RESUMEN
UNLABELLED: Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. IMPORTANCE: HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus, and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L particles, revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.
Asunto(s)
Antígenos CD/metabolismo , Células Dendríticas/virología , Regulación de la Expresión Génica/inmunología , Herpesvirus Humano 1/metabolismo , Evasión Inmune/inmunología , Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Virión/fisiología , Análisis de Varianza , Cartilla de ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Microscopía Electrónica , Transporte de Proteínas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Antígeno CD83RESUMEN
Mature dendritic cells (mDCs) are the most potent antigen-presenting cells known today, as they are the only antigen-presenting cells able to induce naïve T-cells. Therefore, they play a crucial role during the induction of effective antiviral immune responses. Interestingly, the surface molecule CD83 expressed on mDCs is targeted by several viruses. As CD83 has been shown to exert co-stimulatory functions on mDCs, its downmodulation represents a viral immune escape mechanism. Mechanistically, it has been shown that herpes simplex virus type 1 infection leads to proteasomal degradation of CD83, resulting in a strongly diminished T-cell stimulatory capacity of the infected mDC. Previous data suggest that the viral immediate-early protein ICP0 (infected-cell protein 0) plays an important role in this process. In the present study, we showed that ICP0 is sufficient to induce CD83 degradation in the absence of any other viral factor. However, the mechanism of ICP0-mediated CD83 degradation is not yet understood. Here, we provide evidence that ubiquitination of lysine residues is, despite the published E3 ubiquitin ligase activity of ICP0, not necessary for CD83 degradation. This finding was underlined by the observation that expression of an ICP0 mutant lacking the E3 ubiquitin ligase domain in mDCs still induced CD83 degradation. Finally, inhibition of E1 activating enzyme using the specific inhibitor 4[4-(5-nitro-furan-2-ylmethylene)-3.5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester did not prevent CD83 degradation. Taken together, our data provide strong evidence that ICP0 alone induces CD83 degradation independent of its E3 ubiquitin ligase function and of the ubiquitin machinery.
Asunto(s)
Antígenos CD/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/virología , Herpesvirus Humano 1/inmunología , Proteínas Inmediatas-Precoces/inmunología , Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligasas/inmunología , Antígenos CD/química , Antígenos CD/genética , Células Dendríticas/metabolismo , Genes Virales , Células HEK293 , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/genética , Evasión Inmune/genética , Inmunoglobulinas/química , Inmunoglobulinas/genética , Lisina/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutagénesis Sitio-Dirigida , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Antígeno CD83RESUMEN
Excessive macrophage (Mφ) activation results in chronic inflammatory responses or autoimmune diseases. Therefore, identification of novel immune checkpoints on Mφ, which contribute to resolution of inflammation, is crucial for the development of new therapeutic agents. Herein, we identify CD83 as a marker for IL-4 stimulated pro-resolving alternatively activated Mφ (AAM). Using a conditional KO mouse (cKO), we show that CD83 is important for the phenotype and function of pro-resolving Mφ. CD83-deletion in IL-4 stimulated Mφ results in decreased levels of inhibitory receptors, such as CD200R and MSR-1, which correlates with a reduced phagocytic capacity. In addition, CD83-deficient Mφ upon IL-4 stimulation, show an altered STAT-6 phosphorylation pattern, which is characterized by reduced pSTAT-6 levels and expression of the target gene Gata3. Concomitantly, functional studies in IL-4 stimulated CD83 KO Mφ reveal an increased production of pro-inflammatory mediators, such as TNF-α, IL-6, CXCL1 and G-CSF. Furthermore, we show that CD83-deficient Mφ have enhanced capacities to stimulate the proliferation of allo-reactive T cells, which was accompanied by reduced frequencies of Tregs. In addition, we show that CD83 expressed by Mφ is important to limit the inflammatory phase using a full-thickness excision wound healing model, since inflammatory transcripts (e.g. Cxcl1, Il6) were increased, whilst resolving transcripts (e.g. Ym1, Cd200r, Msr-1) were decreased in wounds at day 3 after wound infliction, which reflects the CD83 resolving function on Mφ also in vivo. Consequently, this enhanced inflammatory milieu led to an altered tissue reconstitution after wound infliction. Thus, our data provide evidence that CD83 acts as a gatekeeper for the phenotype and function of pro-resolving Mφ.
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Proteínas de Punto de Control Inmunitario , Interleucina-4 , Animales , Ratones , Macrófagos , Fagocitos , InflamaciónRESUMEN
Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.
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Antígeno CD83 , Macrófagos , Linfocitos T , Humanos , Diferenciación Celular , FenotipoRESUMEN
Rationale: Sepsis, a global health burden, is often complicated by viral infections leading to increased long-term morbidity and mortality. Interleukin-3 (IL-3) has been identified as an important mediator amplifying acute inflammation in sepsis; however, its function in the host response to viral infections during sepsis remains elusive. Objectives: To investigate the role of IL-3 during viral pneumonia in sepsis. Methods: We included septic patients from two different cohorts and used in vitro and in vivo assays. The obtained data were substantiated using a second model (SARS-CoV-2 infections). Measurements and main results: Low plasma IL-3 levels were associated with increased herpes simplex virus (HSV) airway infections in septic patients, resulting in reduced overall survival. Likewise, Il-3-deficient septic mice were more susceptible to pulmonary HSV-1 infection and exhibited higher pulmonary inflammation than control mice. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating plasmacytoid dendritic cells (pDCs) into the airways and by enhancing pDC-mediated T cell activation upon viral stimulation. Interestingly, the ability of IL-3 to improve adaptive immunity was confirmed in patients with SARS-CoV-2 infections. Conclusion: Our study identifies IL-3 as a predictive disease marker for viral reactivation in sepsis and reveals that IL-3 improves antiviral immunity by enhancing the recruitment and the function of pDCs.
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COVID-19 , Sepsis , Animales , Ratones , Antivirales , Células Dendríticas , Interleucina-3 , Pulmón , SARS-CoV-2 , Linfocitos TRESUMEN
Here we show that soluble CD83 induces the resolution of inflammation in an antigen-induced arthritis (AIA) model. Joint swelling and the arthritis-related expression levels of IL-1ß, IL-6, RANKL, MMP9, and OC-Stamp were strongly reduced, while Foxp3 was induced. In addition, we observed a significant inhibition of TRAP+ osteoclast formation, correlating with the reduced arthritic disease score. In contrast, cell-specific deletion of CD83 in human and murine precursor cells resulted in an enhanced formation of mature osteoclasts. RNA sequencing analyses, comparing sCD83- with mock treated cells, revealed a strong downregulation of osteoclastogenic factors, such as Oc-Stamp, Mmp9 and Nfatc1, Ctsk, and Trap. Concomitantly, transcripts typical for pro-resolving macrophages, e.g., Mrc1/2, Marco, Klf4, and Mertk, were upregulated. Interestingly, members of the metallothionein (MT) family, which have been associated with a reduced arthritic disease severity, were also highly induced by sCD83 in samples derived from RA patients. Finally, we elucidated the sCD83-induced signaling cascade downstream to its binding to the Toll-like receptor 4/(TLR4/MD2) receptor complex using CRISPR/Cas9-induced knockdowns of TLR4/MyD88/TRIF and MTs, revealing that sCD83 acts via the TRIF-signaling cascade. In conclusion, sCD83 represents a promising therapeutic approach to induce the resolution of inflammation and to prevent bone erosion in autoimmune arthritis.
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Antígenos CD , Artritis , Inmunoglobulinas , Glicoproteínas de Membrana , Osteólisis , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Antígenos CD/metabolismo , Artritis/metabolismo , Humanos , Inmunoglobulinas/metabolismo , Inflamación/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Osteoclastos/metabolismo , Osteólisis/metabolismo , Receptor Toll-Like 4/metabolismo , Antígeno CD83RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. In a prospective multicentric study, we identify IL-3 as an independent prognostic marker for the outcome during SARS-CoV-2 infections. Specifically, low plasma IL-3 levels is associated with increased severity, viral load, and mortality during SARS-CoV-2 infections. Patients with severe COVID-19 exhibit also reduced circulating plasmacytoid dendritic cells (pDCs) and low plasma IFNα and IFNλ levels when compared to non-severe COVID-19 patients. In a mouse model of pulmonary HSV-1 infection, treatment with recombinant IL-3 reduces viral load and mortality. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating pDCs into the airways by stimulating CXCL12 secretion from pulmonary CD123+ epithelial cells, both, in mice and in COVID-19 negative patients exhibiting pulmonary diseases. This study identifies IL-3 as a predictive disease marker for SARS-CoV-2 infections and as a potential therapeutic target for pulmunory viral infections.
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COVID-19/diagnóstico , Interleucina-3/sangre , Animales , COVID-19/mortalidad , Quimiocina CXCL12/inmunología , Células Dendríticas/citología , Modelos Animales de Enfermedad , Femenino , Alemania , Humanos , Inmunidad Innata , Interferones/sangre , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Linfocitos T/citología , Carga ViralRESUMEN
Herpes simplex viruses not only infect a variety of different cell types, including dendritic cells (DCs), but also modulate important cellular functions in benefit of the virus. Given the relevance of directed immune cell migration during the initiation of potent antiviral immune responses, interference with DC migration constitutes a sophisticated strategy to hamper antiviral immunity. Notably, recent reports revealed that HSV-1 significantly inhibits DC migration in vitro. Thus, we aimed to investigate whether HSV-2 also modulates distinct hallmarks of DC biology. Here, we demonstrate that HSV-2 negatively interferes with chemokine-dependent in vitro migration capacity of mature DCs (mDCs). Interestingly, rather than mediating the reduction of the cognate chemokine receptor expression early during infection, HSV-2 rapidly induces ß2 integrin (LFA-1)-mediated mDC adhesion and thereby blocks mDC migration. Mechanistically, HSV-2 triggers the proteasomal degradation of the negative regulator of ß2 integrin activity, CYTIP, which causes the constitutive activation of LFA-1 and thus mDC adhesion. In conclusion, our data extend and strengthen recent findings reporting the reduction of mDC migration in the context of a herpesviral infection. We thus hypothesize that hampering antigen delivery to secondary lymphoid organs by inhibition of mDC migration is an evolutionary conserved strategy among distinct members of Herpesviridae.
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Movimiento Celular , Células Dendríticas/patología , Células Dendríticas/virología , Herpesvirus Humano 2/patogenicidad , Adhesión Celular , Células Cultivadas , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Herpesvirus Humano 2/inmunología , Humanos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Proteínas Virales/genéticaRESUMEN
Herpes simplex virus type 1 (HSV-1) is a very common human pathogenic virus among the world's population. The lytic replication cycle of HSV-1 is, amongst others, characterized by a tripartite viral gene expression cascade, the assembly of nucleocapsids involving their subsequent nuclear egress, tegumentation, re-envelopment and the final release of progeny viral particles. During productive infection of a multitude of different cell types, HSV-1 generates not only infectious heavy (H-) particles, but also non-infectious light (L-) particles, lacking the capsid. In monocyte-derived mature dendritic cells (mDCs), HSV-1 causes a non-productive infection with the predominant release of L-particles. Until now, the generation and function of L-particles is not well understood, however, they are described as factors transferring viral components to the cellular microenvironment. To obtain deeper insights into the L-particle composition, we performed a mass-spectrometry-based analysis of L-particles derived from HSV-1-infected mDCs or BHK21 cells and H-particles from the latter one. In total, we detected 63 viral proteins in both H- and L-particle preparations derived from HSV-1-infected BHK21 cells. In L-particles from HSV-1-infected mDCs we identified 41 viral proteins which are differentially distributed compared to L-particles from BHK21 cells. In this study, we present data suggesting that L-particles modify mDCs and suppress their T cell stimulatory capacity. Due to the plethora of specific viral proteins incorporated into and transmitted by L-particles, it is tempting to speculate that L-particles manipulate non-infected bystander cells for the benefit of the virus.
RESUMEN
Dendritic cells (DCs) are the guardians of the immune system since they are located in the majority of peripheral tissues. In addition, they are crucial for the induction of an effective immune response based on their unique capacity to stimulate naive T cells. During co-evolution, the human pathogen herpes simplex virus type 1 (HSV-1) has evolved several immune evasion mechanisms in order to subvert the host's immune system especially by targeting DC biology and function. Here we demonstrate that HSV-1 infection influences the IL-6 receptor (IL6R) expression both on protein and mRNA levels in/on human monocyte-derived mature DCs (mDCs). Surprisingly, reduced IL6R expression levels were also observed on uninfected bystander mDCs. Mechanistically, we clearly show that HSV-1-derived non-infectious light (L-) particles are sufficient to trigger IL6R regulation on uninfected bystander mDCs. These L-particles lack the viral DNA-loaded capsid and are predominantly produced during infection of mDCs. Our results show that the deletion of the HSV-1 tegument protein vhs partially rescued the reduced IL6R surface expression levels on/in bystander mDCs. Using a neutralizing antibody, which perturbs the transfer of L-particles to bystander mDCs, was sufficient to rescue the modulation of IL6R surface expression on uninfected bystander mDCs. This study provides evidence that L-particles transfer specific viral proteins to uninfected bystander mDCs, thereby negatively interfering with their IL6R expression levels, however, to a lesser extend compared to H-particles. Due to their immune-modulatory capacity, L-particles represent an elaborated approach of HSV-1-mediated immune evasion.
Asunto(s)
Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno/genética , Receptores de Interleucina-6/genética , Biomarcadores , Efecto Espectador , Células Cultivadas , Células Dendríticas/inmunología , Herpes Simple/genética , Herpes Simple/inmunología , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/aislamiento & purificación , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunomodulación , Monocitos/inmunología , Monocitos/metabolismo , ARN Mensajero/genética , Receptores de Interleucina-6/metabolismo , Replicación ViralRESUMEN
Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.
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Autofagia/fisiología , Células Dendríticas/virología , Electroporación/métodos , Herpesvirus Humano 1/patogenicidad , Monocitos/citología , ARN Interferente Pequeño , Células Dendríticas/fisiología , HumanosRESUMEN
Dendritic cells (DCs) are crucial for the induction of potent antiviral immune responses. In contrast to immature DCs (iDCs), mature DCs (mDCs) are not permissive for infection with herpes simplex virus type 1 (HSV-1). Here, we demonstrate that HSV-1 infection of iDCs and mDCs induces autophagy, which promotes the degradation of lamin A/C, B1, and B2 in iDCs only. This in turn facilitates the nuclear egress of progeny viral capsids and thus the formation of new infectious particles. In contrast, lamin protein levels remain stable in HSV-1-infected mDCs due to an inefficient autophagic flux. Elevated protein levels of KIF1B and KIF2A in mDCs inhibited lamin degradation, likely by hampering autophagosome-lysosome fusion. Therefore, in mDCs, fewer progeny capsids were released from the nuclei into the cytosol, and fewer infectious virions were assembled. We hypothesize that inhibition of autophagic lamin degradation in mDCs represents a very powerful cellular counterstrike to inhibit the production of progeny virus and thus viral spread.
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Cápside/metabolismo , Núcleo Celular , Citosol , Células Dendríticas , Herpesvirus Humano 1/metabolismo , Liberación del Virus/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citosol/metabolismo , Citosol/virología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Herpesvirus Humano 1/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Laminas/genética , Laminas/metabolismo , ProteolisisRESUMEN
Infections with Herpes simplex viruses (HSV) belong to the most common human diseases worldwide, resulting in symptoms ranging from painful, but commonly self-limiting lesions of the orofacial or genital tract to severe infections of the eye or life-threatening generalized infections. Frequent HSV-reactivations at the eye may lead to the development of herpetic stromal keratitis, which is one of the major causes of infectious blindness in developed countries. The vast majority of life-threatening generalized infections occur in immunocompromised individuals, such as transplant recipients or patients suffering from advanced human immunodeficiency virus (HIV) infection with concurrent HSV-reactivation. Over the past decades, Acyclovir (ACV) became the golden standard for the treatment of HSV infections. However, long-term antiviral treatment, as it is required mainly in immunocompromised patients, led to the emergence of resistances towards ACV and other antivirals. Therefore, there is a clear need for the development of new potent antivirals which combine good oral bioavailability and tolerability with low side effects. In the current study we present SC93305 as a novel potent antiviral substance that proved to be highly effective not only against different HSV-1 and HSV-2 strains but also towards ACV- and multi-resistant HSV-1 and HSV-2 isolates. SC93305 shows comparable antiviral activity as reported for ACV and very importantly it does not interfere with the activation of specific immune cells. Here we report that SC93305 does not affect the biological function of dendritic cells (DC), the most potent antigen presenting cells of the immune system to induce antiviral immune responses, nor T cell stimulation or the release of inflammatory cytokines. Thus, SC93305 is a new and promising candidate for the treatment of HSV-1 and HSV-2 infections and in particular also for the inhibition of drug-resistant HSV-1/2 strains.
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Antivirales/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Herpes Simple/virología , Simplexvirus/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Herpes Simple/inmunología , Herpesvirus Humano 1/efectos de los fármacos , Humanos , Inmunidad Celular/inmunología , Inmunidad Humoral , Estructura Molecular , Pruebas de Neutralización , Linfocitos T/inmunología , Linfocitos T/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
It has been shown that herpes simplex virus type 1 (HSV-1) blocks specific immune responses by various mechanisms. Cell lines infected with HSV-1 for instance show a severe impairment of the interferon-gamma (IFN-gamma)-induced signal transducer and activator of transcription 1 (STAT1) signaling pathway. Thus, we examined the influence of HSV-1 infection on IFN-gamma signal transduction in mature dendritic cells (mDCs). In this study, we report the down-regulation of the IFN-gamma receptor alpha chain (IFNGR1) at the mRNA level in HSV-1 infected mDCs. As a consequence, the expression of the IFNGR1 subunit on the cell surface of the infected cell was strongly reduced. Furthermore, we were able to show the inhibition of STAT1 phosphorylation following HSV-1 infection in mDCs, while protein levels of STAT1 remained constant. As a direct downstream effect of STAT1 phosphorylation, the activation of the transcription factor IRF-1 was also clearly inhibited and could no longer be induced by the addition of IFN-gamma. Additional experiments using a virus strain lacking the vhs gene suggested that the mutant virus is more sensitive to IFN-gamma as STAT1 signaling was inhibited to a lesser extent. Infection with a UV-inactivated, replication incompetent virus did not influence the STAT1 signaling pathway at all. In conclusion, we show that HSV-1 blocks IFN-gamma signaling in mDCs, which requires viral gene expression and involves the viral protein vhs.
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Células Dendríticas/metabolismo , Células Dendríticas/virología , Herpesvirus Humano 1/fisiología , Interferón gamma/metabolismo , Transducción de Señal , Humanos , Factor 1 Regulador del Interferón/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1/metabolismo , Receptor de Interferón gammaRESUMEN
Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation.
RESUMEN
As potent antigen-presenting cells, dendritic cells (DCs) are essential for the initiation of effective antiviral immune responses. Viruses and especially herpesviruses, which are able to establish lifelong persistence, exploit several immune evasion mechanisms targeting DC biology. Our group has previously shown that the α-herpesvirus herpes simplex virus type 1 inhibits mature DC (mDC) migration by inducing adhesion via degrading the cellular protein CYTIP (cytohesin-1 interacting protein), an important negative regulator of ß2-integrin activity. In the present study, we extended our analysis to the ß-herpesvirus human cytomegalovirus (HCMV), to investigate whether other herpesviridae also induce such modulations. Indeed, HCMV impairs mDC transwell migration capability following a CCL19-chemokine gradient, despite equivalent expression levels of the cognate chemokine receptor CCR7 at the corresponding time points post-infection. Remarkably, HCMV infection potently induced ß2-integrin activity on mDCs. Furthermore, directly HCMV-infected mDCs, exhibiting viral gene expression, strongly adhere to fibronectin and ICAM-1, in contrast to mDCs lacking infection or viral gene expression. Interestingly, HCMV-positive mDCs display a proteasome-dependent degradation of CYTIP. Contrasting the migration toward CCL19, elevated expression levels of the chemokine receptor CXCR4 in HCMV-infected mDCs were associated with functional CXCL12-chemotaxis under the herein used conditions. In summary, our results show that HCMV shapes mDC adhesion to compromise migration toward CCL19, but retaining CXCL12 responsiveness. Thus, we hypothesize that a preferred migration pattern toward the bone marrow, but not to secondary lymphoid organs, could ultimately cause a failure in the induction of potent antiviral immune responses.
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CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound, full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), consisting of the extracellular domain only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts its function on dendritic cells and additional immune cells. Here, we report the crystal structure of human sCD83 up to a resolution of 1.7Å solved in three different crystal forms. Interestingly, ß-strands C', Câ³, and D that are typical for V-set Ig-domains could not be traced in sCD83. Mass spectrometry analyses, limited proteolysis experiments, and bioinformatics studies show that the corresponding segment displays enhanced main-chain accessibility, extraordinary low sequence conservation, and a predicted high disorder propensity. Chimeric proteins with amino acid swaps in this segment show unaltered immune-suppressive activities in a TNF-α assay when compared to wild-type sCD83. This strongly indicates that this segment does not participate in the biological activity of CD83. The crystal structure of CD83 shows the recurrent formation of dimers and trimers in the various crystal forms and reveals strong structural similarities between sCD83 and B7 family members and CD48, a signaling lymphocyte activation molecule family member. This suggests that CD83 exerts its immunological activity by mixed homotypic and heterotypic interactions as typically observed for proteins present in the immunological synapse.
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Antígenos CD/química , Células Dendríticas/inmunología , Inmunoglobulinas/química , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Antígenos CD/metabolismo , Biomarcadores/química , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Antígeno CD83RESUMEN
Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC). To get insight into the regulation of hCD83 expression, we cloned a 3037 bp fragment up-stream of the translation initiation codon. Deletion mutants were constructed revealing highest promoter activity in the -261 fragment containing four SP1 binding sites and one NF-kappaB element. Electrophoretic mobility shift assays demonstrated the specific interaction of NF-kappaB factors with the NF-kappaB element as well as specific binding of SP1 and SP3 to the SP1 binding site. The hCD83 promoter was inducible by TNF-alpha. This inducibility was strictly dependent on the intact NF-kappaB element.
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Inmunoglobulinas/genética , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas , Antígenos CD , Sitios de Unión , Clonación Molecular , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunoglobulinas/biosíntesis , Glicoproteínas de Membrana/biosíntesis , FN-kappa B/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antígeno CD83RESUMEN
HSV-1 is a very successful representative of the α-herpesvirus family, and â¼ 90% of the population is seropositive for this particular virus. Although the pathogen usually causes the well-known mild lesions on the lips, also, severe infections of the eye or the brain can be observed in rare cases. It is well known, that this virus can efficiently infect the most potent APCs, i.e., the DCs, in their immature and mature state. Although the infection of the iDC has been shown to be productive, infection of mMDDCs is believed to be abortive in the early phase of the viral replication cycle. In line with these findings, no virus particles can be detected in the supernatant of HSV-1-infected mMDDC. In this study, however, we show for the first time that this pathogen completes its replication cycle in mMDDCs. We detected the presence of viral gene transcripts of all three phases of the replication cycle, as well as of late viral proteins, and even the generation of small amounts of progeny virus. Although we could confirm the findings that these particles are not released into the supernatant, surprisingly, the newly generated viral particles can be passed on to Vero cells, as well as to primary keratinocytes in a cell-cell contact-dependent manner. Finally, we provide evidence that the viral gE is involved in the transfer of infectious virus from mMDDCs to other permissive cells.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , Herpes Simple/transmisión , Herpesvirus Humano 1/fisiología , Virión/fisiología , Replicación Viral , Animales , Western Blotting , Adhesión Celular , Comunicación Celular , Movimiento Celular , Chlorocebus aethiops , Células Dendríticas/metabolismo , Herpes Simple/inmunología , Herpes Simple/metabolismo , ARN Mensajero/genética , ARN Viral/genética , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The cell surface protein CD83 belongs to the immunoglobulin super family and is highly expressed on mature dendritic cells (DCs). A membrane bound and a soluble form of CD83 (sCD83) have been described. Previously, the isolation of a purified recombinant sCD83 molecule from bacterial cultures using high pressure liquid chromatography was reported. This recombinant protein reduced DC-mediated T cell proliferation in vitro and displayed an inhibitory effect in the experimental autoimmune encephalomyelitis (EAE) model. When purifying sCD83 from bacteria, however, a lipopolysaccharide fraction is frequently co-isolated with the recombinant sCD83 protein. Moreover, the subsequent separation of sCD83 from contaminating LPS is usually accompanied by a considerable loss of soluble CD83. A further disadvantage of soluble CD83 expression in prokaryotic cells is the lack of functional glycosylation. To overcome these problems, we developed an alternative strategy to express sCD83 in eukaryotic human embryonic kidney (HEK) 293 T cells. Using this system, we showed that recombinant sCD83 was LPS-free and effectively glycosylated with all three asparagine residues at least partially involved. The functionality of the expressed sCD83 protein was examined using the mixed lymphocyte reaction (MLR) assay, demonstrating a reduced DC-mediated T cell proliferation as previously reported for the sCD83 protein purified from E. coli. Thus, a new protocol for efficient eukaryotic expression and purification of sCD83 was established, which might have several advantages compared to prokaryotic expression systems.