Single-cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut

  1. Sergio Triana
  2. Camila Metz Zumaran
  3. Carlos Ramirez
  4. Carmon Kee
  5. Patricio Doldan
  6. Mohammed Shahraz
  7. Daniel Schraivogel
  8. Andreas R. Gschwind
  9. Lars M. Steinmetz
  10. Carl Herrmann
  11. Theodore Alexandrov
  12. Steeve Boulant
  13. Megan L. Stanifer

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Abstract

Objective

Exacerbated pro-inflammatory immune response contributes to COVID-19 pathology. Despite the evidence about SARS-CoV-2 infecting the human gut, little is known about the importance of the enteric phase of SARS-CoV-2 for the viral lifecycle and for the development of COVID-19-associated pathologies. Similarly, it remains unknown whether the innate immune response triggered in this organ to combat viral infection is similar or distinct compared to the one triggered in other organs.

Design

We exploited human ileum- and colon-derived organoids as a non-transformed culture model supporting SARS-CoV-2 infection. We characterized the replication kinetics of SARS-CoV-2 in intestinal epithelial cells and correlated the expression of the viral receptor ACE2 with infection. We performed conventional and targeted single-cell transcriptomics and multiplex single-molecule RNA fluorescence in situ hybridization and used IFN-reporter bioassays to characterize the response of primary human intestinal epithelial cells to SARS-CoV-2 infection.

Results

We identified a subpopulation of enterocytes as the prime target of SARS-CoV-2. We found the lack of positive correlation between susceptibility to infection and the expression of ACE2 and revealed that SARS-CoV-2 downregulates ACE2 expression upon infection. Infected cells activated strong proinflammatory programs and produced interferon, while expression of interferon-stimulated genes was limited to bystander cells due to SARS-CoV-2 suppressing the autocrine action of interferon in infected cells.

Conclusion

Our findings reveal that SARS-CoV-2 curtails the immune response in primary human intestinal epithelial cells to promote its replication and spread and this highlights the gut as a proinflammatory reservoir that should be considered to fully understand SARS-CoV-2 pathogenesis.

Significance of the study

What is already known about this subject?

  • COVID-19 patients have gastrointestinal symptoms which likely correlates with SARS-CoV-2 infection of the intestinal epithelium

  • SARS-CoV-2 replicates in human intestinal epithelial cells.

  • Intestinal organoids are a good model to study SARS-CoV-2 infection of the gastrointestinal tract

  • There is a limited interferon response in human lung epithelial cells upon SARS-CoV-2 infection.

What are the new findings?

  • A specific subpopulation of enterocytes are the prime targets of SARS-CoV-2 infection of the human gut.

  • There is a lack of correlation between ACE2 expression and susceptibility to SARS-CoV-2 infection. SARS-CoV-2 downregulates ACE2 expression upon infection.

  • Human intestinal epithelium cells produce interferon upon SARS-CoV-2 infection.

  • Interferon acts in a paracrine manner to induce interferon stimulated genes that control viral infection only in bystander cells.

  • SARS-CoV-2 actively blocks interferon signaling in infected cells.

How might it impact on clinical practice in the foreseeable future?

  • The absence of correlation between ACE2 levels and susceptibility suggest that medications influencing ACE2 levels (e.g. high blood pressure drugs) will not make patients more susceptible to SARS-CoV-2 infection.

  • The restricted cell tropism and the distinct immune response mounted by the GI tract, suggests that specific cellular restriction/replication factors and organ specific intrinsic innate immune pathways can represent unique therapeutic targets to treat COVD-19 patients by considering which organ is most infected/impacted by SARS-CoV-2.

  • The strong pro-inflammatory signal mounted by the intestinal epithelium can fuel the systemic inflammation observed in COVID-19 patients and is likely participating in the lung specific pathology.

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  1. ScreenIT

    SciScore for 10.1101/2020.10.21.348854: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: This study was carried out in accordance with the recommendations of the University Hospital Heidelberg with informed written consent from all subjects in accordance with the Declaration of Helsinki.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Mouse monoclonal antibody against SARS-CoV NP (Sino biologicals MM05) and mouse monoclonal against J2 (scions) were diluted in phosphate-buffered saline (PBS) at 1/1000 dilution and incubated for 1h at RT.
    SARS-CoV NP
    suggested: (Sino Biological Cat# 40143-MM05, RRID:AB_2827977)
    J2
    suggested: (US Biological Cat# U1000-87M, RRID:AB_2210756)
    Secondary antibody (anti-mouse CW800) and DNA dye Draq5 (Abcam) were diluted 1/10,000 in blocking buffer and incubated for 1 h at RT.
    anti-mouse CW800
    suggested: None
    Draq5
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral infections: Media was removed from cells and 106 pfu of SARS-CoV-2 (as determined in Vero cells) was added to cells for 1 hour at 37°C.
    Vero
    suggested: None
    In-cell western: 20,000 Vero E6 cells were seeded per well into a 96-well dish 24 hours prior to infection.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Targeted single-cell RNA-sequencing: For targeted scRNAseq, outer and inner primers for targeted amplification were designed using an R package for primer design described in [24] and available through Bioconductor (http://bioconductor.org/packages/release/bioc/html/TAPseq.html).
    Bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)
    In summary, following demultiplexing by sample, sequencing data were processed following the workflow provided by Drop-seq tools (v. 1.13, http://mccarrolllab.org/dropseq/) with STAR (v.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    A custom script (Python v. 3.6.6) was used to filter for chimeric reads with a transcripts-per-transcript (TPT) cutoff of 0.25, and UMI observations were converted to transcript counts.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Differential expression tests were performed using MAST [47].
    MAST
    suggested: (MAST, RRID:SCR_016340)
    Subsequently, genes whose mRNAs were found to be differentially expressed were subjected to a gene set overrepresentation analysis using the EnrichR package in R.
    EnrichR
    suggested: (Enrichr, RRID:SCR_001575)
    Furthermore signalling pathways enrichment was calculated using PROGENy.
    PROGENy
    suggested: (PROGENY, RRID:SCR_006647)
    Further brightness and contrast adjustments were performed using Fiji.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    To measure the single cell fluorescent intensity for the ACE2, ISG15 and SARS-CoV-2 probes, a pipeline using CellProfiler 3.1.9 was developed.
    CellProfiler
    suggested: None
    Finally, the SARS-CoV2 mean intensity signal was plotted against the normalized ACE mean intensity signal or the ISG15 mean intensity signal using GraphPad Prism Version 6.0
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    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 10.1101/2020.10.21.348854 on bioRxiv