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1.  Limited Type I Interferons and Plasmacytoid Dendritic Cells during Neonatal Respiratory Syncytial Virus Infection Permit Immunopathogenesis upon Reinfection 
Journal of Virology  2014;88(16):9350-9360.
ABSTRACT
Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action.
IMPORTANCE Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design.
doi:10.1128/JVI.00818-14
PMCID: PMC4136292  PMID: 24920801
2.  SUBVERSION OF PULMONARY DENDRITIC CELL FUNCTION BY PARAMYXOVIRUS INFECTIONS1 
Lower respiratory tract infections caused by the paramyxoviruses human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are characterized by short-lasting virus-specific immunity and often long term airway morbidity, both of which may be the result of alterations in the antigen presenting function of the lung which follow these infections. In this study, we investigated whether hMPV and RSV experimental infections alter the phenotype and function of dendritic cells (DC) subsets which are recruited to the lung. Characterization of lung DC trafficking demonstrated a differential recruitment of plasmacytoid DC (pDC), conventional DC (cDC) and interferon-producing killer DC (IKDC) to the lung and draining lymph nodes after hMPV and RSV infection. In vitro infection of lung DC indicated that in pDC, production of IFN-α, TNF-α, and CCL5 was induced only by hMPV while CCL3 and CCL4 were induced by both viruses. In cDC, a similar repertoire of cytokines was induced by hMPV and RSV, except for IFN-β, which was not induced by RSV. The function of lung pDC was altered following hMPV or RSV infection in vivo, as we demonstrated a reduced capacity of lung pDC to produce IFN-α as well as other cytokines including IL-6, TNF-α, CCL2, CCL3 and CCL4 in response to TLR9 agonist. Moreover, we observed an impaired capacity of cDC from infected mice to present Ag to CD4+ T cells, an effect that lasted beyond the acute phase of infection. Our findings suggest that acute paramyxovirus infections can alter the long term immune function of pulmonary DC.
doi:10.4049/jimmunol.0802262
PMCID: PMC2865244  PMID: 19234204
Dendritic cells; Lung; Viral; Cytokines; Cell trafficking
3.  Whole Blood Gene Expression Profiles to Assess Pathogenesis and Disease Severity in Infants with Respiratory Syncytial Virus Infection 
PLoS Medicine  2013;10(11):e1001549.
In this study, Mejias and colleagues found that specific blood RNA profiles of infants with RSV LRTI allowed for specific diagnosis, better understanding of disease pathogenesis, and better assessment of disease severity.
Please see later in the article for the Editors' Summary
Background
Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity.
Methods and Findings
This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%–98%]) and specificity (98% [95% CI 88%–99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (<6 mo). We identified a genomic score that significantly correlated with outcomes of care including a clinical disease severity score and, more importantly, length of hospitalization and duration of supplemental O2.
Conclusions
Blood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for biomarker discovery and identification of potential therapeutic or preventive targets, and demonstrates that large microarray datasets can be translated into a biologically meaningful context and applied to the clinical setting.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lower respiratory tract infections (LRTIs)—bacterial and viral infections of the lungs and airways (the tubes that take oxygen-rich air to the lungs)—are major causes of illness and death in children worldwide. Pneumonia (infection of the lungs) alone is responsible for 14% of all child deaths. The leading cause of viral LTRIs in children is respiratory syncytial virus (RSV), which is readily transmitted from person to person by direct contact with nasal fluids or airborne droplets. Almost all children have an RSV infection before their second birthday, but most have only minor symptoms similar to those of a common cold and are cared for at home. Unfortunately, some children develop more serious conditions when they become infected with RSV, such as pneumonia or bronchiolitis (swelling and mucus build-up in the bronchioles, the smallest air passages in the lungs). These children have to be admitted to the hospital for supportive care—there is no specific treatment for RSV infection—such as the provision of supplemental oxygen.
Why Was This Study Done?
The lack of a treatment (and of a vaccine) for RSV is largely due to our incomplete understanding of the cellular events and reactions, including the host immune response, that occur during the development of an RSV infection (disease pathogenesis). Moreover, based on physical examination and available diagnostic tools, it is impossible to predict which children infected with RSV will develop a serious condition that requires hospitalization and which ones can be safely nursed at home. Here, the researchers use microarrays to analyze the global host response to acute RSV LTRI in infants, to define gene expression patterns that are specific to RSV infection rather than infection with other common respiratory viruses, and to identify biomarkers that indicate the severity of RSV infection. “Microarray” analysis allows researchers to examine gene expression patterns (“RNA transcriptional profiles”) in, for example, whole blood; a biomarker is a molecule whose level in bodily fluids or tissues indicates how a disease might develop and helps with patient classification.
What Did the Researchers Do and Find?
The researchers compared the RNA transcriptional profile in whole blood taken from children less than two years old hospitalized with RSV, human rhinovirus, or influenza virus infection (rhinovirus and influenza are two additional viral causes of LRTI), and from healthy infants. Using “statistical group comparisons,” they identified more than 2,000 transcripts that were differentially expressed in blood from 45 infants with RSV infection and from 14 healthy matched controls. Genes related to interferon function (interferons are released by host cells in response to the presence of disease-causing organisms) and neutrophil function (neutrophils are immune system cells that, like interferons, are involved in the innate immune response, the body's first line of defense against infection) were among the most overexpressed genes in infants infected with RSV. Genes regulating T and B cells (components of the adaptive immune response, the body's second-line of defense against infection) were among the most underexpressed genes. This specific transcriptional profile, which was validated in three additional groups of infants, accurately distinguished between infants infected with RSV and those infected with human rhinovirus or influenza virus. Finally, a “molecular distance to health” score (a numerical score that quantifies the transcriptional perturbation associated with an illness) was correlated with the clinical disease severity score of the study participants, with how long they needed supplemental oxygen, and with their duration of hospitalization.
What Do These Findings Mean?
These findings suggest that it might be possible to use whole blood RNA transcriptional profiles to distinguish between infants infected with RSV and those with other viruses that commonly cause LRTI. Moreover, if these findings can be replicated in more patients (including non-hospitalized children), gene expression profiling might provide a strategy for triaging patients with RSV infections when they first present to an emergency department and for monitoring clinical changes during the course of the infection, particularly given the development of molecular tools that might soon enable the “real time” acquisition of transcriptional profiles in the clinical setting. Finally, although certain aspects of the study design limit the accuracy and generalizability of the study's findings, these data provide new insights into the pathogenesis of RSV infection and open new avenues for the discovery of biomarkers for RSV infection and for the identification of therapeutic and preventative targets.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001549.
This study is further discussed in a PLOS Medicine Perspective by Peter Openshaw
The US Centers for Disease Control and Prevention provides information about RSV infection
The US National Heart, Lung, and Blood Institute provides information about the respiratory system and about RSV infections
The UK National Health Service Choices website provides information about bronchiolitis
The British Lung Foundation also provides information on RSV and on bronchiolitis
MedlinePlus provides links to other resources about RSV infections and about pneumonia (in English and Spanish); the MedlinePlus encyclopedia has a page on bronchiolitis (in English and Spanish)
PATH is an international non-profit organization investigating new RSV vaccines
doi:10.1371/journal.pmed.1001549
PMCID: PMC3825655  PMID: 24265599
4.  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
5.  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
6.  Exposure of neonates to Respiratory Syncytial Virus is critical in determining subsequent airway response in adults 
Respiratory Research  2006;7(1):107.
Background
Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis in infants and the elderly. Furthermore, epidemiological data suggest that RSV infection during infancy is a potent trigger of subsequent wheeze and asthma development. However, the mechanism by which RSV contributes to asthma is complex and remains largely unknown. A recent study indicates that the age of initial RSV infection is a key factor in determining airway response to RSV rechallenge. We hypothesized that severe RSV infection during neonatal development significantly alters lung structure and the pulmonary immune micro-environment; and thus, neonatal RSV infection is crucial in the development of or predisposition to allergic inflammatory diseases such as asthma.
Methods
To investigate this hypothesis the present study was conducted in a neonatal mouse model of RSV-induced pulmonary inflammation and airway dysfunction. Seven-day-old mice were infected with RSV (2 × 105 TCID50/g body weight) and allowed to mature to adulthood. To determine if neonatal RSV infection predisposed adult animals to enhanced pathophysiological responses to allergens, these mice were then sensitized and challenged with ovalbumin. Various endpoints including lung function, histopathology, cytokine production, and cellularity in bronchoalveolar lavage were examined.
Results
RSV infection in neonates alone led to inflammatory airway disease characterized by airway hyperreactivity, peribronchial and perivascular inflammation, and subepithelial fibrosis in adults. If early RSV infection was followed by allergen exposure, this pulmonary phenotype was exacerbated. The initial response to neonatal RSV infection resulted in increased TNF-α levels in bronchoalveolar lavage. Interestingly, increased levels of IL-13 and mucus hyperproduction were observed almost three months after the initial infection with RSV.
Conclusion
Neonatal RSV exposure results in long term pulmonary inflammation and exacerbates allergic airways disease. The early increase in TNF-α in the bronchoalveolar lavage implicates this inflammatory cytokine in orchestrating these events. Finally, the data presented emphasize IL-13 and TNF-α as potential therapeutic targets for treating RSV induced-asthma.
doi:10.1186/1465-9921-7-107
PMCID: PMC1563465  PMID: 16893457
7.  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
8.  Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses 
Journal of Virology  2013;87(24):13466-13479.
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)2 form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.
doi:10.1128/JVI.01741-13
PMCID: PMC3838285  PMID: 24089561
9.  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
10.  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
11.  Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies  
PLoS ONE  2013;8(11):e81695.
Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14+ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.
doi:10.1371/journal.pone.0081695
PMCID: PMC3841124  PMID: 24303065
12.  Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus 
Journal of Virology  2005;79(9):5507-5515.
Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.
doi:10.1128/JVI.79.9.5507-5515.2005
PMCID: PMC1082779  PMID: 15827165
13.  Plasmacytoid Dendritic Cells in the Tumor Microenvironment: Immune Targets for Glioma Therapeutics12 
Neoplasia (New York, N.Y.)  2012;14(8):757-770.
Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma (glioblastoma multiforme [GBM]) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of in vivo phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM.
PMCID: PMC3431182  PMID: 22952428
14.  Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10. 
Journal of Virology  1994;68(8):5321-5325.
In previous studies, children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency than did controls during subsequent natural RSV infection. In earlier efforts to develop an animal model for this phenomenon, extensive pulmonary histopathology developed in FI-RSV-immunized cotton rats and mice subsequently challenged with RSV. In mice, depletion of CD4+ T cells at the time of RSV challenge completely abrogated this histopathology. Furthermore, the predominant cytokine mRNA present in lungs of FI-RSV-immunized mice during subsequent infection with RSV was that characteristically secreted by Th2 T cells, namely interleukin-4 (IL-4). In the present studies, we sought to determine the relative contributions of gamma interferon (IFN-gamma), IL-2, IL-4, and IL-10 to the lymphocytic infiltration into the lungs observed following RSV challenge of mice previously immunized with FI-RSV. Mice previously immunized with FI-RSV or infected with RSV were depleted of IFN-gamma, IL-2, IL-4, or IL-10 immediately before RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels was quantified. The phenomenon of pulmonary-histopathology potentiation by FI-RSV was reproduced in the present study, thereby allowing us to investigate the effect of cytokine depletion on the process. Simultaneous depletion of both IL-4 and IL-10 completely abrogated pulmonary histopathology in FI-RSV-immunized mice. Depletion of IL-4 alone significantly reduced bronchiolar, though not perivascular, histopathology. Depletion of IL-10 alone had no effect. Depletion of IFN-gamma, IL-2, or both together had no effect on the observed histopathology. These data indicate that FI-RSV immunization primes for a Th2-, IL-4-, and IL-10-dependent inflammatory response to subsequent RSV infection. It is possible that this process played a role in enhanced disease observed in infants and children immunized with FI-RSV.
PMCID: PMC236482  PMID: 8035532
15.  Pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of CD4+ T cells. 
Journal of Virology  1992;66(12):7444-7451.
In previous studies, it was observed that children immunized with a formalin-inactivated respiratory syncytial virus vaccine (FI-RSV) developed severe pulmonary disease with greater frequency during subsequent natural RSV infection than did controls. During earlier efforts to develop an animal model of this phenomenon, enhanced pulmonary histopathology was observed after intranasal RSV challenge of FI-RSV-immunized cotton rats. Progress in understanding the immunologic basis for these observations has been hampered by the lack of reagents useful in manipulating the immune response of the cotton rat. This problem prompted us to reinvestigate the characteristics of immunity to RSV in the mouse. In the present studies, extensive pulmonary histopathology was observed in FI-RSV-immunized or RSV-infected BALB/c mice upon RSV challenge, and studies to determine the relative contributions of CD4+ or CD8+ T cells to this process were undertaken. Mice previously immunized with FI-RSV or infected with RSV were depleted of CD4+, CD8+, or both T-cell subsets immediately prior to RSV challenge, and the magnitude of inflammatory cell infiltration around bronchioles and pulmonary blood vessels and into alveolar spaces was quantified. The magnitude of infiltration at each anatomic site in previously FI-RSV-immunized or RSV-infected, nondepleted animals was similar, indicating that this is not a relevant model for enhanced disease. However, the effect of T-cell subset depletion on pulmonary histopathology following RSV challenge was very different between the two groups. Depletion of CD4+ T cells completely abrogated pulmonary histopathology in FI-RSV-immunized mice, whereas it had a much smaller effect on mice previously infected with RSV. FI-RSV-immunized or RSV-infected animals depleted of CD8+ T cells had only a modest reduction of pulmonary histopathology. In addition, RSV infection induced high levels of major histocompatibility complex class I-restricted cytotoxic T-cell activity, whereas FI-RSV immunization induced a low level. These data indicate that immunization with FI-RSV induces a cellular immune response different from that induced by RSV infection, which likely played a role in enhanced disease observed in infants and children.
PMCID: PMC240452  PMID: 1433525
16.  Differential Response of Dendritic Cells to Human Metapneumovirus and Respiratory Syncytial Virus 
Dendritic cells (DCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract. Human metapneumovirus (hMPV) is a recently identified pathogen and like its better known relative, respiratory syncytial virus (RSV), has been increasingly recognized as a major cause of respiratory morbidity in infants and in elderly persons. In the present study, we examined susceptibility as well as cellular responses of human DCs to hMPV compared with RSV. Monocyte-derived DCs (moDCs) were susceptible to infection by both viruses, but only RSV was able to induce a productive infection with release of viral progeny. Despite the fact that viral infection resulted in phenotypic maturation of moDCs, as shown by the upregulation of cell surface markers and antigen-presenting molecules (MHC I and II, CD80, CD83, CD86, CD38), RSV-infected moDCs showed a severely impaired capacity to stimulate CD4+ T cell proliferation. Compared with hMPV, RSV was a more potent inducer of inflammatory and immunomodulatory cytokines, including TNF-α, IL-6, IL-1β, IL-10, and IL-12p70 in both moDCs and plasmacytoid dendritic cells (pDCs). On the other hand, hMPV, but not RSV, was able to trigger production of IFN-α by moDCs, while both viruses strongly induced IFN-α in pDCs. Finally, both viruses strikingly suppressed IFN-α production by moDCs or pDCs stimulated with synthetic dsRNA and CpG-ODN, respectively. The findings provide novel evidence that RSV and hMPV differentially activate human DCs and may use distinct mechanisms to interfere with the host innate and adaptive immune responses.
doi:10.1165/rcmb.2005-0287OC
PMCID: PMC2644197  PMID: 16284360
dendritic cells; respiratory syncytial virus; human metapneumovirus; interferon type I; innate immunity
17.  Primary Human mDC1, mDC2, and pDC Dendritic Cells Are Differentially Infected and Activated by Respiratory Syncytial Virus 
PLoS ONE  2011;6(1):e16458.
Respiratory syncytial virus (RSV) causes recurrent infections throughout life. Vaccine development may depend upon understanding the molecular basis for induction of ineffective immunity. Because dendritic cells (DCs) are critically involved in early responses to infection, their interaction with RSV may determine the immunological outcome of RSV infection. Therefore, we investigated the ability of RSV to infect and activate primary mDCs and pDCs using recombinant RSV expressing green fluorescent protein (GFP). At a multiplicity of infection of 5, initial studies demonstrated ∼6.8% of mDC1 and ∼0.9% pDCs were infected. We extended these studies to include CD1c−CD141+ mDC2, finding mDC2 infected at similar frequencies as mDC1. Both infected and uninfected cells upregulated phenotypic markers of maturation. Divalent cations were required for infection and maturation, but maturation did not require viral replication. There is evidence that attachment and entry/replication processes exert distinct effects on DC activation. Cell-specific patterns of RSV-induced maturation and cytokine production were detected in mDC1, mDC2, and pDC. We also demonstrate for the first time that RSV induces significant TIMP-2 production in all DC subsets. Defining the influence of RSV on the function of selected DC subsets may improve the likelihood of achieving protective vaccine-induced immunity.
doi:10.1371/journal.pone.0016458
PMCID: PMC3030580  PMID: 21297989
18.  Systemic Dendritic Cell Mobilization Associated with Administration of FLT3 Ligand to SIV- and SHIV-Infected Macaques 
AIDS Research and Human Retroviruses  2009;25(12):1313-1328.
Abstract
Reports indicate that myeloid and plasmacytoid dendritic cells (mDCs and pDCs), which are key effector cells in host innate immune responses, can be infected with HIV-1 and are reduced in number and function during the chronic phase of HIV disease. Furthermore, it was recently demonstrated that a sustained loss of mDCs and pDCs occurs in SIV-infected macaques. Since loss of functional DC populations might impair innate immune responses to opportunistic microorganisms and neoplastic cells, we explored whether inoculation of naive and SIV- or SHIV-infected pigtailed macaques with the hematopoietic cytokine FLT3-ligand (FLT3-L) would expand the number of mDCs and pDCs in vivo. After the macaques received supraphysiologic doses of FLT3-L, mDCs, pDCs, and monocytes increased up to 45-fold in blood, lymph nodes, and bone marrow (BM), with DC expansion in the BM preceding mobilization in blood and lymphoid tissues. FLT3-L also increased serum levels of IL-12, at least transiently, and elicited higher surface expression of HLA-DR and the activation markers CD25 and CD69 on NK and T cells. During and after treatment of infected animals, APCs increased in number and were activated; however, CD4+ T cell numbers, virion RNA, and anti-SIV/SHIV antibody titers remained relatively stable, suggesting that FLT3-L might be a safe modality to expand DC populations and provide therapeutic benefit during chronic lentivirus infections.
doi:10.1089/aid.2009.0053
PMCID: PMC2828165  PMID: 20001520
19.  Regulation of Porcine Plasmacytoid Dendritic Cells by Cytokines 
PLoS ONE  2013;8(4):e60893.
Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN’s, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role.
doi:10.1371/journal.pone.0060893
PMCID: PMC3620061  PMID: 23577175
20.  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
21.  Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection 
BMC Immunology  2014;15(1):41.
Background
Respiratory syncytial virus (RSV) infects the lung epithelium where it stimulates the production of numerous host cytokines that are associated with disease burden and acute lung injury. Characterizing the host cytokine response to RSV infection, the regulation of host cytokines and the impact of neutralizing an RSV-inducible cytokine during infection were undertaken in this study.
Methods
A549, primary human small airway epithelial (SAE) cells and wild-type, TIR-domain-containing adapter-inducing interferon-β (Trif) and mitochondrial antiviral-signaling protein (Mavs) knockout (KO) mice were infected with RSV and cytokine responses were investigated by ELISA, multiplex analysis and qPCR. Neutralizing anti-leukemia inhibitory factor (LIF) IgG or control IgG was administered to a group of wild-type animals prior to RSV infection.
Results and discussion
RSV-infected A549 and SAE cells release a network of cytokines, including newly identified RSV-inducible cytokines LIF, migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, CXCL12 and stem cell growth factor beta (SCGF-β). These RSV-inducible cytokines were also observed in the airways of mice during an infection. To identify the regulation of RSV inducible cytokines, Mavs and Trif deficient animals were infected with RSV. In vivo induction of airway IL-1β, IL-4, IL-5, IL-6, IL-12(p40), IFN-γ, CCL2, CCL5, CCL3, CXCL1, IP-10/CXCL10, IL-22, MIG/CXCL9 and MIF were dependent on Mavs expression in mice. Loss of Trif expression in mice altered the RSV induction of IL-1β, IL-5, CXCL12, MIF, LIF, CXCL12 and IFN-γ. Silencing of retinoic acid–inducible gene-1 (RIG-I) expression in A549 cells had a greater impact on RSV-inducible cytokines than melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and Trif expression. To evaluate the role of LIF in the airways during RSV infection, animals were treated with neutralizing anti-LIF IgG, which enhanced RSV pathology observed with increased airspace protein content, apoptosis and airway hyperresponsiveness compared to control IgG treatment.
Conclusions
RSV infection in the epithelium induces a network of immune factors to counter infection, primarily in a RIG-I dependent manner. Expression of LIF protects the lung from lung injury and enhanced pathology during RSV infection.
Electronic supplementary material
The online version of this article (doi:10.1186/s12865-014-0041-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12865-014-0041-4
PMCID: PMC4189665  PMID: 25277705
Respiratory syncytial virus; Immune response; Pathogen recognition receptors; Gene expression
22.  Differential Type I Interferon Induction by Respiratory Syncytial Virus and Influenza A Virus In Vivo▿  
Journal of Virology  2007;81(18):9790-9800.
Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-α/β induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-α/β production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-α/β in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.
doi:10.1128/JVI.00530-07
PMCID: PMC2045394  PMID: 17626092
23.  Plasmacytoid Dendritic Cells Promote Host Defense Against Acute Pneumovirus Infection via the TLR7-MyD88-Dependent Signaling Pathway 
Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12 and IL-6. However, RSV-infected pDCs are refractory to TLR7-mediated activation. Here, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7-signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type (WT) but not TLR7- or myeloid differentiation protein 88 (MyD88)-deficient mice, PVM inoculation led to a marked infiltration of pDCs and increased expression of type I, II and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8+ T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient but not TLR7-deficient pDC to TLR7-gene-deleted mice recapitulated the antiviral responses observed in WT mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.
doi:10.4049/jimmunol.1002635
PMCID: PMC3404606  PMID: 21482736
24.  Refining the Balance of Attenuation and Immunogenicity of Respiratory Syncytial Virus by Targeted Codon Deoptimization of Virulence Genes 
mBio  2014;5(5):e01704-14.
ABSTRACT
Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in children for which there is no licensed vaccine. Live-attenuated RSV vaccines are the most clinically advanced in children, but achieving an optimal balance of attenuation and immunogenicity is challenging. One way to potentially retain or enhance immunogenicity of attenuated virus is to mutate virulence genes that suppress host immune responses. The NS1 and NS2 virulence genes of the RSV A2 strain were codon deoptimized according to either human or virus codon usage bias, and the resulting recombinant viruses (dNSh and dNSv, respectively) were rescued by reverse genetics. RSV dNSh exhibited the desired phenotype of reduced NS1 and NS2 expression. RSV dNSh was attenuated in BEAS-2B and primary differentiated airway epithelial cells but not in HEp-2 or Vero cells. In BALB/c mice, RSV dNSh exhibited a lower viral load than did A2, and yet it induced slightly higher levels of RSV-neutralizing antibodies than did A2. RSV A2 and RSV dNSh induced equivalent protection against challenge strains A/1997/12-35 and A2-line19F. RSV dNSh caused less STAT2 degradation and less NF-κB activation than did A2 in vitro. Serial passage of RSV dNSh in BEAS-2B cells did not result in mutations in the deoptimized sequences. Taken together, RSV dNSh was moderately attenuated, more immunogenic, and equally protective compared to wild-type RSV and genetically stable.
IMPORTANCE
Respiratory syncytial virus (RSV) is the leading cause of infant viral death in the United States and worldwide, and no vaccine is available. Live-attenuated RSV vaccines are the most studied in children but have suffered from genetic instability and low immunogenicity. In order to address both obstacles, we selectively changed the codon usage of the RSV nonstructural (NS) virulence genes NS1 and NS2 to the least-used codons in the human genome (deoptimization). Compared to parental RSV, the codon-deoptimized NS1/NS2 RSV was attenuated in vitro and in mice but induced higher levels of neutralizing antibodies and equivalent protection against challenge. We identified a new attenuating module that retains immunogenicity and is genetically stable, achieved through specific targeting of nonessential virulence genes by codon usage deoptimization.
doi:10.1128/mBio.01704-14
PMCID: PMC4173764  PMID: 25249281
25.  Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques 
PLoS Pathogens  2014;10(1):e1003915.
IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα+ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα+ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα− production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67+-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67+-pDC precursors, none of these being IFNα+ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors.
Author Summary
Chronic immune activation is a characteristic of HIV infection and a key contributor to CD4 T-cell depletion and progression to AIDS. Persistent up-regulation of interferon-induced genes (ISG) is associated with chronic immune activation and is a molecular signature of the progression of SIV infection in non-human-primate models. Nevertheless, the type and tissue compartmentalization of IFN-I-producing cells at different stages of infection, and the details of the involvement of IFN-I in sustaining chronic immune activation remain elusive. Using the cynomolgus macaque model of progressive SIV infection, we demonstrate in vivo that plasmacytoid dendritic cells (pDC) are major contributors to IFNα production in lymphoid tissues and, most importantly, that this production rapidly shrinks after primary infection. IFNα production rapidly decreased as a consequence of both activation-induced exhaustion of pDC, and their replacement by pDC precursors with no IFNα production ability. Our data indicate that pDC renewal contributes to the rapid contraction of pDC-derived IFNα production during primary infection, which may favor the transition from acute-to-chronic infection by limiting the efficacy of innate immunity.
doi:10.1371/journal.ppat.1003915
PMCID: PMC3907389  PMID: 24497833

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