevented the paralysis associated with EAE almost completely. In addition, when applied in an early therapeutic setting CNI-1493 also reduced the clinical EAE symptoms. In summary, we show for the first time, that in addition to the earlier reported effects on macrophages, CNI-1493 also influences the function and biology of DC. Since DC are the only antigen-presenting cells (APC) known today to be able to prime naive T cells, the findings reported herein are highly relevant for the therapeutic application of CNI-1493.