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The Secretome Profiling of a Pediatric Airway Epithelium Infected with hRSV Identified Aberrant Apical/Basolateral Trafficking and Novel Immune Modulating (CXCL6, CXCL16, CSF3) and Antiviral (CEACAM1) Proteins*

  • Olivier Touzelet
    Affiliations
    Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK

    Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry & Biomedical Sciences, Queens University Belfast, Belfast BT9 7BL, UK
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  • Lindsay Broadbent
    Affiliations
    Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry & Biomedical Sciences, Queens University Belfast, Belfast BT9 7BL, UK
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  • Stuart D. Armstrong
    Affiliations
    Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK

    NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, L69 7BE, UK
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  • Waleed Aljabr
    Affiliations
    Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK

    Biomedical Research Administration, Research Centre, King Fahad Medical City, P.O. Box 59046 Riyadh 11252, Saudi Arabia
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  • Elaine Cloutman-Green
    Affiliations
    Microbiology, Virology and Infection Control, Level 4 Camelia Botnar Laboratory, Great Ormond Street Hospital, London WC1N 3JH, UK
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  • Ultan F. Power
    Correspondence
    To whom correspondence may be addressed: Centre for Experimental Medicine, School of Medicine, Dentistry & Biomedical Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, BT9 7BL, UK. Tel.: + 44 (0)28 90972285;
    Affiliations
    Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry & Biomedical Sciences, Queens University Belfast, Belfast BT9 7BL, UK
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  • Julian A. Hiscox
    Correspondence
    To whom correspondence may be addressed: Chair in Infection and Global Health, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool, L3 5RF, UK. Tel.: + 44 (0)151 795 0222;
    Affiliations
    Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK

    NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, L69 7BE, UK

    Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore 138648, Singapore
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  • Author Footnotes
    * This work was supported by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at the University of Liverpool in partnership with Public Health England (PHE) and Liverpool School of Tropical Medicine (LSTM) (S.D.A. and J.A.H.). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health, Public Health England. The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental material.
Open AccessPublished:February 19, 2020DOI:https://doi.org/10.1074/mcp.RA119.001546
      The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

      Graphical Abstract

      The epithelial lining fluid is essential in regulating the homeostasis of the airways. Apical secretions contain proteins associated with anti-oxidative, anti-protease, anti-microbial, and anti-inflammatory functions (
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      Analysis of the proteome of human airway epithelial secretions.
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      The abbreviations used are:
      COPD
      chronic obstructive pulmonary disease
      RSV
      respiratory syncytial virus
      HAE
      human airway epithelial model
      WD-PBECs
      well-differentiated pediatric bronchial epithelial cells
      BALF
      bronchoalveolar lavage fluid
      NPA
      nasopharyngeal aspirate
      CF
      cystic fibrosis
      ALRI
      airway lower respiratory tract infection.
      1The abbreviations used are:COPD
      chronic obstructive pulmonary disease
      RSV
      respiratory syncytial virus
      HAE
      human airway epithelial model
      WD-PBECs
      well-differentiated pediatric bronchial epithelial cells
      BALF
      bronchoalveolar lavage fluid
      NPA
      nasopharyngeal aspirate
      CF
      cystic fibrosis
      ALRI
      airway lower respiratory tract infection.
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      hRSV is a major cause of severe acute lower respiratory tract infection (ALRI) in young infants, the immunocompromised and the elderly. It has a high burden in children <5 years old, with ∼34 million new ALRI episodes each year and up to 200,000 deaths (
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      ,
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      ). RSV infection of HAE cultures recapitulates many of the features associated with hRSV infection in vivo (reviewed in (
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      Respiratory syncytial virus interaction with human airway epithelium.
      )). However, the impact of hRSV infection of the airway epithelium on the overall protein secretome is unknown. Therefore, the HAE model provides an ideal platform to address the proteome of airway secretions, thereby providing extensive information on the molecular changes associated with infection. Importantly, HAE cultures provide easily accessible apical and basolateral sides, thereby allowing analysis of airway secretions in both directions, in contrast to NPAs or BALF, which, for practical reasons can only inform changes associated with the apical-facing epithelial surface.
      This study evaluated the impact of hRSV on airway epithelium secretions from pediatric HAE cultures, defined as well-differentiated pediatric primary bronchial epithelial cells (WD-PBECs), by characterizing the protein composition of the apical and basolateral secretomes upon infection relative to mock-infected control cultures, using quantitative proteomics (LC-MS/MS). Importantly, novel proteins associated with RSV infection were identified, including proteins with immune modulating or antiviral activities. Furthermore, our HAE findings reflected changes in NPAs from hRSV-infected infants.

      DISCUSSION

      The proteome of airway secretions was unknown in the context of an acute respiratory virus infections. This study determined the impact of hRSV on the protein composition of airway epithelium secretions. For this purpose, WD-PBECs were infected with a clinical isolate of hRSV (BT2a), or mock-infected, and the apical and basolateral secretomes were profiled using quantitative proteomics.
      The data indicated that the directional nature of the airway secretome at homeostasis (
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      • Johnson E.
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      • Zurko J.
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      • Hathout Y.
      • Rose M.C.
      Directional secretomes reflect polarity-specific functions in an in vitro model of human bronchial epithelium.
      ) was markedly disrupted by hRSV. This was primarily evidenced by the altered polarity of secretion of a large panel of proteins (Fig. 4), encompassing multiple biological processes, and was indicative of a potentially dysfunctional epithelial barrier. Indeed, such epithelial leakage resembles the increased paracellular permeability observed in HAE cultures originating from asthmatic patients (
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      • Coyle P.V.
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      Relative respiratory syncytial virus cytopathogenesis in upper and lower respiratory tract epithelium.
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      Ciliary dyskinesia is an early feature of respiratory syncytial virus infection.
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      CXCR6 is expressed on T cells in both T helper type 1 (Th1) inflammation and allergen-induced Th2 lung inflammation but is only a weak mediator of chemotaxis.
      ) and in asthmatic patients (
      • Besnard A.-G.
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      ). In addition, whereas MUC13 exacerbated the responsiveness of intestinal epithelial cell lines to multiple TLR ligands, leading to increased IL-8 and TNF-α production (
      • Sheng Y.H.
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      MUC1 and MUC13 differentially regulate epithelial inflammation in response to inflammatory and infectious stimuli.
      ), its expression was only up-regulated in NPAs from patients with severe hRSV-associated disease, in contrast to milder cases (
      • Van Den Kieboom C.H.
      • Ahout I.M.L.
      • Zomer A.
      • Brand K.H.
      • De Groot R.
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      Nasopharyngeal gene expression, a novel approach to study the course of respiratory syncytial virus infection.
      ). Overall, our data suggest that CXCL6, CXCL16, CSF3 and, to a lesser extent, SECTM1 and MUC13, may constitute important but previously unidentified drivers/modulators of neutrophil infiltration and recruitment and/or activation of Th2 cells to the lungs during acute hRSV infection.
      In addition to identifying novel hRSV-induced mediators associated with recruitment of immunopathogenic immune cells, this work also highlighted direct cytophysiological and antiviral responses of airway epithelium to hRSV infection. For example, BST2 and CEACAM1, which have known antiviral properties against HIV, influenza virus or hMPV, were specifically induced upon hRSV infection (
      • Vitenshtein A.
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      ,
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      ,
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      ) and in the case of CEACAM1 this correlated with previous data in A549 and undifferentiated primary bronchial epithelial cells (
      • Avadhanula V.
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      • Devincenzo J.P.
      • Wang Y.
      • Webby R.J.
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      Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner.
      ). Importantly, this is the first description of the involvement of CEACAM1 in anti-hRSV defense (Fig. 8). Like BST2, CEACAM1 is normally a membrane-associated protein, but its secretion may be explained by soluble isoforms of the protein (
      • Terahara K.
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      • Taguchi F.
      • Igarashi O.
      • Nochi T.
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      ). Active cleavage of native CEACAM1 from cell surfaces is also possible, especially in such a protease-rich environment. In this study, CEACAM1 was identified as a novel secreted factor with antiviral properties against hRSV in the epithelium environment. Another novel observation was the loss of detection of two membrane transporters, SLC2A2 and ATPV6E6, which are involved in maintaining cation/Ca2+ homeostasis (Fig. 5A and supplemental Table S8). Indeed, efficient mucociliary clearance relies essentially on coordinated cilia movement, which is partly controlled by tight regulation of intracellular and extracellular levels of Ca2+ (
      • Schmid A.
      • Salathe M.
      Ciliary beat co-ordination by calcium.
      ,
      • Droguett K.
      • Rios M.
      • Carreño D.V.
      • Navarrete C.
      • Fuentes C.
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      An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.
      ). Altering the expression/secretion of these host factors suggests that hRSV might affect Ca2+ levels, which in turn could lead to dysregulation of cilia beating. This is consistent with the ability of hRSV to induce cilia beating dyskinesia, at least in the HAE model (
      • Smith C.M.
      • Kulkarni H.
      • Radhakrishnan P.
      • Rutman A.
      • Bankart M.J.
      • Williams G.
      • Hirst R.A.
      • Easton A.J.
      • Andrew P.W.
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      Ciliary dyskinesia is an early feature of respiratory syncytial virus infection.
      ). A reduced mucociliary clearance may indeed exacerbate hRSV-associated pathologies because it drives the transport of inhaled particles and pathogens trapped in the mucus out of the airways and toward the oropharynx.
      In conclusion, global analysis of the apical and basolateral secretomes following RSV infection revealed novel interactions between hRSV and the airway epithelium. It also identified novel anti-hRSV innate immune responses with potential for therapeutic intervention. The application of proteomics to our hRSV/WD-PBEC model, therefore, provided extensive new insights into hRSV/host interactions and the rationale for therapeutic target discovery.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Perez-Riverol Y.
      • Csordas A.
      • Bai J.
      • Bernal-Llinares M.
      • Hewapathirana S.
      • Kundu D.J.
      • Inuganti A.
      • Griss J.
      • Mayer G.
      • Eisenacher M.
      • Pérez E.
      • Uszkoreit J.
      • Pfeuffer J.
      • Sachsenberg T.
      • Yilmaz Ş
      • Tiwary S.
      • Cox J.
      • Audain E.
      • Walzer M.
      • Jarnuczak A.F.
      • Ternent T.
      • Brazma A.
      • Vizcaíno J.A.
      The PRIDE database and related tools and resources in 2019: Improving support for quantification data.
      ) partner repository with the data set identifier PXD013661. Data can be accessed at https://www.ebi.ac.uk/pride/.

      Acknowledgments

      We are grateful to The Centre for Proteome Research, University of Liverpool, for access to MS instrumentation, and to Prof. Paul Yeo (Durham University, UK) for providing the monoclonal antibody anti-hRSV M (M024).

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