Cutting edge: progesterone regulates IFN-alpha production by plasmacytoid dendritic cells

Use of the progesterone (Pg) birth control depot medroxyprogesterone acetate (DMPA) increases a woman's risk for sexually transmitted infection with HIV or HSV-2 via unknown mechanisms. Plasmacytoid dendritic cells (pDCs) are circulating and tissue-resident sentinels capable of making large quantities of IFN-alpha upon recognizing viruses through TLRs 7 and 9. In this study, we show that Pg inhibits TLR9-induced IFN-alpha production by human and mouse pDCs and that DMPA impairs TLR9- and virus-induced IFN-alpha production by pDCs in mice, providing a potential explanation for how DMPA impairs innate antiviral immunity in women. Pg failed to inhibit the Mda-5 pathway of IFN-alpha induction in dendritic cells, suggesting that Pg regulates select antiviral DC programs. This may occur through selective blockade of IFN regulatory factor-7 activation, a novel steroid action. Thus, through inhibition of TLR-mediated IFN-alpha production by pDCs, Pg may regulate antiviral immunity.     

Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.     

Cutting edge: selective usage of chemokine receptors by plasmacytoid dendritic cells

The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are virtually unknown. In this study, we show that myeloid (M-DCs) and plasmacytoid (P-DCs) DCs isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed by blood M-DCs and P-DCs, with the exception of CCR7, is similar. However, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by CD40 ligation, the receptors for inflammatory chemokines are down-regulated, and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation.     

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.     

Cutting edge: proangiogenic properties of alternatively activated dendritic cells

Angiogenesis plays an important role in tissue remodeling and repair during the late phase of inflammation. In the present study, we show that human dendritic cells (DC) that matured in the presence of anti-inflammatory molecules such as calcitriol, PGE2, or IL-10 (alternatively activated DC) selectively secrete the potent angiogenic cytokine vascular endothelial growth factor (VEGF) isoforms VEGF165 and VEGF121. No VEGF production was observed in immature or classically activated DC. Also, the capacity to produce VEGF was restricted to the myeloid DC subset. When implanted in the chick embryo chorioallantoic membrane, alternatively activated DC elicit a marked angiogenic response, which is inhibited by neutralizing anti-VEGF Abs and by the VEGFR-2 inhibitor SU5416. Therefore, alternatively activated DC may contribute to the resolution of the inflammatory reaction by promoting VEGF-induced angiogenesis.     

Receptor cross-linking on human plasmacytoid dendritic cells leads to the regulation of IFN-alpha production

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.     

Nitric oxide inhibits IFN-alpha production of human plasmacytoid dendritic cells partly via a guanosine 3',5'-cyclic monophosphate-dependent pathway

NO, a free radical gas, is known to be critically involved not only in vascular relaxation but also in host defense. Besides direct bactericidal effects, NO has been shown to inhibit Th1 responses and modulate immune responses in vivo, although the precise mechanism is unclear. In this study, we examined the effect of NO on human plasmacytoid dendritic cells (pDCs) to explore the possibility that NO might affect innate as well as adaptive immunity through pDCs. We found that NO suppressed IFN-alpha production of pDCs partly via a cGMP-dependent mechanism, which was accompanied by down-regulation of IFN regulatory factor 7 expression. Furthermore, treatment of pDCs with NO decreased production of IL-6 and TNF-alpha and up-regulated OX40 ligand expression. In accordance with these changes, pDCs treated with NO plus CpG-oligodeoxynucleotide AAC-30 promoted differentiation of naive CD4(+) T cells into a Th2 phenotype. Moreover, pDCs did not express inducible NO synthase even after treatment with AAC-30, LPS, and several cytokines. These results suggest that exogenous NO and its second messenger, cGMP, alter innate as well as adaptive immune response through modulating the functions of pDCs and may be involved in the pathogenesis of certain Th2-dominant allergic diseases.     

Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production

BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches.     

Cutting edge: Expression of functional CD137 receptor by dendritic cells

Interaction between dendritic cells (DCs) and T cells is a prerequisite for the initiation of a T cell response. The molecular nature of this interaction remains to be fully characterized. We report in this work that freshly isolated mouse splenic DCs and bone marrow-derived DCs express CD137 on the cell surface and in soluble form. Triggering CD137 increased the secretion of IL-6 and IL-12 from DCs. More importantly, infusion of an agonistic mAb to CD137 into naive mice enhanced the ability of DCs to stimulate T cell proliferation in response to both alloantigens and a nominal Ag in vitro. This enhancement of DC function is not mediated through activation of T cells, because the effect was also observed in RAG-1 knockout mice that lack T cells. Our findings implicate CD137 as an important receptor involved in the modulation of DC function.     

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.     

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).     

Antigen targeting to plasmacytoid dendritic cells via Siglec-H inhibits Th cell-dependent autoimmunity

Plasmacytoid dendritic cells (PDCs) have been shown to present Ags and to contribute to peripheral immune tolerance and to Ag-specific adaptive immunity. However, modulation of adaptive immune responses by selective Ag targeting to PDCs with the aim of preventing autoimmunity has not been investigated. In the current study, we demonstrate that in vivo Ag delivery to murine PDCs via the specifically expressed surface molecule sialic acid binding Ig-like lectin H (Siglec-H) inhibits Th cell and Ab responses in the presence of strong immune stimulation in an Ag-specific manner. Correlating with sustained low-level MHC class II-restricted Ag presentation on PDCs, Siglec-H-mediated Ag delivery induced a hyporesponsive state in CD4(+) T cells leading to reduced expansion and Th1/Th17 cell polarization without conversion to Foxp3(+) regulatory T cells or deviation to Th2 or Tr1 cells. Siglec-H-mediated delivery of a T cell epitope derived from the autoantigen myelin oligodendrocyte glycoprotein to PDCs effectively delayed onset and reduced disease severity in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis by interfering with the priming phase without promoting the generation or expansion of myelin oligodendrocyte glycoprotein-specific Foxp3(+) regulatory T cells. We conclude that Ag delivery to PDCs can be harnessed to inhibit Ag-specific immune responses and prevent Th cell-dependent autoimmunity.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

Anti-BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha production of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) in human blood are the main source of virus-induced interferon (IFN)-alpha. They exhibit a lineage-negative phenotype but all express BDCA-4, which is homologous to the neuronal receptor neuropilin-1. Specific staining with anti-BDCA-4 antibody is used for positive isolation of PDC from blood by magnetic cells sorting. Here, it is demonstrated that these positively selected PDC showed reduced or completely abolished IFN-alpha release compared to unstained PDC, which were negatively selected by magnetic depletion of lineage-positive blood mononuclear cells. In addition, treatment of these unstained PDC with anti-BDCA-4 mAb also resulted in at least two-fold lower or reduced virus-induced IFN-alpha production. It is shown that the antibody not only affects cell survival or block virus attachment but also reduces IFN-alpha release induced by non-viral CpG oligodeoxynucleotides. In conclusion, data suggest an immunoregulatory role for BDCA-4 on PDC as demonstrated for IFN-alpha response to virus.     

Cutting edge: peripheral neuropeptides attract immature and arrest mature blood-derived dendritic cells

Dendritic cells (DC) are highly motile and play a key role in mediating immune responses in various tissues and lymphatic organs. We investigated locomotion of mononuclear cell-derived DC at different maturation stages toward gradients of sensory neuropeptides in vitro. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, secretin, and secretoneurin induced immature DC chemotaxis comparable to the potency of RANTES, whereas substance P and macrophage-inflammatory protein-3beta stimulated immature cell migration only slightly. Checkerboard analyses revealed a true chemotactic response induced by neuropeptides. Upon maturation of DC, neuropeptides inhibited spontaneous, macrophage-inflammatory protein-3beta- and 6Ckine-induced cell migration. Maturation-dependent changes in migratory behavior coincided with distinct neuropeptide-induced signal transduction in DC. Peripheral neuropeptides might guide immature DC to peripheral nerve fibers where high concentrations of these peptides can arrest the meanwhile matured cells. It seems that one function of sensory nerves is to fasten DC at sites of inflammation.     

CpG-A-induced monocyte IFN-gamma-inducible protein-10 production is regulated by plasmacytoid dendritic cell-derived IFN-alpha

Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) are known for inducing a Th1 cytokine/chemokine environment, but the mechanisms regulating this have been unclear. Recent studies have defined two classes of CpG ODN, CpG-A ODN that induce plasmacytoid dendritic cells (pDC) to secrete very high levels of IFN-alpha, and CpG-B ODN that induce only low levels of IFN-alpha production, but strongly activate B cells. We now demonstrate that a CpG-A ODN directly activates pDC secretion of IFN-alpha and other soluble factors that secondarily induce purified monocytes to secrete high levels of the Th1-promoting chemokine IFN-gamma-inducible protein-10 (IP-10). Cell contact between the monocytes and pDC is not required for this interaction. IFN-alpha is necessary, but only partially sufficient, for this indirect CpG-induced monocyte IP-10 production. Although CpG ODN induce human PBMC to make only very slight amounts of IFN-gamma, we find that these low concentrations synergize with IFN-alpha for inducing monocyte production of IP-10. These studies provide a better understanding of the mechanisms through which CpG ODN create a Th1-like environment.     

Cutting edge: murine dendritic cells require IL-15R alpha to prime NK cells

NK cells protect hosts against viral pathogens and transformed cells, and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma, despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast, IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition, blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally, presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming.     

Cutting edge: Antioxidative properties of myeloid dendritic cells: protection of T cells and NK cells from oxygen radical-induced inactivation and apoptosis

Dendritic cells (DCs) communicate with nonadaptive and adaptive lymphocytes on multiple levels. Efficient DC-lymphocyte interactions require that lymphocytes remain viable and functional also under conditions of oxidative stress, such as in microbial infection or in the malignant microenvironment. For this study, we exposed human T and NK cells to oxidants delivered either by autologous phagocytes or in the form of exogenous hydrogen peroxide. In accordance with earlier studies, these lymphocytes became dysfunctional and subsequently apoptotic. The presence of myeloid DCs efficiently rescued T cells (CD4+ and CD8+) and NK cells from oxidant-induced inactivation and apoptosis. The mechanism of the myeloid DC-mediated lymphocyte protection was, at least in part, explained by the capacity of the myeloid DCs to neutralize extracellular oxygen radicals, which, in turn, was reversible upon coincubation with a catalase inhibitor. Our results are suggestive of a novel aspect of DC-lymphocyte interaction that may have implications for lymphocyte function in inflamed tissue.