CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection

  1. Abigail Vanderheiden
  2. Jeronay Thomas
  3. Allison L. Soung
  4. Meredith E. Davis-Gardner
  5. Katharine Floyd
  6. Fengzhi Jin
  7. David A. Cowan
  8. Kathryn Pellegrini
  9. Adrian Creanga
  10. Amarendra Pegu
  11. Alexandrine Derrien-Colemyn
  12. Pei-Yong Shi
  13. Arash Grakoui
  14. Robyn S. Klein
  15. Steven E. Bosinger
  16. Jacob E. Kohlmeier
  17. Vineet D. Menachery
  18. Mehul S. Suthar

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Abstract

SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.03.442538: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All experiments adhered to the guidelines approved by the Emory University Institutional Animal Care and Committee.
    Euthanasia Agents: Quantification of infectious virus: At the indicated day post infection, mice were euthanized via isoflurane overdose and lung tissue was collected in Omni-Bead ruptor tubes filled with 1% FBS-HBSS.
    Sex as a biological variableAll mice used in these experiments were females between 8-12 weeks of age.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following antibodies were used in this study: CD45:PE (Biolegend, Clone: 30-F11), CD45.2:BV605 (Biolegend, Clone: 104), CD11b:BUV395 BD Biosciences, 440c), I-A/I-E:AF700 (Biolegend, M5/114.15.2), CD11c:BUV737 (Biolegend, N418), CD26: PE-Cy7 (Biolegend, H194-112), CD172a:BV510 (Biolegend, P84), XCR1:AF647 (Biolegend, Zet), CD64: PerCpCy5.5 (Biolegend, X54-5/7.1), F4/80:FITC (Biolegend, BM8), H2kb:BUV805 (BD Biosciences, AF6-88.5), CD86:PE-Dazzle594 (Biolegend, GL1), Live/Dead Ghost Dye:R780 (Tonbo), CD3:APC-Cy7 (Biolegend, 145-2C11), CD19:APC-Cy7 (BD Biosciences, 1D3), NK1.1 (Biolegend, PK136), Ly6C:BV785 (Biolegend, Hk1.4), Ly6G:BV650 (Biolegend, 1A8), SiglecF:BV421 (BD Biosciences, E50-2440).
    CD45.2:BV605
    suggested: None
    I-A/I-E:AF700
    suggested: None
    CD26
    suggested: (BD Biosciences Cat# 749316, RRID:AB_2873690)
    PE-Cy7
    suggested: None
    CD172a:BV510
    suggested: None
    CD64
    suggested: (BD Biosciences Cat# 742023, RRID:AB_2871319)
    PerCpCy5.5
    suggested: (BD Biosciences Cat# 565364, RRID:AB_2869666)
    F4/80:FITC
    suggested: (Bio-Rad Cat# MCA497FA, RRID:AB_567113)
    CD86:PE-Dazzle594
    suggested: None
    CD3:APC-Cy7
    suggested: None
    CD19:APC-Cy7
    suggested: None
    NK1.1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viruses and cells: VeroE6 cells were obtained from ATCC (clone E6, ATCC, #CRL-1586) and cultured in complete DMEM medium consisting of 1x DMEM (VWR, #45000-304), 10% FBS, 25mM HEPES Buffer (Corning Cellgro), 2mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    VeroE6-TMPRSS2 cells and VeroE6-TMPRSS2-hACE2 cells were cultured in complete DMEM in the presence of Gibco Puromycin 10mg/mL (#A11138-03).
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    To perform plaque assays, 10-fold dilutions of viral supernatant in serum free DMEM (VWR, #45000-304) were overlaid on VeroE6-TMPRSS2-hACE2 cells monolayers and adsorbed for 1 hour at 37°C.
    VeroE6-TMPRSS2-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    This virus was then passaged 20 times in Balb/c mice followed by deep sequencing which identified 3 additional acquired mutations (S T417N, S H655Y, and E E8V).
    Balb/c
    suggested: None
    Infection of mice with MA-SARS-CoV-2: C57BL/6J and Ccr2-/- mice were purchased from Jackson Laboratories or bred in-house at the Yerkes National Primate Research Center rodent facility at Emory University.
    C57BL/6J
    suggested: None
    Ccr2-/-
    suggested: None
    Recombinant DNA
    SentencesResources
    VERO E6 cells (ATCC CRL-1586) were transfected with pCAGGS plasmid in which chicken actin gene promoter drives the expression of an open reading frame comprising Puromycin N-acetyl transferase, GSG linker, 2A self-cleaving peptide of thosea asigna virus (T2A), human transmembrane serine protease 2 (TMPRSS2).
    pCAGGS
    suggested: RRID:Addgene_18926)
    Software and Algorithms
    SentencesResources
    Infection of mice with MA-SARS-CoV-2: C57BL/6J and Ccr2-/- mice were purchased from Jackson Laboratories or bred in-house at the Yerkes National Primate Research Center rodent facility at Emory University.
    Jackson Laboratories
    suggested: None
    Gene set enrichment analysis (GSEA) was conducted using ranked gene list produced with Seurat FindMarkers function (comparing CoV2 samples with mock samples) and Genelist were obtained from: MsigDB (hallmarks) and https://www.nature.com/articles/s41422-020-00455-9#MOESM1 (macrophage suppressive and hyperinflammatory).
    Gene set enrichment analysis
    suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)

    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 10.1101/2021.05.03.442538v1 on bioRxiv