Toll-Like Receptor 9 in Plasmacytoid Dendritic Cells Fails To Detect Parvoviruses

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.     

Regulation of Porcine Plasmacytoid Dendritic Cells by Cytokines

Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN’s, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role.     

关于多发性硬化中浆样树突状细胞的研究进展

多发性硬化是中枢神经系统炎症性自身免疫性疾病的典型代表,以白质脱髓鞘为主要特征。浆样树突状细胞,是专职抗原提呈细胞,是固有免疫和适应性免疫的桥梁,在启动初级免疫应答和维持免疫耐受中发挥了重要作用。由于浆样树突状细胞可以产生大量的细胞因子,特别是Ⅰ型干扰素,所以它与抗炎、免疫调节联系紧密。而目前Ⅰ型干扰素(β)被认为是治疗多发性硬化的有效的免疫调节剂。本文就浆样树突状细胞的来源、特性及其在固有免疫、适应性免疫及免疫耐受中的作用机制进行系统归纳整理,并就其未来发展前景做一简单介绍,为进一步探索免疫调节新机制和寻求多发性硬化新的治疗靶点提供理论依据和基础。     

Human Plasmacytoid Dendritic Cells Sense Lymphocytic Choriomeningitis Virus-Infected Cells In Vitro

We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/β) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).     

Role for Plasmacytoid Dendritic Cells in the Immune Control of Recurrent Human Herpes Simplex Virus Infection

Plasmacytoid dendritic cells (pDC) are an important component of the innate immune response, producing large amounts of alpha interferon in response to viral stimulation in vitro. Under noninflammatory conditions, pDC are not found in the skin and are restricted in location to the blood and lymph nodes. Therefore, their role in mucosal and cutaneous herpes simplex virus (HSV) infection has not been well-defined. In this study we show a role for human pDC in the immune response to HSV infection. First, by confocal microscopy we showed that pDC infiltrate the dermis of recurrent genital herpes simplex lesions at early and late phases, often at the dermo-epidermal junction. We then showed that pDC in vitro are resistant to HSV infection despite expressing the entry receptors CD111, CD112, and HVE-A. Within the lesions, pDC were found closely associated with CD3⁺ lymphocytes and NK cells, especially those which were activated (CD69⁺). Furthermore, these HSV-exposed pDC were able to stimulate virus-specific autologous T-lymphocyte proliferation. We conclude from this work that pDC may contribute to the immune control of recurrent herpes virus infection in vivo.     

Plasmacytoid Dendritic Cells Capture and Cross-Present Viral Antigens from Influenza-Virus Exposed Cells

Among the different subsets of dendritic cells (DC), plasmacytoid dendritic cells (PDC) play a unique role in secreting large amounts of type I interferons upon viral stimulation, but their efficiency as antigen-presenting cells has not been completely characterized. We show here, by flow cytometry, with human primary blood PDC and with a PDC cell line, that PDC display poor endocytic capacity for soluble or cellular antigens when compared to monocyte-derived myeloid DC. However, immature PDC efficiently take up cellular material from live influenza-exposed cells, subsequently mature and cross-present viral antigens very efficiently to specific CD8+ T cells. Therefore, during viral infection PDC not only secrete immunomodulatory cytokines, but also recognize infected cells and function as antigen cross-presenting cells to trigger the anti-viral immune response.     

Plasmacytoid Dendritic Cells and Infected Cells Form an Interferogenic Synapse Required for Antiviral Responses

1. Download : Download high-res image (250KB)
2. Download : Download full-size image

     

Type III IFNs Are Produced by and Stimulate Human Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be "professional" type I IFN-producing cells and produce 10- to 100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR7 and TLR9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA, as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using Abs to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α, whereas another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpG A; the cells that coexpressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Ab cross-linking of CD4 or CD303 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and -α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and -λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.     

Inability of Plasmacytoid Dendritic Cells To Directly Lyse HIV-Infected Autologous CD4+ T Cells despite Induction of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-α) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4⁺ T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-α/β were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-α upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV⁺ subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4⁺ T cells from viremic HIV⁺ subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-α, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV⁺ CD4⁺ T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4⁺ T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-α in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4⁺ T cells.Human immunodeficiency virus (HIV) infection is associated with chronic immune activation, progressive immune suppression, and deletion of memory adaptive responses, resulting in increased susceptibility to opportunistic infections (23, 51, 52). Loss of CD4⁺ T cells is the hallmark of HIV infection, with multiple mechanisms proposed as contributing to this loss (activation-induced cell death, direct cytopathic effect, immune cells, and death receptor-mediated apoptosis induction) (reviewed in references 33 and 34). One of the most puzzling observations in AIDS pathogenesis has been the progressive depletion of bystander T cells, especially in lymphoid tissues (25, 33, 34, 55). While antiretroviral therapy (ART) initiated in the early stages of HIV infection, when CD4⁺ T-cell counts are high (>500 cells/μl), may prevent the destruction of lymph node (LN) tissue and the massive depletion of CD4⁺ T lymphocytes by decreasing the rate of virally induced apoptosis (20), a persistent, albeit decreased, level of apoptosis of peripheral blood CD4⁺ and CD8⁺ T cells is seen in ART-treated HIV⁺ subjects despite long-term viral suppression (36).A member of the tumor necrosis factor (TNF) family, TNF-related apoptosis-inducing ligand (TRAIL), has been shown to be involved in HIV-1-associated T-cell apoptosis (33, 34). TRAIL (soluble or membrane bound) induces apoptosis upon binding to death receptor 4 (DR4; also named TRAIL-R1) or DR5 (also named TRAIL-R2, TRICK2, or Killer/DR5).On the basis of the in vitro observation that alpha interferon (IFN-α) and interferon regulator factor 7 (IRF-7) are increased in plasmacytoid dendritic cells (PDC) exposed to HIV-1 (40), the hypothesis that PDC activation by HIV-1 is responsible for an increased level of IFN-α throughout chronic disease has been proposed. It has also been proposed that the activation of the PDC compartment by HIV-1 participates in the initial immune activation following acute infection and contributes to CD4⁺ T-cell depletion by inducing, through IFN-α, the production of TRAIL, which mediates apoptosis of DR5-expressing CD4⁺ T cells following HIV-1 infection (37, 38, 40). However, several lines of evidence question the direct involvement of PDC in the loss of T cells during HIV infection, as PDC numbers are depleted during chronic HIV infection and PDC remaining in circulation are functionally impaired (10). Recent data show that circulating PDC in HIV-infected subjects, although unable to secrete IFN-α after Toll-like receptor (TLR)-mediated activation, constitutively express an increased level of IFN-α mRNA, indicating that during HIV infection PDC are activated yet impaired (71). Rodriguez et al. demonstrated the prevention of spontaneous apoptosis of CD4⁺ and CD8⁺ T cells by IFN-α (63), a major product of PDC following HIV-1 stimulation (3, 28). In addition, Audige et al. (2) showed that blockade of IFN-α and IFN-α receptor during in vitro HIV infection of CD4⁺ T cells isolated from human tonsils did not prevent apoptosis or TRAIL production, suggesting a lack of a central link between IFN-α production and apoptosis of tonsillar CD4⁺ T cells in HIV-1 infection. These data are also consistent with the observation that, in the human peripheral blood lymphocyte-transplanted SCID mouse (hu-PBL-SCID) model, IFN-α efficiently increases the survival of CD4⁺ T cells (49). Thus, controversy remains on the role of IFN-α as an indirect or direct inducer of apoptosis of CD4⁺ T cells through PDC/TRAIL induction, whereas the possibility that IFN-α acts as an antiviral agent by controlling HIV-1 replication and thus reducing virally mediated T-cell loss appears to be supported by several studies (reviewed in references 26, 47, and 58). In this regard, endogenous IFN-α produced by PDC has been shown to play an important role in controlling HIV infection in the human thymus (35), upregulating host antiviral factors such as APOBEC (1, 32, 44, 70) and stimulating NK cell-mediated cytotoxic activity against autologous HIV-infected targets (72).In this report, we investigated the in vivo correlates of viremia in chronically infected subjects by studying the relationship between therapy interruption-associated viremia and plasma IFN-α and TRAIL levels. We also tested in vitro the functional outcome of HIV-1-activated PDC in terms of their ability to mediate lysis of primary autologous CD4 T cells (infected or not with HIV-1), compared to indirect PDC-mediated lysis effects on the NK-dependent antiviral cytotoxic response.     

Plasmacytoid Dendritic Cells Are Crucial in Bifidobacterium adolescentis-Mediated Inhibition of Yersinia enterocolitica Infection

In industrialized countries bacterial intestinal infections are commonly caused by enteropathogenic Enterobacteriaceae. The interaction of the microbiota with the host immune system determines the adequacy of an appropriate response against pathogens. In this study we addressed whether the probiotic Bifidobacterium adolescentis is protective during intestinal Yersinia enterocolitica infection. Female C57BL/6 mice were fed with B. adolescentis, infected with Yersinia enterocolitica, or B. adolescentis fed and subsequently infected with Yersinia enterocolitica. B. adolescentis fed and Yersinia infected mice were protected from Yersinia infection as indicated by a significantly reduced weight loss and splenic Yersinia load when compared to Yersinia infected mice. Moreover, protection from infection was associated with increased intestinal plasmacytoid dendritic cell and regulatory T-cell frequencies. Plasmacytoid dendritic cell function was investigated using depletion experiments by injecting B. adolescentis fed, Yersinia infected C57BL/6 mice with anti-mouse PDCA-1 antibody, to deplete plasmacytoid dendritic cells, or respective isotype control. The B. adolescentis-mediated protection from Yersinia dissemination to the spleen was abrogated after plasmacytoid dendritic cell depletion indicating a crucial function for pDC in control of intestinal Yersinia infection. We suggest that feeding of B. adolescentis modulates the intestinal immune system in terms of increased plasmacytoid dendritic cell and regulatory T-cell frequencies, which might account for the B. adolescentis-mediated protection from Yersinia enterocolitica infection.     

Identification of Toll-Like Receptor 9 as Parapoxvirus Ovis-Sensing Receptor in Plasmacytoid Dendritic Cells

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and co-stimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.     

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.     

Tumour-Derived Reg3A Educates Dendritic Cells to Promote Pancreatic Cancer Progression

As a pancreatic inflammatory marker, regenerating islet-derived protein 3A (Reg3A) plays a key role in inflammation-associated pancreatic carcinogenesis by promoting cell proliferation, inhibiting apoptosis, and regulating cancer cell migration and invasion. This study aimed to reveal a novel immuno-regulatory mechanism by which Reg3A modulates tumour-promoting responses during pancreatic cancer (PC) progression. In an in vitro Transwell system that allowed the direct co-culture of human peripheral blood-derived dendritic cells (DCs) and Reg3A-overexpressing/ silenced human PC cells, PC cell-derived Reg3A was found to downregulate CD80, CD83 and CD86 expression on educated DCs, increase DC endocytic function, inhibit DC-induced T lymphocyte proliferation, reduce IL-12p70 production, and enhance IL-23 production by DCs. The positive effect of tumour-derived Reg3A-educated human DCs on PC progression was demonstrated in vivo by intraperitoneally transferring them into PC-implanted severe combined immunodeficiency (SCID) mice reconstituted with human T cells. A Reg3A-JAK2/STAT3 positive feedback loop was identified in DCs educated with Reg3A. In conclusion, as a tumour-derived factor, Reg3A acted to block the differentiation and maturation of the most important antigen-presenting cells, DCs, causing them to limit their potential anti-tumour responses, thus facilitating PC escape and progression.     

Effect of Natalizumab Treatment on Circulating Plasmacytoid Dendritic Cells: A Cross-Sectional Observational Study in Patients with Multiple Sclerosis

Objectives

Dendritic cells (DCs) serve a critical role both in promoting and inhibiting adaptive immunity. The goal of this study was to investigate the effect of natalizumab (NTZ) treatment on DC numbers, phenotype, and function in patients with multiple sclerosis (MS).

Methods

Frequency and phenotype of myeloid and plasmacytoid DCs (MDCs and PDCs, respectively) were analyzed in blood from two separate cohorts of untreated, interferon-treated, or NTZ-treated MS patients. In addition, PDCs were stimulated with CpG-containing oligonucleotides or co-cultured with homologous T cells in the presence or absence of NTZ in vitro to determine functional effects of NTZ treatment.

Results

We observed that NTZ treatment was associated with a 25–50% reduction in PDC frequency in peripheral blood as compared to untreated MS patients, while the frequency of MDCs was unchanged. PDCs in NTZ-treated patients displayed a mature, activated phenotype with increased expression of HLA-DR, TLR9, CCR7, IL-6 and IL-12. In contrast, in vitro treatment with NTZ did not increase markers of PDC activation or their ability to induce T cell differentiation.

Conclusion

Our study shows that NTZ treatment is associated with a reduced frequency of PDCs in the peripheral circulation, but that PDCs in NTZ-treated individuals display an activated phenotype. Taken together the data suggests that transmigration of activated PDCs is preferentially affected by blockade of integrin α4 leading to an increased frequency of activated PDCs in blood.     

Plasmacytoid Dendritic Cells Contribute to Systemic but Not Local Antiviral Responses to HSV Infections

Plasmacytoid dendritic cells (pDC) produce type I interferons (IFN-I) and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV) infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.