In vitro infection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response

  1. Matthew A. Schaller
  2. Yamini Sharma
  3. Zadia Dupee
  4. Duy Nguyen
  5. Juan Uruena
  6. Ryan Smolchek
  7. Julia C. Loeb
  8. Tiago N. Machuca
  9. John A. Lednicky
  10. David J. Odde
  11. Robert F. Campbell
  12. W. Gregory Sawyer
  13. Borna Mehrad

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Abstract

Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the in vitro study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 16 donors, most of whom had risk factors for severe illness from COVID-19. Cryopreserved tissues preserved 90% of cell viability and contained heterogeneous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectible with HCoV-OC43 and SARS-CoV-2 coronavirus strains, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL-6, CXCL8 and IFNβ in response to SARS-CoV-2 infection. Treatment of tissues with dexamethasone and the experimental drug, N-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted antiviral activity, suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the in vitro study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.

Importance

The current biological systems for the study of COVID-19 are in vitro systems that differ from the human lung in many respects, and animal hosts to which the virus is not adapted. We developed another alternative for studying pathogenesis and drug susceptibility of SARS-CoV-2 in a cryopreserved bank of human lung tissues. We consider the importance of this work to relate to the practical use of this culture system as a repeatable and scalable approach that allows for the study of an important infection in relevant tissues.

The tissue bank highlights the heterogeneous response to SARS-CoV-2 infection and treatment, which allows researchers to investigate why treatments work in some donors but not others.

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  1. ScreenIT

    SciScore for 10.1101/2021.01.20.427541: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics statement: The study was performed in accordance with the Declaration of Helsinki, under a protocol approved by the University of Florida Institutional Review Board (IRB202000920) after written informed consent from participants.
    Consent: Ethics statement: The study was performed in accordance with the Declaration of Helsinki, under a protocol approved by the University of Florida Institutional Review Board (IRB202000920) after written informed consent from participants.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The antibodies used in this study include E-cadherin, polyclonal nuclear capsid and spike SARS-CoV-2 antibodies, and HCoV-OC43 antibodies (see table 1).
    E-cadherin
    suggested: None
    HCoV-OC43
    suggested: None
    When staining for ZO-1, samples were incubated with primary rabbit anti-ZO-1 antibody overnight at 4°C, followed by washing and incubating with conjugated secondary antibodies (A-21433; Invitrogen) against the appropriate species for 3h at room temperature.
    anti-ZO-1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral cultures: HCoV-OC43 (strain HCoV-OC43-JAL-1) and SARS-CoV-2 (strain UF-1) viruses were propagated in Vero-E6 cell line (ATCC® CRL-1586™) in advanced DMEM (Gibco™, 12491015; Thermo Fisher) supplemented with 10% HyClone™ Defined low antibody, heat-inactivated, gamma-irradiated fetal bovine serum (SH30070.03IR2540; Cytiva), 1% L-alanine and L-glutamine supplement (GlutaMAX™, 35050061; Gibco™), and 1% penicillin/streptomycin mixture (17-602E; Lonza™ BioWhittaker™), at 37°C in 5% CO2 in 75 cm2 flasks.
    Vero-E6
    suggested: None
    104 PFU was used for infection to enable a direct comparison between Vero cell cultures and lung microtissues infected with SARS-CoV-2.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    Viral cultures: HCoV-OC43 (strain HCoV-OC43-JAL-1) and SARS-CoV-2 (strain UF-1) viruses were propagated in Vero-E6 cell line (ATCC® CRL-1586™) in advanced DMEM (Gibco™, 12491015; Thermo Fisher) supplemented with 10% HyClone™ Defined low antibody, heat-inactivated, gamma-irradiated fetal bovine serum (SH30070.03IR2540; Cytiva), 1% L-alanine and L-glutamine supplement (GlutaMAX™, 35050061; Gibco™), and 1% penicillin/streptomycin mixture (17-602E; Lonza™ BioWhittaker™), at 37°C in 5% CO2 in 75 cm2 flasks.
    HCoV-OC43
    suggested: None
    Software and Algorithms
    SentencesResources
    Flow cytometry data was then analyzed using FlowJo X (BD biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Data were analyzed using Prism software (version 9.0 for Mac, GraphPad, San Diego, CA, USA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    While each of these technologies has its strengths and limitations, all are limited by the need for fresh tissues. Our study demonstrates that polyampholyte-based cryopreservation media can be used to preserve normal and diseased lung tissues in large batches. After cryopreservation, thawed microtissues displayed excellent viability, were metabolically active, and contained the major cell populations of the human lung. Our data is in agreement with previous studies demonstrating the advantages of using polyampholyte-based media as a cryopreservative (8, 9). A notable finding in our study was that the human tissues tested displayed similar infectability after inoculation with a given SARS-CoV-2 dose, as measured by expression of viral nucleocapsid proteins, suggesting that inter-individual variability in infection is not attributable to the susceptibility of host respiratory tissues to infection (Figures 3A-B). In contrast, infection with a given virus inoculum resulted in dramatic inter-individual heterogeneity in the cytokine responses of the infected tissues (Figures 3C-E). In this context, multiple studies have described the variability in host susceptibility to COVID-19 infection, using clinical outcomes, cytokine responses, and duration and extent of viral shedding as readouts, leading to the identification of acquired polymorphisms as risk factors for severe disease (31–37). Our data adds to this literature by assessing the effect of a uniform viral inoculum between hos...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.01.20.427541 on bioRxiv