Herpes simplex virus type 2 induces rapid cell death and functional impairment of murine dendritic cells in vitro

Dendritic cells (DC) are critical for stimulation of naive T cells. Little is known about the effect of herpes simplex virus type 2 (HSV-2) infection on DC structure or function or if the observed effects of HSV-1 on human DC are reproduced in murine DC. Here, we demonstrate that by 12 h postinfection, wild-type (wt) HSV-2 (186) abortively infected murine bone marrow-derived DC and induced early cell death compared to UV-inactivated HSV-2 or mock-infected DC. HSV-2-induced loss of DC viability was more rapid than that induced by HSV-1 and was due, in part, to apoptosis, as shown by TEM, caspase-3 activation, and terminal deoxynucleotidyl transferase-mediated dCTP biotin nick end labeling. HSV induced type-specific changes in the murine DC immunophenotype. At 12 h postinfection, wt HSV-2 upregulated DC major histocompatibility complex (MHC) class II expression, and in contrast to UV-inactivated HSV-2, downregulated expression of MHC class I, but it had no effect on surface CD40, CD80, or CD86. Wt HSV-1 (MC-1) induced only CD40 upregulation. More-profound effects on the DC immunophenotype were observed in HSV-2-infected neonatal DC. Wt HSV of either serotype impaired murine DC-induced T-cell alloproliferation and lipopolysaccharide-induced DC interleukin-12 secretion. Thus, there are marked differences in the levels of HSV-induced cytolysis in DC according to the HSV serotype, although HSV-2 displays immunomodulatory effects on the DC immunophenotype and function similar to those of HSV-1.     

CD1 antigen presentation by human dendritic cells as a target for herpes simplex virus immune evasion

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying activation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down-regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus.     

Herpes simplex virus infection of human dendritic cells induces apoptosis and allows cross-presentation via uninfected dendritic cells

HSV efficiently infects dendritic cells (DCs) in their immature state and induces down-regulation of costimulatory and adhesion molecules. As in mice, HSV infection of human DCs also leads to their rapid and progressive apoptosis, and we show that both early and late viral proteins contribute to its induction. Because topical HSV infection is confined to the epidermis, Langerhans cells are expected to be the major APCs in draining lymph nodes. However, recent observations in murine models show T cell activation to be mediated by nonepidermal DC subsets, suggesting cross-presentation of viral Ag. In this study we provide an explanation for this phenomenon, demonstrating that HSV-infected apoptotic DCs are readily phagocytosed by uninfected bystander DCs, which, in turn, stimulate virus-specific CD8+ T cell clones.     

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.     

Production and function of activin A in human dendritic cells

Activin A, a member of the transforming growth factor-beta superfamily, has a role in tissue repair and inflammation. In our previous studies, we identified by immunohistochemistry DC-SIGN(+) dendritic cells as a source of activin A in vivo. The present study was aimed at investigating activin A production by dendritic cells (DC) in vitro and its function. Here we demonstrate that monocyte-derived DC (Mo-DC) released abundant levels of activin A during the maturation process induced by TLR agonists, bacteria (B. henselae, S. thyphimurium), TNF and CD40L. Activin A was also induced in monocyte-derived Langerhans cells (LC) and in blood myeloid DC by LPS and/or CD40L stimulation, but not in blood plasmacytoid DC following stimulation with influenza virus. Activin A production by DC was selectively down-regulated by anti-inflammatory molecules such as dexamethasone or IL-10. Neutralization of endogenous activin A using its inhibitor follistatin, or the addition of exogenous activin A during LPS maturation did not affect Mo-DC maturation marker expression, cytokine release or allostimulatory function. However, Mo-DC matured with LPS in the presence of exogenous activin A displayed a higher FITC-dextran uptake, similar to that of immature DC. Moreover, activin A promoted monocyte differentiation to DC and reversed the inhibitory effects of IL-6 on DC differentiation of monocytes. These findings demonstrate that different subsets of DC release activin A, a cytokine that promotes DC generation, and affects the ability of mature DC to take up antigens (Ags).     

Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell,Epi-cDC2s,via different entry pathways

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.     

Respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate CD4 T cells

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in children, the elderly, and immune-compromised individuals. CD4 and CD8 T cells play a crucial role in the elimination of RSV from the infected lung, but T cell memory is not sufficient to completely prevent reinfections. The nature of the adaptive immune response depends on innate immune reactions initiated after interaction of invading pathogens with host APCs. For respiratory pathogens myeloid dendritic cell (DC) precursors that are located underneath the epithelial cell layer lining the airways may play a crucial role in primary activation of T cells and regulating their functional potential. In this study, we investigated the role of human monocyte-derived DC in RSV infection. We showed that monocyte-derived DC can be productively infected, which results in maturation of the DC judged by the up-regulation of CD80, CD83, CD86, and HLA class II molecules. However, RSV infection of DC caused impaired CD4 T cell activation characterized by a lower T cell proliferation and ablation of cytokine production in activated T cells. The suppressive effect was caused by an as yet unidentified soluble factor produced by RSV-infected DC.     

Foot-and-mouth disease virus exhibits an altered tropism in the presence of specific immunoglobulins, enabling productive infection and killing of dendritic cells

Foot-and-mouth disease virus (FMDV) causes an acute vesicular disease of farm animals. The development of successful control strategies is limited by an incomplete understanding of the immune response to FMDV. Dendritic cells (DC) mediate the induction of immunity to pathogens, but their role in FMDV infection of cattle is uncharacterized. Bovine monocyte-derived DC (moDC) were exposed to integrin-binding and cell culture-adapted strains of FMDV in vitro. MoDC were not largely susceptible to infection by integrin-binding FMDV but were susceptible to culture-adapted virus. Binding specific antibodies to integrin-binding FMDV at neutralizing or subneutralizing IgG concentrations significantly enhanced infection via CD32 (FcγR). Monocytes also expressed CD32 but were nonsusceptible to FMDV immune complex (IC) infection, indicating a requirement for additional factors involved in cellular susceptibility. Infection of moDC by the FMDV IC was productive and associated with high levels of cell death. Infected moDC were unable to efficiently stimulate FMDV-specific CD4(+) memory T cells, but exposing moDC to IC containing inactivated FMDV resulted in significantly increased T cell stimulation. Thus, neutralized FMDV concurrently loses its ability to infect susceptible cells while gaining the capacity to infect immune cells. This represents a change in the tropism of FMDV that could occur after the onset of the antibody response. We propose that IC could dynamically influence the anti-FMDV immune response and that this may explain why the early immune response to FMDV has evolved toward T cell independence in vivo. Moreover, we propose that DC targeting could prove useful in the development of effective vaccines against FMDV.     

Accelerated dendritic cell differentiation from migrating Ly6C(lo) bone marrow monocytes in early dermal West Nile virus infection

No study has investigated the participation of Ly6C(+) monocytes in the earliest phase of skin infection with the mosquito-borne West Nile virus. In a novel murine model mimicking natural dermal infection, CCL2-dependent bone marrow (BM)-derived monocyte migration, differentiation into Ly6C(+) dendritic cells (DC), and accumulation around dermal deposits of infected fibroblasts by day 1 postinfection were associated with increasing numbers of monocyte-derived TNF/inducible NO synthase-producing DC by day 2 postinfection in draining auricular lymph nodes (ALN). Adoptive transfer demonstrated simultaneous migration of bone marrow-derived Ly6C(lo) monocytes to virus-infected dermis and ALN, where they first become Ly6C(hi) DC within 24 h and then Ly6C(lo) DC by 72 h. DC migration from the infected dermis to the ALN derived exclusively from Ly6C(lo) BM monocytes. This demonstrates that Ly6C(hi) and Ly6C(lo) BM-derived monocytes have different fates in vivo and suggests that BM may be a reservoir of preinflammatory monocytes for rapid deployment as inflammatory DC during virus infection.     

Langerhans cells require MyD88-dependent signals for Candida albicans response but not for contact hypersensitivity or migration

Langerhans cells (LC) are a subset of skin-resident dendritic cells (DC) that reside in the epidermis as immature DC, where they acquire Ag. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli, including epicutaneous sensitization with hapten and skin infection with Candida albicans. Mature LC migrate to the skin-draining LN, where they present Ag to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL-18 on LC. In addition, TLR ligands are present in C. albicans, and hapten sensitization produces endogenous TLR ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88(fl) mice in which LC are insensitive to IL-1 family members and most TLR ligands. LC migration in the steady state, after hapten sensitization and postinfection with C. albicans, was unaffected. Contact hypersensitivity in Langerin-Cre MyD88(fl) mice was similarly unaffected. Interestingly, in response to C. albicans infection, these mice displayed reduced proliferation of Ag-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of costimulatory molecules was intact on LC, but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration, but is required for the full activation and function of LC in the setting of fungal infection.     

A novel viral system for generating antigen-specific T cells

Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with approximately 50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-alpha treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-alpha, and IFN-alpha. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-alpha-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.     

Human immunodeficiency virus type 1 replication in dendritic cell-T-cell cocultures is increased upon incorporation of host LFA-1 due to higher levels of virus production in immature dendritic cells

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.     

Human BDCA-1-positive blood dendritic cells differentiate into phenotypically distinct immature and mature populations in the absence of exogenous maturational stimuli: differentiation failure in HIV infection

Current immunological opinion holds that myeloid dendritic cell (mDC) precursors migrate from the blood to the tissues, where they differentiate into immature dermal- and Langerhans-type dendritic cells (DC). Tissue DC require appropriate signals from pathogens or inflammatory cytokines to mature and migrate to secondary lymphoid tissue. We show that purified blood mDC cultured in vitro with GM-CSF and IL-4, but in the absence of added exogenous maturation stimuli, rapidly differentiate into two maturational and phenotypically distinct populations. The major population resembles immature dermal DC, being positive for CD11b, CD1a, and DC-specific ICAM-3-grabbing nonintegrin. They express moderate levels of MHC class II and low levels of costimulatory molecules. The second population is CD11b(-/low) and lacks CD1a and DC-specific ICAM-3-grabbing nonintegrin but expresses high levels of MHC class II and costimulatory molecules. Expression of CCR7 on the CD11b(-/low) population and absence on the CD11b(+) cells further supports the view that these cells are mature and immature, respectively. Differentiation into mature and immature populations was not blocked by polymyxin B, an inhibitor of LPS. Neither population labeled for Langerin, E-cadherin, or CCR6 molecules expressed by Langerhans cells. Stimulation of 48-h cultured DC with LPS, CD40L, or poly(I:C) caused little increase in MHC or costimulatory molecule expression in the CD11b(-/low) DC but caused up-regulated expression in the CD11b(+) cells. In HIV-infected individuals, there was a marked decrease in the viability of cultured blood mDC, a failure to differentiate into the two populations described for normal donors, and an impaired ability to stimulate T cell proliferation.     

Significant virus replication in Langerhans cells following application of HIV to abraded skin: relevance to occupational transmission of HIV

The cellular events that occur following occupational percutaneous exposure to HIV have not been defined. In this study, we studied relevant host cellular and molecular targets used for acquisition of HIV infection using split-thickness human skin explants. Blockade of CD4 or CCR5 before R5 HIV application to the epithelial surface of skin explants completely blocked subsequent HIV transmission from skin emigrants to allogeneic T cells, whereas preincubation with C-type lectin receptor inhibitors did not. Immunomagnetic bead depletion studies demonstrated that epithelial Langerhans cells (LC) accounted for >95% of HIV dissemination. When skin explants were exposed to HIV variants engineered to express GFP during productive infection, GFP+ T cells were found adjacent to GFP+ LC. In three distinct dendritic cell (DC) subsets identified among skin emigrants (CD1a+langerin+DC-specific intercellular adhesion molecule grabbing non-integrin (SIGN)- LC, CD1a+langerin-DC-SIGN- dermal DC, and CD1a-langerin-DC-SIGN+ dermal macrophages), HIV infection was detected only in LC. These results suggest that productive HIV infection of LC plays a critical role in virus dissemination from epithelium to cells located within subepithelial tissue. Thus, initiation of antiretroviral drugs soon after percutaneous HIV exposure may not prevent infection of LC, which is likely to occur rapidly, but may prevent or limit subsequent LC-mediated infection of T cells.     

Dendritic Cells Transmit Human Immunodeficiency Virus Type 1 to Monocytes and Monocyte-Derived Macrophages

Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4⁺ T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4⁺ cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83⁻ DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83⁺ DC (maDC); this finding is in contrast to data previously obtained with CD4⁺ T cells. Third, maturation from imDC to maDC efficiently silenced expression of β₂-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.     

登革病毒对人树突状细胞感染性的研究

探讨登革病毒对人树突状细胞(DC)的感染性。人外周新鲜血常规分离单核细胞,经细胞因子GMCSF、IL4诱导培养成DC,通过形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型(DV2)感染DC,于作用后6h、24h、48h、72h、96h分别收集上清液和细胞,甲基纤维素微量空斑试验测定病毒滴度,间接免疫荧光法检测细胞上病毒抗原表达,透射电镜观察病毒在细胞内的定位。病毒感染后6h即可在培养上清中测出病毒,病毒滴度在48h达到高峰,以后逐渐下降。间接免疫荧光法证明感染的DC胞浆及胞膜上携带病毒抗原。透射电镜下在病毒感染48h后DC胞浆内可见大量病毒颗粒。树突状细胞是登革病毒感染的靶细胞,病毒可感染DC并产生大量病毒颗粒,可能在其发病机制中起重要作用。     

DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.