[Show abstract][Hide abstract] ABSTRACT: Innate immune recognition is an important early event in the host response to herpes simplex virus-1 (HSV-1) infection. Dendritic cells (DC) play an important sentinel role in this recognition. Previous studies have shown that monocyte-derived DC (MDDC) respond to HSV-1 by up-regulation of costimulatory molecules and type I IFN release, but the molecular targets on the virus recognized by the DC have not been defined. In this study we show that MDDC recognize and respond to the four essential viral glycoproteins, gB, gD, and gHgL, independent of other viral proteins or nucleic acids. DC recognition of these four glycoproteins leads to the up-regulation of CD40, CD83, CD86, and HLA-DR and to the production of IFN-alpha and IL-10, but not IL-12p70. Glutaraldehyde-fixation and nonfunctional gH mutants were used to show that recognition of glycoproteins does not require membrane fusion. The nature of the recognition event was probed further by transfecting glycoproteins individually or in combination, by blocking individual proteins with Abs, or by using mutant gD constructs unable to bind to their known cognate receptors. Unexpectedly, MDDC responses were found to require expression of all four glycoproteins. Furthermore, gD mutants that cannot bind nectin-1 and/or herpesvirus entry mediator can still induce DC maturation. Finally, although HSV-1 can signal via the TLR2 receptor, this receptor does not mediate recognition of glycoproteins. Thus, the complex of the four essential HSV-1 entry glycoproteins on the cell surface can provide a target for innate immune recognition of this virus.
The Journal of Immunology 07/2008; 180(11):7525-36. DOI:10.4049/jimmunol.180.11.7525 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) sense the presence of conserved microbial structures in their local microenvironment via specific pattern recognition receptors (PRRs). This leads to a programme of changes, which include migration and activation, and enables them to induce adaptive T cell immunity. Mitogen-activated protein kinases (MAPKs) are implicated in this response, but the pathways leading from PRR ligation to MAPK activation, and hence DC activation, are not fully understood. Recent studies in the nervous system have suggested that the mixed lineage kinase (MLK) family of MAPK kinase kinase proteins may be involved as an intermediary step between PRRs and MAPKs. Therefore, in this study, we have used a well-established DC model to explore the role of MLKs in these cells. Messenger RNA for MLKs 2, 3, 4 and DLK and protein for MLKs 2, 3 and DLK are found in DC. DC activation in response to model PRR ligands, such as LPS or poly (I:C), is accompanied by phosphorylation of MLK3. In contrast, another known PRR ligand, zymosan, induces little MLK3 phosphorylation. Inhibition of MLK activity using a pharmacological inhibitor, CEP11004, blocks p38 and Jun N-terminal kinase (JNK) MAPK activation in response to LPS and poly (I:C), but not zymosan. The inhibition is associated with a block in DC activation as measured by cell-surface marker expression and cytokine secretion. Thus, MLKs are expressed in DC, and are implicated in DC activation, and the involvement of MLKs appears to be selective, depending on the nature of the DC stimulus.
International Immunology 09/2007; 19(8):923-33. DOI:10.1093/intimm/dxm050 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herpes Simplex Virus-1 is a common infectious agent, but the precise detail of entry and infection of cells has only now begun to be clarified. Four viral surface glycoproteins (gB, gD, gH and gL) are required. This review summarises the known structure and function of each of these essential viral envelope glycoproteins, and explores what is known about their close cooperation with each other in mediating cellular membrane fusion. It is suggested that, following gD binding to one of its entry receptors, membrane fusion is mediated by gB and the heterodimer gH/gL. Significantly, these four entry glycoproteins also play a key role in the interaction between HSV and the host immune system. The glycoproteins serve an important role as targets of adaptive immunity. However, recent studies have demonstrated that the same proteins also play a key role in initiating the early innate immune response to HSV. Understanding the complex functions of these HSV proteins may be essential for successful development of vaccines for HSV.
Reviews in Medical Virology 05/2007; 17(3):205-15. DOI:10.1002/rmv.531 · 5.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The central role of dendritic cells (DC) in the initiation of immune responses requires these cells to be able to determine the degree of danger in their microenvironment. Abrogating the activity of type I interferon (IFN) secreted after lipopolysaccharide (LPS) stimulation of DC inhibits CD86 and human leucocyte antigen-DR (HLA-DR) upregulation at a low LPS concentration. At a higher concentration of LPS, while changes in surface phenotype are not dependent on type I IFN, this cytokine is required for maximal secretion of interleukin-12 (IL-12) and tumour necrosis factor-alpha (TNFalpha) by DC. Thus, the secretion and autocrine activity of type I IFN after Toll-like receptor stimulation enables DC to orchestrate a hierarchical maturation response with regard to changes in surface phenotype and secretion of cytokines. In addition, the activation of nuclear factor-kappaB and p38 pathways in DC can occur either in an additive fashion when DC are exposed to dual stimulation or can be activated in discrete phases over time when DC are exposed to LPS alone. The differential activation of these pathways provides a mechanism for DC to integrate the activation by multiple stimuli and thus amplify responses to pathogen infection.
[Show abstract][Hide abstract] ABSTRACT: The maturation state of dendritic cells (DC) is regulated by a variety of factors. These include ligands expressed by T cells, such as members of the TNF superfamily. Recent studies have highlighted the role of one such molecule, LIGHT, as a positive regulator of DC biology, promoting the maturation of these cells through the activation of NF-kappaB pathways. In addition, HSV-1 envelope glycoproteins can also bind the LIGHT receptor, herpes virus entry mediator (HVEM), and activate similar downstream signalling pathways in DC. The consequence of this host-viral interaction may be a novel pathway of viral immune evasion.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are potent antigen-presenting cells that are critical in the initiation of immune responses to control and/or eliminate viral infections. Recent studies have investigated the effects of virus infection on the biology of DC. This review summarizes these changes, focusing on both the DC parameters affected and the viral factors involved. In addition, the central role of DC biology in the pathogenesis of several viral families, including herpesviruses, paramyxoviruses and retroviruses, is explored. The field of pathogen recognition by DC is addressed, focusing on its role in protecting the host from viral infection, as well as the ability of viruses to exploit such host receptor ligation and signalling to their replicative advantage. The hypothesis is proposed that virus and host have evolved a symbiotic relationship to ensure both viral transmission and host survival.
International Journal of Experimental Pathology 09/2005; 86(4):187-204. DOI:10.1111/j.0959-9673.2005.00440.x · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) sense infection in their local microenvironment and respond appropriately in order to induce T cell immunity. This response is mediated in part via the mitogen-activated protein kinase (MAPK) pathways. Hydrogen peroxide is present frequently in the inflammatory DC milieu and is known to activate MAPK. Therefore this study examines the role of hydrogen peroxide, both alone and in combination with lipopolysaccharide (LPS), in the regulation of activation of two key MAPK, p38 and JNK, regulation of phenotype, and regulation of apoptosis in human monocyte-derived DC. At low concentrations, hydrogen peroxide activates p38, but does not alter DC phenotype. At higher concentrations, hydrogen peroxide activates both p38 and JNK. Activation of JNK, which is associated with inhibition of tyrosine phosphatases in DC, is linked to the induction of DC apoptosis. An upstream JNK inhibitor (CEP11004) and a competitive JNK inhibitor (SP600125) both partially protected the DC from the proapoptotic effects of hydrogen peroxide. Unexpectedly, hydrogen peroxide and LPS synergize in inducing JNK activation and DC apoptosis. JNK-mediated apoptosis may limit damaging immune responses against neoepitopes generated by modification of self-antigens by reactive oxygen species present at sites of inflammation.
Free Radical Biology and Medicine 07/2005; 38(12):1637-52. DOI:10.1016/j.freeradbiomed.2005.02.022 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Impaired T-cell reactivity to HCV, a hallmark of inefficient adaptive immunity, is believed to be responsible for the high propensity of HCV to cause chronic infection. Dendritic cells are the most potent antigen-presenting cells and many viruses affect various dendritic cell functions. Data suggest that such changes induced by HCV may have an important role in viral persistence. HCV has been shown to bind to dendritic cells, although viral replication within these cells occurs at a very low level. Dendritic cells from people with chronic HCV infection are impaired in their capacity to stimulate T cells. This impairment may be a consequence of changes in the expression of major histocompatibility complex and costimulatory molecules on its surface, as well as in the production of cytokines such as interleukin 12. In addition, hepatic dendritic cells may be affected by the tolerogenic microenvironment of the liver, possibly generating dendritic cells that promote regulatory T cells, which suppress the cellular immune response mounted against HCV.
[Show abstract][Hide abstract] ABSTRACT: The relationship between phagocytic capacity and morphology of dendritic cells (DCs) has not been investigated previously. Therefore, in order to approach this question, we have developed a novel assay, which is described here. The model of dendritic cells (DCs) used was based upon cytokine-induced differentiation of peripheral blood mononuclear cells, followed by culture on a fibronectin substratum. Under these conditions, standard current methods of quantifying phagocytosis are not applicable, as they rely upon flow cytometric analysis of fluid phase cells; and for adherent cells, quantitative efficiency of uptake is very difficult to measure. Furthermore, for both fluid phase and adherent cells, it is difficult to discriminate between internal and externally bound probe, and degradation of internalised probes can lead to artefacts. Therefore, in this study, these technical issues have been overcome by a simple and flexible assay. Phycoerythrin (PE)-conjugated antibodies are used to target microbeads to the DCs. Following an appropriate incubation period, secondary staining with fluorescein isothiocyanate (FITC)-conjugated antibody allows discrimination between internal and externally bound beads. Microscopic visualisation allows individual beads to be studied easily and thus phagocytosis quantified, whilst permitting parallel examination of morphological parameters. In particular, the relationship between bead uptake and the nature and distribution of the dendritic processes can be evaluated.
[Show abstract][Hide abstract] ABSTRACT: Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-kappaB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.
The Journal of Immunology 10/2004; 173(6):4108-19. DOI:10.4049/jimmunol.173.6.4108 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infection with herpes simplex virus remains a significant cause of disease. The host immune system plays an important role in containing viral replication, and there has been considerable progress in defining which components of immunity are key to the resolution of infection. Nevertheless, effective immunoprophylaxis or immunotherapy has not yet been achieved.
Recent work has focused on understanding the early events leading to the herpes simplex virus-specific immune response, in particular on the role of antigen-presenting dendritic cells. Herpes simplex virus has evolved a number of ways of interfering with antigen presentation by dendritic cells, thus presumably impeding or delaying the host immune response. Nevertheless, herpes simplex virus triggers strong cellular and humoral immunity. The ability of dendritic cells to take up dead or dying infected cells and cross-present them to cognate T cells may be the key to resolving this apparent paradox. Interaction between dendritic cell subsets, and particularly the virus-induced release of type I interferons may be essential to drive efficient antigen cross-presentation and subsequent T-cell activation.
A greater understanding of the importance of dendritic cells in driving viral immunity, and of the ligands that activate these cells and the cytokines they secrete, has provided novel vaccination strategies. The delivery of immunomodulatory genes together with viral antigens, for example by DNA vaccination, may harness the full potential of dendritic cells, and achieve the goal of effective immunological control of herpes simplex virus.
Current Opinion in Infectious Diseases 07/2004; 17(3):199-203. DOI:10.1097/00001432-200406000-00005 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) respond to danger signals from tissue injury by amplifying their immune-inducing capacity. In the cancer context, this may lead to in vivo antitumor synergism between DCs and DNA-damaging chemotherapeutic agents. Neither the interaction between DCs and dying tumor cells nor whether different ways of inducing cell injury can deliver danger signals of different strength to DCs nor the potential role of damaged DNA as a danger signal has been studied rigorously. Here we report that coculture of immature DCs with tumor cells treated with the alkylating agents melphalan and chlorambucil leads to enhanced autologous and allogeneic T-cell activation, up-regulation of surface expression of MHC and costimulatory molecules, and increased interleukin (IL)-12 secretion. Exposure of the same DCs to tumor cells killed by cytarabine or by freeze-thaw (primary necrosis) resulted in significantly less T-cell proliferation and IL-12 production, indicating that DCs are able to sense and respond differentially to the mode of cell death. Exposure of DCs to DNA purified from tumor cells treated with alkylating agents also increased their T-cell-stimulating capacity, expression of CD86, and IL-12 secretion, supporting the hypothesis that the activating effects of tumor cells are linked to the nature of the DNA damage. This is the first study that shows that DCs respond differentially to killed tumor cells, depending upon the mechanism of DNA damage and consequent cell death.
Cancer Research 09/2003; 63(16):5143-50. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several lines of evidence suggest that dendritic cells (DCs), the most potent antigen-presenting cells known, play a role in the immunological control of herpes simplex virus (HSV) infections. HSV infection of DCs induced submaximal maturation, but DCs failed to mature further in response to lipopolysaccharide (LPS). LPS induced interleukin (IL)-12 secretion, and the induction of primary and secondary T cell responses were impaired by infection. Ultimately, DC infection resulted in delayed, asynchronous apoptotic cell death. However, infected DCs induced HSV recall responses in some individuals. Furthermore, soluble factors secreted by DCs after infection induced DC maturation and primed for IL-12 secretion after LPS stimulation. These data support a pathogenetic model of HSV infection, in which initial delay in the generation of immune responses to HSV at peripheral sites is mediated by disruption of DC function but is overcome by bystander DC maturation and cross-presentation of HSV antigens.
The Journal of Infectious Diseases 02/2003; 187(2):165-78. DOI:10.1086/367675 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously.
Methods: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO4, and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. Results: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis.
Conclusions: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering–activation–apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.