Activated interstitial macrophages are a predominant target of viral takeover and focus of inflammation in COVID-19 initiation in human lung

  1. Timothy Ting-Hsuan Wu
  2. Kyle J. Travaglini
  3. Arjun Rustagi
  4. Duo Xu
  5. Yue Zhang
  6. Leonid Andronov
  7. SoRi Jang
  8. Astrid Gillich
  9. Roozbeh Dehghannasiri
  10. Giovanny Martínez-Colón
  11. Aimee Beck
  12. Daniel Dan Liu
  13. Aaron J. Wilk
  14. Maurizio Morri
  15. Winston L. Trope
  16. Rob Bierman
  17. Irving L. Weissman
  18. Joseph B. Shrager
  19. Stephen R. Quake
  20. Christin S. Kuo
  21. Julia Salzman
  22. W. E. Moerner
  23. Peter S. Kim
  24. Catherine A. Blish
  25. Mark A. Krasnow

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Early stages of deadly respiratory diseases such as COVID-19 have been challenging to elucidate due to lack of an experimental system that recapitulates the cellular and structural complexity of the human lung while allowing precise control over disease initiation and systematic interrogation of molecular events at cellular resolution. Here we show healthy human lung slices cultured ex vivo can be productively infected with SARS-CoV-2, and the cellular tropism of the virus and its distinct and dynamic effects on host cell gene expression can be determined by single cell RNA sequencing and reconstruction of “infection pseudotime” for individual lung cell types. This revealed that the prominent SARS-CoV-2 target is a population of activated interstitial macrophages (IMs), which as infection proceeds accumulate thousands of viral RNA molecules per cell, comprising up to 60% of the cellular transcriptome and including canonical and novel subgenomic RNAs. During viral takeover of IMs, there is cell-autonomous induction of a pro-fibrotic program ( TGFB1 , SPP1 ), and an inflammatory program characterized by the early interferon response, chemokines ( CCL2 , 7, 8 , 13, CXCL10 ) and cytokines ( IL6, IL10) , along with destruction of cellular architecture and formation of dense viral genomic RNA bodies revealed by super-resolution microscopy. In contrast, alveolar macrophages (AMs) showed neither viral takeover nor induction of a substantial inflammatory response, although both purified AMs and IMs supported production of infectious virions. Spike-dependent viral entry into AMs was neutralized by blockade of ACE2 or Sialoadhesin/CD169, whereas IM entry was neutralized only by DC-SIGN/CD209 blockade. These results provide a molecular characterization of the initiation of COVID-19 in human lung tissue, identify activated IMs as a prominent site of viral takeover and focus of inflammation and fibrosis, and suggest therapeutic targeting of the DC-SIGN/CD209 entry mechanism to prevent IM infection, destruction and early pathology in COVID-19 pneumonia. Our approach can be generalized to define the initiation program and evaluate therapeutics for any human lung infection at cellular resolution.

Article activity feed

  1. Version published to 10.1101/2022.05.10.491266v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.05.10.491266: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Tissue samples obtained from surgical resections were obtained under a protocol approved by the Stanford University Human Subjects Research Compliance Office (IRB 15166) and informed consent was obtained from each patient before surgery.
    IRB: The research protocol for donor samples was approved by the DNW’s internal ethics committee (Research project STAN-19-104) and the medical advisory board, as well as by the Institutional Review Board at Stanford University which determined that this project does not meet the definition of human subject research as defined in federal regulations 45 CFR 46.102 or 21 CFR 50.3.
    Sex as a biological variableCase 1 was a male organ donor aged 62.
    RandomizationThe experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
    BlindingThe experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
    Power AnalysisNo statistical methods were used to predetermine sample size.
    Cell Line AuthenticationContamination: Cell lines: VeroE6 cells were obtained from ATCC as mycoplasma-free stocks and maintained in supplemented DMEM (DMEM (Dulbecco’s Modified Eagle Medium) (Thermo 11885-092) with 1X L-glut (Thermo SH30034),

    Table 2: Resources

    Antibodies
    SentencesResources
    AlexaFluor plus secondary antibodies (488 plus, anti-mouse, Invitrogen A32723; 750, anti-rabbit, Invitrogen A21039) were used at 1:1,000.
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A32723, RRID:AB_2633275)
    anti-rabbit
    suggested: (Innovative Research Cat# A21039, RRID:AB_1500687)
    The following primary antibodies were used at 1:100: CD68 (mouse, Abcam ab955), RAGE (rabbit, Abcam ab216329).
    CD68
    suggested: (Abcam Cat# ab955, RRID:AB_307338)
    The “EPCAM-CD31-” flowthrough was collected and stained with CD206 antibodies conjugated to biotin (Miltenyi 130-095-214), washed twice with MACS buffer, then stained with Anti-Biotin MicroBeads (Miltenyi 130-090-485) and passed through an LS MACS column on a MidiMACS Separator magnet, designated “EPCAM-CD31-CD206+”.
    CD206
    suggested: (Miltenyi Biotec Cat# 130-095-214, RRID:AB_10827698)
    Anti-Biotin
    suggested: (Miltenyi Biotec Cat# 130-090-485, RRID:AB_244365)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: VeroE6 cells were obtained from ATCC as mycoplasma-free stocks and maintained in supplemented DMEM (DMEM (Dulbecco’s Modified Eagle Medium) (Thermo 11885-092) with 1X L-glut (Thermo SH30034),
    VeroE6
    suggested: None
    HeLa/ACE2/TMPRSS2 cells were a generous gift from Dr. Jesse Bloom at the Fred Hutchinson Cancer Research Center.
    HeLa/ACE2/TMPRSS2
    suggested: JCRB Cat# JCRB1835, RRID:CVCL_B3LV)
    Plaque assay: VeroE6 or VeroE6/TMPRSS2 cells were plated at 4.5-5 x 105 cells/well in a standard 12-well tissue culture plate (Falcon) one day prior to infection.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    , Spike pseudotyped lentiviruses encoding a nanoluciferase-tdTomato reporter were produced in HEK-293T cells (5 × 106 cells per 10-cm culture dish) by co-transfection of a 5-plasmid system as described previously58.
    HEK-293T
    suggested: None
    On day 1, mAbs were serially diluted in D10 medium and then mixed with lentivirus (diluted in D10 medium, supplemented with polybrene (Sigma-Aldrich, TR1003), 1:1000, v/v) for 1 hr before being transferred to HeLa-ACE2/TMPRSS2 cells.
    HeLa-ACE2/TMPRSS2
    suggested: JCRB Cat# JCRB1835, RRID:CVCL_B3LV)
    Recombinant DNA
    SentencesResources
    Based on the original lentiviral backbone plasmid (pHAGE-Luc2-IRES-ZsGreen, Addgene 164432), we replaced the Luc2-IRES-ZsGreen reporter with a cassette encoding H2B fused to Nanoluciferase (Promega) to minimize background luminescence, followed by a T2A self-cleaving peptide, and tdTomato fluorescent protein using gBlock synthesis (Integrated DNA Technologies).
    pHAGE-Luc2-IRES-ZsGreen
    suggested: None
    The 5-plasmid system includes a packaging vector (pHAGE-H2B-NanoLuc-T2A-tdTomato), a plasmid encoding full-length Spike with a 21-residue deletion on the C-terminus (pHDM SARS-CoV-2-SpikeΔ21), and three helper plasmids (pHDM-Hgpm2, pHDM-Tat1b, and pRC-CMV_Rev1b).
    pHAGE-H2B-NanoLuc-T2A-tdTomato
    suggested: None
    pHDM-Tat1b
    suggested: RRID:Addgene_164442)
    pRC-CMV_Rev1b
    suggested: None
    Software and Algorithms
    SentencesResources
    Viral pseudotime analysis: For viral pseudotime analysis, computations were performed in R using the Seurat package (v3).
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    We used STAR v.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    2.7.5a as the aligner and aligned fastq files from all infections to our custom built Human GRCh38 (GENCODE v29) and SARS-CoV-2 WA1 (GenBank: MN985325.1) reference.
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    Micrographs were acquired with laser scanning confocal fluorescence microscopy (Leica Stellaris 8) and processed with ImageJ and Imaris (version 9.2.0, Oxford Instruments).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    Arcplots depicting number of subgenomic junctions was plotted using a custom Python function (available on Github).
    Python
    suggested: (IPython, RRID:SCR_001658)

    Results from OddPub: Thank you for sharing your code and data.

    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 10.1101/2022.05.10.491266v1 on bioRxiv