Infection of dendritic cells with herpes simplex virus type 1 induces rapid degradation of CYTIP, thereby modulating adhesion and migration.

Immune responses require spatial and temporal coordinated interactions between different cell types within distinct microenvironments. This dynamic interplay depends on the competency of the involved cells, predominantly leukocytes, to actively migrate to defined sites of cellular encounters in various tissues. Because of their unique capacity to transport antigen from the periphery to secondary lymphoid tissues for the activation of naive T cells, dendritic cells (DCs) play a key role in the initiation and orchestration of adaptive immune responses. Therefore, pathogen-mediated interference with this process is a very effective way of immune evasion. CYTIP (cytohesin-interacting protein) is a key regulator of DC motility. It has previously been described to control LFA-1 deactivation and to regulate DC adherence. CYTIP expression is up-regulated during DC maturation, enabling their transition from the sessile to the motile state. Here, we demonstrate that on infection of human monocyte-derived DCs with herpes simplex virus type 1 (HSV-1), CYTIP is rapidly degraded and as a consequence ß-2 integrins, predominantly LFA-1, are activated. Furthermore, we show that the impairment of migration in HSV-1-infected DCs is in part the result of this increased integrin-mediated adhesion. Thus, we propose a new mechanism of pathogen-interference with central aspects of leukocyte biology.

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.

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.

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.