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1.  Peptidoglycan Recognition Protein Pglyrp1 Enhances Experimental Asthma by Promoting Th2 and Th17 and Limiting Regulatory T Cell and Plasmacytoid Dendritic Cell Responses 
Asthma is a common inflammatory disease involving crosstalk between innate and adaptive immunity. We reveal that antibacterial innate immunity protein, peptidoglycan recognition protein 1 (Pglyrp1), is involved in the development of allergic asthma. Pglyrp1−/− mice developed less severe asthma than wild type (WT) mice following sensitization with house dust mite (HDM) allergen. HDM-sensitized Pglyrp1−/− mice, compared with WT mice, had diminished: bronchial hyper-responsiveness (lung airway resistance); numbers of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid and lungs; inflammatory cell infiltrates in the lungs around bronchi, bronchioles, and pulmonary arteries and veins; lung remodeling (mucin-producing goblet cell hyperplasia and metaplasia and smooth muscle hypertrophy and fibrosis); levels of IgE, eotaxins, IL-4, IL-5, and IL-17 in the lungs; and numbers of Th2 and Th17 cells and expression of their marker genes in the lungs. The mechanism underlying this decreased sensitivity of Pglyrp1−/− mice to asthma was increased generation and activation of CD8α+β+ and CD8α+β− plasmacytoid dendritic cells (pDC) and increased recruitment and activity of regulatory T (Treg) cells in the lungs. In vivo depletion of pDC in HDM-sensitized Pglyrp1−/− mice reversed the low responsive asthma phenotype of Pglyrp1−/− mice to resemble the more severe WT phenotype. Thus, Pglyrp1−/− mice efficiently control allergic asthma by up-regulating pDC and Treg cells in the lungs, whereas in WT mice Pglyrp1 is proinflammatory and decreases pDC and Treg, and increases pro-asthmatic Th2 and Th17 responses. Blocking Pglyrp1 or enhancing pDC in the lungs may be beneficial for prevention and treatment of asthma.
doi:10.4049/jimmunol.1202675
PMCID: PMC3608703  PMID: 23420883
2.  Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance 
Lung-resident antigen-presenting macrophages promote tolerance to inhaled antigens via the induction of regulatory T cells.
Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MØs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3+ iTreg cells is unclear. Here, we show that lung-resident tissue MØs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3+ Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MØs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MØs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MØs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma.
doi:10.1084/jem.20121849
PMCID: PMC3620360  PMID: 23547101
3.  Endotoxin Augments Myeloid Dendritic Cell Influx into the Airways in Patients with Allergic Asthma 
Rationale: Epidemiologic studies have shown that exacerbation of asthma is modulated by environmental endotoxin. High levels of endotoxin are associated with asthma symptoms and the current use of asthma medication. However, the underlying mechanisms by which endotoxin modulates asthma are not completely understood.
Objectives: The aim of the study was to test whether endotoxin enhances the response of individuals with allergic asthma to allergen, and to determine if this interaction is associated with increased numbers of antigen-presenting cells in the airways.
Methods: Seventeen subjects with mild allergic asthma underwent segmental challenge with allergen, endotoxin, and the combination of both in three different lung segments via bronchoscopy. The cellular influx including monocytes, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs), as well as the level of cytokines, were assessed in bronchoalveolar lavage fluid obtained 24 hours after segmental challenge. Monocytes, mDCs, and pDCs were isolated and their capacity to induce T cell proliferation was determined.
Measurements and Main Results: Endotoxin enhanced the cellular response to allergen. The combination of allergen and endotoxin resulted in increased numbers of total cells, lymphocytes, neutrophils, eosinophils, monocytes, and mDCs, as well as increased levels of lipopolysaccharide-binding protein, IL-1α, IL-6, and tumor necrosis factor–α in the bronchoalveolar lavage fluid compared with allergen alone. Isolated mDCs but not pDCs induced a strong T cell proliferation in vitro.
Conclusions: Endotoxin augments the allergic inflammation in the lungs of individuals with asthma, and induces an enhanced influx of monocytes and functionally active antigen-presenting mDCs into the respiratory tract.
doi:10.1164/rccm.200706-870OC
PMCID: PMC2427055  PMID: 18388357
dendritic cells; monocytes; bronchoalveolar lavage; endobronchial allergen challenge; mixed lymphocyte culture test
4.  The Role of Plasmacytoid and Myeloid Dendritic Cells in Induction of Asthma in a Mouse Model and the Effect of a TLR9 Agonist on Dendritic Cells 
Purpose
To determine the role of plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) in priming effector T cells to induce allergy, and to evaluate the effect of immunostimulatory sequences (ISS, TLR9 agonist) on dendritic cells.
Methods
Cultured mDC and pDC with/without ISS were injected intratracheally into sensitized Balb/C mice. Mice were sacrificed, and then pulmonary function tests, bronchoalveolar lavage (BAL), cell counts, and cytokine levels were evaluated. Migration of dendritic cells was also evaluated after ISS administration.
Results
In mice injected with mDC, airway hyperresponsiveness, eosinophil counts, and Th2 cytokine levels in BAL increased with increasing numbers of mDC injected. However, in mice injected with pDC, none of these changed, suggesting poor priming of T cells by pDC. In addition, mDC pulsed with ISS inhibited asthmatic reactions, and ISS administration inhibited migration of DC to the lung.
Conclusions
We suggest that pDC played a limited role in priming T cells in this asthma model and that mDC played a major role in inducing asthma. In addition, ISS inhibited migration of DC to the lung.
doi:10.4168/aair.2011.3.3.199
PMCID: PMC3121062  PMID: 21738886
Plasmacytoid dendritic cell; myeloid dendritic cell; allergy; immunostimulatory sequence; TLR9; migration
5.  Expression of High-Affinity IgE Receptor on Human Peripheral Blood Dendritic Cells in Children 
PLoS ONE  2012;7(2):e32556.
Background
In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.
Methods
Following informed consent of subjects (n = 27, aged 12–188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19− ILT7− CD1c+; pDC: CD19− ILT7+ CD1c−). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.
Results
Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.
Conclusions
In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC.
doi:10.1371/journal.pone.0032556
PMCID: PMC3285694  PMID: 22384272
6.  Plasmacytoid Dendritic Cells Prevent Cigarette Smoke and Chlamydophila pneumoniae–Induced Th2 Inflammatory Responses 
Smoking promotes the development of allergic asthma and pneumonia. Chlamydophila pneumoniae lung infection is associated with an increased risk for asthma, inducing an immune response regulated by dendritic cells (DCs). This study sought to determine whether exposure to cigarette smoke modulates the functional activity of CD11c-positive DCs in the lung, with and without concomitant C. pneumoniae infection. Bone marrow–derived DCs (BMDCs) were exposed in vitro to cigarette smoke extract (CSE) and/or live C. pneumoniae (Cpn), and then adoptively transferred intratracheally into wild-type mice. Although CSE plus Cpn appeared to exert an additive effect on the production of Th2 cytokines in vitro, we did not see this effect in vivo. However, the adoptive transfer of DCs pulsed with both CSE and C. pneumoniae into the lungs of naive mice led to an influx of plasmacytoid DCs (pDCs) that suppressed the Th2 skewing ability of the transferred BMDCs. The depletion of pDCs by antibody restored the Th2 skewing ability of the BMDCs. The expression of indoleamine-2,3-dioxygenase in the lung was reduced after the depletion of pDCs, and blocking IFN-α in vitro prevented the ability of pDCs to inhibit the Th2 responses induced by myeloid DCs (mDCs), suggesting their potential involvement in the mechanism of altered polarization. In conclusion, exposure to cigarette smoke skews C. pneumoniae–induced mDCs responses toward a Th2 bias in the lung, which is prevented by pDCs. We propose that pDCs may play a major role in the immunosuppressive lung environment in smokers with C. pneumoniae infection.
doi:10.1165/rcmb.2009-0224OC
PMCID: PMC2951872  PMID: 19901344
dendritic cells; Chlamydia pneumoniae; bacterial pneumonia; cigarette smoke exposure
7.  Allergen Uptake, Activation, and IL-23 Production by Pulmonary Myeloid DCs Drives Airway Hyperresponsiveness in Asthma-Susceptible Mice 
PLoS ONE  2008;3(12):e3879.
Maladaptive, Th2-polarized inflammatory responses are integral to the pathogenesis of allergic asthma. As regulators of T cell activation, dendritic cells (DCs) are important mediators of allergic asthma, yet the precise signals which render endogenous DCs “pro-asthmatic”, and the extent to which these signals are regulated by the pulmonary environment and host genetics, remains unclear. Comparative phenotypic and functional analysis of pulmonary DC populations in mice susceptible (A/J), or resistant (C3H) to experimental asthma, revealed that susceptibility to airway hyperresponsiveness is associated with preferential myeloid DC (mDC) allergen uptake, and production of Th17-skewing cytokines (IL-6, IL-23), whereas resistance is associated with increased allergen uptake by plasmacytoid DCs. Surprisingly, adoptive transfer of syngeneic HDM-pulsed bone marrow derived mDCs (BMDCs) to the lungs of C3H mice markedly enhanced lung IL-17A production, and rendered them susceptible to allergen-driven airway hyperresponsiveness. Characterization of these BMDCs revealed levels of antigen uptake, and Th17 promoting cytokine production similar to that observed in pulmonary mDCs from susceptible A/J mice. Collectively these data demonstrate that the lung environment present in asthma-resistant mice promotes robust pDC allergen uptake, activation, and limits Th17-skewing cytokine production responsible for driving pathologic T cell responses central to the development of allergen-induced airway hyperresponsiveness.
doi:10.1371/journal.pone.0003879
PMCID: PMC2586658  PMID: 19060952
8.  Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells In Vitro and In Vivo 
Background
Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro.
Methods & Findings
Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production
Conclusions
This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.
Author Summary
Dengue is an important endemic tropical disease to which there are no specific therapeutics or approved vaccines. Currently several aspects of pathophysiology remain incompletely understood. A crucial cellular population for viral infections, the plasmacytoid dendritic cells (pDCs) was analyzed in this study. The authors found an in vivo association between the activation state of pDCs and the disease outcome. Membrane TNF-related apoptosis inducing ligand (TRAIL) expressing pDCs, representing activated pDCs, were found in higher frequency in milder cases of dengue than severe cases or healthy individuals. Detection of antiviral cytokine interferon-alpha (IFN-α) and soluble TRAIL positively correlated with pDC activation. Dengue virus (DENV) serotype-2 was able to directly activate pDCs in vitro. Under DENV stimulation TRAIL was relocalized from intracellular to pDC plasma membrane and IFN-α was highly produced. The authors suggest an endocytosis-dependent pathway for DENV-induced pDC activation. It is also highlighted here a role for exogenous IFN-α and pDCs in reducing viral replication in monocytes, one of DENV main target cells. These findings may contribute in the future to the establishment of good prognostic immune responses together with clinical manifestations/warning signs.
doi:10.1371/journal.pntd.0002257
PMCID: PMC3675005  PMID: 23755314
9.  Differential Activation of Human Monocyte-Derived and Plasmacytoid Dendritic Cells by West Nile Virus Generated in Different Host Cells▿  
Journal of Virology  2007;81(24):13640-13648.
Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-α) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNVVero) was a potent inducer of IFN-α secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNVVero to induce IFN-α in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-α production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-α when stimulated with WNV grown in mosquito cells (WNVC7/10), while mDCs responded similarly to WNVVero or WNVC7/10. Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNVVero but not WNVC7/10, while interleukin-8 was produced in greater amounts by mDCs infected with WNVC7/10 than in those infected with WNVVero. These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections.
doi:10.1128/JVI.00857-07
PMCID: PMC2168853  PMID: 17913823
10.  Dendritic cell subsets in human bronchoalveolar lavage fluid after segmental allergen challenge 
Thorax  2006;62(2):168-175.
Background
Dendritic cells control pulmonary immune reactions. Characteristics of dendritic cells in human bronchoalveolar lavage fluid (BALF) after allergen challenge are unknown.
Methods
7 patients with allergic asthma (median 23 years, range 19–25 years) underwent segmental challenge and were lavaged 10 min and 24 h after challenge. Dendritic cell subsets and surface markers in BALF and in peripheral blood were analysed using four‐colour flow cytometry.
Results
Plasmacytoid dendritic cells (pDCs, median 0.06%, range 0.01–0.08%) and myeloid dendritic cells (mDCs, median 0.47%, range 0.27–0.87%) were detectable in BALF from control segments. CD1a‐positive dendritic cells in BALF were identified as a subpopulation of mDCs. Both pDCs (median 0.56%, range 0.09–1.83%) and mDCs (median 1.82%, range 0.95–2.29%) increased significantly in BALF 24 h (p = 0.018 compared with the control segments for pDCs and mDCs), but not 10 min, after allergen challenge. The percentage increase in pDCs was higher than that of mDCs after allergen challenge, as reflected by an enhanced pDC:mDC ratio after allergen challenge. In peripheral blood, there was a significant decrease in mDCs (p = 0.038) and a trend to a decrease in pDCs (p = 0.068) 24 h after allergen challenge. Analysis of dendritic cell surface molecules showed that after allergen challenge, BALF dendritic cells have a less mature phenotype compared with BALF dendritic cells from control segments.
Conclusion
Using a comprehensive strategy to analyse dendritic cell subsets in human BALF, we have shown for the first time that both myeloid and plasmacytoid dendritic cells accumulate in the airway lumen after allergen challenge in patients with asthma.
doi:10.1136/thx.2006.067793
PMCID: PMC2111237  PMID: 16928719
11.  Innate Immune Responses to Rhinovirus are Reduced by the High-Affinity IgE Receptor in Allergic Asthmatic Children 
Background
Children with allergic asthma have more frequent and severe human rhinovirus (HRV)-induced wheezing and asthma exacerbations through unclear mechanisms.
Objective
To determine whether increased high-affinity IgE receptor (FcεRI) expression and cross-linking impairs innate immune responses to HRV, particularly in allergic asthmatic children.
Methods
Peripheral blood mononuclear cells (PBMC) were obtained from 44 children and surface expression of FcεRI on plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), monocytes, and basophils was assessed using flow cytometry. Cells were also incubated with rabbit anti-human IgE to cross-link FcεRI, followed by stimulation with HRV-16, and interferon (IFN)-α and -λ1 were measured by Luminex. The relationships among FcεRI expression and cross-linking, HRV-induced IFN-α and -λ1 production, and childhood allergy and asthma were subsequently analyzed.
Results
FcεRIα expression on pDCs was inversely associated with HRV-induced IFN- α and IFN-λ1 production. Cross-linking FcεRI prior to HRV stimulation further reduced PBMC IFN-α (47% relative reduction, 95% confidence interval [CI], 32–62%, p<0.0001) and IFN-λ1 (81% relative reduction, 95% CI, 69–93%, p<0.0001) secretion. Allergic asthmatic children had higher surface expression of FcεRIα on pDCs and mDCs when compared to non-allergic non-asthmatic children. Further, after FcεRI cross-linking, allergic asthmatic children had significantly lower HRV-induced IFN responses than allergic non-asthmatics (IFN-α, p=0.004; IFN-λ1, p=0.02) and non-allergic non-asthmatics (IFN-α, p=0.002; IFN-λ1, p=0.01).
Conclusions
Allergic asthmatic children have impaired innate immune responses to HRV that correlate with increased FcεRI expression on pDCs and are reduced by FcεRI cross-linking. These effects likely increase susceptibility to HRV-induced wheezing and asthma exacerbations.
doi:10.1016/j.jaci.2012.05.023
PMCID: PMC3437329  PMID: 22766097
asthma; allergic; rhinovirus; interferon; FcεRI; IgE receptor; plasmacytoid dendritic cells
12.  Plasmacytoid Dendritic Cells Mediate the Regulation of Inflammatory Type T Cell Response for Optimal Immunity against Respiratory Chlamydia Pneumoniae Infection 
PLoS ONE  2013;8(12):e83463.
Chlamydia pneumoniae (Cpn) infection is a leading cause for a variety of respiratory diseases and has been implicated in the pathogenesis of chronic inflammatory diseases. The regulatory mechanisms in host defense against Cpn infection are less understood. In this study, we investigated the role of plasmacytoid dendritic cells (pDCs) in immune regulation in Cpn respiratory tract infection. We found that in vivo depletion of pDCs increased the severity of infection and lung pathology. Mice depleted of pDC had greater body weight loss, higher lung bacterial burden and excessive tissue inflammation compared to the control mice. Analysis of specific T cell cytokine production pattern in the lung following Cpn infection revealed that pDC depleted mice produced significantly higher amounts of inflammatory cytokines, especially TNF-α, but lower IL-10 compared to the controls. In particular, pDC depleted mice showed pathogenic T cell responses characterized by inflammatory type-1 (CD8 and CD4) and inflammatory Th2 cell responses. Moreover, pDC depletion dramatically reduced CD4 regulatory T cells (Tregs) in the lungs and draining lymph nodes. Furthermore, pDC-T cell co-culture experiments showed that pDCs isolated from Cpn infected mice were potent in inducing IL-10 producing CD4 Tregs. Together, these findings provide in vivo evidence for a critical role of pDCs in homeostatic regulation of immunity during Cpn infection. Our findings highlight the importance of a ‘balanced’ immune response for host protective immunity and preventing detrimental immunopathology during microbial infections.
doi:10.1371/journal.pone.0083463
PMCID: PMC3873288  PMID: 24386207
13.  Spherical Lactic Acid Bacteria Activate Plasmacytoid Dendritic Cells Immunomodulatory Function via TLR9-Dependent Crosstalk with Myeloid Dendritic Cells 
PLoS ONE  2012;7(4):e32588.
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.
doi:10.1371/journal.pone.0032588
PMCID: PMC3323594  PMID: 22505996
14.  Abnormalities in circulating plasmacytoid dendritic cells in patients with systemic lupus erythematosus 
Arthritis Research & Therapy  2010;12(4):R137.
Introduction
Dendritic cells (DCs) are capable of inducing immunity or tolerance. Previous studies have suggested plasmacytoid DCs (pDCs) are pathogenic in systemic lupus erythematosus (SLE). However, the functional characteristics of directly isolated peripheral circulating blood pDCs in SLE have not been evaluated previously.
Methods
Peripheral blood pDCs from 62 healthy subjects and 58 SLE patients were treated with apoptotic cells derived from polymorphonuclear cells (PMNs). Antigen loaded or unloaded pDCs were then co-cultured with autologous or allogenous T cells. Changes in T cell proliferation, cell surface CD25 expression, intracellular Foxp3 expression and cytokine production were evaluated. pDCs that had captured apoptotic PMNs (pDCs + apoPMNs were also studied for their cytokine production (interferon (IFN)-alpha, interleukin (IL)-6, IL-10, IL-18) and toll like receptor (TLR) expression.
Results
Circulating pDCs from SLE patients had an increased ability to stimulate T cells when compared with control pDCs. Using allogenous T cells as responder cells, SLE pDCs induced T cell proliferation even in the absence of apoptotic PMNs. In addition, healthy pDCs + apoPMNs induced suppressive T regulatory cell features with increased Foxp3 expression in CD4 + CD25 + cells while SLE pDCs + apoPMNs did not. There were differences in the cytokine profile of pDCs that had captured apoptotic PMNs between healthy subjects and patients with SLE. Healthy pDCs + apoPMNs showed decreased production of IL-6 but no significant changes in IL-10 and IL-18. These pDCs + apoPMNs also showed increased mRNA transcription of TLR9. On the other hand, while SLE pDCs + apoPMNs also had decreased IL-6, there was decreased IL-18 mRNA expression and persistent IL-10 protein synthesis. In addition, SLE pDCs lacked TLR9 recruitment.
Conclusions
We have demonstrated that peripheral circulating pDCs in patients with SLE were functionally abnormal. They lacked TLR9 expression, were less capable of inducing regulatory T cell differentiation and had persistent IL-10 mRNA expression following the capture of apoptotic PMNs. We suggest circulating pDCs may be pathogenically relevant in SLE.
doi:10.1186/ar3075
PMCID: PMC2945027  PMID: 20618924
15.  Tolerogenic and Activatory Plasmacytoid Dendritic Cells in Autoimmunity 
Plasmacytoid dendritic cells (pDCs) are a particular subset of DCs that link innate and adaptive immunity. They are responsible for the substantial production of type 1 interferon (IFN-I) in response to viral RNA or DNA through activation of TLR7 and 9. Furthermore, pDCs present antigens (Ag) and induce naïve T cell differentiation. It has been demonstrated that pDCs can induce immunogenic T cell responses through differentiation of cytotoxic CD8+ T cells and effector CD4+ T cells. Conversely, pDCs exhibit strong tolerogenic functions by inducing CD8+ T cell deletion, CD4+ T cell anergy, and Treg differentiation. However, since IFN-I produced by pDCs efficiently activates and recruits conventional DCs, B cells, T cells, and NK cells, pDCs also indirectly affect the nature and the amplitude of adaptive immune responses. As a consequence, the precise role of Ag-presenting functions of pDCs in adaptive immunity has been difficult to dissect in vivo. Additionally, different experimental procedures led to conflicting results regarding the outcome of T cell responses induced by pDCs. During the development of autoimmunity, pDCs have been shown to play both immunogenic and tolerogenic functions depending on disease, disease progression, and the experimental conditions. In this review, we will discuss the relative contribution of innate and adaptive pDC functions in modulating T cell responses, particularly during the development of autoimmunity.
doi:10.3389/fimmu.2013.00059
PMCID: PMC3589693  PMID: 23508732
plasmacytoid dendritic cells; type-I IFNs; antigen-presentation; tolerance; autoimmunity
16.  Elevated myeloid: plasmacytoid dendritic cell ratio associates with late, but not early liver rejection in children induced with, anti-human thymocyte globulin1 
Transplantation  2009;88(4):589-594.
Background
Dendritic cells (DC) play an important role in the induction and regulation of immune responses.
Methods
Myeloid CD11c+DC (MDC), which may have inflammatory functions, and plasmacytoid CD123+ DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, cross-sectionally, once, in 48 children, and longitudinally (pre-transplant, and at days 1–60, 61–200, 201–400 post transplant) in 30 children following liver transplantation (LTx). All children received 53/25 cadaveric/live donor liver allografts with rabbit anti-human thymocyte globulin (rATG) induction, and steroid-free Tacrolimus therapy. Rejectors in both groups were those children (n=35), who experienced biopsy-proven acute cellular rejection (ACR) within 60 days of DC monitoring.
Results
Among rejectors in the longitudinal and cross-sectional cohorts, the MDC: PDC ratio was higher, and was associated with decreased PDC frequencies. Logistic regression analysis, leave-one out cross-validation, and receiver operating characteristic analysis applied to 30 cross-sectional subjects revealed that an MDC:PDC ratio 1.78 was associated with rejector status with sensitivity/specificity of 76.9/88.2%. Sensitivity and specificity were replicated in the 18 remaining cross-sectional subjects (88.8 and 78.8%, respectively), but not in longitudinally-monitored subjects, during the early, 60-day period after LTx (30.76 and 62.50%, respectively). A significant negative correlation was observed between Tacrolimus whole blood concentrations and PDC frequencies (Spearman r = −0.370, p=0.005) in 48 cross-sectional subjects in whom DC subsets were monitored 1–3 years after LTx, but not during the early post-LTx period.
Conclusion
We conclude that an elevated MDC: PDC ratio associates with liver graft rejection, which occurs after first year in children induced with rATG.
doi:10.1097/TP.0b013e3181b11f12
PMCID: PMC2731691  PMID: 19696644
dendritic cell subsets; liver transplantation; pediatric; anti-thymocyte globulin
17.  Comparative analysis of circulating dendritic cell subsets in patients with atopic diseases and sarcoidosis 
Respiratory Research  2013;14(1):29.
Background
Dendritic cells (DCs) are professional antigen-presenting cells that play a crucial role in the initiation and modulation of immune responses. Human circulating blood DCs are divided into two major subsets: myeloid DCs (mDCs); and plasmacytoid DCs (pDCs). Furthermore, mDCs are subdivided into two subsets: Th1-promoting mDCs (mDC1s); and Th2-promoting mDCs (mDC2s). Although CD1a, CD1c, and CD141 are generally used for classifying mDC subsets, their adequacy as a specific marker remains unclear. We performed this study to compare circulating mDC, pDC, mDC1, and mDC2 subsets between Th1- and Th2-mediated diseases using CD1a and CD141, and to analyze the adequacy of CD1a and CD141 as a marker for mDC1s and mDC2s, respectively.
Methods
Thirty patients with sarcoidosis, 23 patients with atopic diseases, such as atopic bronchial asthma, and 23 healthy subjects as controls were enrolled in this study. Peripheral blood DC subsets were analyzed with flow cytometry according to expressions of CD11c, CD123, CD1a, and CD141. For functional analysis, we measured interleukin (IL) 12p40 levels produced by the sorted mDC subsets.
Results
The sarcoidosis group showed decreased total DC (P < 0.05) and mDC counts (P < 0.05) compared to controls. The atopy group showed decreased CD1a+mDC count (P < 0.05), and increased CD1a-mDC count (P < 0.05) compared to controls. CD141+mDC count in the atopy group was higher than controls (P < 0.05). Sorted CD1a+mDCs produced higher levels of IL-12p40 than CD1a-mDCs (P = 0.025) and CD141+mDCs (P = 0.018).
Conclusions
We conclude that decreased count of CD1a+mDC and increased count of CD141+mDC may reflect the Th2-skewed immunity in atopic diseases. The results of IL-12 levels produced by the sorted mDC subsets suggested the adequacy of CD1a and CD141 as a marker for mDC1 and mDC2, respectively, in vivo.
doi:10.1186/1465-9921-14-29
PMCID: PMC3599330  PMID: 23497225
Dendritic cells; Peripheral blood; Sarcoidosis; Myeloid DC (mDC); CD1a; CD141
18.  Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells 
PLoS ONE  2011;6(6):e20784.
Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.
doi:10.1371/journal.pone.0020784
PMCID: PMC3112152  PMID: 21695198
19.  Major Depletion of Plasmacytoid Dendritic Cells in HIV-2 Infection, an Attenuated Form of HIV Disease 
PLoS Pathogens  2009;5(11):e1000667.
Plasmacytoid dendritic cells (pDC) provide an important link between innate and acquired immunity, mediating their action mainly through IFN-α production. pDC suppress HIV-1 replication, but there is increasing evidence suggesting they may also contribute to the increased levels of cell apoptosis and pan-immune activation associated with disease progression. Although having the same clinical spectrum, HIV-2 infection is characterized by a strikingly lower viremia and a much slower rate of CD4 decline and AIDS progression than HIV-1, irrespective of disease stage. We report here a similar marked reduction in circulating pDC levels in untreated HIV-1 and HIV-2 infections in association with CD4 depletion and T cell activation, in spite of the undetectable viremia found in the majority of HIV-2 patients. Moreover, the same overexpression of CD86 and PD-L1 on circulating pDC was found in both infections irrespective of disease stage or viremia status. Our observation that pDC depletion occurs in HIV-2 infected patients with undetectable viremia indicates that mechanisms other than direct viral infection determine the pDC depletion during persistent infections. However, viremia was associated with an impairment of IFN-α production on a per pDC basis upon TLR9 stimulation. These data support the possibility that diminished function in vitro may relate to prior activation by HIV virions in vivo, in agreement with our finding of higher expression levels of the IFN-α inducible gene, MxA, in HIV-1 than in HIV-2 individuals. Importantly, serum IFN-α levels were not elevated in HIV-2 infected individuals. In conclusion, our data in this unique natural model of “attenuated” HIV immunodeficiency contribute to the understanding of pDC biology in HIV/AIDS pathogenesis, showing that in the absence of detectable viremia a major depletion of circulating pDC in association with a relatively preserved IFN-α production does occur.
Author Summary
Infection by HIV-2, the second AIDS-associated virus, is considered a unique natural model of attenuated HIV disease. HIV-2 infected individuals exhibit much lower levels of circulating virus (viremia) and progress to AIDS at slower rates than HIV-1 infected patients. In this study, we characterized for the first time blood plasmacytoid dendritic cells (pDC), important mediators between innate and acquired immunity, in HIV-2 infection. We observed a profound reduction in circulating pDC levels in HIV-2 infected patients, even in those with undetectable viremia, to levels similar to those found in HIV-1 infection. Moreover, we documented a more differentiated pDC phenotype in both infected cohorts relative to healthy individuals. Despite these similarities between HIV-1 and HIV-2 infections, pDC from HIV-2 patients with undetectable viremia exhibited, upon in vitro stimulation, a better-preserved ability to produce interferon-α (IFN-α), an important anti-viral cytokine with potential to stimulate other immune cells. Overall, our data suggest that the presence of virus in circulation, although not critical for the reduction in pDC number, appears to be central for the impairment of their function. This study of pDC in HIV-2 infection fills a gap in the understanding of their potential role in HIV/AIDS pathogenesis.
doi:10.1371/journal.ppat.1000667
PMCID: PMC2773933  PMID: 19936055
20.  Plasmacytoid dendritic cells prime IL-10–producing T regulatory cells by inducible costimulator ligand 
Although there is evidence for distinct roles of myeloid dendritic cells (DCs [mDCs]) and plasmacytoid pre-DCs (pDCs) in regulating T cell–mediated adaptive immunity, the concept of functional DC subsets has been questioned because of the lack of a molecular mechanism to explain these differences. In this study, we provide direct evidence that maturing mDCs and pDCs express different sets of molecules for T cell priming. Although both maturing mDCs and pDCs upregulate the expression of CD80 and CD86, only pDCs upregulate the expression of inducible costimulator ligand (ICOS-L) and maintain high expression levels upon differentiation into mature DCs. High ICOS-L expression endows maturing pDCs with the ability to induce the differentiation of naive CD4 T cells to produce interleukin-10 (IL-10) but not the T helper (Th)2 cytokines IL-4, -5, and -13. These IL-10–producing T cells are T regulatory cells, and their generation by ICOS-L is independent of pDC-driven Th1 and Th2 differentiation, although, in the later condition, some contribution from endogenous IL-4 cannot be completely ruled out. Thus, in contrast to mDCs, pDCs are poised to express ICOS-L upon maturation, which leads to the generation of IL-10–producing T regulatory cells. Our findings demonstrate that mDC and pDCs are intrinsically different in the expression of costimulatory molecules that drive distinct types of T cell responses.
doi:10.1084/jem.20061660
PMCID: PMC2118437  PMID: 17200410
21.  Modulation of respiratory dendritic cells during Klebsiella pneumonia infection 
Respiratory Research  2013;14(1):91.
Background
Klebsiella pneumoniae is a leading cause of severe hospital-acquired respiratory tract infections and death but little is known regarding the modulation of respiratory dendritic cell (DC) subsets. Plasmacytoid DC (pDC) are specialized type 1 interferon producing cells and considered to be classical mediators of antiviral immunity.
Method
By using multiparameter flow cytometry analysis we have analysed the modulation of respiratory DC subsets after intratracheal Klebsiella pneumonia infection.
Results
Data indicate that pDCs and MoDC were markedly elevated in the post acute pneumonia phase when compared to mock-infected controls. Analysis of draining mediastinal lymph nodes revealed a rapid increase of activated CD103+ DC, CD11b+ DC and MoDC within 48 h post infection. Lung pDC identification during bacterial pneumonia was confirmed by extended phenotyping for 120G8, mPDCA-1 and Siglec-H expression and by demonstration of high Interferon-alpha producing capacity after cell sorting. Cytokine expression analysis of ex vivo-sorted respiratory DC subpopulations from infected animals revealed elevated Interferon-alpha in pDC, elevated IFN-gamma, IL-4 and IL-13 in CD103+ DC and IL-19 and IL-12p35 in CD11b+ DC subsets in comparison to CD11c+ MHC-class IIlow cells indicating distinct functional roles. Antigen-specific naive CD4+ T cell stimulatory capacity of purified respiratory DC subsets was analysed in a model system with purified ovalbumin T cell receptor transgenic naive CD4+ responder T cells and respiratory DC subsets, pulsed with ovalbumin and matured with Klebsiella pneumoniae lysate. CD103+ DC and CD11b+ DC subsets represented the most potent naive CD4+ T helper cell activators.
Conclusion
These results provide novel insight into the activation of respiratory DC subsets during Klebsiella pneumonia infection. The detection of increased respiratory pDC numbers in bacterial pneumonia may indicate possible novel pDC functions with respect to lung repair and regeneration.
doi:10.1186/1465-9921-14-91
PMCID: PMC3848864  PMID: 24044871
Klebsiella pneumonia; Pneumonia; Plasmacytoid dendritic cells
22.  Complement Component 3C3 and C3a Receptor Are Required in Chitin-Dependent Allergic Sensitization to Aspergillus fumigatus but Dispensable in Chitin-Induced Innate Allergic Inflammation 
mBio  2013;4(2):e00162-13.
ABSTRACT
Levels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3−/− and C3aR−/− mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells.
IMPORTANCE
Asthma is one of the fastest growing chronic illnesses worldwide. Chitin, a ubiquitous polymer in our environment and a key component in the cell wall of fungal spores and the exoskeletons of insects, parasites, and crustaceans, triggers innate allergic inflammation. However, there is little understanding of how chitin is initially recognized by mammals and how early recognition of chitin affects sensitization to environmental allergens and development of allergic asthma. The complement system is evolutionarily one of the oldest facets of the early or innate warning systems in mammals. We studied whether and how complement components influence the recognition of chitin and shape the downstream sensitization toward fungal allergens. We show here that complement recognition of chitin plays a critical role in shaping the behavior of dendritic cells, which in turn regulate the function of T cells that mediate allergic responses to fungi.
doi:10.1128/mBio.00162-13
PMCID: PMC3622928  PMID: 23549917
23.  Role of plasmacytoid dendritic cell subsets in allergic asthma 
Allergy  2013;68(6):695-701.
Plasmacytoid dendritic cells (pDCs) are major type-I interferon producing cells that play important roles in antiviral immunity and tolerance induction. These cells share a common DC progenitor with conventional DCs and Fms-like tyrosine kinase-3 ligand is essential for their development. Several subsets of pDCs have been identified to date including CCR9+, CD9+ and CD2+ pDCs. Recently, three subsets of pDCs were described namely, CD8α−β−, CD8α+β− and CD8α+β+ subsets. Interestingly, CD8α+β− and CD8α+β+ but not CD8α−β− pDCs were shown to have tolerogenic effects in experimentally induced allergic asthma. These tolerogenic effects were shown to be mediated by the generation of FOXP3+ regulatory T cells through retinoic acid and the induction of retinaldehyde dehydrogenase enzymes. These newly described subsets of pDCs show high potentials for novel therapeutic approaches for the treatment of allergic diseases. In this review, we will address the new progress in our understanding of pDC biology with respect to allergic disease in particular allergic asthma.
doi:10.1111/all.12166
PMCID: PMC3693732  PMID: 23662841
plasmacytoid dendritic cells; allergy; tolerance; pDCs; Allergic asthma
24.  Plasmacytoid Dendritic Cells Mediate Oral Tolerance 
Immunity  2008;29(3):464-475.
SUMMARY
Oral tolerance prevents oral sensitization to dietary antigens (Ags), including proteins and haptens, and development of delayed-type hypersensitivity (DTH) responses. We showed here that plasmacytoid dendritic cells (pDCs) prevented oral T cell priming and were responsible for systemic tolerance to CD4+ and CD8+ T cell-mediated DTH responses induced by Ag feeding. Systemic depletion of pDCs prevented induction of tolerance by antigen feeding. Transfer of oral Ag-loaded liver pDCs to naive recipient mice induced Ag-specific suppression of CD4+ and CD8+ T cell responses to protein and hapten, respectively. Liver is a site of oral Ag presentation, and pDCs appeared to induce anergy or deletion of Ag-specific T cells in the liver relatively rapidly via a CD4+ T cell-independent mechanism. These data demonstrate that oral tolerance relies on Ag presentation by pDC to T cells and suggest that pDC could represent a key therapeutic target for intestinal and systemic inflammatory diseases.
doi:10.1016/j.immuni.2008.06.017
PMCID: PMC3545652  PMID: 18789731
25.  Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice 
The Journal of Clinical Investigation  2009;119(12):3723-3738.
The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC–driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC–driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10–dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.
doi:10.1172/JCI39717
PMCID: PMC2786798  PMID: 19907079

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