Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 spike protein

  1. Hung Caohuy
  2. Ofer Eidelman
  3. Tinghua Chen
  4. Ognoon Mungunsukh
  5. Qingfeng Yang
  6. Nathan I. Walton
  7. Bette S. Pollard
  8. Sara Khanal
  9. Shannon Hentschel
  10. Catalina Florez
  11. Andrew S. Herbert
  12. Harvey B. Pollard

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Abstract

SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air–liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.

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  1. Version published to 10.1038/s41598-024-66473-4
  2. Version published to 10.1101/2022.01.18.476803v3 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2022.01.18.476803: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    CFTR in the immuno-precipitated protein complexes was analyzed by western blotting using anti-CFTR antibodies, clones UNC 570 and UNC 660, development of which were which were funded by the Cystic Fibrosis Foundation, and purchased by us from the University of North Carolina, Chapel Hill, NC).
    anti-CFTR
    suggested: None
    Recombinant proteins and antibodies: Recombinant proteins produced in human T293 cells were obtained as follows: Recombinant (exodomain) Human Angiotensin Converting Enzyme 2 (ACE2) (Cat # 230-30165; lot 04U0620TW) was purchased from RayBiotech (Peachtree Cornewrs, GA, 30092)
    Human Angiotensin Converting Enzyme 2
    suggested: (Abcam Cat# ab78512, RRID:AB_1565849)
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant proteins and antibodies: Recombinant proteins produced in human T293 cells were obtained as follows: Recombinant (exodomain) Human Angiotensin Converting Enzyme 2 (ACE2) (Cat # 230-30165; lot 04U0620TW) was purchased from RayBiotech (Peachtree Cornewrs, GA, 30092)
    T293
    suggested: RRID:CVCL_T293)
    Software and Algorithms
    SentencesResources
    Statistics: To determine the significance of changes in kinetic parameters of ACE2 binding to SARS-CoV-2 Spike mutants we applied least-squares regression to the linearized Eadie-Hoffstee plot of the binding data, and determined the statistical significance of the difference between the slopes (i.e., KD’s) and between the intercepts (i.e., Bmax) using R or Stata statistical packages.
    Stata
    suggested: (Stata, RRID:SCR_012763)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    This study has limitations. First, it is a limitation of our study that we do not yet understand how ACE2 binds to CFTR. Nonetheless, we know experimentally that they do bind together, both in epithelia and in basal cells. Second, it is a limitation that we do not yet understand the nature of the apparent damage or instability inflicted on CFTR by the Spike:ACE2:CFTR interaction. Nonetheless, the classical impermeant biotin experiment indicates this must happen. Third, we do not yet know which cells in the model epithelia are experimentally responsible for the CFTR and ACE2 activities. However, the basal cells possess both ACE2 and CFTR and share some activities with the epithelia. Furthermore, secretory cells, which dominate airway CFTR expression in native superficial epithelia [75], are present in the differentiated BCi-NS1.1 epithelia [39], as is ACE2 [38]. We suggest that substantively addressing these questions would be clearly beyond the experimental scope of the present study and look forward to addressing them in the future.

    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: We did not find any issues relating to colormaps.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  4. Version published to 10.1101/2022.01.18.476803v2 on bioRxiv
  5. Version published to 10.1101/2022.01.18.476803v1 on bioRxiv