RESUMO
The structure of soil aggregates plays an important role for the turnover of particulate organic matter (POM) and vice versa. Analytical approaches usually do not disentangle the continuous re-organization of soil aggregates, caught between disintegration and assemblage. This led to a lack of understanding of the mechanistic relationship between aggregation and organic matter dynamics in soils. In this study, we took advantage of a process-based mechanistic model that describes the interaction between the dynamic (re-)arrangement of soil aggregates, based on dynamic image analysis data of wet-sieved aggregates, to analyze the turnover of POM, and simultaneous soil surface interactions in a spatially and temporally explicit way. Our novel modeling approach enabled us to unravel the temporal development of aggregate sizes, organic carbon (OC) turnover of POM, and surface coverage as affected by soil texture, POM input, and POM decomposition rate comparing a low and high clay soil (18% and 33% clay content). Our results reveal the importance of the dynamic re-arrangement of soil structure on POM-related turnover of OC in soils. Firstly, aggregation was largely determined by the POM input fostering aggregates through additional gluing joints outweighing soil texture at lower decomposition rate, whereas at higher decomposition rate, soil texture had a higher influence leading to larger aggregates in the high clay soil. Secondly, the POM storage increased with clay content, showing that surface interactions may delay the turnover of OC into CO2 . Thirdly, we observed a structural priming effect in which the increased input of POM induced increased structural re-arrangement stimulating the mineralization of old POM. This work highlights that the dynamic re-arrangement of soil aggregates has important implications for OC turnover and is driven by underlying surface interactions where temporary gluing spots stabilize larger aggregates.
Assuntos
Carbono , Solo , Carbono/química , Argila , Solo/químicaRESUMO
OBJECTIVE: The objective of this in vitro study was to investigate fracture load, fracture types, and impact of chewing simulation of human molars restored with 3D printed indirect polyetheretherketone (PEEK) inlays and compare these with milled indirect PEEK inlays, direct resin composite fillings, and sound teeth. MATERIALS AND METHODS: A total of 112 molars with form congruent class I cavities were restored with (n = 16/group) 3D printed indirect PEEK inlays via fused layer manufacturing (FLM): (1) Essentium PEEK (ESS), (2) KetaSpire PEEK MS-NT1 (KET), (3) VESTAKEEP i4 G (VES), (4) VICTREX PEEK 450G (VIC), (5) milled indirect PEEK inlays JUVORA Dental Disc 2 (JUV), and (6) direct resin composite fillings out of Tetric EvoCeram (TET). Sound teeth (7) acted as positive control group. Half of the specimens of each group (n = 8) were treated in a chewing simulator combined with thermal cycling (1.2 million × 50 N; 12,000 × 5 °C/55 °C). Fracture load and fracture types of all molars were determined. Statistical analyses using Kolmogorov-Smirnov test and two-way ANOVA with partial eta squared (ηp2) followed by Scheffé post hoc test, chi square test and Weibull modulus m with 95% confidence interval were computed (p < 0.05). RESULTS: ESS and TET demonstrated the lowest fracture load with a minimum of 956 N, whereas sound molars showed the highest values of up to 2981 N. Chewing simulation indicated no impact (p = 0.132). With regard to Weibull modulus, KET presented a lower value after chewing simulation than JUV, whereas TET had the highest value without chewing simulation. All indirect restorations revealed a tooth fracture (75-100%), direct resin composite fillings showed a restoration fracture (87.5%), and 50% of the sound teeth fractured completely or had cusp fractures. CONCLUSIONS: All 3D printed and milled indirect PEEK inlays as well as the direct resin composite fillings presented a higher fracture load than the expected physiological and maximum chewing forces. CLINICAL RELEVANCE: 3D printing of inlays out of PEEK via FLM provided promising results in mechanics, but improvements in terms of precision and esthetics will be required to be practicable in vivo to represent an alternative dental material.
Assuntos
Impressão Tridimensional , Benzofenonas , Resinas Compostas , Análise do Estresse Dentário , Estética Dentária , Humanos , Restaurações Intracoronárias , Cetonas , Teste de Materiais , Polietilenoglicóis , Polímeros , Cimentos de Resina , Fraturas dos DentesRESUMO
OBJECTIVES: The aim of this study was to investigate the effect of artificial aging on the Martens parameters of different 3D printed and milled polyaryletherketon (PAEK) materials. METHODS: In total 120 specimens of 4 different polyetheretherketon (PEEK) materials (Essentium PEEK, KetaSpire PEEK MS-NT1, VICTREX PEEK 450G and VESTAKEEP i4 G) were additively manufactured via fused layer manufacturing (FLM) in either horizontal or vertical directions (n=15 per group). 75 specimens were milled out of prefabricated PAEK blanks from the materials breCAM.BioHPP, Dentokeep, JUVORA Dental Disc 2 and Ultaire AKP (=15 per group). Martens hardness (HM), indentation hardness (HIT) and indentation modulus (EIT) were determined initially and longitudinally after thermocycling (5-55°C, 10,000x) and autoclaving (134°C, 2bar). In each case, the surface topography of the specimens was examined for modifications using a light microscope. Data were analysed with Kolmogorov-Smirnov test, univariate ANOVA followed by post-hoc Scheffé test with partial eta squared (ηp2), Kruskal-Wallis-, Mann-Whitney-U-, Friedman- and Wilcoxon-Test. A value of p<0.05 was considered as significant. RESULTS: Milled specimens showed higher Martens parameters than printed ones (p<0.001). Artificial aging had a negative effect on the measured parameters (p<0.001). Horizontally printed specimens presented higher Martens parameters than vertically printed ones, regardless of material and aging process (p<0.001). Essentium PEEK and breCAM.BioHPP showed the highest and VICTREX PEEK 450G as well as Ultaire AKP the lowest values of all investigated PAEK materials initially, after thermocycling and after autoclaving (p<0.001). Microscopic examinations showed that artificial aging did not cause any major modifications of the materials. SIGNIFICANCE: Additively manufactured PEEK materials showed lower Martens parameters than milled ones, whereas horizontally printed specimens presented higher values than vertically printed ones. Artificial aging had a negative effect on the Martens parameters, but not on the surface topography.
Assuntos
Mustelidae , Envelhecimento , Animais , Dureza , Teste de Materiais , Impressão TridimensionalRESUMO
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.
Assuntos
Células da Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Eletroporação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Hipóxia Celular , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipídeos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fenótipo , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Quimiocinas CC/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Herpesvirus Humano 1/fisiologia , Receptores CCR7/metabolismo , Receptores de Prostaglandina E/metabolismo , Quimiotaxia , Humanos , Prostaglandinas E/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4RESUMO
CD83 is one of the most characteristic cell surface markers for fully matured dendritic cells (DCs). In their function as antigen presenting cells they induce T-cell mediated immune responses. In this review we provide an overview on well described and proposed functions of this molecule as well as on very recent insights and new hypothesis. Already the CD83 messenger RNA processing differs remarkably from the processing of other cellular mRNAs: instead of the usual TAP mRNA export pathway, the CD83 mRNA is exported by the specific CRM1-mediated pathway, utilized only by a minority of cellular mRNAs. On the protein level, two different isoforms of CD83 exist: a membrane-bound and a soluble form. The isoforms are generated by different subsets of cells, including DCs, T-cells and B-cells, and also differ in their biological function. While the membrane-bound CD83 is of immune stimulatory capacity, activates T-cells and is important for the generation of thymocytes, the soluble CD83 has the opposite effect and has an immune inhibitory capacity. Due to its immune inhibitory function, CD83 has great potential for treatment of autoimmune diseases, for organ transplantations, and for immunotherapy, just to name a few examples. Moreover, some viruses prevent recognition by the host's immune system by specifically targeting CD83 surface expression.
Assuntos
Antígenos CD/fisiologia , Células Dendríticas/fisiologia , Imunoglobulinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Animais , Antígenos CD/genética , Autoimunidade/fisiologia , Células Dendríticas/citologia , Humanos , Imunidade Celular/fisiologia , Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Linfócitos T/fisiologia , Antígeno CD83RESUMO
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.
Assuntos
Células Dendríticas/fisiologia , Eletroporação/métodos , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Movimento Celular , Células Dendríticas/imunologia , Inativação Gênica , Humanos , Imunofenotipagem , Lamina Tipo A/imunologia , Teste de Cultura Mista de Linfócitos , RNA/química , RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The appropriate prediction of the fate of the contaminant is an essential step when evaluating the risk of severe groundwater pollutions-in particular in the context of natural attenuation. We numerically study the reactive transport of phenanthrene at the field scale in a multilayer soil profile based on experimental data. The effect of carrier facilitation by dissolved organic carbon is emphasized and incorporated in the model. Previously published simulations are restricted to the saturated zone and/or to homogeneous soil columns at the laboratory scale. A numerical flow and transport model is extended and applied to understand and quantify the relevant processes in the case of a strongly sorbing hydrophobic organic compound that is subject to carrier facilitation in the unsaturated zone. The contaminant migration is investigated on long- and short-term time scales and compared to predictions without carrier facilitation. The simulations demonstrate the importance of carrier facilitation and suggest strongly to take this aspect into account. By carrier facilitation breakthrough times at the groundwater level decreased from 500 to approximately 8 years and concentration peaks increased by two orders of magnitude in the long-term simulation assuming a temporary spill in an initially unpolluted soil with a non-sorbing carrier.
Assuntos
Simulação por Computador , Fenantrenos/análise , Poluentes do Solo/análise , Solo/análise , Movimentos da Água , Poluentes Químicos da Água/análise , Carbono/química , Modelos Teóricos , Compostos Orgânicos/química , Porosidade , Solubilidade , Fatores de TempoRESUMO
Dendritic cells (DC) and macrophages (MΦ) play a pivotal role in antimicrobial defense, in the regulation of immune responses, and in maintaining tissue homeostasis. The analysis of DC and MΦ function relies on primary cells albeit these cells are known to be difficult to transfect. This makes the use of small interfering RNA (siRNA) for targeted manipulation of gene expression by RNA interference difficult. In the following chapter, we provide a detailed protocol for the successful transfer of siRNA via electroporation into a defined population of mouse bone marrow-derived MΦ or DC that does not cause toxicity to the myeloid cells or nonspecific alterations of their biological functions. Factors that influence the transfection and knockdown rate will be highlighted.
Assuntos
Células da Medula Óssea/citologia , Células Dendríticas/metabolismo , Eletroporação/métodos , Macrófagos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Animais , Células Dendríticas/citologia , Técnicas de Silenciamento de Genes , Macrófagos/citologia , CamundongosRESUMO
Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1-3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein:RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway.
Assuntos
ADP-Ribosil Ciclase 1/genética , Proteínas ELAV/genética , RNA Mensageiro/genética , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Primers do DNA , Humanos , Ativação Linfocitária , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Herpes Simples/transmissão , Herpesvirus Humano 1/fisiologia , Vírion/fisiologia , Replicação Viral , Animais , Western Blotting , Adesão Celular , Comunicação Celular , Movimento Celular , Chlorocebus aethiops , Células Dendríticas/metabolismo , Herpes Simples/imunologia , Herpes Simples/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Mature dendritic cells (mDCs) are the most potent antigen presenting cells within the human immune system known today. However, several viruses, including herpes simplex virus type 1 (HSV-1) have developed numerous immune escape mechanisms, such as the avoidance of peptide presentation through the major histocompatibility complex (MHC) class I to CD8(+) cytotoxic T-cells. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome, a multicatalytic protease complex. Upon exposure to IFN-gamma, the constitutive proteasome is partially replaced by the immunoproteasome, which contains the IFN-gamma-inducible subunits LMP2, MECL1 and LMP7. In this study, we report the downregulation of LMP7 on mRNA level in HSV-1 infected mDCs. Interestingly, this reduction was not vhs-mediated since using a virus strain lacking the vhs gene we obtained similar results. However, on protein level, LMP7-expression was not affected, which is probably due the high stability of the LMP7 protein. Also the incorporation of LMP7 into the immunoproteasome was not affected by HSV-1. However, for the in vivo situation, in which DC reside for a prolonged time period in peripheral tissues, the reduced LMP7-mRNA level could be of biological importance, since the virus could escape/hide from immune system of the host and establish latency processes.
Assuntos
Células Dendríticas/enzimologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Complexos Multienzimáticos/metabolismo , Células Cultivadas , Regulação para Baixo , Herpes Simples/virologia , Humanos , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/análiseRESUMO
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.
Assuntos
Células Dendríticas/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/metabolismo , Tristetraprolina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/virologia , Humanos , Fosforilação , Tristetraprolina/genética , Regulação para CimaRESUMO
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.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipopolissacarídeos/farmacologia , Animais , Diferenciação Celular , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , RNA Interferente Pequeno/genética , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Células Dendríticas/efeitos dos fármacos , Ativação Linfocitária , Glicoproteínas de Membrana/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Linfócitos T/imunologia , Antígenos CD/genética , Células Dendríticas/imunologia , Eletroporação , Humanos , Imunoglobulinas/genética , Ativação Linfocitária/genética , Glicoproteínas de Membrana/genética , Monócitos/imunologia , Antígeno CD83RESUMO
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.
Assuntos
Antígenos CD/biossíntese , Células Dendríticas/citologia , Herpesvirus Humano 1/metabolismo , Imunoglobulinas/biossíntese , Glicoproteínas de Membrana/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Separação Celular , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Herpes Simples/imunologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Cinética , Leucócitos Mononucleares/virologia , Transfecção , Ubiquitina-Proteína Ligases/metabolismo , Antígeno CD83RESUMO
Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.
Assuntos
Antígenos CD/biossíntese , Antígenos de Superfície/fisiologia , Regulação da Expressão Gênica , Imunoglobulinas/biossíntese , Carioferinas/fisiologia , Glicoproteínas de Membrana/biossíntese , Proteínas de Ligação a RNA/fisiologia , RNA/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ácidos Graxos Insaturados/farmacologia , Inativação Gênica , Genes Reporter , Vetores Genéticos , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Células Jurkat , Cinética , Luciferases/metabolismo , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Reação em Cadeia da Polimerase , Ligação Proteica , Biossíntese de Proteínas , Transporte Proteico , RNA/química , RNA/metabolismo , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína Exportina 1 , Antígeno CD83RESUMO
Recently, we reported that soluble CD83 has a strong immunosuppressive activity in vitro as well as in vivo. Sequence alignment of CD83 between different species revealed the presence of five cysteines in the extracellular Ig-domain of the protein. This opens up the possibility that four cysteines are involved in the formation of two intramolecular disulfide bonds and a possible involvement of the remaining fifth cysteine in the formation of an intermolecular covalent disulfide bond, leading to the dimerization of the extracellular protein domains. Using recombinant mutational analyses, where the fifth cytosine at amino acid position 129 was mutated to a serine, we could prove that the fifth cysteine residue was indeed necessary for the dimerization. Functional analyses revealed that the mutant protein inhibited almost completely the upregulation of CD83-expression during DC maturation. Furthermore, the functional activity of the mutant protein was investigated using MLR assays and we could show that the mutant soluble CD83 protein inhibited DC-mediated allogeneic T-cell stimulation in vitro.
Assuntos
Imunoglobulinas/química , Glicoproteínas de Membrana/química , Sequência de Aminoácidos , Animais , Antígenos CD , Western Blotting , Proliferação de Células , Separação Celular , Clonagem Molecular , Bases de Dados como Assunto , Células Dendríticas/citologia , Dimerização , Dissulfetos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imunoglobulinas/biossíntese , Glicoproteínas de Membrana/biossíntese , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Software , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Linfócitos T/imunologia , Regulação para Cima , Antígeno CD83RESUMO
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.
Assuntos
Células Dendríticas/imunologia , Herpesvirus Humano 1/imunologia , Diferenciação Celular , Movimento Celular , Células Dendríticas/citologia , Regulação para Baixo , Herpes Simples/imunologia , Humanos , Mutação , RNA Mensageiro/análise , Receptores CCR7 , Receptores CXCR4/análise , Receptores CXCR4/genética , Receptores de Quimiocinas/análise , Receptores de Quimiocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases , Fatores de Tempo , Proteínas Virais/genéticaRESUMO
Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA-/- knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.