Electroporation of siRNA into mouse bone marrow-derived macrophages and dendritic cells.

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.

Functional characterization of the HuR:CD83 mRNA interaction.

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.

Herpes simplex virus type I (HSV-1) replicates in mature dendritic cells but can only be transferred in a cell-cell contact-dependent manner.

HSV-1 is a very successful representative of the α-herpesvirus family, and ∼ 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.

Herpes simplex virus type I infection of mature dendritic cells leads to reduced LMP7-mRNA-expression levels.

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.

HSV-1 upregulates the ARE-binding protein tristetraprolin in a STAT1- and p38-dependent manner in mature dendritic cells.

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.

Small interfering RNA (siRNA) delivery into murine bone marrow-derived dendritic cells by electroporation.

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.

Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function.

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.

Infection of human dendritic cells with herpes simplex virus type 1 dramatically diminishes the mRNA levels of the prostaglandin E(2) receptors EP2 and EP4.

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.

Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome.

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.

CD83 knockdown in monocyte-derived dendritic cells by small interfering RNA leads to a diminished T cell stimulation.

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.

CD83: an update on functions and prospects of the maturation marker of dendritic cells.

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.

Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation.

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.

Expression of CD83 is regulated by HuR via a novel cis-active coding region RNA element.

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.

Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated migration.

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.

CD83 is a dimer: Comparative analysis of monomeric and dimeric isoforms.

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.

Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression.

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.