Mechanical control of innate immune responses against viral infection revealed in a human Lung Alveolus Chip

  1. Haiqing Bai
  2. Longlong Si
  3. Amanda Jiang
  4. Chaitra Belgur
  5. Roberto Plebani
  6. Crystal Oh
  7. Melissa Rodas
  8. Atiq Nurani
  9. Sarah Gilpin
  10. Rani K. Powers
  11. Girija Goyal
  12. Rachelle Prantil- Baun
  13. Donald E. Ingber

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Abstract

Mechanical forces associated with breathing play a fundamental role in lung development and disease but the molecular pathways remain largely unknown. Here, we used a mechanically actuatable Human Lung Alveolus Chip that recapitulates human lung alveolar type I and type II cell differentiation, alveolar-capillary interface formation, and genome-wide gene expression profiles characteristic of the distal lung to investigate the role of physical forces associated with cyclic breathing motions in lung innate immune responses to viral infection. When the mechanically active Alveolus Chips are infected with the influenza H3N2 virus, a cascade of host responses is elicited on-chip, including increased production of cytokines and expression of inflammation-associated genes in pulmonary epithelial and endothelial cells, resulting in enhanced recruitment of circulating immune cells as occurs during viral infection in vivo. Surprisingly, studies carried out in parallel with static chips revealed that physiological breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells. This is mediated at least in part through upregulation of S100 calcium-binding protein A7 (S100A7), which binds to the Receptor for Advanced Glycation End Products (RAGE), an inflammatory mediator that is most highly expressed in the lung alveolus in vivo . This mechano-immunological control mechanism is further supported by the finding that existing RAGE inhibitor drugs can suppress the production of inflammatory cytokines in response to influenza virus infection in this model. S100A7-RAGE interactions and modulation of mechanical ventilation parameters could therefore serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.26.441498: (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
    Antibody Cytokine analysis: Vascular effluents from Alveolus Chips were collected and analyzed for a panel of cytokines and chemokines, including IL-6, IL-8, IP-10, TNF-α, RANTES, S100A8/A9, and GM-CSF using custom ProcartaPlex assay kits (Invitrogen).
    IL-6
    suggested: None
    IL-8
    suggested: None
    IP-10
    suggested: None
    TNF-α
    suggested: None
    S100A8/A9
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    OC43 coronavirus (VR-1558) was obtained from the ATCC and expanded in HCT-8 cells (ATCC) as previously described 59.
    HCT-8
    suggested: None
    Culture and transfection of A549 cells: A549 cells (ATCC CCL-185) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and penicillin-streptomycin (Life Technologies).
    A549
    suggested: None
    Confluent MDCK cell monolayers in 12-well plate were washed with PBS, inoculated with 1 mL of 10-fold serial dilutions of influenza virus samples for 1 hour at 37℃, and then overlaid with 1 mL of DMEM (Gibco) supplemented with 1.5% low melting point agarose (Sigma-Aldrich) and 2 µg/mL TPCK-treated trypsin (Sigma- Aldrich).
    MDCK
    suggested: None
    Recombinant DNA
    SentencesResources
    pCMV6-XL5 empty plasmid (PCMV6XL5) and S100A7 plasmid were purchased from Origene (#SC122639).
    pCMV6-XL5
    suggested: None
    S100A7
    suggested: None
    Software and Algorithms
    SentencesResources
    Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome using the STAR aligner v.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Unique gene hit counts were calculated by using feature Counts from the Subread package v.
    Subread
    suggested: (Subread, RRID:SCR_009803)
    1.5.2 followed by differential expression analysis using DESeq2.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Volcano plots were generated using the EnhancedVolcano R package.
    EnhancedVolcano
    suggested: (EnhancedVolcano, RRID:SCR_018931)
    Heatmaps and scatter plots were generated using the ggplot2 R package.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    RNA-sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under the accession code xxx.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    Fluorescence imaging was conducted using a confocal laser-scanning microscope (SP5 X MP DMI-6000, Germany) and image processing was done using the ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Endotoxin-free plasmid purification was performed by Genewiz.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Graphing and statistical comparison of the data were performed using Prism 9 (GraphPad Software).
    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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:
    • 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

    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 10.1101/2021.04.26.441498 on bioRxiv