DENV-infected Dendritic Cells Research Articles

TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity.

Possible risk mediators in primary dengue virus (DenV) infection that favor secondary DenV infection to life-threatening dengue hemorrhagic fever (DHF) and shock syndrome (DSS) via antibody-dependent enhancement (ADE) have not yet been described. Here, DenV infection enhanced the expression of inflammatory mediators and activation molecules in dendritic cells (DCs) through TLR2/MyD88 pathway. TLR2 appeared to facilitate DenV infection in DCs that were less permissive than macrophages for viral replication. In experiments using separate evaluations of DenV-infected and uninfected bystander DCs, infected DCs showed impaired maturation accompanied with TLR2-dependent production of inflammatory cytokines, by which uninfected bystander DCs showed increased expression of co-stimulatory molecules. Differential phosphorylation of MAPK and STAT3 was also detected between DenV-infected and uninfected DCs. Furthermore, DenV infection stimulated Th2-polarized humoral and cellular immunity against foreign and DenV Ag via TLR2/MyD88 pathway, and DenV-infected DCs were revealed to facilitate Th2-biased immune responses in TLR2-dependent manner. TLR2/MyD88-mediated Th2-biased Ab responses to primary DenV infection increased the infectivity of secondary homotypic or heterotypic DenV via ADE. Collectively, these results indicate that TLR2/MyD88 pathway in DC-priming receptors can drive Th2-biased immune responses during primary DenV infection, which could favor secondary DenV infection to DHF/DSS via ADE.

RIG-I-like Receptor Triggering by Dengue Virus Drives Dendritic Cell Immune Activation and TH1 Differentiation.

Dengue virus (DENV) causes 400 million infections annually and is one of several viruses that can cause viral hemorrhagic fever, which is characterized by uncontrolled immune activation resulting in high fever and internal bleeding. Although the underlying mechanisms are unknown, massive cytokine secretion is thought to be involved. Dendritic cells (DCs) are the main target cells of DENV, and we investigated their role in DENV-induced cytokine production and adaptive immune responses. DENV infection induced DC maturation and secretion of IL-1β, IL-6, and TNF. Inhibition of DENV RNA replication abrogated these responses. Notably, silencing of RNA sensors RIG-I or MDA5 abrogated DC maturation, as well as cytokine responses by DENV-infected DCs. DC maturation was induced by type I IFN responses because inhibition of IFN-α/β receptor signaling abrogated DENV-induced DC maturation. Moreover, DENV infection of DCs resulted in CCL2, CCL3, and CCL4 expression, which was abrogated after RIG-I and MDA5 silencing. DCs play an essential role in TH cell differentiation, and we show that RIG-I and MDA5 triggering by DENV leads to TH1 polarization, which is characterized by high levels of IFN-γ. Notably, cytokines IL-6, TNF, and IFN-γ and chemokines CCL2, CCL3, and CCL4 have been associated with disease severity, endothelial dysfunction, and vasodilation. Therefore, we identified RIG-I and MDA5 as critical players in innate and adaptive immune responses against DENV, and targeting these receptors has the potential to decrease hemorrhagic fever in patients.

Dengue virus infection induces interferon-lambda1 to facilitate cell migration.

A marked increase in the rate of dengue virus (DENV) infection has resulted in more than 212 deaths in Taiwan since the beginning of 2015, mostly from fatal outcomes such as dengue hemorrhagic fever and dengue shock syndrome. The pathogenic mechanisms of these fatal manifestations are poorly understood. Cytokines induce an overwhelming immune reaction and thus have crucial roles. Interferon-lambda (IFN-λ), a newly identified IFN subtype, has antiviral effects, but its immunologic effects in DENV infection have not been investigated. In the present study, we show that DENV infection preferentially induced production of IFN-λ1 in human dendritic cells (DCs) and human lung epithelial cells. Virus nonstructural 1 (NS1) glycoprotein was responsible for the effect. DENV-induced production of IFN-λ1 was dependent on signaling pathways involving toll-like receptor (TLR)-3, interferon regulation factor (IRF)-3, and nuclear factor-kappaB (NF-κB). Blocking interaction between IFN-λ1 and its receptor IFN-λR1 through siRNA interference reduced DENV-induced DC migration towards the chemoattractants CCL19 and CCL21, by inhibiting CCR7 expression. Furthermore, IFN-λ1 itself induced CCR7 expression and DC migration. Our study presents the first evidence of the mechanisms and effects of IFN-λ1 induction in DENV-infected DCs and highlights the role of this cytokine in the immunopathogenesis of DENV infection.

The combination of type I IFN, TNF-α, and cell surface receptor engagement with dendritic cells enables NK cells to overcome immune evasion by dengue virus.

Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC). NK cells responded by cytokine production and the lysis of target cells. Notably, in the absence of significant monokine production by DenV-infected DC, it was the combination of type I IFNs and TNF-α produced by DenV-infected DC that was important for stimulating the IFN-γ and cytotoxic responses of NK cells. Cell-bound factors enhanced NK cell IFN-γ production. In particular, reduced HLA class I expression was observed on DenV-infected DC, and IFN-γ production was enhanced in licensed/educated NK cell subsets. NK-DC cell contact was also identified as a requirement for a cytotoxic response, and there was evidence for both perforin/granzyme as well as Fas/Fas ligand-dependent pathways of killing by NK cells. In summary, our results have uncovered a previously unappreciated role for the combined effect of type I IFNs, TNF-α, and cell surface receptor-ligand interactions in triggering the antidengue response of primary human NK cells.

Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing

Dengue virus (DENV) is the cause of a potentially life-threatening disease that affects millions of people worldwide. The lack of a small animal model that mimics the symptoms of DENV infection in humans has slowed the understanding of viral pathogenesis and the development of therapies and vaccines. Here, we investigated the use of humanized "bone marrow liver thymus" (BLT) mice as a model for immunological studies and assayed their applicability for preclinical testing of antiviral compounds. Human immune system (HIS) BLT-NOD/SCID mice were inoculated intravenously with a low-passage, clinical isolate of DENV-2, and this resulted in sustained viremia and infection of leukocytes in lymphoid and nonlymphoid organs. In addition, DENV infection increased serum cytokine levels and elicited DENV-2-neutralizing human IgM antibodies. Following restimulation with DENV-infected dendritic cells, in vivo-primed T cells became activated and acquired effector function. An adenosine nucleoside inhibitor of DENV decreased the circulating viral RNA when administered simultaneously or 2 days postinfection, simulating a potential treatment protocol for DENV infection in humans. In summary, we demonstrate that BLT mice are susceptible to infection with clinical DENV isolates, mount virus-specific adaptive immune responses, and respond to antiviral drug treatment. Although additional refinements to the model are required, BLT mice are a suitable platform to study aspects of DENV infection and pathogenesis and for preclinical testing of drug and vaccine candidates. IMPORTANCE Infection with dengue virus remains a major medical problem. Progress in our understanding of the disease and development of therapeutics has been hampered by the scarcity of small animal models. Here, we show that humanized mice, i.e., animals engrafted with components of a human immune system, that were infected with a patient-derived dengue virus strain developed clinical symptoms of the disease and mounted virus-specific immune responses. We further show that this mouse model can be used to test preclinically the efficacy of antiviral drugs.

Impairment of CD4+ T Cell Polarization by Dengue Virus–Infected Dendritic Cells

The production of type I interferon alpha/beta (IFN-α/β) is crucial to viral clearance during dengue virus (DENV) infection; however, in vitro-infected dendritic cells (DCs) exhibit a decreased capacity to respond to IFN-α/β stimulation, and antigen-presenting cells (APCs) isolated from patients with acute DENV infection exhibit defects in T cell priming. In order to ascertain the stimulatory capacity of primary human monocyte-derived DCs infected with wild-type DENV isolates, representing a range of genotypes and disease outcomes, we cocultured infected DCs with allogeneic-naive CD4(+) T cells. The gene expression patterns of IFN-α/β sensitive genes were quantitated to determine if the infected DCs displayed a blunted IFN-α/β response. DENV-infected DCs induced the initial proliferation of naive CD4(+) T cells but they remained nonpolarized in effector function. The expression of IFN-α/β-stimulated genes was downregulated, revealing that the inhibition of IFN-α/β signaling is conserved among endemic DENV serotype 2 strains. The failure of naive CD4(+) T cells to differentiate into IFN gamma-producing effector T cells when primed by DENV-infected DCs cannot be explained solely by a block in IFN-α/β signaling, suggesting that the ability of DENV to evade the early host response is multifaceted.

Inhibition of the Type I Interferon Response in Human Dendritic Cells by Dengue Virus Infection Requires a Catalytically Active NS2B3 Complex

Dengue virus (DENV) is the most prevalent arthropod-borne human virus, able to infect and replicate in human dendritic cells (DCs), inducing their activation and the production of proinflammatory cytokines. However, DENV can successfully evade the immune response in order to produce disease in humans. Several mechanisms of immune evasion have been suggested for DENV, most of them involving interference with type I interferon (IFN) signaling. We recently reported that DENV infection of human DCs does not induce type I IFN production by those infected DCs, impairing their ability to prime naive T cells toward Th1 immunity. In this article, we report that DENV also reduces the ability of DCs to produce type I IFN in response to several inducers, such as infection with other viruses or exposure to Toll-like receptor (TLR) ligands, indicating that DENV antagonizes the type I IFN production pathway in human DCs. DENV-infected human DCs showed a reduced type I IFN response to Newcastle disease virus (NDV), Sendai virus (SeV), and Semliki Forest virus (SFV) infection and to the TLR3 agonist poly(I:C). This inhibitory effect is DENV dose dependent, requires DENV replication, and takes place in DENV-infected DCs as early as 2 h after infection. Expressing individual proteins of DENV in the presence of an IFN-alpha/beta production inducer reveals that a catalytically active viral protease complex is required to reduce type I IFN production significantly. These results provide a new mechanism by which DENV evades the immune system in humans.

Dengue virus infection promotes translocation of high mobility group box 1 protein from the nucleus to the cytosol in dendritic cells, upregulates cytokine production and modulates virus replication

High mobility group box 1 (HMGB1) protein functions in regulation of transcription, cellular activation and pro-inflammatory responses. However, the potential role of HMGB1 during viral infection has not been investigated. This study attempted to elucidate whether the HMGB1-mediated inflammatory response contributes to the pathogenesis of dengue virus (DENV) infection. Our data showed that HMGB1 was released at low DENV infection levels (m.o.i. of 1) under non-necrotic conditions by human dendritic cells (DCs). When DENV-infected DCs were co-cultured with autologous T cells, there was increased production of HMGB1 by both cell types. HMGB1 regulated tumour necrosis factor alpha, interleukin (IL)-6, IL-8 and alpha interferon secretion in DENV-infected DCs. Additionally, increased HMGB1 production was associated with reduced DENV replication titres in DCs. These results suggest that HMGB1 production influences DENV infection in susceptible hosts.