Goblet Cell Hyperplasia Increases SARS-CoV-2 Infection in COPD

  1. Jaspreet K. Osan
  2. Sattya N. Talukdar
  3. Friederike Feldmann
  4. Beth Ann DeMontigny
  5. Kailey Jerome
  6. Kristina L. Bailey
  7. Heinz Feldmann
  8. Masfique Mehedi

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Abstract

SARS-CoV-2 has become a major problem across the globe, with approximately 50 million cases and more than 1 million deaths and currently no approved treatment or vaccine. Chronic obstructive pulmonary disease (COPD) is one of the underlying conditions in adults of any age that place them at risk for developing severe illness associated with COVID-19. We established an airway epithelium model to study SARS-CoV-2 infection in healthy and COPD lung cells. We found that both the entry receptor ACE2 and the co-factor transmembrane protease TMPRSS2 are expressed at higher levels on nonciliated goblet cell, a novel target for SARS-CoV-2 infection. We observed that SARS-CoV-2 infected goblet cells and induced syncytium formation and cell sloughing. We also found that SARS-CoV-2 replication was increased in the COPD airway epithelium likely due to COPD associated goblet cell hyperplasia. Our results reveal goblet cells play a critical role in SARS-CoV-2 infection in the lung.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.11.379099: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationAt least two random fields were selected per sample and imaged.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The Transwell inserts were then incubated with the following primary antibodies (Abs) (alone or in combination) in IF washing buffer overnight at 4°C: anti-ZO-1 rabbit polyclonal (1:200) (Thermo Fisher Scientific), anti-E-cadherin rabbit monoclonal (1:200) (Cell Signaling Technologies), anti-MUC5B rabbit monoclonal (1:500) (Atlas Antibodies), anti-MUC5AC mouse polyclonal (1:100) (Abnova), anti-MUC5AC rabbit monoclonal (1:200) (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal (1:500) (Cell Signaling Technologies), anti-TMPRSS2 mouse monoclonal (1:50) (Santa Cruz Biotechnology), anti-SARS CoV-2 nucleocapsid mouse monoclonal (1:10) (Thermo Fisher Scientific) and anti-ACE2 mouse monoclonal (1:20) (R&D Systems).
    anti-ZO-1
    suggested: (SICGEN Cat# AB0054-200, RRID:AB_2333162)
    anti-E-cadherin
    suggested: (Thermo Fisher Scientific Cat# 13-1800, RRID:AB_2533004)
    anti-MUC5B
    suggested: (LSBio (LifeSpan Cat# LS-C9123-100, RRID:AB_11204778)
    anti-acetyl-alpha-tubulin
    suggested: (Affinity Biosciences Cat# DF2982, RRID:AB_2840961)
    anti-TMPRSS2
    suggested: None
    anti-SARS
    suggested: None
    anti-ACE2
    suggested: None
    After incubation with blocking buffer, the tissue was immediately incubated with the following primary antibodies overnight at 4°C in a humidified, light-protected chamber: SARS-CoV-2 N (1:50) (Thermo Fischer Scientific) for the detection of SARS-CoV-2 nucleoprotein (N), SARS-CoV-2 spike (S) protein (1:100) (Thermo Fischer Scientific) for the detection of SARS-CoV2 S protein, acetylated-alpha-tubulin (1:500) (Cell Signaling Technologies) for the staining of ciliated cells, MUC5AC (1:500) (Cell Signaling Technologies) and MUC5B (1:500) (Atlas Antibodies) for the staining of goblet cells, and P63 (1:100) (Abcam Inc.) for the staining of basal cells.
    SARS-CoV2 S protein, acetylated-alpha-tubulin
    suggested: None
    MUC5AC
    suggested: (Leica Biosystems Cat# NCL-MUC-5AC, RRID:AB_442113)
    P63
    suggested: None
    The next day, the slides were washed three times with PBST and then incubated for 45 minutes with anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific) and anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific) secondary antibodies in a humidified, light-protected chamber.
    anti-mouse
    suggested: None
    anti-rabbit
    suggested: None
    ACE2 was detected by Western blotting using anti-ACE2 goat polyclonal antibody (R&D Systems) and corresponding donkey anti-goat IRDye 800 secondary antibodies (Li-Cor Biosciences).
    anti-goat
    suggested: None
    For the loading control, alpha-tubulin was detected by anti-alpha-tubulin mouse monoclonal antibody (Sigma-Aldrich) and the corresponding goat anti-mouse IRDye 680 secondary antibody (Li-Cor Biosciences).
    anti-alpha-tubulin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Tissue culture infective dose 50 (TCID50): Vero cells were seeded on 96-well plates to confluency, and SARS-CoV-2 endpoint titration was performed.
    Vero
    suggested: None
    We also followed a similar approach for the extraction of RNA from A549 cells.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Software and Algorithms
    SentencesResources
    The cells were maintained in airway epithelial cell (AEC) growth medium (PromoCell) containing AEC supplements (PromoCell), 2% penicillin/streptomycin (Thermos Fisher Scientific), and 1% amphotericin B (Thermos Fisher Scientific) (complete AEC medium) at 37°C in an incubator with 5% CO2.
    Thermos Fisher Scientific
    suggested: None
    The images were processed with Imaris software version 9.5.1 (Oxford Instruments Group) and used for the conversion of Z-stack images (.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    We used nine independent images to quantify the total cell number based on F-actin (Texas Red channel) and goblet cells using the Alexa Flour 647 channel (anti-MUC5B) with the Fiji multipointer option.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One of the limitations in the ALI culture research is passaging of primary NHBE cells may impact on their ability to differentiate into airway epithelium. We could demonstrate that primary NHBE cells obtained after four passages without using any additional supplements can still be differentiated into human airway epithelium (Rayner et al., 2019). In a separate study, we confirmed that passaging NHBE cells up to four times has insignificant effect on the whole-genome transcriptome by comparing transcriptome profiles of each passage cells (data not shown). The ability to expand primary cells that also form fully differentiated mucociliary epithelium reduces repeat sample collections from patients where samples are difficult and limited, such as infants and deceased patients (Rayner et al., 2019; Wolf et al., 2017). Our results demonstrate primary NHBE cells either from healthy or a COPD patient can be passaged up to four times and that normal epithelial phenotypic features are maintained in passaged primary NHBE cells. One emergent question is why the human-to-human transmission of SARS-CoV-2 is much higher compared to SARS-CoV-1, although both viruses share ACE2 as cell surface receptor and use TMPRSS2 to facilitate their entry into the host cell (Hoffmann et al., 2020; Lukassen et al., 2020). The SARS-CoV-2 spike protein has an additional furin cleavage site that is absent in SARS-CoV-1, and it is hypothesized that furin cleavage facilitates human-to-human transmission (Cout...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 40, 42, 43, 44, 46 and 48. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.11.11.379099 on bioRxiv