Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control

ABSTRACT Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-β-, IFN-λ-, and NF-κB-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells. IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.

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Fimbria-Mediated Enhanced Attachment of NontypeableHaemophilus influenzae to Respiratory Syncytial Virus-Infected Respiratory Epithelial Cells

Infection and Immunity ◽

10.1128/iai.67.1.187-192.1999 ◽

1999 ◽

Vol 67 (1) ◽

pp. 187-192 ◽

Cited By ~ 62

Author(s):

Zili Jiang ◽

Nobuo Nagata ◽

Edgar Molina ◽

Lauren O. Bakaletz ◽

Hal Hawkins ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

A549 Cells ◽

Respiratory Epithelial Cells ◽

Nthi Strain ◽

Soluble Mediator ◽

Isogenic Mutant Strain ◽

Rsv Infection ◽

Syncytial Virus ◽

Respiratory Epithelial

ABSTRACT Respiratory syncytial virus (RSV) infection is known to predispose children to otitis media and sinusitis due to bacteria such as nontypeable Haemophilus influenzae (NTHI). In this study, we investigated the role of NTHI surface outer membrane protein P5-homologous fimbriae (P5-fimbriae) in attachment to RSV-exposed A549 epithelial cells. Analysis by fluorescence flow cytometry showed that a live P5-fimbriated NTHI strain (NTHIF+) attached to a higher proportion of RSV-exposed A549 cells than to control cells (mean, 68% for RSV versus 29% for control; P = 0.008), while attachment of the P5-fimbriae-deficient isogenic mutant strain (NTHIF−) was significantly lower than in control cells and rose only slightly following RSV exposure (mean, 17% for RSV versus 10% for control, P = 0.229). Attachment of NTHIF+ did not correlate with the amount of RSV antigen expressed by A549 cells. Furthermore, paraformaldehyde-fixed NTHIF+ also demonstrated an enhanced binding to RSV-exposed cells. Observations by transmission electronic microscopy showed that the mean number of bacteria attached per 100 RSV-exposed A549 cells was higher for NTHIF+ than NTHIF− (99 versus 18; P < 0.001). No intracellular bacteria were identified. UV-irradiated conditioned supernatants collected from RSV-infected A549 cultures (UV-cRSV) also enhanced the attachment of NTHIF+ to A549, suggesting the presence of a preformed soluble mediator(s) in UV-cRSV that enhances the expression of receptors for P5-fimbriae on A549 cells. In summary, RSV infection significantly enhances NTHI attachment to respiratory epithelial cells. P5-fimbria is the critical appendage of NTHI that participates in this attachment. In clinical settings, blocking of the P5-fimbria-mediated attachment of NTHIF+ by passive or active immunity may reduce the morbidity due to NTHI during RSV infection.

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Synergistic Upregulation of Interleukin-8 Secretion from Pulmonary Epithelial Cells by Direct and Monocyte-Dependent Effects of Respiratory Syncytial Virus Infection

Journal of Virology ◽

10.1128/jvi.74.18.8425-8433.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8425-8433 ◽

Cited By ~ 26

Author(s):

Lynette H. Thomas ◽

Melissa I. Y. Wickremasinghe ◽

Mike Sharland ◽

Jon S. Friedland

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

A549 Cells ◽

Interleukin 8 ◽

Respiratory Epithelial Cells ◽

Necrosis Factor Alpha ◽

Leukocyte Influx ◽

Rsv Infection ◽

Syncytial Virus ◽

Respiratory Epithelial

ABSTRACT Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 ± 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 ± 2,160 pg/ml; RSV alone, 12,170 ± 300 pg/ml; RSV-CM plus RSV, 27,040 ± 5,260 pg/ml; P < 0.05). RSV-CM induced degradation of IκBα within 5 min but did not affect IκBβ. RSV-CM activated transient nuclear binding of NF-κB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-κB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-κB-dependent, NF-IL6-requiring mechanism.

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Central Role of the NF-κB Pathway in theScgb1a1-Expressing Epithelium in Mediating Respiratory Syncytial Virus-Induced Airway Inflammation

Journal of Virology ◽

10.1128/jvi.00441-18 ◽

2018 ◽

Vol 92 (11) ◽

Cited By ~ 11

Author(s):

Bing Tian ◽

Jun Yang ◽

Yingxin Zhao ◽

Teodora Ivanciuc ◽

Hong Sun ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cell ◽

Epithelial Cells ◽

Lower Respiratory Tract ◽

Pol Ii ◽

Neutrophilic Inflammation ◽

Rsv Infection ◽

Syncytial Virus ◽

Epithelial Cell Population

ABSTRACTLower respiratory tract infection with respiratory syncytial virus (RSV) produces profound inflammation. Despite an understanding of the role of adaptive immunity in RSV infection, the identity of the major sentinel cells initially triggering inflammation is controversial. Here we evaluate the role of nonciliated secretoglobin (Scgb1a1)-expressing bronchiolar epithelial cells in RSV infection. Mice expressing a tamoxifen (TMX)-inducible Cre recombinase-estrogen receptor fusion protein (CreERTM) knocked into theScgb1a1locus were crossed with mice that harbor aRelAconditional allele (RelAfl), with loxP sites flanking exons 5 to 8 of the Rel homology domain. TheScgb1a1CreERTM/+× RelAfl/flmouse is aRelAconditional knockout (RelACKO) of a nonciliated epithelial cell population enriched in the small bronchioles. TMX-treated RelACKOmice have reduced pulmonary neutrophilic infiltration and impaired expression and secretion of NF-κB-dependent cytokines in response to RSV. In addition, RelACKOmice had reduced expression levels of interferon (IFN) regulatory factor 1/7 (IRF1/7) and retinoic acid-inducible gene I (RIG-I), components of the mucosal IFN positive-feedback loop. We demonstrate that RSV replication induces RelA to complex with bromodomain-containing protein 4 (BRD4), a cofactor required for RNA polymerase II (Pol II) phosphorylation, activating the atypical histone acetyltransferase (HAT) activity of BRD4 required for phospho-Ser2 Pol II formation, histone H3K122 acetylation, and cytokine secretionin vitroandin vivo. TMX-treated RelACKOmice have less weight loss and reduced airway obstruction/hyperreactivity yet similar levels of IFN-γ production despite higher levels of virus production. These data indicate that the nonciliatedScgb1a1-expressing epithelium is a major innate sensor for restricting RSV infection by mediating neutrophilic inflammation and chemokine and mucosal IFN production via the RelA-BRD4 pathway.IMPORTANCERSV infection is the most common cause of infant hospitalizations in the United States, resulting in 2.1 million children annually requiring medical attention. RSV primarily infects nasal epithelial cells, spreading distally to produce severe lower respiratory tract infections. Our study examines the role of a nonciliated respiratory epithelial cell population in RSV infection. We genetically engineered a mouse that can be selectively depleted of the NF-κB/RelA transcription factor in this subset of epithelial cells. These mice show an impaired activation of the bromodomain-containing protein 4 (BRD4) coactivator, resulting in reduced cytokine expression and neutrophilic inflammation. During the course of RSV infection, epithelial RelA-depleted mice have reduced disease scores and airway hyperreactivity yet increased levels of virus replication. We conclude that RelA-BRD4 signaling in nonciliated bronchiolar epithelial cells mediates neutrophilic airway inflammation and disease severity. This complex is an attractive target to reduce the severity of infection.

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Respiratory Syncytial Virus-Induced Host IFN Signaling Differs Between A549 and BEAS-2B Epithelial Cell Lines

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2014.12.964 ◽

2015 ◽

Vol 135 (2) ◽

pp. AB9

Author(s):

Philippa Hillyer ◽

Rachel E. Shepard ◽

Megan Uehling ◽

Faruk Sheik ◽

Cindy Luongo ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cell ◽

Cell Lines ◽

Epithelial Cell Lines ◽

Syncytial Virus

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β2-integrin LFA1 mediates airway damage following neutrophil transepithelial migration during respiratory syncytial virus infection

European Respiratory Journal ◽

10.1183/13993003.02216-2019 ◽

2020 ◽

Vol 56 (2) ◽

pp. 1902216 ◽

Cited By ~ 2

Author(s):

Jenny Amanda Herbert ◽

Yu Deng ◽

Pia Hardelid ◽

Elisabeth Robinson ◽

Luo Ren ◽

...

Keyword(s):

Viral Load ◽

Respiratory Syncytial Virus ◽

Epithelial Cell ◽

Epithelial Cells ◽

Cell Damage ◽

Antiviral Treatment ◽

Beat Frequency ◽

Antiviral Drug ◽

Rsv Infection ◽

Syncytial Virus

Respiratory syncytial virus (RSV) bronchiolitis is the most common cause of infant hospital admissions, but there is limited understanding of the mechanisms of disease, and no specific antiviral treatment. Using a novel in vitro primary transepithelial neutrophil migration model and innovative imaging methods, we show that RSV infection of nasal airway epithelium increased neutrophil transepithelial migration and adhesion to infected epithelial cells, which is associated with epithelial cell damage and reduced ciliary beat frequency, but also with a reduction in infectious viral load.Following migration, RSV infection results in greater neutrophil activation, degranulation and release of neutrophil elastase into the airway surface media compared to neutrophils that migrated across mock-infected nasal epithelial cells. Blocking of the interaction between the ligand on neutrophils (the β2-integrin LFA-1) for intracellular adhesion molecule (ICAM)-1 on epithelial cells reduced neutrophil adherence to RSV-infected cells and epithelial cell damage to pre-infection levels, but did not reduce the numbers of neutrophils that migrated or prevent the reduction in infectious viral load.These findings have provided important insights into the contribution of neutrophils to airway damage and viral clearance, which are relevant to the pathophysiology of RSV bronchiolitis. This model is a convenient, quantitative preclinical model that will further elucidate mechanisms that drive disease severity and has utility in antiviral drug discovery.

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Respiratory syncytial virus activates Rab5a to suppress IRF1-dependent IFN-λ production, subverting the antiviral defense of airway epithelial cells

Journal of Virology ◽

10.1128/jvi.02333-20 ◽

2021 ◽

Author(s):

Shi Mo ◽

Wei Tang ◽

Jun Xie ◽

Sisi Chen ◽

Luo Ren ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Antiviral Immunity ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Novel Therapeutics ◽

Potential Target ◽

Rsv Infection ◽

Syncytial Virus ◽

Respiratory Epithelial

The limited antiviral options and lack of an effective vaccine against human respiratory syncytial virus (RSV) highlight the need for a novel antiviral therapy. One alternative is to identify and target the host factors required for viral infection. Here, using RNA interference to knock down Rab proteins, we provide multiple lines of evidence that Rab5a is required for RSV infection: (a) Rab5a is upregulated both in RSV-A2-infected A549 cells and RSV-A2-challenged BALB/c mice’s airway epithelial cells at early infection phase; (b) shRNA-mediated knockdown of Rab5a is associated with reduced lung pathology in RSV A2 challenged mice; (c) Rab5a expression is correlated with disease severity of RSV infection of infants. Knockdown of Rab5a increases IFN-λ (lambda) production by mediating IRF1 nuclear translocation. Our results highlight a new role for Rab5a in RSV infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity, which suggests that Rab5a is a potential target for novel therapeutics against RSV infection. Importance This study highlights the important role of Rab5a in RSV infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity and attenuates inflammation of the airway, which suggests that Rab5a is a powerful potential target for novel therapeutics against RSV infection.

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Incomplete Regulation of NF-κB by IκBα during Respiratory Syncytial Virus Infection in A549 Cells

Journal of Virology ◽

10.1128/jvi.73.5.4502-4507.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4502-4507 ◽

Cited By ~ 15

Author(s):

Michael A. Fiedler ◽

Kara Wernke-Dollries

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

A549 Cells ◽

Inhibitory Effect ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Rsv Infection ◽

Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) infection of airway epithelial cells results in persistent NF-κB activation and NF-κB-mediated interleukin-8 production. Previous studies in airway epithelial cells demonstrated that tumor necrosis factor alpha (TNF-α)-induced NF-κB activation is transient due to regulation by IκBα. However, during RSV infection, IκBα has only a partial inhibitory effect on NF-κB activation. Studies presented here demonstrate that neither increased IκBα production which occurs as a result of RSV-induced NF-κB activation nor inhibition of proteasome-mediated IκBα degradation results in a reversal of RSV-induced NF-κB activation. Thus, while manipulation of IκBα results in reversal of TNF-α-induced NF-κB activation, manipulation of IκBα does not result in a reversal of RSV-induced NF-κB activation.

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Respiratory Syncytial Virus Replication Is Promoted by Autophagy-Mediated Inhibition of Apoptosis

Journal of Virology ◽

10.1128/jvi.02193-17 ◽

2018 ◽

Vol 92 (8) ◽

Cited By ~ 21

Author(s):

Miao Li ◽

Jian Li ◽

Ruihong Zeng ◽

Jianling Yang ◽

Jianguo Liu ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Virus Replication ◽

Cell Apoptosis ◽

Related Gene ◽

Respiratory Epithelial Cells ◽

Host Interactions ◽

Rsv Infection ◽

Syncytial Virus ◽

Respiratory Epithelial

ABSTRACT Respiratory syncytial virus (RSV) is the main cause of acute lower respiratory tract infection (ALRI) in children worldwide. Virus-host interactions affect the progression and prognosis of the infection. Autophagy plays important roles in virus-host interactions. Respiratory epithelial cells serve as the front line of host defense during RSV infection, However, it is still unclear how they interact with RSV. In this study, we found that RSV induced autophagy that favored RSV replication and exacerbated lung pathology in vivo . Mechanistically, RSV induced complete autophagy flux through reactive oxygen species (ROS) generation and activation of the AMP-activated protein kinase/mammalian target of rapamycin (AMPK-MTOR) signaling pathway in HEp-2 cells. Furthermore, we evaluated the functions of autophagy in RSV replication and found that RSV replication was increased in HEp-2 cells treated with rapamycin but decreased remarkably in cells treated with 3-methylademine (3-MA) or wortmannin. Knockdown key molecules in the autophagy pathway with short hairpinp RNA (shRNA) against autophagy-related gene 5 ( ATG5 ), autophagy-related gene 7 ( ATG7 ), or BECN1/Beclin 1 or treatment with ROS scavenger N-acetyl- l -cysteine (NAC) and AMPK inhibitor (compound C) suppressed RSV replication. 3-MA or sh ATG5/BECN1 significantly decreased cell viability and increased cell apoptosis at 48 hours postinfection (hpi). Blocking apoptosis with Z-VAD-FMK partially restored virus replication at 48 hpi. Those results provide strong evidence that autophagy may function as a proviral mechanism in a cell-intrinsic manner during RSV infection. IMPORTANCE An understanding of the mechanisms that respiratory syncytial virus utilizes to interact with respiratory epithelial cells is critical to the development of novel antiviral strategies. In this study, we found that RSV induces autophagy through a ROS-AMPK signaling axis, which in turn promotes viral infection. Autophagy favors RSV replication through blocking cell apoptosis at 48 hpi. Mechanistically, RSV induces mitophagy, which maintains mitochondrial homeostasis and therefore decreases cytochrome c release and apoptosis induction. This study provides a novel insight into this virus-host interaction, which may help to exploit new antiviral treatments targeting autophagy processes.

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Differential Mucin Expression by Respiratory Syncytial Virus and Human Metapneumovirus Infection in Human Epithelial Cells

Mediators of Inflammation ◽

10.1155/2015/347292 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Ma. Del Rocío Baños-Lara ◽

Boyang Piao ◽

Antonieta Guerrero-Plata

Keyword(s):

Immune Response ◽

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Viral Infections ◽

Human Metapneumovirus ◽

Human Epithelial Cells ◽

Mucin Expression ◽

Rsv Infection ◽

Syncytial Virus ◽

Tract Disease

Mucins (MUC) constitute an important component of the inflammatory and innate immune response. However, the expression of these molecules by respiratory viral infections is still largely unknown. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two close-related paramyxoviruses that can cause severe low respiratory tract disease in infants and young children worldwide. Currently, there is not vaccine available for neither virus. In this work, we explored the differential expression of MUC by RSV and hMPV in human epithelial cells. Our data indicate that the MUC expression by RSV and hMPV differs significantly, as we observed a stronger induction of MUC8, MUC15, MUC20, MUC21, and MUC22 by RSV infection while the expression of MUC1, MUC2, and MUC5B was dominated by the infection with hMPV. These results may contribute to the different immune response induced by these two respiratory viruses.

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