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1.  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
2.  Novel immune genes associated with excessive inflammatory and antiviral responses to rhinovirus in COPD 
Respiratory Research  2013;14(1):15.
Background
Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls.
Methods
Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication.
Results
COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-β/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-β expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines.
Conclusions
COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD.
doi:10.1186/1465-9921-14-15
PMCID: PMC3570361  PMID: 23384071
COPD; Immune response; Viral infection; Gene expression
3.  Ribavirin Treatment Up-Regulates Antiviral Gene Expression via the Interferon-Stimulated Response Element in Respiratory Syncytial Virus-Infected Epithelial Cells 
Journal of Virology  2003;77(10):5933-5947.
Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. RSV replication is a potent activator of the epithelial-cell genomic response, influencing the expression of a spectrum of cellular pathways, including proinflammatory chemokines of the CC, CXC, and CX3C subclasses. Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nontoxic antiviral agent currently licensed for the treatment of severe RSV lower respiratory tract infections. Because ribavirin treatment reduces the cytopathic effect in infected cells, we used high-density microarrays to investigate the hypothesis that ribavirin modifies the virus-induced epithelial genomic response to replicating virus. Ribavirin treatment administered in concentrations of 10 to 100 μg/ml potently inhibited RSV transcription, thereby reducing the level of RSV N transcripts to ∼13% of levels in nontreated cells. We observed that in both the absence and the presence of ribavirin, RSV infection induced global alterations in the host epithelial cell, affecting ∼49% of the ∼6,650 expressed genes detectable by the microarray. Ribavirin influences the expression of only 7.5% of the RSV-inducible genes (total number of genes, 272), suggesting that the epithelial-cell genetic program initiated by viral infection is independent of high-level RSV replication. Hierarchical clustering of the ribavirin-regulated genes identified four expression patterns. In one group, ribavirin inhibited the expression of the RSV-inducible CC chemokines MIP-1α and -1β, which are important in RSV-induced pulmonary pathology, and interferon (IFN), a cytokine important in the mucosal immune response. In a second group, ribavirin further up-regulated a set of RSV- and IFN-stimulated response genes (ISGs) encoding antiviral proteins (MxA and p56), complement products, acute-phase response factors, and the STAT and IRF transcription factors. Because IFN-β expression itself was reduced in the ribavirin-treated cells, we further investigated the mechanism for up-regulation of the IFN-signaling pathway. Enhanced expression of IFI 6-16, IFI 9-27, MxA/p78, STAT-1α, STAT-1β, IRF-7B, and TAP-1-LMP2 transcripts were independently reproduced by Northern blot analysis. Ribavirin-enhanced TAP-1-LMP2 expression was a transcriptional event where site mutations of the IFN-stimulated response element (ISRE) blocked RSV and ribavirin-inducible promoter activity. Furthermore, ribavirin up-regulated the transcriptional activity of a reporter gene selectively driven by the ISRE. In specific DNA pull-down assays, we observed that ribavirin enhanced RSV-induced STAT-1 binding to the ISRE. We conclude that ribavirin potentiates virus-induced ISRE signaling to enhance the expression of antiviral ISGs, suggesting a mechanism for the efficacy of combined treatment with ribavirin and IFN in other chronic viral diseases.
doi:10.1128/JVI.77.10.5933-5947.2003
PMCID: PMC154027  PMID: 12719586
4.  Critical Role of Constitutive Type I Interferon Response in Bronchial Epithelial Cell to Influenza Infection 
PLoS ONE  2012;7(3):e32947.
Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.
doi:10.1371/journal.pone.0032947
PMCID: PMC3292582  PMID: 22396801
5.  The Synthetic Bacterial Lipopeptide Pam3CSK4 Modulates Respiratory Syncytial Virus Infection Independent of TLR Activation 
PLoS Pathogens  2010;6(8):e1001049.
Respiratory syncytial virus (RSV) is an important cause of acute respiratory disease in infants, immunocompromised subjects and the elderly. However, it is unclear why most primary RSV infections are associated with relatively mild symptoms, whereas some result in severe lower respiratory tract infections and bronchiolitis. Since RSV hospitalization has been associated with respiratory bacterial co-infections, we have tested if bacterial Toll-like receptor (TLR) agonists influence RSV-A2-GFP infection in human primary cells or cell lines. The synthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), the prototype ligand for the heterodimeric TLR1/TLR2 complex, enhanced RSV infection in primary epithelial, myeloid and lymphoid cells. Surprisingly, enhancement was optimal when lipopeptides and virus were added simultaneously, whereas addition of Pam3CSK4 immediately after infection had no effect. We have identified two structurally related lipopeptides without TLR-signaling capacity that also modulate RSV infection, whereas Pam3CSK4-reminiscent TLR1/2 agonists did not, and conclude that modulation of infection is independent of TLR activation. A similar TLR-independent enhancement of infection could also be demonstrated for wild-type RSV strains, and for HIV-1, measles virus and human metapneumovirus. We show that the effect of Pam3CSK4 is primarily mediated by enhanced binding of RSV to its target cells. The N-palmitoylated cysteine and the cationic lysines were identified as pivotal for enhanced virus binding. Surprisingly, we observed inhibition of RSV infection in immortalized epithelial cell lines, which was shown to be related to interactions between Pam3CSK4 and negatively charged glycosaminoglycans on these cells, which are known targets for binding of laboratory-adapted but not wild-type RSV. These data suggest a potential role for bacterial lipopeptides in enhanced binding of RSV and other viruses to their target cells, thus affecting viral entry or spread independent of TLR signaling. Moreover, our results also suggest a potential application for these synthetic lipopeptides as adjuvants for live-attenuated viral vaccines.
Author Summary
Respiratory syncytial virus (RSV) infections are an important cause of hospitalization of infants during the winter season. However, RSV is often not the only detectable pathogen, but co-infections with respiratory bacteria are common. It has been hypothesized that this results from epithelial damage caused by the virus, facilitating colonization by pathogenic bacteria such as Streptococcus pneumoniae. However, an inverse order of events is not impossible: bacterial infections may activate respiratory epithelial cells through TLR signaling, resulting in increased susceptibility to virus infections. We tested this hypothesis by screening bacterial TLR agonists for their capacity to modulate RSV infection in different cell types, and identified the lipopeptide and prototype TLR1/2 agonist Pam3CSK4 as an enhancer of RSV infections. However, to our surprise this proved independent of TLR activation, but was mediated by enhancement of binding between virus and target cell. Two structurally related lipopeptides unable to stimulate TLR responses were identified that enhanced infections with RSV, but also with other enveloped viruses including HIV-1, human metapneumovirus, and measles virus. We speculate that bacterial infections may influence the pathogenesis of virus infections by facilitating binding to target cells.
doi:10.1371/journal.ppat.1001049
PMCID: PMC2924323  PMID: 20808895
6.  MicroRNA-221 Modulates RSV Replication in Human Bronchial Epithelium by Targeting NGF Expression 
PLoS ONE  2012;7(1):e30030.
Background
Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells.
Methodology
Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication.
Principal Findings
RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity.
Conclusions/Significance
Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of exogenous miRNAs to the airways may provide a new strategy for future antiviral therapies based on RNA interference.
doi:10.1371/journal.pone.0030030
PMCID: PMC3260191  PMID: 22272270
7.  Thymic stromal lymphopoietin is induced by respiratory syncytial virus–infected airway epithelial cells and promotes a type 2 response to infection 
Background
Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis and asthma exacerbation.
Objective
The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection.
Methods
Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air–liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection.
Results
Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-κB. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology.
Conclusion
These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.
doi:10.1016/j.jaci.2012.07.031
PMCID: PMC4284103  PMID: 22981788
TSLP; RSV; asthma; epithelium; TH2
8.  Oncolytic targeting of androgen-sensitive prostate tumor by the respiratory syncytial virus (RSV): consequences of deficient interferon-dependent antiviral defense 
BMC Cancer  2011;11:43.
Background
Oncolytic virotherapy for cancer treatment utilizes viruses for selective infection and death of cancer cells without any adverse effect on normal cells. We previously reported that the human respiratory syncytial virus (RSV) is a novel oncolytic virus against androgen-independent PC-3 human prostate cancer cells. The present study extends the result to androgen-dependent prostate cancer, and explores the underlying mechanism that triggers RSV-induced oncolysis of prostate cancer cells.
Methods
The oncolytic effect of RSV on androgen-sensitive LNCaP human prostate cancer cells and on androgen-independent RM1 murine prostate cancer cells was studied in vitro in culture and in vivo in a xenograft or allograft tumor model. In vitro, cell viability, infectivity and apoptosis were monitored by MTT assay, viral plaque assay and annexin V staining, respectively. In vivo studies involved virus administration to prostate tumors grown in immune compromised nude mice and in syngeneic immune competent C57BL/6J mice. Anti-tumorogenic oncolytic activity was monitored by measuring tumor volume, imaging bioluminescent tumors in live animals and performing histopathological analysis and TUNEL assay with tumors
Results
We show that RSV imposes a potent oncolytic effect on LNCaP prostate cancer cells. RSV infectivity was markedly higher in LNCaP cells compared to the non-tumorigenic RWPE-1 human prostate cells. The enhanced viral burden led to LNCaP cell apoptosis and growth inhibition of LNCaP xenograft tumors in nude mice. A functional host immune response did not interfere with RSV-induced oncolysis, since growth of xenograft tumors in syngeneic C57BL/6J mice from murine RM1 cells was inhibited upon RSV administration. LNCaP cells failed to activate the type-I interferon (IFNα/β)-induced transcription factor STAT-1, which is required for antiviral gene expression, although these cells could produce IFN in response to RSV infection. The essential role of IFN in restricting infection was further borne out by our finding that neutralizing IFN activity resulted in enhanced RSV infection in non-tumorigenic RWPE-1 prostate cells.
Conclusions
We demonstrated that RSV is potentially a useful therapeutic tool in the treatment of androgen-sensitive and androgen-independent prostate cancer. Moreover, impaired IFN-mediated antiviral response is the likely cause of higher viral burden and resulting oncolysis of androgen-sensitive prostate cancer cells.
doi:10.1186/1471-2407-11-43
PMCID: PMC3038980  PMID: 21276246
9.  Healthy but not RSV-infected lung epithelial cells profoundly inhibit T cell activation 
Thorax  2008;64(4):283-290.
Background:
Respiratory viruses, including respiratory syncytial virus (RSV), can cause asthma exacerbations and bronchiolitis. Both conditions are associated with enhanced cognate immune responses and inflammation and reduced immune regulation. Lung epithelial cells (LECs) can contribute to antiviral and allergic immune responses while gut epithelial cells can inhibit effector T cell responses. A study was performed to determine whether healthy LECs regulate antigen-specific T cell responses and if this regulation is lost during RSV infection.
Methods:
LA4 cells, a murine LEC line, infected with RSV or primary murine LECs were co-cultured with ovalbumin-specific T cell receptor transgenic CD4+ T cells from DO11.10 mice and ovalbumin-pulsed bone marrow-derived dendritic cells (DC) to assess T cell proliferation by flow cytometry and cytokine production.
Results:
The presence of LECs abrogated DC-induced T cell proliferation and significantly reduced T cell cytokine release. These effects of LECs were predominantly contact-dependent, primarily affected T cells directly and were partly mediated by transforming growth factor β. Soluble factors and DC-mediated effects also contributed to T cell inhibition. RSV infection of LECs reduced their inhibitory capacity in an infection dose-dependent manner. This was independent of proinflammatory cytokines released by infected LECs, but in part due to Toll-like receptor activation and to infection-induced cell death.
Conclusion:
Healthy LECs are potent inhibitors of T cell activation, but this regulatory function is lost after RSV infection. These findings suggest a central role for LECs in maintaining the tolerogenic environment of healthy lungs. Loss of this regulatory capacity after viral infection may allow development of excessive cognate immune responses and pulmonary inflammation.
doi:10.1136/thx.2007.094870
PMCID: PMC2655656  PMID: 18710906
10.  Human Influenza Is More Effective than Avian Influenza at Antiviral Suppression in Airway Cells 
Airway epithelial cells are the initial site of infection with influenza viruses. The innate immune responses of airway epithelial cells to infection are important in limiting virus replication and spread. However, relatively little is known about the importance of this innate antiviral response to infection. Avian influenza viruses are a potential source of future pandemics; therefore, it is critical to examine the effectiveness of the host antiviral system to different influenza viruses. We used a human influenza (H3N2) and a low-pathogenic avian influenza (H11N9) to assess and compare the antiviral responses of Calu-3 cells. After infection, H3N2 replicated more effectively than the H11N9 in Calu-3 cells. This was not due to differential expression of sialic acid residues on Calu-3 cells, but was attributed to the interference of host antiviral responses by H3N2. H3N2 induced a delayed antiviral signaling and impaired type I and type III IFN induction compared with the H11N9. The gene encoding for nonstructural (NS) 1 protein was transfected into the bronchial epithelial cells (BECs), and the H3N2 NS1 induced a greater inhibition of antiviral responses compared with the H11N9 NS1. Although the low-pathogenic avian influenza virus was capable of infecting BECs, the human influenza virus replicated more effectively than avian influenza virus in BECs, and this was due to a differential ability of the two NS1 proteins to inhibit antiviral responses. This suggests that the subversion of human antiviral responses may be an important requirement for influenza viruses to adapt to the human host and cause disease.
doi:10.1165/rcmb.2010-0157OC
PMCID: PMC3135850  PMID: 20705938
influenza; innate immune response; antiviral; IFN
11.  Respiratory Syncytial Virus Infection Sensitizes Cells to Apoptosis Mediated by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand 
Journal of Virology  2003;77(17):9156-9172.
Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease worldwide, especially in the pediatric population. For viruses in general, apoptotic death of infected cells is a mechanism for reducing virus replication. Apoptosis can also be an important factor in augmenting antigen presentation and the host immune response. We examined apoptosis in response to RSV infection of primary small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 cell lines. The primary cells and the A549 cell line gave generally similar responses, indicating their appropriateness as models in contrast to HEp-2 cells. With the use of RNase protection assays with probes representing 33 common apoptosis factors, we found strong transcriptional activation of both pro- and antiapoptotic factors in response to RSV infection, which were further studied at the protein level and by functional assays. In particular, RSV infection strongly up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors death receptor 4 (DR4) and DR5. Furthermore, RSV-infected cells became highly sensitive to apoptosis induced by exogenous TRAIL. These findings suggest that RSV-infected cells in vivo are susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4+ cells that bear membrane-bound TRAIL. RSV infection also induced several proapoptotic factors of the Bcl-2 family and caspases 3, 6, 7, 8, 9, and 10, representing both the death receptor- and mitochondrion-dependent apoptotic pathways. RSV also mediated the strong induction of antiapoptotic factors of the Bcl-2 family, especially Mcl-1, which might account for the delayed induction of apoptosis in RSV-infected cells in the absence of exogenous induction of the TRAIL pathway.
doi:10.1128/JVI.77.17.9156-9172.2003
PMCID: PMC187410  PMID: 12915532
12.  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
13.  Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-κB 
mBio  2012;3(6):e00220-12.
ABSTRACT
Respiratory syncytial virus (RSV) is the most common viral cause of severe lower respiratory tract illness in infants and children. The virus replicates in polarized epithelial cells in the airway and, to a lesser extent, infects airway antigen-presenting cells, such as dendritic cells (DCs). RSV possesses a number of expressed genes that antagonize the effect of type I interferons and other related host factor pathways that inhibit replication efficiency. Virus infection alters host gene transcription and the translation of host transcripts through specific antagonism of the function of host proteins, through induction of RNA stress granules, and through induction of altered patterns of host gene expression. In healthy cells, microRNAs (miRNAs) regulate gene expression by targeting the noncoding region of mRNA molecules to cause silencing or degradation of transcripts. It is not known whether or not RSV infection alters the level of microRNAs in cells. We profiled the pattern of expression of host cell microRNAs in RSV-infected epithelial cells or DCs and found that RSV did alter microRNA expression but in a cell-type-specific manner. The studies showed that let-7b was upregulated in DCs, while let-7i and miR-30b were upregulated in epithelial cells in a process that required viral replication. Interestingly, we found that the RSV nonstructural genes NS1 and NS2 antagonized the upregulation of let-7i and miR-30b. RSV appears to manipulate host cell gene expression through regulation of expression of miRNAs related to the interferon response. The data suggest a new mechanism of virus-host cell interactions for paramyxoviruses.
IMPORTANCE
Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract illness in infants and children. The human innate immune response inhibits RSV replication early after inoculation, principally through the effect of substances called interferons. The virus, however, has developed several mechanisms for counteracting the host innate immune response. It is not known whether or not RSV infection alters the expression of host microRNAs, which are short RNA sequences that are posttranscriptional regulators. This paper shows that RSV does induce unique patterns of microRNA expression related to the NF-κB pathway or interferon pathways. The microRNA profiles differed depending on the cell type that was infected, airway cell or antigen-presenting cell. Interestingly, the virus appears to counteract the microRNA response by expressing nonstructural viral genes in the cell that reduce microRNA induction. The data suggest a new way in which paramyxoviruses regulate the host cell response to infection.
doi:10.1128/mBio.00220-12
PMCID: PMC3529541  PMID: 23249809
14.  Rhinovirus-Induced IL-1β Release from Bronchial Epithelial Cells Is Independent of Functional P2X7 
Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor–dependent inflammatory responses, largely through P2X7. Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X7 to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X7 messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X7 was observed in BECs, and IL-1β was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1β after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2′-3′-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1β release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1β release further. BEC processing of pro–IL-1β to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X7 in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing.
doi:10.1165/rcmb.2011-0267OC
PMCID: PMC3488691  PMID: 22493010
P2X7; IL-1β; human rhinovirus; respiratory epithelium; danger signaling
15.  Neonatal Calf Infection with Respiratory Syncytial Virus: Drawing Parallels to the Disease in Human Infants 
Viruses  2012;4(12):3731-3753.
Respiratory syncytial virus (RSV) is the most common viral cause of childhood acute lower respiratory tract infections. It is estimated that RSV infections result in more than 100,000 deaths annually worldwide. Bovine RSV is a cause of enzootic pneumonia in young dairy calves and summer pneumonia in nursing beef calves. Furthermore, bovine RSV plays a significant role in bovine respiratory disease complex, the most prevalent cause of morbidity and mortality among feedlot cattle. Infection of calves with bovine RSV shares features in common with RSV infection in children, such as an age-dependent susceptibility. In addition, comparable microscopic lesions consisting of bronchiolar neutrophilic infiltrates, epithelial cell necrosis, and syncytial cell formation are observed. Further, our studies have shown an upregulation of pro-inflammatory mediators in RSV-infected calves, including IL-12p40 and CXCL8 (IL-8). This finding is consistent with increased levels of IL-8 observed in children with RSV bronchiolitis. Since rodents lack IL-8, neonatal calves can be useful for studies of IL-8 regulation in response to RSV infection. We have recently found that vitamin D in milk replacer diets can be manipulated to produce calves differing in circulating 25-hydroxyvitamin D3. The results to date indicate that although the vitamin D intracrine pathway is activated during RSV infection, pro-inflammatory mediators frequently inhibited by the vitamin D intacrine pathway in vitro are, in fact, upregulated or unaffected in lungs of infected calves. This review will summarize available data that provide parallels between bovine RSV infection in neonatal calves and human RSV in infants.
doi:10.3390/v4123731
PMCID: PMC3528288  PMID: 23342375
Bovine respiratory syncytial virus; human respiratory syncytial virus; innate immunity; adaptive immunity; vaccine
16.  Fibroblast-myofibroblast transition is differentially regulated by bronchial epithelial cells from asthmatic children 
Respiratory Research  2015;16(1):21.
Background
Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs.
Methods
BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ2 in BEC-HLF co-cultures using monoclonal antibody inhibition.
Results
Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone.
Conclusion
These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.
doi:10.1186/s12931-015-0185-7
PMCID: PMC4333174
Air-liquid interface culture; Airway remodeling; Asthma; Bronchial epithelial cells; Cell culture; Fibroblasts; Myofibroblasts; α-smooth muscle actin; TGFβ2
17.  Regulation of STAT signaling in mouse bone marrow derived dendritic cells by respiratory syncytial virus 
Virus research  2011;156(0):127-133.
Background/aims
Dendritic cells (DCs) act as a portal for virus invasion as well as potent antigen-presenting cells (APCs) involved in the antiviral host response. Interferons (IFNs) are produced in response to bacterial and viral infection and activate innate immune responses to efficiently counteract and remove pathogenic invaders. Respiratory syncytial virus (RSV) could inhibit IFN-mediated signaling pathway in epithelial cells; however, the effects of RSV on IFN signaling in the dendritic cells (DCs) are still unknown.
Methods
Mouse bone marrow derived DCs (BMDCs) were mock or infected with RSV at different multiplicity of infection (MOI) for 24 h, and then treated with different cytokines such as interferon-β (IFN-β), IFN-γ or interleukin-10 (IL-10). The mRNA expression of RSV nonstructural protein-1 (NS-1) and NS-2 was detected by RT-PCR. The expression of Janus family kinase-signal transducer and activator of transcription (JAK/STAT) signaling proteins was assessed by immunoblotting assays. The nuclear localization of specific signaling proteins was determined by immunofluorescence assay.
Results
Increasing amounts of NS-1 or NS-2 mRNA expression in BMDCs were observed with infected RSV at increasing MOI, suggesting BMDCs were permissive for viral gene expression. Further examination of the IFN-β signaling cascade showed RSV infection increased the total cellular levels of STAT1 and STAT2 in BMDCs, but impaired the IFN-β-dependent phosphorylation and nuclear localization of STAT1 and STAT2. The inhibitory effects of RSV on STAT1 and STAT2 phosphorylation and translocation were abolished by UV inactivation. In contrast, RSV did not inhibit the IFN-γ-stimulated STAT1 phosphorylation and nuclear localization. IL-10-stimulated STAT3 phosphorylation was also unaffected by RSV.
Conclusions
As well as RSV inhibiting STAT protein levels through degradation mechanisms in epithelial cells, these findings demonstrate that RSV also can specifically inhibit the type I interferon response in BMDCs through regulation of STAT1 and STAT2 phosphorylation and nuclear translocation.
doi:10.1016/j.virusres.2011.01.007
PMCID: PMC3726278  PMID: 21255624
Bone marrow derived dendritic cells; Respiratory syncytial virus; Type I interferon; STAT; Signal pathway
18.  A Simian Virus 5 (SV5) P/V Mutant Is Less Cytopathic than Wild-Type SV5 in Human Dendritic Cells and Is a More Effective Activator of Dendritic Cell Maturation and Function 
Journal of Virology  2006;80(7):3416-3427.
Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI−) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI− infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI− mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-α and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+ T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.
doi:10.1128/JVI.80.7.3416-3427.2006
PMCID: PMC1440371  PMID: 16537609
19.  NGF Is an Essential Survival Factor for Bronchial Epithelial Cells during Respiratory Syncytial Virus Infection 
PLoS ONE  2009;4(7):e6444.
Background
Overall expression of neurotrophins in the respiratory tract is upregulated in infants infected by the respiratory syncytial virus (RSV), but it is unclear where (structural vs. inflammatory cells, upper vs. lower airways) and why, these changes occur. We analyzed systematically the expression of neurotrophic factors and receptors following RSV infection of human nasal, tracheal, and bronchial epithelial cells, and tested the hypothesis that neurotrophins work as innate survival factors for infected respiratory epithelia.
Methodology
Expression of neurotrophic factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF) and receptors (trkA, trkB, p75) was analyzed at the protein level by immunofluorescence and flow cytometry and at the mRNA level by real-time PCR. Targeted siRNA was utilized to blunt NGF expression, and its effect on virus-induced apoptosis/necrosis was evaluated by flow cytometry following annexin V/7-AAD staining.
Principal Findings
RSV infection was more efficient in cells from more distal (bronchial) vs. more proximal origin. In bronchial cells, RSV infection induced transcript and protein overexpression of NGF and its high-affinity receptor trkA, with concomitant downregulation of the low-affinity p75NTR. In contrast, tracheal cells exhibited an increase in BDNF, trkA and trkB, and nasal cells increased only trkA. RSV-infected bronchial cells transfected with NGF-specific siRNA exhibited decreased trkA and increased p75NTR expression. Furthermore, the survival of bronchial epithelial cells was dramatically decreased when their endogenous NGF supply was depleted prior to RSV infection.
Conclusions/Significance
RSV infection of the distal airway epithelium, but not of the more proximal sections, results in overexpression of NGF and its trkA receptor, while the other p75NTR receptor is markedly downregulated. This pattern of neurotrophin expression confers protection against virus-induced apoptosis, and its inhibition amplifies programmed cell death in the infected bronchial epithelium. Thus, pharmacologic modulation of NGF expression may offer a promising new approach for management of common respiratory infections.
doi:10.1371/journal.pone.0006444
PMCID: PMC2715860  PMID: 19649262
20.  Retinoic Acid-Inducible Gene I Mediates Early Antiviral Response and Toll-Like Receptor 3 Expression in Respiratory Syncytial Virus-Infected Airway Epithelial Cells▿  
Journal of Virology  2006;81(3):1401-1411.
Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respiratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I “knockdown” significantly inhibited early nuclear factor-κB (NF-κB) and interferon response factor 3 (IRF3) activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-β), interferon-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expression levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18 h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-κB binding but did inhibit IFN-β, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that TLR3 knockdown significantly reduced NF-κB/RelA transcription by its ability to block the activating phosphorylation of NF-κB/RelA at serine residue 276. We further found that TLR3 induction following RSV infection was regulated by RIG-I-dependent IFN-β secreted from infected airway epithelial cells and was mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways controlling NF-κB activation.
doi:10.1128/JVI.01740-06
PMCID: PMC1797494  PMID: 17108032
21.  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
22.  Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation 
PLoS ONE  2014;9(7):e102368.
Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63+ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.
doi:10.1371/journal.pone.0102368
PMCID: PMC4102526  PMID: 25033192
23.  Local IL-17A Potentiates Early Neutrophil Recruitment to the Respiratory Tract during Severe RSV Infection 
PLoS ONE  2013;8(10):e78461.
Respiratory syncytial virus (RSV) bronchiolitis triggers a strong innate immune response characterized by excessive neutrophil infiltration which contributes to RSV induced pathology. The cytokine IL-17A enhances neutrophil infiltration into virus infected lungs. IL-17A is however best known as an effector of adaptive immune responses. The role of IL-17A in early immune modulation in RSV infection is unknown. We aimed to elucidate whether local IL-17A facilitates the innate neutrophil infiltration into RSV infected lungs prior to adaptive immunity. To this end, we studied IL-17A production in newborns that were hospitalized for severe RSV bronchiolitis. In tracheal aspirates we measured IL-17A concentration and neutrophil counts. We utilized cultured human epithelial cells to test if IL-17A regulates RSV infection-induced IL-8 release as mediator of neutrophil recruitment. In mice we investigated the cell types that are responsible for early innate IL-17A production during RSV infection. Using IL-17A neutralizing antibodies we tested if IL-17A is responsible for innate neutrophil infiltration in mice. Our data show that increased IL-17A production in newborn RSV patient lungs correlates with subsequent neutrophil counts recruited to the lungs. IL-17A potentiates RSV-induced production of the neutrophil-attracting chemokine IL-8 by airway epithelial cells in vitro. Various lung-resident lymphocytes produced IL-17A during early RSV infection in Balb/c mice, of which a local population of CD4 T cells stood out as the predominant RSV-induced cell type. By removing IL-17A during early RSV infection in mice we showed that IL-17A is responsible for enhanced innate neutrophil infiltration in vivo. Using patient material, in vitro studies, and an animal model of RSV infection, we thus show that early local IL-17A production in the airways during RSV bronchiolitis facilitates neutrophil recruitment with pathologic consequences to infant lungs.
doi:10.1371/journal.pone.0078461
PMCID: PMC3806820  PMID: 24194936
24.  SELENIUM DEFICIENCY ALTERS EPITHELIAL CELL MORPHOLOGY AND RESPONSES TO INFLUENZA 
Free radical biology & medicine  2007;42(12):1826-1837.
It is unknown whether nutritional deficiencies affect the morphology and function of structural cells, such as epithelial cells, and modify the susceptibility to viral infections. We developed an in vitro system of differentiated human bronchial epithelial cells (BEC) grown either under selenium adequate (Se+) or selenium deficient (Se-) conditions, to determine whether selenium deficiency impairs host defense responses at the level of the epithelium. Se- BECs had normal SOD activity, but decreased activity of the selenium-dependent enzyme GPX1. Interestingly, catalase activity was also decreased in Se- BECs. Both Se- and Se+ BECs differentiated into a mucociliary epithelium; however, Se- BEC demonstrated increased mucus production and increased Muc5AC mRNA levels. This effect was also seen in Se+ BEC treated with 3-aminotriazole, and inhibitor of catalase activity, suggesting an association between catalase activity and mucus production. Both Se- and Se+ were infected with influenza A/Bangkok/1/79 and examined 24 hours post-infection. Influenza-induced IL-6 production was greater while influenza-induced IP-10 production was lower in Se- BECs. In addition, influenza-induced apoptosis was greater in Se- BEC as compared to the Se+ BECs. These data demonstrate that selenium deficiency has a significant impact on the morphology and influenza-induced host defense responses in human airway epithelial cells.
doi:10.1016/j.freeradbiomed.2007.03.017
PMCID: PMC2048669  PMID: 17512462
Influenza; selenium; bronchial epithelial cells; in vitro
25.  Alpha/Beta Interferon Receptor Signaling Amplifies Early Proinflammatory Cytokine Production in the Lung during Respiratory Syncytial Virus Infection 
Journal of Virology  2014;88(11):6128-6136.
ABSTRACT
Type I interferons (IFNs) are produced early upon virus infection and signal through the alpha/beta interferon (IFN-α/β) receptor (IFNAR) to induce genes that encode proteins important for limiting viral replication and directing immune responses. To investigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways, we examined their importance in early lung responses to infection with respiratory syncytial virus (RSV). IFNAR1-deficient (IFNAR1−/−) mice displayed increased lung viral load and weight loss during RSV infection. As expected, expression of IFN-inducible genes was markedly reduced in the lungs of IFNAR1−/− mice. Surprisingly, we found that the levels of proinflammatory cytokines and chemokines in the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling. Furthermore, low levels of proinflammatory cytokines were also detected in the lungs of IFNAR1−/− mice challenged with noninfectious innate immune stimuli such as selected Toll-like receptor (TLR) agonists. Finally, recombinant IFN-α was sufficient to potentiate the production of inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli. Thus, in addition to its well-known role in antiviral resistance, type I IFN receptor signaling acts as a central driver of early proinflammatory responses in the lung. Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases characterized by enhanced inflammatory cytokine production.
IMPORTANCE The initial response to viral infection is characterized by the production of interferons (IFNs). One group of IFNs, the type I IFNs, are produced early upon virus infection and signal through the IFN-α/β receptor (IFNAR) to induce proteins important for limiting viral replication and directing immune responses. Here we examined the importance of type I IFNs in early responses to respiratory syncytial virus (RSV). Our data suggest that type I IFN production and IFNAR receptor signaling not only induce an antiviral state but also serve to amplify proinflammatory responses in the respiratory tract. We also confirm this conclusion in another model of acute inflammation induced by noninfectious stimuli. Our findings are of relevance to human disease, as RSV is a major cause of infant bronchiolitis and polymorphisms in the IFN system are known to impact disease severity.
doi:10.1128/JVI.00333-14
PMCID: PMC4093897  PMID: 24648449

Results 1-25 (1164441)