pDC Function Research Articles

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.

New role for antigen-presenting activated pDCs in promoting Th17 cells and impacting antitumor immunity

Plasmacytoid dendritic cells (pDCs) are not only potent inflammatory cytokine producers but also function as antigen-presenting cells (APCs). We have shown that vaccination using CpG-B activated tumor antigen (Ag) presenting pDCs induce Th17 cells that promote intratumoral immune cell recruitment, including antitumor cytotoxic T lymphocytes CTLs. Therefore, strategies targeting both innate and adaptive pDC functions may improve antitumor T-cell immunity.

Differential long noncoding RNA signatures in activated plasmacytoid dendritic cells (IRM6P.659)

Abstract Long noncoding RNAs (lncRNAs) represent a novel class of RNA which act through diverse mechanisms to exert control over a wide range of cellular processes. Through control of transcription, post-transcriptional regulation, or cellular signaling events, lncRNAs can function as potent modulators of innate immune responses, however very few lncRNAs have been investigated. We utilized whole exome sequencing of plasmacytoid dendritic cells (pDC) treated with IFN (interferon)-β, TLR7 agonist (toll like receptor, R848), and R848 with type I IFN blockade to identify 910 noncoding transcripts which are differentially regulated by IFN signaling and TLR7 activation. Through dissection of the stimulatory events controlling lncRNA expression, we have identified subsets of type I IFN- and TLR7-induced or suppressed lncRNAs. We hypothesize that many of these transcripts participate in the orchestration of innate and adaptive antiviral immunity through their modulation of pDC function. Dysregulation of these lncRNAs could significantly alter pDC maturation, IFN production, antigen presentation, turnover or localization, events which would be consequential during viral infection or IFN-driven autoimmune disease.

IFN-β Therapy Regulates TLR7-Mediated Response in Plasmacytoid Dendritic Cells of Multiple Sclerosis Patients Influencing an Anti-Inflammatory Status.

Plasmacytoid dendritic cells (pDCs) display altered immune-phenotype in multiple sclerosis (MS) patients and are found actively recruited in postmortem MS brain lesions, implying that their immune regulation may represent an important aspect of MS pathogenesis. Because of the reported Toll-like receptor 7 (TLR7) implication in autoimmunity, in this study we characterized how IFN-β therapy impacts on pDC activation to TLR7 triggering in MS patients, aspect only poorly investigated so far. In vivo IFN-β administration regulates pDC functions in TLR7-treated peripheral blood mononuclear cell (PBMC) cultures differently from what is observed in isolated cells, suggesting that IFN-β may activate inhibitory mechanisms in MS peripheral blood involved in turning off pDC response to dampen the ongoing inflammation. Indeed, IL-10, a key regulatory cytokine found increased upon TLR7 stimulation in in vivo IFN-β-exposed PBMCs, directly reduced pDC-mediated IFN-α production. IFN-β therapy also shaped T-cell responses by decreasing TLR7-induced pDC maturation and inducing T-cell inhibitory molecules. Accordingly, raised pDC-induced IL-27 and decreased IL-23 expression, together with high IL-10 level, contribute to inhibit Th17 cell differentiation. Our study uncovered a role for IFN-β in the regulation of TLR7-mediated pDC responses in MS toward an anti-inflammatory phenotype opening new opportunities to better understand mechanisms of action of this drug in controlling MS immunopathogenesis.

Toll-like receptor 9 dependent interferon-α release is impaired in severe asthma but is not associated with exacerbation frequency

Patients with asthma and chronic obstructive pulmonary disease (COPD) are susceptible to exacerbations, often caused by microbial pathogens. We hypothesised that intracellular Toll-like receptor (TLR) function in blood mononuclear cells (PBMCs) from these subjects would be impaired and that this impairment is related to exacerbation frequency. PBMCs stimulated with a TLR-9 agonist (but not TLR-3 or 7/8) produced significantly less IFN-α in asthma (26 [3–696]pg/ml) compared to control (943 [164–1651]) and COPD (597 [127–1186]) subjects (p=0.0019) but this was not related to the number of exacerbations per year in asthma or COPD. In COPD, IFN-α levels were related to KCO (% predicted) in COPD (r=−0.41, p=0.01). IFN-α was derived from plasmacytoid dendritic cells (pDCs) and their frequency was lower in asthma compared to control subjects (control 0.48% [0.33–0.64] versus asthma 0.29% [0.13–0.34], p=0.019) whereas pDC function per se was not significantly impaired between groups. The mechanism underlying reduced IFN-α production and the clinical consequences in severe asthma remains to be established.

Clinical profiles of circulating plasmacytoid dendritic cells in chronic hepatitis B patients in response to pegylated-interferon alfa-2a treatment

To investigate the changes in circulating plasmacytoid dendritic cells (pDCs) in patients with chronic hepatitis B (CHB) during the course of treatment with pegylated-interferon alfa-2s (peg-IFNa-2a) and to determine the correlations with therapeutic response. Forty-one patients with CHB who were receiving peg-IFNa-2a antiviral treatment for 48 weeks were enrolled in the study.Expression of the Toll-like receptor 9 (TLR9) on and frequency and functionality of the pDCs were analyzed at treatment weeks 0, 2, 12, 24, 36 and 48. All patients exhibited an initially rapid decrease in the numbers of circulating pDCs and showed CpG-induced endogenous IFNa production within the first 2 weeks of treatment.Subsequently, all responders displayed a continuous increase in pDC numbers as well as functionality, both of which peaked around week 12 of treatment; in addition, these treatment responses were accompanied by significantly increased levels of type 1 T helper cytokines (P less than 0.05), which did not occur in the non-responders. pDCs are involved in the initial therapeutic immune response stimulated by peg-IFNa-2a treatment.Recovery of blood pDC number and functionality may represent a predictor of favorable response to peg-IFNa-2a antiviral treatment in patients with CHB.

The plasmacytoid dendritic cell as the Swiss army knife of the immune system: molecular regulation of its multifaceted functions.

Plasmacytoid dendritic cells (pDC) have been regarded as the "professional type I IFN-producing cells" of the immune system following viral recognition that relies on the expression of TLR7 and TLR9. Furthermore, pDC link the innate and adaptive immune systems via cytokine production and Ag presentation. More recently, their ability to induce tolerance and cytotoxicity has been added to their "immune skills." Such a broad range of actions, resembling the diverse functional features of a Swiss army knife, requires strong and prompt molecular regulation to prevent detrimental effects, including autoimmune pathogenesis or tumor escape. Over the last decades, we and other investigators have started to unravel some aspects of the signaling pathways that regulate the various functions of human pDC. In this article, we review aspects of the molecular regulatory mechanisms to control pDC function in light of their multifaceted roles during immunity, autoimmunity, and cancer.

S-15 : Cytokine-driven loss of plasmacytoid dendritic cell function in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5 + CD19 + B cells in the peripheral blood, and in primary and secondary lymphoid organs. A major complication associated with CLL is severe recurrent infections, which are often fatal. Vulnerability to infection is due to a wide variety of immunological defects, yet the initiating events of immunodeficiency in CLL are unclear. Using CLL patient samples and a mouse model of CLL, we have discovered that plasmacytoid dendritic cells (pDCs), which function underpins the activity of effector immune cells critical for anti-viral immunity and anti-tumor responses, are reduced in number and functionally impaired in progressive CLL. As a result, the levels of interferon alpha (IFNa) production, a cytokine critical for immunity, are markedly reduced. Lower pDC numbers with impaired IFNa production was due to the decreased expression of FMS-like tyrosine kinase 3 receptor (Flt3) and Toll-like receptor 9 (TLR9), respectively. Reduced Flt3 expression was reversed using inhibitors of TGF-b and TNF, an effect correlating with a reduction in tumor load. Defects in pDC numbers and function offer new insight into mechanisms underpinning the profound immunodeficiency affecting CLL patients and provide a potentially novel avenue for restoring immunocompetency in CLL.

Genetic evidence for the role of plasmacytoid dendritic cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of antibodies to self-nucleic acids, immune complex deposition, and tissue inflammation such as glomerulonephritis. Innate recognition of self-DNA and -RNA and the ensuing production of cytokines such as type I interferons (IFNs) contribute to SLE development. Plasmacytoid dendritic cells (pDCs) have been proposed as a source of pathogenic IFN in SLE; however, their net contribution to the disease remains unclear. We addressed this question by reducing gene dosage of the pDC-specific transcription factor E2-2 (Tcf4), which causes a specific impairment of pDC function in otherwise normal animals. We report that global or DC-specific Tcf4 haplodeficiency ameliorated SLE-like disease caused by the overexpression of the endosomal RNA sensor Tlr7. Furthermore, Tcf4 haplodeficiency in the B6.Sle1.Sle3 multigenic model of SLE nearly abolished key disease manifestations including anti-DNA antibody production and glomerulonephritis. Tcf4-haplodeficient SLE-prone animals showed a reduction of the spontaneous germinal center reaction and its associated gene expression signature. These results provide genetic evidence that pDCs are critically involved in SLE pathogenesis and autoantibody production, confirming their potential utility as therapeutic targets in the disease.

Human plasmacytoid dendritic cells regulate IFN-α production through activation-induced splicing of IL-18Rα.

IFN-α production by pDCs regulates host protection against viruses and is implicated in autoimmune pathology. Human pDCs express high levels of IL-18R, but little is known of its role in pDC function. We report that IL-18R signaling negatively regulates IFN-α production through activation-induced splicing of IL-18Rα in human pDCs. Our data reveal two distinct isoforms of IL-18Rα in human pDCs: the known, full-length receptor (IL-18Rα1) and a novel, truncated variant (IL-18Rα2), which functions as a molecular decoy that competitively inhibits the canonical IL-18Rα1/IL-18Rβ signaling pathway. Whereas NK cells and pDCs both express IL-18Rα1, pDCs express significantly higher levels of IL-18Rα2, resulting in differential responses of these populations to IL-18. Flu exposure increases IL-18Rα1 expression in pDCs, and the blocking of IL-18R enhances pDC production of IFN-α and IP-10; thus, pDCs use activation-induced splicing to regulate IFN-α production in response to flu. These data demonstrate that IL-18R modulates IFN-α release by human pDCs and suggest that IL-18R signaling may represent a promising therapeutic target.

Impaired Interferon-Alpha Production by Plasmacytoid Dendritic Cells after Cord Blood Transplantation in Children: Implication for Post-transplantation Toll-Like Receptor Ligand–Based Immunotherapy

Plasmacytoid dendritic cells (pDCs) initiate both innate and adaptive immune responses, making them attractive targets for post-transplantation immunotherapy, particularly after cord blood transplantation (CBT). Toll-like receptor (TLR) agonists are currently studied for pDC stimulation in various clinical settings. Their efficacy depends on pDC number and functionality, which are unknown after CBT. We performed a longitudinal study of pDC reconstitution in children who underwent bone marrow transplantation (BMT) and single-unit CBT. Both CBT and unrelated BMT patients received antithymocyte globulin as part of their graft-versus-host disease prophylaxis regimen. pDC blood counts were higher in CBT patients than in healthy volunteers from 2 to 9 months after transplantation, whereas they remained lower in BMT patients. We showed that cord blood progenitors gave rise in vitro to a 500-fold increase in functional pDCs over bone marrow counterparts. Upon stimulation with a TLR agonist, pDCs from both CBT and BMT recipients upregulated T cell costimulatory molecules, whereas interferon-alpha (IFN-α) production was impaired for 9 months after CBT. TLR agonist treatment is thus not expected to induce IFN-α production by pDCs after CBT, limiting its immunotherapeutic potential. Fortunately, in vitro production of large amounts of functional pDCs from cord blood progenitors paves the way for the post-transplantation adoptive transfer of pDCs.

Increased IFN-α-producing plasmacytoid dendritic cells (pDCs) in human Th1-mediated type 1 diabetes: pDCs augment Th1 responses through IFN-α production.

Increasing evidence suggests that type 1 IFN (IFN-αβ) is associated with pathogenesis of Th1-mediated type 1 diabetes (T1D). A major source of IFN-αβ is plasmacytoid dendritic cells (pDCs). In this study, we analyzed peripheral blood pDC numbers and functions in at-risk, new-onset, and established T1D patients and controls. We found that subjects at risk for T1D and new-onset and established T1D subjects possessed significantly increased pDCs but similar number of myeloid DCs when compared with controls. pDC numbers were not affected by age in T1D subjects but declined with increasing age in control subjects. It was demonstrated that IFN-α production by PBMCs stimulated with influenza viruses was significantly higher in T1D subjects than in controls, and IFN-α production was correlated with pDC numbers in PBMCs. Of interest, only T1D-associated Coxsackievirus serotype B4 but not B3 induced majority of T1D PBMCs to produce IFN-α, which was confirmed to be secreted by pDCs. Finally, in vitro studies demonstrated IFN-α produced by pDCs augmented Th1 responses, with significantly greater IFN-γ-producing CD4(+) T cells from T1D subjects. These findings indicate that increased pDCs and their IFN-αβ production may be associated with this Th1-mediated autoimmune disease, especially under certain viral infections linked to T1D pathogenesis.

Plasmacytoid Dendritic Cells in Melanoma: Can We Revert Bad into Good?

Tumor-infiltrating plasmacytoid dendritic cells (pDCs) promote an immunosuppressive milieu that drives tumor growth in melanoma. This phenomenon typically results from the lack of appropriate pDC activation signals in the tumor microenvironment, but it is also actively controlled by tumor cells, which have evolved strategies to inhibit type I IFN production by pDCs. In this issue, Camisaschi et al. identify a new mechanism in which tumors avoid type I IFN production by triggering LAG-3-dependent activation of pDCs. Combination therapies that restore pDC functionality and trigger innate activation to produce type I IFN should be envisaged to induce effective antitumor immunity.

OP0102 Effect of Immunoglobulin E on Plasmacytoid Dendritic Cells in Systemic Lupus Erythematosus Physiopathology through Interferon-Alpha Downregulation

BackgroundPlasmacytoid dendritic cells (pDCs) are dendritic cells characterized by their unique ability to produce large amounts of type I interferon (IFNalpha/beta) upon Toll-like receptor (TLR) 7/9 triggering. Blood type I...

Histone deacetylase inhibitor valproic acid affects plasmacytoid dendritic cells phenotype and function

ObjectivePlasmacytoid dendritic cells (PDC) represent a rare subset of dendritic cells specialized in the production of type I IFN in response to microbial pathogens. Recent data suggested that histone deacetylase (HDAC) inhibitors possess potent immunomodulatory properties both in vitro and in vivo. In this study, we assayed the ability of the HDAC inhibitor, valproic acid (VPA), to influence the phenotype and functional properties of human PDC isolated from peripheral blood. Methods and resultsWe showed that VPA inhibited the production of IFN-α and the proinflammatory cytokines TNF-α and IL-6 by CpG-activated PDC. VPA also affected the phenotype of PDC by reducing the expression of costimulatory molecules induced by CpG activation. Moreover, VPA reduced the capacity of CpG-stimulated PDC to promote CD4+ T cell proliferation and IFN-γ production, while enhancing the proportion of IL-10 positive T cells. ConclusionThese results suggest that HDAC inhibition by VPA alters essential human PDC functions, highlighting the need for monitoring immune functions in cancer patients receiving HDAC inhibitors, but also making these drugs attractive therapies in inflammatory, and autoimmune diseases implicating PDC.

Cytokine-driven loss of plasmacytoid dendritic cell function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+CD19+ B cells in the peripheral blood, and in primary and secondary lymphoid organs. A major complication associated with CLL is severe recurrent infections, which are often fatal. Vulnerability to infection is due to a wide variety of immunological defects, yet the initiating events of immunodeficiency in CLL are unclear. Using CLL patient samples and a mouse model of CLL, we have discovered that plasmacytoid dendritic cells (pDCs), which underpin the activity of effector immune cells critical for anti-viral immunity and anti-tumor responses, are reduced in number and functionally impaired in progressive CLL. As a result, the levels of interferon alpha (IFNα) production, a cytokine critical for immunity, are markedly reduced. Lower pDC numbers with impaired IFNα production was due to the decreased expression of FMS-like tyrosine kinase 3 receptor (Flt3) and Toll-like receptor 9 (TLR9), respectively. Reduced Flt3 expression was reversed using inhibitors of TGF-β and TNF, an effect correlating with a reduction in tumor load. Defects in pDC numbers and function offer a new insight into mechanisms underpinning the profound immunodeficiency affecting CLL patients and provide a potentially novel avenue for restoring immuno-competency in CLL.

Functional limitations of plasmacytoid dendritic cells limit type I interferon, T cell responses and virus control in early life.

Infant mortality from viral infection remains a major global health concern: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8+ T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC), which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8) is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.

Plasmacytoid dendritic cells and autoimmune inflammation

Plasmacytoid dendritic cells (pDC) are a sub-population of dendritic cells (DC) that produce large amounts of type I interferon (IFN) in response to nucleic acids that bind and activate toll-like-receptor (TLR)9 and TLR7. Type I IFN can regulate the function of B, T, DC, and natural killer (NK) cells and can also alter the residence time of leukocytes within lymph nodes. Activated pDC can also function as antigen presenting cells (APC) and have the potential to prime and differentiate T cells into regulatory or inflammatory effector cells, depending on the context. In this review we discuss pDC ontogeny, function, trafficking, and activation. We will also examine how pDC can potentially be involved in regulating immune responses in the periphery as well as within the central nervous system (CNS) during multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE).

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg ). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (-64%) but TLR-9-induced IFN-α secretion only slightly (-20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4(+) forkhead box protein 3 (FoxP3)(+) regulatory T cells, but did not affect the generation of suppressive CD8(+) CD38(+) lymphocyte activation gene (LAG)-3(+) Treg . In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4(+) T cell proliferation and induce CD4(+) FoxP3(+) regulatory T cell generation.

Environmental alkylphenols modulate cytokine expression in plasmacytoid dendritic cells.

BackgroundAlkylphenols, such as nonylphenol (NP) and 4-octylphenol (4-OP), have the potential to disturb immune system due to their weak estrogen-like activity, an effect with potential serious public health impact due to the worldwide distribution of these substances. Plasmacytoid dendritic cells (PDCs) can secrete large amounts of type I IFNs and are critical in immune regulation. However, there has been limited study about the influence of alkylphenols on the function of pDCs.ObjectiveThe aim of this study was to examine the effect of alkylphenols on pDC functions in vitro and in vivo and then further explored the involved signaling pathways and epigenetic changes.MethodsCirculating pDCs from human peripheral blood mononuclear cells were treated with alkylphenols with or without CpG stimulation. Alkylphenol-associated cytokine responses, signaling events, histone modifications and viral activity were further examined. In NP-exposed mice, the effect of NP on splenic pDC function and allergic lung inflammation were also assessed.ResultsThe results showed that NP increased the expression of TNF-α, but suppressed IL-10 production in the range of physiological doses, concomitant with activation of the MKK3/6-p38 signaling pathway and enhanced levels of acetylated histone 3 as well as histone 4 at the TNFA gene locus. Further, in CpG-stimulated pDCs, NP suppressed type I IFNs production, associated with down-regulation of IRF-7 and MKK1/2-ERK-Elk-1 pathways and led to the impaired anti-enterovirus 71 activity in vitro. Additionally, splenic pDCs from NP-exposed mice showed similar cytokine changes upon CpG stimulation under conditions relevant to route and level of exposure in humans. NP treatment also enhanced allergic lung inflammation in vivo.ConclusionAlkylphenols may influence pDCs’ functions via their abilities to induce expression of a pro-inflammatory cytokine, TNF-α, and to suppress regulatory cytokines, including IL-10, IFN-α and IFN-β, suggesting the potential impact of endocrine disrupting chemicals on immune regulation.