Plasmacytoid dendritic cells (pDCs) mediate type I interferon (IFN-I) responses to viruses that are recognized through the Toll-like receptor 7 (TLR7) or TLR9 signaling pathway. However, it is unclear how pDCs regulate the antiviral responses via innate and adaptive immune cells. We generated diphtheria toxin receptor transgenic mice to selectively deplete pDCs by administration of diphtheria toxin. pDC-depleted mice were challenged with viruses known to activate pDCs. In murine cytomegalovirus (MCMV) infection, pDC depletion reduced early IFN-I production and augmented viral burden facilitating the expansion of natural killer (NK) cells expressing the MCMV-specific receptor Ly49H. During vesicular stomatitis virus (VSV) infection, pDC depletion enhanced early viral replication and impaired the survival and accumulation of virus-specific cytotoxic T lymphocytes. We conclude that pDCs mediate early antiviral IFN-I responses and influence the accrual of virus-specific NK or CD8+ T cells in a virus-dependent manner.
Plasmacytoid dendritic cells (pDCs) represent a unique and crucial immune cell population capable of producing large amounts of type I interferons (IFNs) in response to viral infection. The function of pDCs as the professional type I IFN-producing cells is linked to their selective expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the endosomal compartments. Type I IFNs produced by pDCs not only directly inhibit viral replication but also play an essential role in linking the innate and adaptive immune system. The aberrant activation of pDCs by self nucleic acids through TLR signaling and the ongoing production of type I IFNs do occur in some autoimmune diseases. Therefore, pDC may serve as an attractive target for therapeutic manipulations of the immune system to treat viral infectious diseases and autoimmune diseases.
plasmacytoid dendritic cells; type I interferon; TLR7; TLR9; antiviral immunity; autoimmune diseases
Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.
Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.
A persistent type I interferon (IFN) response is thought to be important in driving immune activation and progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. Plasmacytoid dendritic cells (pDC) produce copious amounts of type I IFN upon virus exposure through engagement of Toll-like receptor (TLR) 7 and TLR9 and thus may be central players in the etiology of immune activation. We used a dual antagonist of TLR7 and TLR9 to selectively block the response of pDC but not other mononuclear phagocytes prior to and for 8 weeks following simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major, but not exclusive, producers of IFN-α that mediate a marked but transient IFN-α response in lymph nodes in the acute phase of infection. TLR7 and TLR9 antagonist prevented this IFN-α production without suppressing pDC recruitment. Nevertheless, TLR7 and TLR9 blockade did not impact expression of IFN-stimulated genes or decrease the activation of T cells, the hallmarks of immune activation. The findings indicate that TLR7 and TLR9-driven activation of pDC is unlikely to be a major contributor to immune activation in the early stages of immunodeficiency virus infections and suggest that therapeutic strategies aimed at targeting pDC and IFN-α production may not reduce HIV-associated immunopathology.
Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-α responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC's type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.
PLSCR1; TLR9; IRF7; pDC; IFN-α signaling
Cutaneous injury in mice drives transient TLR7- and TLR9-mediated production of type I interferon by plasmacytoid dendritic cells, which is required for re-epithelialization of the skin.
Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/β)–producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.
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.
Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.
Plasmacytoid dendritic cells (pDCs) are the professional interferon (IFN)-producing cells of the immune system. pDCs specifically express Toll-like receptor (TLR)7 and TLR9 molecules and produce massive amounts of type I IFN by sensing microbial nucleic acids via TLR7 and TLR9. Here we report that protein kinase C and casein kinase substrate in neurons (PACSIN) 1, is specifically expressed in human and mouse pDCs. Knockdown of PACSIN1 by short hairpin RNA (shRNA) in a human pDC cell line significantly inhibited the type I IFN response of the pDCs to TLR9 ligand. PACSIN1-deficient mice exhibited normal levels of conventional DCs and pDCs, demonstrating that development of pDCs was intact although PACSIN1-deficient pDCs showed reduced levels of IFN-α production in response to both cytosine guanine dinucleotide (CpG)-oligonucleotide (ODN) and virus. In contrast, the production of proinflammatory cytokines in response to those ligands was not affected in PACSIN1-deficient pDCs, suggesting that PACSIN1 represents a pDC-specific adaptor molecule that plays a specific role in the type I IFN signaling cascade.
Interferon; Plasmacytoid dendritic cells (pDCs); TLR9
Production of type I interferon in response to virus infection reduces plasmacytoid dendritic cell numbers, thus creating a negative feedback loop.
Plasmacytoid dendritic cells (pDCs) specialize in the secretion of type I interferons (IFN-I) and thus are considered critical mediators of antiviral responses. We recently reported that pDCs have a very early but limited and transient capacity to curtail viral infections. Additionally, pDC numbers are not sustained in human infections caused by Hepatitis B or C viruses (HBV and HCV) and HIV. Thus, the numbers and/or function of pDCs appear to be regulated during the course of viral infection. In this study, we show that splenic pDCs are reduced in vivo during several systemic viral infections and after administration of synthetic toll-like receptor ligands. We demonstrate that IFN-I, regardless of the source, contributes to this decline and mediates pDC death via the intrinsic apoptosis pathway. These findings demonstrate a feedback control mechanism by which IFN-I modulates pDC numbers, thus fine-tuning systemic IFN-I response to viruses. IFN-I–mediated control of pDCs may explain the loss of pDCs during human infections caused by HBV, HCV, or HIV and has important therapeutic implications for settings in which IFN-I is used to treat infections and autoimmune diseases.
Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.
Immunomodulators that induce local endogenous interferon-alpha (IFN-α) production by plasmacytoid dendritic cells (pDCs) may offer new strategies for the treatment of patients chronically infected with the hepatitis C virus (HCV). However, such an approach may be compromised if reports are true that IFN-α production by pDCs from patients with chronic HCV (cHCV) is profoundly impaired. To address the question of pDC dysfunction in cHCV more definitively, in the present study a panel of four prototypic synthetic agonists of toll-like receptor 7 (TLR7) or TLR9 were administered in vitro to pDCs purified from cHCV patients and from normal uninfected donors and their responses compared in terms of not only IFN-α production but also the global expression of other cytokines and phenotypic maturation. Plasmacytoid DCs from uninfected donors produced substantial levels of IFN-α in response to three of the four agonists and yet only one TLR9 agonist, a class C CpG oligodeoxynucleotide (ODN), induced robust IFN-α production by pDCs from cHCV patients. Proinflammatory cytokine production and phenotypic maturation in response to all four agonists was equivalent in infected and uninfected pDCs. These data point to a profound but selective defect in IFN-α production by pDCs from cHCV donors. Nonetheless, a class C CpG ODN successfully induced robust IFN-α production, suggesting that this class of TLR9 agonist may have utility as a future immunotherapeutic for the treatment of chronic HCV infection.
CpG; dendritic cell; hepatitis C; interferon-α; plasmacytoid; toll-like receptor
Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-α and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-α production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-α production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1–positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-α from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1–positive endosomes accompanied by the loss of IFN-α production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-α and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-α response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.
Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12 and IL-6. However, RSV-infected pDCs are refractory to TLR7-mediated activation. Here, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7-signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type (WT) but not TLR7- or myeloid differentiation protein 88 (MyD88)-deficient mice, PVM inoculation led to a marked infiltration of pDCs and increased expression of type I, II and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8+ T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient but not TLR7-deficient pDC to TLR7-gene-deleted mice recapitulated the antiviral responses observed in WT mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.
Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-α/β induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-α/β production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-α/β in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.
Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-α in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with herpes simplex virus type 2 (HSV-2), a viral pathogen that activates TLR9. We found that impaired IFN-α production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective upregulation of interferon regulatory factor 7 (IRF-7), a key adaptor in the type I IFN pathway, as compared to younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-α defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.
Rodent; viral infection; dendritic cell
Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.
We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.
Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.
These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.
Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-α in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-α production was abolished in Irak-1–deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-κB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1–deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-α induction in pDCs.
Plasmacytoid dendritic cells (pDC) are a specialized sensor of viral and bacterial nucleic acids and a major producer of IFN-α that promotes host defense by priming both innate and acquired immune responses. Although synthetic Toll-like receptor (TLR) ligands, pathogenic bacteria and viruses activate pDC, there is limited investigation of non-pathogenic microbiota that are in wide industrial dietary use, such as lactic acid bacteria (LAB). In this study, we screened for LAB strains, which induce pDC activation and IFN-α production using murine bone marrow (BM)-derived Flt-3L induced dendritic cell culture. Microbial strains with such activity on pDC were absent in a diversity of bacillary strains, but were observed in certain spherical species (Lactococcus, Leuconostoc, Streptococcus and Pediococcus), which was correlated with their capacity for uptake by pDC. Detailed study of Lactococcus lactis subsp. lactis JCM5805 and JCM20101 revealed that the major type I and type III interferons were induced (IFN-α, -β, and λ). IFN-α induction was TLR9 and MyD88-dependent; a slight impairment was also observed in TLR4-/- cells. While these responses occurred with purified pDC, IFN-α production was synergistic upon co-culture with myeloid dendritic cells (mDC), an interaction that required direct mDC-pDC contact. L. lactis strains also stimulated expression of immunoregulatory receptors on pDC (ICOS-L and PD-L1), and accordingly augmented pDC induction of CD4+CD25+FoxP3+ Treg compared to the Lactobacillus strain. Oral administration of L. lactis JCM5805 induced significant activation of pDC resident in the intestinal draining mesenteric lymph nodes, but not in a remote lymphoid site (spleen). Taken together, certain non-pathogenic spherical LAB in wide dietary use has potent and diverse immunomodulatory effects on pDC potentially relevant to anti-viral immunity and chronic inflammatory disease.
While plasmacytoid dendritic cells (pDCs), a natural type I interferon (IFN) producing cell type, are regarded as critical for innate immunity to viruses, their role in defense against fungal infections remains unknown. We examined the interactions of pDCs with hyphae of the invasive human fungal pathogen Aspergillus fumigatus. Human pDCs spread over hyphae and inhibited their growth. Antifungal activity was retained in pDC lysates, did not require direct fungal contact, and was partially reversed by zinc. Incubation with hyphae resulted in pDC cytotoxicity, partly due to fungal gliotoxin secretion. Following hyphal stimulation, pDCs released proinflammatory cytokines via a TLR9-independent mechanism. Pulmonary challenge of mice with A. fumigatus resulted in a substantial influx of pDCs into lungs and pDC-depleted mice were hypersusceptible to invasive aspergillosis. These data demonstrate the antifungal activity of pDCs against A. fumigatus and establish their non-redundant role in host defenses against invasive aspergillosis in vivo.
Recognition of nucleic acids by TLR9 expressed by human plasmacytoid dendritic cells (PDC) plays a key role in the defense against viral infections. Upon microbial pathogen stimulation, PDC secret large amounts of type I interferon and attend thereby both innate and adaptive immune mechanisms. Unmethylated CpG motifs, which are an integral part of bacterial or viral DNA are used in vitro and in vivo to activate the TLR9 pathway, while inhibitory oligodeoxynucleotide (iODN) are capable to depress TLR9 signaling. In this study we show that TTAGGG motif containing iODN efficiently block the TLR9 signaling in terms of herpes simplex virus (HSV) induced type I interferon production by PDC. However, iODN as well as control ODN still promote PDC maturation with upregulated expression of costimulatory molecules, major histocompatibility complex molecules and other signs for PDC maturation. Furthermore, iODN and control ODN incubated PDC show increased T cell stimulatory functions. Co-culture experiments with autologous T cells indicate that iODN treated PDC induce more CD4+CD25+Foxp3+ T regulatory cells from naïve CD4+ T cells and preincubation of HSV stimulated PDC with iODN upregulated T cells’ IFN-γ production. These data indicate that iODN while blocking type I interferon production by PDC, modify PDC activation and maturation as well as T cell priming and stimulation. The knowledge about the different functions of iODN on PDC elucidated here might be crucial for immunotherapeutic strategies in which iODN motifs are used to prevent the interaction of CpG-DNA with TLR9 to calm down specific immunological responses, since our data show that iODN might not only have inhibitory functions but moreover be effective activators of immune cells.
Inhibitory oligodeoxynucleotide; dendritic cell; cell activation; cell surface molecules; cytokines
Type I interferons (IFN) and dendritic cells (DC) share an overlapping history, with rapidly accumulating evidence for vital roles for both production of type 1 IFN by DC and the interaction of this IFN both with DC and components of the innate and adaptive immune responses. Within the innate immune response, the plasmacytoid DC (pDC) are the “professional” IFN producing cells, expressing specialized toll-like receptors (TLR7 and -9) and high constitutive expression of IRF-7 that allow them to respond to viruses with rapid and extremely robust IFN production; following activation and production of IFN, the pDC subsequently mature into antigen presenting cells that help to shape the adaptive immune response. However, like most cells in the body, the myeloid or conventional DC (mDC or cDC) also produce type I IFNs, albeit typically at a lower level than that observed with pDC, and this IFN is also important in innate and adaptive immunity induced by these classic antigen presenting cells. These two major DC subsets and their IFN products interact both with each other as well as with NK cells, monocytes, T helper cells, T cytotoxic cells, T regulatory cells and B cells to orchestrate the early immune response. This review will discuss some of the converging history of DC and IFN as well as mechanisms for IFN induction in DC and the effects of this IFN on the developing immune response.
dendritic cells; plasmacytoid dendritic cells; type I interferon; IFN
Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-α/β in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8α+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-α/β regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8α+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-α/β, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.
plasmacytoid dendritic cell; interferon α/β; murine cytomegalovirus; antigen presentation; CD8 T lymphocyte
IFNA1 (interferon alpha) is a key cytokine regulating the activity of numerous immune cells. Plasmacytoid dendritic cells (pDCs) as natural interferon-producing cells play critical roles as sensors of pathogens and link innate to adaptive immunity. CpG motifs within DNA sequences activating toll-like receptor 9 (TLR9) are the main stimuli eliciting IFNA1 secretion from pDCs. Adrenergic substances are capable of differentially modulating the response from various immune cells. Hence, the aim of this study was to examine how adrenoceptor stimulation influences TLR9-induced IFNA1 secretion from human pDCs.
PBMCs generated from human whole blood and pDCs enriched from buffy coats were stimulated with LPS and CpG-ODN 2336 in the presence or absence of epinephrine and different adrenoceptor antagonists. Secretion of TNF and IFNA1 was measured by ELISA. Flow cytometry was used to determine efficacy of pDC enrichment and adrenoceptor expression of PBMC subsets. The influence of modified IFNA1 secretion on NK cell activity was evaluated using a colorimetric tumor cell lysis assay.
TLR9-induced IFNA1 secretion as well as TLR4-induced TNF secretion from PBMCs was dose-dependently attenuated by coincubation with epinephrine. Combination with different specific adrenoceptor antagonists revealed that this effect was mediated by the adrenoceptor β2 (ADRB2). Since flow cytometric analysis could exclude the presence of ADRB2 on pDCs, highly enriched pDCs lacked any visible impact of adrenoceptor stimulation on TLR9-induced IFNA1 release. Combination of pDCs with PBMCs restored the effect, even when they were separated by a permeable membrane. Suppression of TLR9-mediated IFNA1 secretion from PBMCs by adrenoceptor stimulation reduced the lytic activity of NK cells on K562 tumor cells.
We provide insights into the underlying mechanisms of the interrelation between immune responses and pharmacological agents widely used in clinical practice. Our results have implications for the future treatment of human patients, in which the endogenous immune response plays a pivotal role, such as during viral infections, inflammatory diseases and cancers.
HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC) functions, including response to Toll-Like Receptor (TLR) ligands. Because TLR responsiveness may affect a host's response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC) activation of naïve T-cells in HIV+ subjects.
Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848). We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression), cytokine secretion (IFN-alpha and IL-6), and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.
MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.
These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.