Activation Of Plasmacytoid Dendritic Cells Research Articles

MyD88-Dependent Immune Activation Mediated by Human Immunodeficiency Virus Type 1-Encoded Toll-Like Receptor Ligands

Immune activation is a major characteristic of human immunodeficiency virus type 1 (HIV-1) infection and a strong prognostic factor for HIV-1 disease progression. The underlying mechanisms leading to immune activation in viremic HIV-1 infection, however, are not fully understood. Here we show that, following the initiation of highly active antiretroviral therapy, the immediate decline of immune activation is closely associated with the reduction of HIV-1 viremia, which suggests a direct contribution of HIV-1 itself to immune activation. To propose a mechanism, we demonstrate that the single-stranded RNA of HIV-1 encodes multiple uridine-rich Toll-like receptor 7/8 (TLR7/8) ligands that induce strong MyD88-dependent plasmacytoid dendritic cell and monocyte activation, as well as accessory cell-dependent T-cell activation. HIV-1-encoded TLR ligands may, therefore, directly contribute to the immune activation observed during viremic HIV-1 infection. These data provide an initial rationale for inhibiting the TLR pathway to directly reduce the chronic immune activation induced by HIV-1 and the associated immune pathogenesis.

Toll-like receptor 9–dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

Regulation of TLR9 dependent DNA Immune complex mediated cell activation by High Mobility Group Box Protein 1 (HMGB1) and Receptor for Advanced Glycation End products (RAGE) (128.35)

Abstract Stimulation of endosomal associated TLR9 by DNA plays an important role in activation of plasmacytoid dendritic cells (pDCs) and autoreactive B cells and may contribute to the pathogenesis of systemic lupus erythematosus (SLE). HMGB1 is a DNA binding protein that is liberated either from cells undergoing necrosis or following cytokine stimulation. Here we show that HMGB1 forms a high affinity complex with CpG-A oligodeoxynucleotides and augments the production of IFN-a from murine pDCs. We also show that HMGB1 is indeed a component of naturally formed DNA containing chromatin complexes. IFNa induced by either the exogenously formed HMGB1/CpG-A complex or naturally formed complex is TLR9 dependent and is inhibited by HMGB1 and RAGE antagonists. The complex without HMGB1 induces significantly less IFNa. HMGB1-RAGE interactions are required for activation of autoreactive B cells following stimulation with DNA immune complexes and are critical in the regulation of type I interferon gene induction by DNA complexes present in lupus plasma. Our data provide a novel mechanism by which HMGB1 confers potent stimulatory activity to DNA through a RAGE dependent mechanism and may contribute to the pathogenesis of lupus.

Antagonizing HMGB1 inhibits proteinuria in a murine model of lupus-like disease (131.24)

Abstract High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts like an inflammatory cytokine when it is released by dying or stimulated cells. Elevated levels are detected at sites of inflammation in a number of autoimmune disorders including SLE. In addition, HMGB1 has been shown to contribute to the activation of plasmacytoid dendritic cells as well as B cells, cell types that are implicated in the pathogenesis of SLE. To address the role of HMGB1 in experimental lupus, we investigated the protective efficacy of a fully humanized mAb (IA-4, which recognizes both mouse and human HMGB1) in an IFNα-accelerated lupus model. In this model, adenovirus IFNα-treated NZB/W F1 mice exhibit 90–100% proteinuria incidence at 4–6 weeks post-treatment. The functionality of IA-4 has been demonstrated in vitro by its ability to inhibit rHMGB1-induced IL-6 secretion in human PBMC (IC50 = 0.9 nM). Prophylactic administration of the anti-HMGB1 mAb IA-4 at 10 mg/kg significantly inhibited the proteinuria severity score at seven weeks (65% inhibition; p<0.05 compared to an isotype control). Consistent with the proteinuria data, histopathological assessment of the kidney also revealed protection with IA-4 treatment. Despite its protection in nephritis, IA-4 treatment showed no detectable effect on serum levels of IFN-α and autoantibodies against dsDNA or SSA/Ro. Taken together, these data suggest that HMGB1 is a critical effector molecule in lupus nephritis and that anti-HMGB1 treatment may provide a potential therapeutic benefit for the treatment of SLE.

Human plasmacytoid dendritic cells regulate immune responses to Epstein-Barr virus (EBV) infection and delay EBV-related mortality in humanized NOD-SCID mice

AbstractEpstein-Barr virus (EBV) is associated with posttransplant lymphoproliferative disease (PTLD), which is a leading cause of cancer death in recipients of transplants. We investigated the role of plasmacytoid dendritic cells (PDCs) in the development of EBV infection and the onset of lymphoproliferative disease (LPD) in humanized NOD-SCID mice and studied the effect of EBV on PDC function. NOD-SCID mice reconstituted with PDC-depleted peripheral blood mononuclear cells (PBMCs) from EBV IgG+ human donors had significantly enhanced mortality from disseminated EBV infection (median survival, 43 days) compared to PBMC-only mice (median survival, 72 days; log-rank P < .05). Mice reconstituted with PDC-enriched PBMCs challenged with EBV exhibited delayed mortality from EBV-LPD (median survival, 80 days) compared to PBMC-only mice challenged with EBV (median survival, 50 days; log-rank P < .05). EBV-stimulated pDCs produced interferon α (IFN-α) and promoted the activation of natural killer cells and IFN-γ–producing CD3+T cells. PDC activation of CD3+T cells in response to EBV stimulation was dependent on cell-to-cell contact, in part mediated by toll-like receptor 9 (TLR-9) signaling that was inhibited by chloroquine and TLR-9 inhibitory CpG. Thus, PDCs play an important role in anti-EBV cellular immune responses that may be targets for manipulation in novel strategies for the treatment of PTLD.

Activation of Plasmacytoid Dendritic Cells with TLR9 Agonists Initiates Invariant NKT Cell-Mediated Cross-Talk with Myeloid Dendritic Cells

CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-alpha. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles.

Plasmacytoid dendritic cell activation by foot‐and‐mouth disease virus requires immune complexes

Natural IFN-producing cells (NIPC), also called plasmacytoid dendritic cells, represent an essential component of the innate immune defense against infection. Despite this, not much is known about the pathways involved in their activation by non-enveloped viruses. The present study demonstrates that the non-enveloped foot-and-mouth disease virus (FMDV) cannot stimulate IFN-alpha responses in NIPC, unless complexed with FMDV-specific immunoglobulins. Stimulation of NIPC with such immune complexes employs FcgammaRII ligation, leading to strong secretion of IFN-alpha. In contrast to the stimulation of NIPC by many enveloped viruses, FMDV induction of IFN-alpha production requires live virus. It is necessary for the virus to initiate its replicative cycle. Moreover, it is an abortive replication, as witnessed by the decrease of dsRNA levels and viral titers with time post infection. Sensitivity of the NIPC stimulation to wortmannin and chloroquin, but not leupeptin, indicates an essential role for the pre-lysosomal stage endosomal compartment. In conclusion, the present study demonstrates that immune complexes provide the means for a non-interferogenic virus to induce IFN-alpha responses by NIPC. This indicates an important link between NIPC and antibodies in immune responses against non-enveloped viruses such as FMDV.

Human CD4+CD25high Regulatory T Cells Modulate Myeloid but Not Plasmacytoid Dendritic Cells Activation

Human CD4(+)CD25(+) regulatory T cells (Treg) play an essential role in the prevention of autoimmune diseases. However, the mechanisms of immune suppression and the spectrum of cells they target in vivo remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they have been shown to inhibit the Ag-presenting functions of macrophage- and monocyte-derived dendritic cells (DC). We have now studied the maturation of human blood-derived myeloid DC and plasmacytoid DC activated with TLR ligands in the presence of Treg. Preactivated Treg suppressed strongly TLR-triggered myeloid DC maturation, as judged by the blocking of costimulatory molecule up-regulation and the inhibition of proinflammatory cytokines secretion that resulted in poor Ag presentation capacity. Although IL-10 played a prominent role in inhibiting cytokines secretion, suppression of phenotypic maturation required cell-cell contact and was independent of TGF-beta and CTLA-4. In contrast, the acquisition of maturation markers and production of cytokines by plasmacytoid DC triggered with TLR ligands were insensitive to regulatory T cells. Therefore, human Treg may enlist myeloid, but not plasmacytoid DC for the initiation and the amplification of tolerance in vivo by restraining their maturation after TLR stimulation.

Differential expansion, activation and effector functions of conventional and plasmacytoid dendritic cells in mouse tissues transiently infected with Listeria monocytogenes

Dendritic cells (DC) are crucial in generating immunity to infection. Here we characterize changes in DC in terms of number, activation and effector functions, focusing on conventional DC (cDC) and plasmacytoid DC (pDC), in Listeria-infected mice. Kinetic studies showed a subset- and tissue-specific expansion of cDC and upregulation of CD80 and CD86 on splenic and mesenteric lymph node (MLN) cDC after intragastric infection. Expansion of pDC was more prolonged than cDC, and pDC upregulated CD86 and MHC-II, but not CD80, in both the spleen and MLN. cDC were an important source of IL-12 but not TNF-alpha during infection, while pDC made neither of these cytokines. Instead other CD11c(int) cells produced these cytokines. Using five-colour flow cytometry and double intracellular cytokine staining, we detected phenotypically similar CD11c(int)CD11b(+)Gr1(+) cells with distinct capacities to produce TNF-alpha/IL-12 or TNF-alpha/iNOS (inducible nitric oxide synthase) in Listeria-infected tissues. IL-12p70 was also produced by sorted CD11c(hi) and CD11c(int)CD11b(+)Gr1(+) cells. Furthermore, production of TNF-alpha, iNOS and IL-12 was differentially dependent on cellular localization of the bacteria. Cytosol-restricted bacteria induced TNF-alpha and iNOS-producing cells, albeit at lower frequency than wild-type bacteria. In contrast, IL-12 was induced only with wild-type bacteria. These data provide new insight into the relative abundance and function of distinct CD11c-expressing populations during the early stage of Listeria infection.

TLR9 Activation Is Defective in Common Variable Immune Deficiency

Common variable immune deficiency (CVID) is a primary immune deficiency characterized by low levels of serum immune globulins, lack of Ab, and reduced numbers of CD27+ memory B cells. Although T, B, and dendritic cell defects have been described, for the great majority, genetic causes have not been identified. In these experiments, we investigated B cell and plasmacytoid dendritic cell activation induced via TLR9, an intracellular recognition receptor that detects DNA-containing CpG motifs from viruses and bacteria. CpG-DNA activates normal B cells by the constitutively expressed TLR9, resulting in cytokine secretion, IgG class switch, immune globulin production, and potentially, the preservation of long-lived memory B cells. We found that CpG-DNA did not up-regulate expression of CD86 on CVID B cells, even when costimulated by the BCR, or induce production of IL-6 or IL-10 as it does for normal B cells. TLR9, found intracytoplasmically and on the surface of oligodeoxynucleotide-activated normal B cells, was deficient in CVID B cells, as was TLR9 mRNA. TLR9 B cell defects were not related to proportions of CD27+ memory B cells. CpG-activated CVID plasmacytoid dendritic cells did not produce IFN-alpha in normal amounts, even though these cells contained abundant intracytoplasmic TLR9. No mutations or polymorphisms of TLR9 were found. These data show that there are broad TLR9 activation defects in CVID which would prevent CpG-DNA-initiated innate immune responses; these defects may lead to impaired responses of plasmacytoid dendritic cells and loss of B cell function.

Virus or TLR Agonists Induce TRAIL-Mediated Cytotoxic Activity of Plasmacytoid Dendritic Cells

Among dendritic cells, plasmacytoid dendritic cells (PDC) represent a functionally distinct lineage. Regarding innate immunity, PDC secrete large amounts of type I IFN upon viral exposure or stimulation by microbial products such as unmethylated CpG-motif containing oligo-DNA due to their selective expression of TLR7 and TLR9. We asked whether they could acquire cytotoxic functions during the early phases of infection or after activation with TLR7 or TLR9 agonists. In the present study, we describe a human PDC cell line called GEN2.2, derived from leukemic PDC, that shares most of the phenotypic and functional features of normal PDC. We show that after contact with the influenza virus, GEN2.2, as well as normal PDC, acquires TRAIL and killer activity against TRAIL-sensitive target cells. Moreover, we show that activation of GEN2.2 cells by CpG-motif containing oligo-DNA or R848 also induces TRAIL and endows them with the ability to kill melanoma cells. Therefore, PDC may represent a major component of innate immunity that could participate to the clearance of infected cells and tumor cells. This phenomenon could be relevant for the efficacy of TLR7 or TLR9 agonists in the therapy of infectious disease and cancer.

Human CD4+ CD25high Regulatory T Cells Inhibit Myeloid but Not Plasmacytoid Dendritic Cells Activation.

CD4+CD25+ regulatory T cells (Treg) are essential negative regulators of immune responses. However, the mechanisms of immune suppression and the spectrum of cells they target remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they might also affect antigen presenting cells (APC).Here, we studied the maturation of human myeloid (mDC) and plasmacytoid (pDC) dendritic cells activated with Toll-like receptor (TLR) ligands in the presence of CD4+ CD25high regulatory T cells in vitro. T cells and DC subsets were purified from normal human peripheral blood. LPS, CpG ODN 2216 and R-848 were used to trigger the maturation of mDC, pDC or both through TLR4, TLR9 and TLR7/8 respectively. Preactivated CD4+ CD25high Treg had no effect on the maturation of pDC. Conversely, they strongly suppressed TLR-triggered mDC costimulatory molecules up-regulation, pro-inflammatory cytokines secretion and their antigen presentation capacity, as opposed to conventionnal T cells (Tconv). At a ratio of 3 Treg for 1 DC, the percentage of mDC acquiring CD80 was reduced 5 fold (from 75% to 16%) while the Mean Fluorescence Intensity was decreased by approximately 65% for CD80 and 35% for CD86 after LPS stimulation and by 50% and 20% after R-848 stimulation. Furthermore, Treg dramatically decreased the secretion of IL-12p40, TNF-α, and IL-6 by mDC (95%, 93% and 50% average inhibition respectively) after LPS activation and to a lesser but still significant extent (38%, 35%, and 38% average inhibition respectively) after R848 stimulation. Finally, we found that Treg-conditionned mDC had a reduced ability to trigger naïve T cell proliferation in a mixed leukocyte reaction.Suppression of mDC activation by Treg appeared to require cell-cell contact. Moreover, the inhibition of pro-inflammatory cytokines secretion, but not of phenotypic maturation, was almost completely restored using an anti-IL10 receptor monoclonal antibody, but not anti-TGFβ nor anti-CTLA-4 blocking antibodies. Those data suggest that Treg prevent the co-stimulatory molecules up-regulation on mDC through contact dependent mechanisms, while the modulation of cytokines secretion appears to be largely mediated by IL-10.Overall, our results provide the first evidence of a direct inhibition of human mDC but not pDC maturation by CD4+ CD25high Treg. Therefore, by restraining the maturation of mDC, human Treg may enlist those cells for the initiation and the amplification of tolerance in vivo.

HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration

Plasmacytoid dendritic cells (PDC), natural type-1 interferon (IFN) producing cells, could play a role in the innate anti-HIV immune response. Previous reports indicated that PDC IFN production is induced by HIV. Our results show a more robust IFN induction when purified PDC (>95%) were exposed to HIV-infected cells. This effect was not observed with non-viable cells, DNA, and RNA extracted from infected cells, and viral proteins. The response was blocked by anti-CD4 and neutralizing anti-gp120 antibodies as well as soluble CD4. IFN induction by HIV-infected cells was also prevented by low-dose chloroquine, which inhibits endosomal acidification. PDC IFN release resulted in reduced HIV production by infected CD4+ cells, supporting an anti-HIV activity of PDC. Stimulated CD4+ cells induced PDC activation and maturation; markers for PDC migration (CCR7) were enhanced by HIV-infected CD4+ cells only. This latter finding could explain the decline in circulating PDC in HIV-infected individuals.

Activation of plasmacytoid dendritic cells

Four years after the discovery of mouse plasmacytoid dendritic cells (pDC), pDC are still very much an 'enigmatic' cell type. It is clear that pDC are potent producers of type I IFN in response to viral, bacterial and even mammalian nucleotides. The role that they play in vivo before and after activation is still under scrutiny. This review concentrates on the pathways to activation of pDC, examining the activating ligands, receptors and signalling molecules that are known to be involved, and the relevance of these activation pathways to human disease.

Activation of liver plasmacytoid dendritic cells enhances their immunostimulatory capacity

Kingham, Peter T. MD; Pillarisetty, Venu MD; Katz, Steven MD; Chaudhry, Umer MD; DeMatteo, Ronald P. MD Author Information

Type I interferon dependence of plasmacytoid dendritic cell activation and migration

Differential expression of Toll-like receptor (TLR) by conventional dendritic cells (cDCs) and plasmacytoid DC (pDCs) has been suggested to influence the type of immune response induced by microbial pathogens. In this study we show that, in vivo, cDCs and pDCs are equally activated by TLR4, -7, and -9 ligands. Type I interferon (IFN) was important for pDC activation in vivo in response to all three TLR ligands, whereas cDCs required type I IFN signaling only for TLR9- and partially for TLR7-mediated activation. Although TLR ligands induced in situ migration of spleen cDC into the T cell area, spleen pDCs formed clusters in the marginal zone and in the outer T cell area 6 h after injection of TLR9 and TLR7 ligands, respectively. In vivo treatment with TLR9 ligands decreased pDC ability to migrate ex vivo in response to IFN-induced CXCR3 ligands and increased their response to CCR7 ligands. Unlike cDCs, the migration pattern of pDCs required type I IFN for induction of CXCR3 ligands and responsiveness to CCR7 ligands. These data demonstrate that mouse pDCs differ from cDCs in the in vivo response to TLR ligands, in terms of pattern and type I IFN requirement for activation and migration.

Plasmacytoid dendritic cell (pDC) activation by CpG-DNA inhibits IL-3-dependent cytokine secretion by human basophils: The role of IFN-alpha

RATIONALE: To further investigate the inhibitory effects of Type I interferons on basophil function by testing whether pDC's activated by immunomodulatory CpG-DNA inhibit IL-13 and IL-4 secreted by basophils under conditions of IL-3 priming.

Imiquimod Treatment In Vivo Induces Massive Skin Recruitment and Activation of Plasmacytoid Dendritic Cells in a Disease-Independent Manner

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Dendritic Cells and Vaccines

IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences

AbstractImmunostimulatory sequences (ISS) are short oligonucleotides containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides that stimulate innate immune responses through Toll-like receptor-9 on B cells and plasmacytoid dendritic cell (PDC) precursors. The anti-inflammatory cytokine interleukin (IL)-10 is predicted to be a potent inhibitor of many of the activities described for ISS, and this may impact the use of ISS in disease states characterized by elevated IL-10. As the activities of ISS on PDCs are central to many clinical applications of ISS, we have studied the effects of IL-10 on PDC stimulation by 3 distinct classes of ISS. IL-10 inhibited cytokine production and survival of ISS-activated PDCs; however, IL-12 induction was much more sensitive to inhibition than interferon (IFN)-α induction. Within the PDC population are cells that respond to ISS by producing either IL-12 or IFN-α but not both cytokines. IL-12-producing PDCs require costimulation through CD40 and appear more mature than IFN-α-producing PDCs. The 3 distinct classes of ISS differed with respect to induction of PDC maturation and T-cell priming capacity. IL-10 regulated PDC activation but did not inhibit the subsequent T-cell-priming ability of PDCs already activated by ISS. (Blood. 2003;102:4487-4492)