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1.  ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia 
PLoS Pathogens  2011;7(11):e1002358.
Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23−/− mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23−/− mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.
Author Summary
Infections of the lower respiratory tract by single-stranded RNA viruses represent a major health problem worldwide. Animal models indicate that the severity of infections caused by these viruses is due essentially to an excessive primary immune response of the host, rather than the direct cytopathogenicity of the viruses. Plasmacytoid dendritic cells have been reported to play an important role in anti-viral immunity, but the factors responsible for the recruitment of these cells to the infected lung were unknown. This study depicts the roles of the G protein-coupled receptor ChemR23 in the recruitment of plasmacytoid dendritic cells and anti-viral immunity in a mouse model of acute viral pneumonia. The data also highlight the role of ChemR23 in dampening the lung inflammatory response. This latter effect is independent of pDC recruitment but involves non-leukocytic cells. This observation is of particular interest considering the established role of airway endothelial and epithelial cells in the immune responses following bacterial, viral and fungal infections. Our results suggest therefore that the chemerin/ChemR23 system might be considered as a target for anti-viral and anti-inflammatory therapies.
doi:10.1371/journal.ppat.1002358
PMCID: PMC3207933  PMID: 22072972
2.  Flt3L-dependence helps define an uncharacterized subset of murine cutaneous dendritic cells 
Skin-derived dendritic cells (DC) are potent antigen presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DC carry antigens and constitutively migrate to the skin draining lymph nodes (LN). In mice, Langerin-CD11b− dermal DC are a low-frequency, heterogeneous, migratory DC subset that traffic to LN (Langerin-CD11b-migDC). Here, we build on the observation that Langerin-CD11b− migDC are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice which accumulate migDC, demonstrate these cells are cutaneous residents. Langerin-CD11b-Flt3L responsive DC are largely CD24(+) and CX3CR1low and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11bmigDC present antigen with equal efficiency to other DC subsets ex vivo including classical CD8α cDC and Langerin+CD103+ dermal DC. Finally, transcriptome analysis suggests a close relationship to other skin DC, and a lineage relationship to other classical DC. This work demonstrates that Langerin- CD11b− dermal DC, a previously overlooked cell subset, may be an important player in the cutaneous immune environment.
doi:10.1038/jid.2013.515
PMCID: PMC3994898  PMID: 24288007
3.  Unique Type I Interferon Responses Determine the Functional Fate of Migratory Lung Dendritic Cells during Influenza Virus Infection 
PLoS Pathogens  2011;7(11):e1002345.
Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103+ DCs and CD11bhigh DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103+ DCs allow the virus to replicate to significantly higher levels than do the CD11bhigh DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11bhigh DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103+ DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11bhigh DCs. The attenuated IFNAR signaling by CD103+ DCs correlates with their described superior antigen presentation capacity for naïve CD8+ T cells when compared to CD11bhigh DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The “interferon-resistant” CD103+ DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination.
Author Summary
Migratory lung dendritic cells (DCs) control the initiation of the adaptive immune responses to influenza virus by expanding virus-specific T cells in draining lymph nodes (MLNs) that will subsequently clear the pathogen from the respiratory tract. Here we demonstrate that both subsets of lung DCs, CD103+ DCs and CD11bhigh DCs become infected by influenza virus in vivo and migrate to the MLNs, but only CD103+ DCs support productive virus replication. Enhanced virus replication in CD103+ DCs compared to CD11bhigh DCs was responsible for their superior antigen presentation efficacy for naïve CD8+ T cells and originated from a difference in sensitivity of the two DC populations to type I interferon (I-IFN). These data show that in contrast to most other immune cell types, DCs can become productively infected with influenza virus and I-IFN operates as a master regulator controlling which DC subset will present antigen during a viral infection. A deeper understanding of basic innate and adaptive immune response mechanisms regulated by I-FN may lead to the development of cutting edge therapies and improve vaccine efficacy against influenza and other viruses.
doi:10.1371/journal.ppat.1002345
PMCID: PMC3207893  PMID: 22072965
4.  Plasmacytoid dendritic cell depletion leads to an enhanced mononuclear phagocyte response in lungs of mice with lethal influenza virus infection 
Plasmacytoid dendritic cells (pDCs) have been implicated both in the control and pathogenesis of influenza virus infection. We demonstrate that pDC depletion has marked effects on the response of mononuclear phagocytes, including conventional DCs (cDCs) and macrophages, to lethal influenza virus infection. Infection of mice lacking pDCs through antibody-mediated depletion resulted in substantially increased accumulation of mononuclear phagocytes and their progenitors in lungs compared to non-treated controls. pDC ablation resulted in a 5- to 35-fold enhancement of intracellular TNF-α and IL-6 production from inflammatory cDCs and exudate macrophages. Purified pulmonary cDCs and macrophages cultured from pDC-depleted mice produced significantly elevated levels of pro-inflammatory cytokines and chemokines compared to pDC-intact counterparts. Elimination of pDCs resulted in decreased lung IFN-α and an immediate and transient reduction in lung virus burden but did not impact disease outcome. These data reveal a suppressive effect of pDCs on the inflammatory response to influenza virus infection in the lung.
doi:10.1016/j.cimid.2012.01.012
PMCID: PMC3368063  PMID: 22421538
innate immunity; cellular immunity; mucosal immunity; dendritic cell; macrophage
5.  Plasmacytoid Dendritic Cells Play a Role for Effective Innate Immune Responses during Chlamydia pneumoniae Infection in Mice 
PLoS ONE  2012;7(10):e48655.
Plasmacytoid dendritic cells (pDCs) are known for their robust antiviral response and their pro-tolerance effects towards allergic diseases and tissue engraftments. However, little is known about the role pDCs may play during a bacterial infection, including pulmonary Chlamydia pneumoniae (CP). In this study, we investigated the role of pDCs during pulmonary CP infection. Our results revealed that depletion of pDCs during acute CP infection in mice results in delayed and reduced lung inflammation, with an early delay in cellular recruitment and significant reduction in early cytokine production in the lungs. This was followed by impaired and delayed bacterial clearance from the lungs which then resulted in a severe and prolonged chronic inflammation and iBALT like structures containing large numbers of B and T cells in these animals. We also observed that increasing the pDC numbers in the lung by FLT3L treatment experimentally results in greater lung inflammation during acute CP infection. In contrast to these results, restimulation of T-cells in the draining lymph nodes of pDC-depleted mice induced greater amounts of proinflammatory cytokines than we observed in control mice. These results suggest that pDCs in the lung may provide critical proinflammatory innate immune responses in response to CP infection, but are suppressive towards adaptive immune responses in the lymph node. Thus pDCs in the lung and the draining lymph node appear to have different roles and phenotypes during acute CP infection and may play a role in host immune responses.
doi:10.1371/journal.pone.0048655
PMCID: PMC3485374  PMID: 23119083
6.  Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice 
PLoS Pathogens  2014;10(7):e1004291.
The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.
Author Summary
Persistent expression of IFN-I is correlated with disease progression in HIV-1 infected humans or SIV-infected monkeys. Thus, persistent pDC activation has been implicated in contributing to AIDS pathogenesis. To define the role of pDC in HIV-1 infection and immunopathogenesis in vivo, we developed a monoclonal antibody that specifically and efficiently depletes human pDC in all lymphoid organs in humanized mice. We discover that pDC are the critical IFN-I producer cells in response to acute HIV-1 infection, because depletion of pDC completely abolished induction of IFN-I or ISG by HIV-1 infection, correlated with elevated level of HIV-1 replication. When pDC were depleted during chronic HIV-1 infection in humanized mice, pDC were still the major IFN-I producing cells in vivo, which contributed to HIV-1 suppression. Despite of higher level of viral replication in pDC-depleted mice, we found that HIV-induced depletion of human T cells and leukocytes was significantly reduced in lymphoid organs, correlated with reduced cell death induction by HIV-1 infection. Our findings demonstrate that pDC play two opposing roles in HIV-1 pathogenesis: they produce IFN-I to suppress HIV-1 replication and induce death of human immune cells to contribute to HIV-induced T cell depletion and immunopathogenesis.
doi:10.1371/journal.ppat.1004291
PMCID: PMC4117636  PMID: 25077616
7.  Cellular Immune Responses to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Infection in Senescent BALB/c Mice: CD4+ T Cells Are Important in Control of SARS-CoV Infection▿  
Journal of Virology  2009;84(3):1289-1301.
We characterized the cellular immune response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection in 12- to 14-month-old BALB/c mice, a model that mimics features of the human disease. Following intranasal administration, the virus replicated in the lungs, with peak titers on day 2 postinfection. Enhanced production of cytokines (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokines (CXCL10, CCL2, CCL3, and CCL5) correlated with migration of NK cells, macrophages, and plasmacytoid dendritic cells (pDC) into the lungs. By day 7, histopathologic evidence of pneumonitis was seen in the lungs when viral clearance occurred. At this time, a second wave of enhanced production of cytokines (TNF-α, IL-6, gamma interferon [IFN-γ], IL-2, and IL-5), chemokines (CXCL9, CXCL10, CCL2, CCL3, and CCL5), and receptors (CXCR3, CCR2, and CCR5), was detected in the lungs, associated with an influx of T lymphocytes. Depletion of CD8+ T cells at the time of infection did not affect viral replication or clearance. However, depletion of CD4+ T cells resulted in an enhanced immune-mediated interstitial pneumonitis and delayed clearance of SARS-CoV from the lungs, which was associated with reduced neutralizing antibody and cytokine production and reduced pulmonary recruitment of lymphocytes. Innate defense mechanisms are able to control SARS-CoV infection in the absence of CD4+ and CD8+ T cells and antibodies. Our findings provide new insights into the pathogenesis of SARS, demonstrating the important role of CD4+ but not CD8+ T cells in primary SARS-CoV infection in this model.
doi:10.1128/JVI.01281-09
PMCID: PMC2812346  PMID: 19906920
8.  Early Pandemic Influenza (2009 H1N1) in Ho Chi Minh City, Vietnam: A Clinical Virological and Epidemiological Analysis 
PLoS Medicine  2010;7(5):e1000277.
Rogier van Doorn and colleagues analyze the initial outbreak, attempts at containment, and establishment of community transmission of pandemic H1N1 influenza in Ho Chi Minh City, Vietnam.
Background
To date, little is known about the initial spread and response to the 2009 pandemic of novel influenza A (“2009 H1N1”) in tropical countries. Here, we analyse the early progression of the epidemic from 26 May 2009 until the establishment of community transmission in the second half of July 2009 in Ho Chi Minh City (HCMC), Vietnam. In addition, we present detailed systematic viral clearance data on 292 isolated and treated patients and the first three cases of selection of resistant virus during treatment in Vietnam.
Methods and Findings
Data sources included all available health reports from the Ministry of Health and relevant health authorities as well as clinical and laboratory data from the first confirmed cases isolated at the Hospital for Tropical Diseases in HCMC. Extensive reverse transcription (RT)-PCR diagnostics on serial samples, viral culture, neuraminidase-inhibition testing, and sequencing were performed on a subset of 2009 H1N1 confirmed cases. Virological (PCR status, shedding) and epidemiological (incidence, isolation, discharge) data were combined to reconstruct the initial outbreak and the establishment of community transmission. From 27 April to 24 July 2009, approximately 760,000 passengers who entered HCMC on international flights were screened at the airport by a body temperature scan and symptom questionnaire. Approximately 0.15% of incoming passengers were intercepted, 200 of whom tested positive for 2009 H1N1 by RT-PCR. An additional 121 out of 169 nontravelers tested positive after self-reporting or contact tracing. These 321 patients spent 79% of their PCR-positive days in isolation; 60% of PCR-positive days were spent treated and in isolation. Influenza-like illness was noted in 61% of patients and no patients experienced pneumonia or severe outcomes. Viral clearance times were similar among patient groups with differing time intervals from illness onset to treatment, with estimated median clearance times between 2.6 and 2.8 d post-treatment for illness-to-treatment intervals of 1–4 d, and 2.0 d (95% confidence interval 1.5–2.5) when treatment was started on the first day of illness.
Conclusions
The patients described here represent a cross-section of infected individuals that were identified by temperature screening and symptom questionnaires at the airport, as well as mildly symptomatic to moderately ill patients who self-reported to hospitals. Data are observational and, although they are suggestive, it is not possible to be certain whether the containment efforts delayed community transmission in Vietnam. Viral clearance data assessed by RT-PCR showed a rapid therapeutic response to oseltamivir.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every year, millions of people catch influenza—a viral infection of the airways—and about half a million people die as a result. These yearly seasonal epidemics occur because small but frequent changes in the influenza virus mean that the immune response produced by infection with one year's virus provides only partial protection against the next year's virus. Sometimes, however, a very different influenza virus emerges to which people have virtually no immunity. Such viruses can start global epidemics (pandemics) and can kill millions of people. Consequently, when the first case of influenza caused by a new virus called pandemic A/H1N1 2009 (2009 H1N1, swine flu) occurred in March 2009 in Mexico, alarm bells rang. National and international public health agencies quickly issued advice about how the public could help to control the spread of the virus and, as the virus spread, some countries banned flights from affected regions and instigated screening for influenza-like illness at airports. However, despite everyone's efforts, the virus spread rapidly and on June 11, 2009 the World Health Organization (WHO) declared that an influenza pandemic was underway.
Why Was This Study Done?
To date, little is known about the spread of and response to 2009 H1N1 in tropical countries. In this study, therefore, the researchers investigate the early progression of the 2009 H1N1 pandemic in Ho Chi Minh City, Vietnam, and the treatment of infected patients. On April 27, 2009, when WHO announced that human-to-human transmission of 2009 H1N1 was occurring, the Vietnamese Ministry of Health mandated airport body temperature scans and symptom questionnaire screening of travelers arriving in Vietnam's international airports. Suspected cases were immediately transferred to in-hospital isolation, screened for virus using a sensitive test called PCR, and treated with the anti-influenza drug oseltamivir if positive. The first case of 2009 H1N1 infection in Vietnam was reported on May 31, 2009 in a student who had returned from the US on May 26, 2009, and, despite these efforts to contain the infection, by the second half of July the virus was circulating in Ho Chi Minh City (community transmission).
What Did the Researchers Do and Find?
The researchers used reports from the Ministry of Health and relevant health authorities and clinical and laboratory data for people infected with 2009 H1N1 and isolated in hospital to reconstruct the initial outbreak and the establishment of community transmission in Ho Chi Minh City. Between April 27 and July 24 2009, three-quarters of a million passengers arriving in the city on international flights were screened at the airport. 200 passenger tested positive for 2009 H1N1 as did 121 nontravelers who were identified during this period after self-reporting illness or through contact tracing. The infected individuals spent 79% of the days when they tested positive for 2009 H1N1 by PCR (days when they were infectious) in isolation; 60% of their PCR-positive days were spent in isolation and treatment. Importantly, travelers and nontravelers spent 10% and 42.2%, respectively, of their potentially infectious time in the community. None of the patients became severely ill but 61% experienced an influenza-like illness. Finally, the average time from starting treatment to clearance of the virus was between 2.6 and 2.8 days for patients who began treatment 1 to 4 days after becoming ill; for those who started treatment on the first day of illness, the average virus clearance time was 2.0 days.
What Do These Findings Mean?
These findings, although limited by missing data, suggest that the strict containment measures introduced early in the 2009 H1N1 pandemic in Ho Chi Minh City may have reduced the circulation of infected people in the community. This reduction in circulation might have delayed the onset of community transmission, suggest the researchers, but because the study was observational, this possibility cannot be proven. However, importantly, these findings show that the containment measures were unable to prevent the eventual establishment of pandemic influenza in Vietnam, presumably because many imported cases were not detected by airport screening. Finally, these findings suggest that in Vietnam, as in other countries, 2009 H1N1 causes a mild disease and that this disease responds quickly to treatment with oseltamivir whenever treatment is started in relation to the onset of illness.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000277.
The US Centers for Disease Control and Prevention provides information about influenza for patients and professionals, including specific information on H1N1 influenza and how to prevent its spread
Flu.gov, a US government website, provides information on H1N1, avian, and pandemic influenza
The World Health Organization provides information on seasonal influenza and has detailed information on H1N1 influenza (in several languages); the WHO Representative Office in Vietnam provides an overview of the current 2009 H1N1 situation in Vietnam
The UK Health Protection Agency provides information on pandemic influenza and on H1N1 influenza
Wikipedia has a timeline of the 2009 H1N1 pandemic (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1000277
PMCID: PMC2872648  PMID: 20502525
9.  Plasmacytoid Dendritic Cells Contribute to Systemic but Not Local Antiviral Responses to HSV Infections 
PLoS Pathogens  2013;9(10):e1003728.
Plasmacytoid dendritic cells (pDC) produce type I interferons (IFN-I) and proinflammatory cytokines in response to viruses; however, their contribution to antiviral immunity in vivo is unclear. In this study, we investigated the impact of pDC depletion on local and systemic antiviral responses to herpes simplex virus (HSV) infections using CLEC4C-DTR transgenic mice. We found that pDC do not appear to influence viral burden or survival after vaginal HSV-2 infection, nor do they seem to contribute to virus-specific CD8 T cell responses following subcutaneous HSV-1 infection. In contrast, pDC were important for early IFN-I production, proinflammatory cytokine production, NK cell activation and CD8 T cell responses during systemic HSV-2 and HSV-1 infections. Our data also indicate that unlike pDC, TLR3-expressing cells are important for promoting antiviral responses to HSV-1 regardless of the route of virus administration.
Author Summary
Herpes simplex viruses (HSV) cause a variety of diseases in human from the common cold sore to more severe illnesses such as pneumonia, herpes simplex keratitis, genital herpes and encephalitis. HSV are large double-stranded DNA viruses that infect epithelial or epidermal cells before establishing a latent infection in sensory neurons. Both innate and adaptive immune responses are necessary for limiting viral replication and maintaining latency. Viral detection through distinct pathogen recognition pathways triggers several signaling cascades that lead to the production of proinflammatory cytokines and type I interferons, which establish inflammation, confer an antiviral state and promote immune responses. Our study provides new insights into the cell types and pathogen recognition pathways involved in antiviral defense to HSV at local and systemic barriers and thus, might facilitate the development of novel strategies to treat HSV infections.
doi:10.1371/journal.ppat.1003728
PMCID: PMC3812046  PMID: 24204273
10.  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
11.  Rapid Influx and Death of Plasmacytoid Dendritic Cells in Lymph Nodes Mediate Depletion in Acute Simian Immunodeficiency Virus Infection 
PLoS Pathogens  2009;5(5):e1000413.
Plasmacytoid dendritic cells (pDC) are essential innate immune system cells that are lost from the circulation in human immunodeficiency virus (HIV)–infected individuals associated with CD4+ T cell decline and disease progression. pDC depletion is thought to be caused by migration to tissues or cell death, although few studies have addressed this directly. We used precise methods of enumeration and in vivo labeling with 5-bromo-2′-deoxyuridine to track recently divided pDC in blood and tissue compartments of monkeys with acute pathogenic simian immunodeficiency virus (SIV) infection. We show that pDC are lost from blood and peripheral lymph nodes within 14 days of infection, despite a normal frequency of pDC in bone marrow. Paradoxically, pDC loss masked a highly dynamic response characterized by rapid pDC mobilization into blood and a 10- to 20-fold increase in recruitment to lymph nodes relative to uninfected animals. Within lymph nodes, pDC had increased levels of apoptosis and necrosis, were uniformly activated, and were infected at frequencies similar to CD4+ T cells. Nevertheless, remaining pDC had essentially normal functional responses to stimulation through Toll-like receptor 7, with half of lymph node pDC producing both TNF-α and IFN-α. These findings reveal that cell migration and death both contribute to pDC depletion in acute SIV infection. We propose that the rapid recruitment of pDC to inflamed lymph nodes in lentivirus infection has a pathologic consequence, bringing cells into close contact with virus, virus-infected cells, and pro-apoptotic factors leading to pDC death.
Author Summary
Plasmacytoid dendritic cells (pDC) are essential components of the innate immune system whose loss from blood is associated with disease progression in human immunodeficiency virus–infected individuals. The mechanism of pDC loss is undefined but is believed to be associated with migration to tissues or cell death. To address this question, we studied pDC kinetics in blood and tissues in the related rhesus macaque monkey model of simian immunodeficiency virus infection. We found that pDC were present in normal numbers in bone marrow but were lost from blood and lymph nodes within 14 days of intravenous infection. Underlying pDC loss was a profound mobilization of pDC from bone marrow into blood and subsequent influx into lymph nodes. In lymph nodes pDC were activated, apoptotic, and frequently infected with virus. Nevertheless, pDC were functionally normal with respect to cytokine production. We conclude that migration and death both contribute to pDC depletion, with influx into lymph nodes bringing cells into an environment favoring their death by infection or apoptosis.
doi:10.1371/journal.ppat.1000413
PMCID: PMC2671605  PMID: 19424421
12.  Interferon-β Pretreatment of Conventional and Plasmacytoid Human Dendritic Cells Enhances Their Activation by Influenza Virus 
PLoS Pathogens  2008;4(10):e1000193.
Influenza virus produces a protein, NS1, that inhibits infected cells from releasing type I interferon (IFN) and blocks maturation of conventional dendritic cells (DCs). As a result, influenza virus is a poor activator of both mouse and human DCs in vitro. However, in vivo a strong immune response to virus infection is generated in both species, suggesting that other factors may contribute to the maturation of DCs in vivo. It is likely that the environment in which a DC encounters a virus would contain multiple pro-inflammatory molecules, including type I IFN. Type I IFN is a critical component of the viral immune response that initiates an antiviral state in cells, primarily by triggering a broad transcriptional program that interferes with the ability of virus to establish infection in the cell. In this study, we have examined the activation profiles of both conventional and plasmacytoid dendritic cells (cDCs and pDCs) in response to an influenza virus infection in the context of a type I IFN-containing environment. We found that both cDCs and pDCs demonstrate a greater activation response to influenza virus when pre-exposed to IFN-β (IFN priming); although, the priming kinetics are different in these two cell types. This strongly suggests that type I IFN functions not only to reduce viral replication in these immune cells, but also to promote greater DC activation during influenza virus infections.
Author Summary
Influenza infection leads to a serious respiratory infection of the lung epithelium. Lying directly below the epithelial cells are immune system sentinels known as dendritic cells. These cells interact with the virus and carry parts of the virus to draining lymph nodes to activate killer T cells. In order to effectively carry out this function, DCs must perceive the presence of a virus using receptors specially adapted for this function. However, when DCs are mixed with influenza virus in the laboratory, no activation occurs because the virus produces a protein called NS1 that blocks the receptors. Yet, patients infected with influenza virus develop a strong adaptive response that leads to recovery from infection. This observation suggests that additional factors must be present that contribute to the activation of the DCs. The most likely contributor is type I interferon, a ubiquitous protein released from many cells upon exposure to virus. In this study, we mixed influenza virus with DCs in the presence of type I interferon and found that this greatly enhanced their activation. Treatment with interferon allowed the DC to bypass the block in activation mediated by the influenza NS1 protein. Our data suggest that the production of type I interferon within an infected patient may endow the DCs with the ability to fully respond to influenza virus.
doi:10.1371/journal.ppat.1000193
PMCID: PMC2568957  PMID: 18974865
13.  Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFNα Response 
PLoS Pathogens  2010;6(6):e1000931.
Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNα and β) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNα production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNα production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNα by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNα induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNα production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNα induction.
Author Summary
Rotaviruses cause severe dehydrating diarrhea and are a leading cause of death in children worldwide. A potent antiviral, interferon-α (IFNα), is rapidly secreted by plasmacytoid dendritic cells (pDCs) in response to viral single-stranded RNA or DNA genomes. Here, we examined the effects of rotavirus on pDCs purified from human blood. We found that very few pDCs supported rotavirus replication, and that pDCs retained similar functionality in response to live or inactivated rotaviruses. While pDCs produced large quantities of IFNα shortly after rotavirus exposure, this was impaired in cells supporting viral replication. We also found that two viral proteins and the rotavirus double-stranded RNA genome were required for the initiation of the pDC IFNα response to rotavirus. Additionally, we found that cleavage of one of these viral proteins, a traditional prerequisite for rotavirus infection in other cell types, was not required for the infection of pDCs or production of IFNα. This may enable the host to rapidly initiate an immune response to rotavirus that subsequently restricts infection to the intestine and contributes to the resolution of disease. Our study provides novel insight into the interaction between rotavirus and the host innate immune response, and also identifies a unique mechanism for the production of IFNα by pDCs.
doi:10.1371/journal.ppat.1000931
PMCID: PMC2880586  PMID: 20532161
14.  A Systems Immunology Approach to Plasmacytoid Dendritic Cell Function in Cytopathic Virus Infections 
PLoS Pathogens  2010;6(7):e1001017.
Plasmacytoid dendritic cell (pDC)-mediated protection against cytopathic virus infection involves various molecular, cellular, tissue-scale, and organism-scale events. In order to better understand such multiscale interactions, we have implemented a systems immunology approach focusing on the analysis of the structure, dynamics and operating principles of virus-host interactions which constrain the initial spread of the pathogen. Using high-resolution experimental data sets coming from the well-described mouse hepatitis virus (MHV) model, we first calibrated basic modules including MHV infection of its primary target cells, i.e. pDCs and macrophages (Mφs). These basic building blocks were used to generate and validate an integrative mathematical model for in vivo infection dynamics. Parameter estimation for the system indicated that on a per capita basis, one infected pDC secretes sufficient type I IFN to protect 103 to 104 Mφs from cytopathic viral infection. This extremely high protective capacity of pDCs secures the spleen's capability to function as a ‘sink’ for the virus produced in peripheral organs such as the liver. Furthermore, our results suggest that the pDC population in spleen ensures a robust protection against virus variants which substantially down-modulate IFN secretion. However, the ability of pDCs to protect against severe disease caused by virus variants exhibiting an enhanced liver tropism and higher replication rates appears to be rather limited. Taken together, this systems immunology analysis suggests that antiviral therapy against cytopathic viruses should primarily limit viral replication within peripheral target organs.
Author Summary
Human infections with highly virulent viruses, such as 1918 influenza or SARS-coronavirus, represent major threats to public health. The initial innate immune responses to such viruses have to restrict virus spread before the adaptive immune responses fully develop. Therefore, it is of fundamental practical importance to understand the robustness and fragility of the early protection against such virus infections mediated by the type I interferon (IFN) response. Because of the inherent complexity of the virus-host system, we have used mathematical modeling to predict the sensitivity of the kinetics and severity of infection to variations in virus and host parameters. Our results suggest that the spleen represents a robust sink system for systemic virus infection and that this system is able to cope with substantial variations in IFN secretion and virus production. However, the system is very fragile to only minor increases in the virus growth rate in peripheral tissues. Collectively, the mathematical approach described in this study allows us to identify the most robust virus and host parameters during early cytopathic virus infection and can serve as a paradigm for systems immunology analyses of multiscale virus-host interaction of many life-threatening cytopathic virus infections.
doi:10.1371/journal.ppat.1001017
PMCID: PMC2908624  PMID: 20661432
15.  Complement Mediated Signaling on Pulmonary CD103+ Dendritic Cells Is Critical for Their Migratory Function in Response to Influenza Infection 
PLoS Pathogens  2013;9(1):e1003115.
Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103+ DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies.
Author Summary
Influenza is a global health problem frequented by epidemics and pandemics. Current vaccines against influenza offer limited protection hence the need for reformulation and repeated vaccination. There is a pressing need to develop newer vaccines that are able to generate T cell response. In order to develop such vaccines, there is a need to understand how T cell responses are generated during influenza infection. Influenza specific T cell responses are generated by the dendritic cells (DCs) in the lung. Upon influenza infection, DCs in the lung carry viral peptides to the draining lymph node (dLN) to initiate an immune response. Thus, migration of DCs from the lung to the dLN is an important step in the initiation of influenza specific T cell response. We now show that activation products of the complement system interact with their receptors on the DCs, which signals for the DCs to migrate from the lung to the dLN. Thus, our results reveal a previously unknown function for complement in mediating lung DC migration during influenza infection and highlight its potential as an adjuvant in novel vaccine strategies.
doi:10.1371/journal.ppat.1003115
PMCID: PMC3542115  PMID: 23326231
16.  Efficient Sensing of Avian Influenza Viruses by Porcine Plasmacytoid Dendritic Cells 
Viruses  2011;3(4):312-330.
H5N1 influenza A virus (IAV) infections in human remain rare events but have been associated with severe disease and a higher mortality rate compared to infections with seasonal strains. An excessive release of pro-inflammatory cytokine together with a greater virus dissemination potential have been proposed to explain the high virulence observed in human and other mammalian and avian species. Among the cells involved in the cytokine storm, plasmacytoid dendritic cells (pDC) could play an important role considering their unique capacity to secrete massive amounts of type I interferon (IFN). Considering the role of IFN as a major component of antiviral responses as well as in priming inflammatory responses, we aimed to characterize the induction of IFN-α release upon infection with IAV originating from various avian and mammalian species in a comparative way. In our porcine pDC model, we showed that the viral components triggering IFN responses related to the ability to hemagglutinate, although virosomes devoid of viral RNA were non-stimulatory. Heat-treatment at 65 °C but not chemical inactivation destroyed the ability of IAV to stimulate pDC. All IAV tested induced IFN-α but at different levels and showed different dose-dependencies. H5 and H7 subtypes, in particular H5N1, stimulated pDC at lower doses when compared to mammalian IAV. At high viral doses, IFN-α levels reached by some mammalian IAV surpassed those induced by avian isolates. Although sialic acid-dependent entry was demonstrated, the α-2,3 or α-2,6 binding specificity alone did not explain the differences observed. Furthermore, we were unable to identify a clear role of the hemagglutinin, as the IFN-α doses-response profiles did not clearly differ when viruses with all genes of identical avian origin but different HA were compared. This was found with IAV bearing an HA derived from either a low, a high pathogenic H5N1, or a human H3. Stimulation of pDC was associated with pDC depletion within the cultures. Taken together and considering the efficient sensing of H5N1 at low dose, pDC on one side may play a role in the cytokine storm observed during severe disease, on the other hand could participate in early antiviral responses limiting virus replication.
doi:10.3390/v3040312
PMCID: PMC3185703  PMID: 21994734
plasmacytoid dendritic cells; influenza A virus; interferon; cytokine storm
17.  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
18.  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
19.  Slc15a4, a Gene Required for pDC Sensing of TLR Ligands, Is Required to Control Persistent Viral Infection 
PLoS Pathogens  2012;8(9):e1002915.
Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.
Author Summary
The immune system consists of two arms aimed at fighting infection. Innate immunity represents the first barrier of defense to swiftly react – within minutes – following intrusion by a pathogen. Adaptive immunity is activated a few days later. Cross-talk between these two systems is critical but the means of communication are not yet fully understood. Plasmacytoid dendritic cells (pDCs) are innate immune cells best recognized for their ability to produce type I interferon (e.g. in response to viral infection.) Evidence for pDCs to modulate the adaptive system in vivo is only recent and still elusive. Using a newly described mouse model named feeble that is characterized by functional deficiency of pDCs, we analysed the role of feeble in the context of acute and chronic viral infection. We found that the feeble mutation affecting pDCs is dispensable for immunity during an acute infection. However our data show that feeble mice failed to control a chronic infection. This was likely due to a reduction in early cytokine secretion and improper activation of adaptive T cells, resulting in virus persistence. Therefore we propose that pDCs are critical for the resolution of chronic infection by linking both arms of immunity.
doi:10.1371/journal.ppat.1002915
PMCID: PMC3441671  PMID: 23028315
20.  Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells 
PLoS Pathogens  2013;9(4):e1003316.
Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.
Author Summary
Hepatitis C Virus (HCV) is the most common bloodborne pathogen for which no vaccine is available. Infection with the virus often leads to persistent (or chronic) infection. Patients with chronic HCV infection can develop progressive liver disease and liver failure, leading to the need for a transplant. It is not fully understood why some people clear the virus and others develop persistent infection. Understanding differences in how patients respond to the virus in the early phases of infection may lead to better treatment of HCV. Here, we use a highly conserved region of the HCV genome to examine innate immunological responses to HCV. We found that plasmacytoid dendritic cells, innate cells keyed to respond with anti-viral interferon proteins, recognize the virus. Additionally, we show that pDCs use RIG-I in the recognition of this virus, which was previously thought to be dispensable in pDCs. The proteins secreted by these cells can control viral replication in a cell-based laboratory system. In cells isolated from healthy donors, we found that fresh human cells can respond in the same manner to the virus as the laboratory strain of cells, and there was a correlation with genetic differences. Our study offers novel insight to how the body recognizes HCV during early infection and host-virus interactions that mediate viral control of this common infection.
doi:10.1371/journal.ppat.1003316
PMCID: PMC3630164  PMID: 23637605
21.  Age-associated impaired plasmacytoid dendritic cell functions lead to decreased CD4 and CD8 T cell immunity 
Age  2010;33(3):363-376.
Increased susceptibility to infections, particularly respiratory viral infections, is a hallmark of advancing age. The underlying mechanisms are not well understood, and there is a scarcity of information regarding the contribution of the innate immune system, which is the first line of defense against infections. In the present study, we have investigated the effect of advancing age on plasmacytoid dendritic cell (PDC) function because they are critical in generating a robust antiviral response via the secretion of interferons (IFN). Our results indicate that PDCs from the aged are impaired in their capacity to secrete IFN-I in response to influenza virus and CPG stimulation. Additionally, we observed a severe reduction in the production of IFN-III, which plays an important role in defense against viral infections at respiratory mucosal surfaces. This reduction in IFN-I and IFN-III were a result of age-associated impaired phosphorylation of transcription factor, IRF-7. Furthermore, aged PDCs were observed to be impaired in their capacity to induce perforin and granzyme in CD8 T cells. Comparison of the antigen-presenting capacity of aged PDC with young PDC revealed that PDCs from aged subjects display reduced capacity to induce proliferation and IFN-gamma secretion in CD4 and CD8 T cells as compared with PDCs from young subjects. In summary, our study demonstrates that advancing age has a profound effect on PDC function at multiple levels and may therefore, be responsible for the increased susceptibility to infections in the elderly.
doi:10.1007/s11357-010-9191-3
PMCID: PMC3168606  PMID: 20953722
Plasmacytoid dendritic cells; Aging; Influenza; Type I interferons; Type III interferons; CD8 cytotoxicity; T cell proliferation
22.  Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells 
PLoS Pathogens  2014;10(10):e1004434.
Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.
Author Summary
Viral recognition by the host often triggers an antiviral state, which suppresses viral spread and imparts adaptive immunity. Like many viruses, dengue virus (DENV) defeats the host-sensing pathway within infected cells. However, in vivo studies have demonstrated a key role of innate immunity in controlling DENV infection. Here we report that sensing of DENV-infected cells by non-permissive innate immune cells, the plasmacytoid dendritic cells (pDCs), triggers a cell-contact- and TLR7-dependent activation of a strong antiviral IFN response. This cell-to-cell sensing involves transmission of viral elements that are clustered at the interface between pDCs and infected cells and is regulated by the actin network. Importantly, we revealed that uncleaved prM surface protein-containing immature particles play a key function in stimulating the innate immune response. These non-infectious immature particles are released by infected cells as a consequence of a suboptimal cleavage site, which is an evolutionarily conserved viral feature that likely favors the export of infectious virus by prevention of premature membrane fusion in the secretory pathway. Therefore our results highlight a conceptually novel trade-off between efficient infectious virus release and the production of IFN-inducing particles. This concept may have broad importance for the many viruses that, like DENV, can disable the pathogen-sensing machinery within infected cells and can release uncleaved glycoprotein-containing non-infectious particles.
doi:10.1371/journal.ppat.1004434
PMCID: PMC4207819  PMID: 25340500
23.  A protective role of murine langerin+ cells in immune responses to cutaneous vaccination with microneedle patches 
Scientific Reports  2014;4:6094.
Cutaneous vaccination with microneedle patches offers several advantages over more frequently used approaches for vaccine delivery, including improved protective immunity. However, the involvement of specific APC subsets and their contribution to the induction of immunity following cutaneous vaccine delivery is not well understood. A better understanding of the functions of individual APC subsets in the skin will allow us to target specific skin cell populations in order to further enhance vaccine efficacy. Here we use a Langerin-EGFP-DTR knock-in mouse model to determine the contribution of langerin+ subsets of skin APCs in the induction of adaptive immune responses following cutaneous microneedle delivery of influenza vaccine. Depletion of langerin+ cells prior to vaccination resulted in substantial impairment of both Th1 and Th2 responses, and decreased post-challenge survival rates, in mice vaccinated cutaneously but not in those vaccinated via the intramuscular route or in non-depleted control mice. Our results indicate that langerin+ cells contribute significantly to the induction of protective immune responses following cutaneous vaccination with a subunit influenza vaccine.
doi:10.1038/srep06094
PMCID: PMC4135340  PMID: 25130187
24.  Both Conventional and Interferon Killer Dendritic Cells Have Antigen-Presenting Capacity during Influenza Virus Infection 
PLoS ONE  2009;4(9):e7187.
Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220+CD11cintNK1.1+ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220− NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.
doi:10.1371/journal.pone.0007187
PMCID: PMC2747012  PMID: 19784375
25.  Alveolar macrophages regulate the induction of primary cytotoxic T-lymphocyte responses during influenza virus infection. 
Journal of Virology  1997;71(12):9450-9457.
Virus-specific cytotoxic T lymphocytes (CTL) are thought to be responsible for the eradication of respiratory influenza virus infections by direct cytolysis of virus-infected epithelial cells. In this study, we provide evidence for a role for alveolar macrophages (AM) in the regulation of pulmonary virus-specific CTL responses. Prior to infection with influenza virus, AM were selectively eliminated in vivo with a liposome-mediated depletion technique, and virus-specific CTL activities of lung and mediastinal lymph node (MLN) cells were assayed ex vivo and compared with those for normal mice. AM depletion resulted in increased primary CTL responses and changed the kinetics of the CTL response. Flow cytometric analysis of lung and MLN cells showed that the percentage of CD8+ cells was not altered after AM depletion and that lung cells from AM-depleted mice had an increased capacity to lyse virus-infected cells. Upon restimulation in vitro, virus-specific CTL activity in lung cells of normal mice was similar to that in lung cells of AM-depleted mice. Furthermore, elimination of AM resulted in increased virus titers in the lung, but virus clearance as a function of time was not affected. Our results show that AM regulate virus-specific CTL responses during respiratory influenza virus infection by removing viral particles, by downregulating the priming and activity of CTL in MLN cells, and by inhibiting the expansion of virus-specific CTL in the lung.
PMCID: PMC230250  PMID: 9371606

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