ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection

  1. Krystal L. Ching
  2. Maren de Vries
  3. Juan Gago
  4. Kristen Dancel-Manning
  5. Joseph Sall
  6. William J. Rice
  7. Clea Barnett
  8. Alireza Khodadadi-Jamayran
  9. Aristotelis Tsirigos
  10. Feng-Xia Liang
  11. Lorna E. Thorpe
  12. Bo Shopsin
  13. Leopoldo N. Segal
  14. Meike Dittmann
  15. Victor J. Torres
  16. Ken Cadwell

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Article activity feed

  1. Version published to 10.1371/journal.pbio.3001754
  2. ScreenIT

    SciScore for 10.1101/2021.12.17.473223: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Surviving subjects signed informed consent to participate in this study while samples and metadata from subjects who died or were incapacitated were de-identified and included in this study.
    IRB: The study protocol was approved by the Institutional Review Board of New York University. Cell Culture and lentivirus production: A549 cells were purchased from ATCC.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Wells were fixed by submerging in 10% formalin for 24 h and washed three times with PBS before analysis by immunofluorescence.

    Table 2: Resources

    Antibodies
    SentencesResources
    Plates were permeabilized with 0.1% Triton-X 100, blocked with 1% BSA/PBS and stained for SARS-CoV-2 N mouse monoclonal SARS-CoV-2 anti-N antibody 1C7 (1:1000, kind gift of Thomas Moran) overnight at 4°C.
    anti-N
    suggested: None
    Cultures were stained with anti-SARS nucleocapsid protein antibody which cross reacts with SARS-CoV-2 N (1:1000, cat. no. 200-401-A50; Rockland) and goat-anti-rabbit Alexa Fluor 488 and DAPI.
    anti-SARS nucleocapsid protein
    suggested: (LSBio (LifeSpan Cat# LS-C75706-1000, RRID:AB_10636665)
    Alexa Fluor 488
    suggested: None
    Flow Cytometry: Exosomes pellets were stained with 100μl of an antibody cocktail in 1X PBS (Corning) containing anti-CD9 (human HI9a, mouse MZ3), anti-CD63 (human H5C6, mouse NVG-2), anti-CD81 antibodies (human 5a6, mouse Eat-2) from Biolegend, and anti-human ACE2 (R&D Biosystems cat no. FABAF9332R) at 1:100 for 1hr at 4°C, rocking.
    anti-CD9
    suggested: None
    anti-CD63
    suggested: None
    anti-CD81
    suggested: None
    anti-human ACE2
    suggested: None
    Grids were permeabilized and blocked with 0.1% Saponin/1% cold-water fish skin gelatin (Perm buffer) for 10 min and first stained with anti-human CD63 (Abcam, ab59479) antibody (1:10) in Perm buffer for 1.5hr.
    anti-human CD63
    suggested: (Abcam Cat# ab59479, RRID:AB_940915)
    Grids were washed 6x with PBS with 0.1% Saponin for 2 min, and then incubated with 18nm gold conjugated anti-mouse antibody (18nm colloidal gold AffiniPure goat anti-mouse IgG (H+L), Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), in Perm buffer for 30min.
    anti-mouse IgG
    suggested: None
    30min
    suggested: None
    The grids were incubated with anti-NP antibody (1:100) in perm buffer for 1.5 hr followed by anti-mouse Fab’ nanogold (1:250
    anti-NP
    suggested: (LSBio (LifeSpan Cat# LS-C145152-100, RRID:AB_10972206)
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549ACE2 cells were generated using lentiviral transduction of a human ACE2 cDNA expressing plasmid (backbone: pLV-EF1a-IRES-Puro (Addgene 85132) as previously described [31].
    A549ACE2
    suggested: None
    1mL lentivirus was used to transduce A549 cells using Polybrene (Millipore).
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Confluent Vero cells were then infected with this exosome-virus mixture.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    The third wash was collected and stored at −80°C for titration by plaque assay on Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Recombinant DNA
    SentencesResources
    A549ACE2 cells were generated using lentiviral transduction of a human ACE2 cDNA expressing plasmid (backbone: pLV-EF1a-IRES-Puro (Addgene 85132) as previously described [31].
    pLV-EF1a-IRES-Puro
    suggested: RRID:Addgene_85132)
    TLR7+ A549 were generated by lentiviral transduction of pcDNA3-TLR7-YFP (a gift from Doug Golenbock (Addgene plasmid # 13022; http://n2t.net/addgene:13022; RRID:Addgene_13022)).
    detected: RRID:Addgene_13022)
    psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260)
    psPAX2
    suggested: RRID:Addgene_12260)
    Software and Algorithms
    SentencesResources
    Micrographs were collected using Leginon [58, 59] at a dose rate of 11.21 e-/Å2/s with a total exposure of 3.50 seconds, for an accumulated dose of 39.23 e-/Å2.
    Leginon
    suggested: (Leginon, RRID:SCR_016731)
    Frames were aligned using Motioncor2 [60].
    Motioncor2
    suggested: (MotionCor2, RRID:SCR_016499)
    Tilt series were collected using SerialEM [61] at a dose rate of 11.21 e-/Å2/s with a total exposure of 0.25 second for an accumulated dose of 2.79 e-/Å2 per tilted image.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Tilt series processing: Frames were aligned using Warp [62], binned to the physical pixel size of 1.408 Å, and constructed into a tilt series stack for further processing.
    Warp
    suggested: (Warp, RRID:SCR_018071)
    Denoised tomograms were inspected using IMOD [65] and clipped to appropriate size.
    IMOD
    suggested: (IMOD, RRID:SCR_003297)
    Movies were made from selected tomograms using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical Analyses: Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, California USA; https://www.graphpad.com/).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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.

  3. Version published to 10.1101/2021.12.17.473223v2 on bioRxiv
  4. Version published to 10.1101/2021.12.17.473223v1 on bioRxiv