Human galectin-9 potently enhances SARS-CoV-2 replication and inflammation in airway epithelial cells

  1. Li Du
  2. Mohamed S Bouzidi
  3. Akshay Gala
  4. Fred Deiter
  5. Jean-Noël Billaud
  6. Stephen T Yeung
  7. Prerna Dabral
  8. Jing Jin
  9. Graham Simmons
  10. Zain Y Dossani
  11. Toshiro Niki
  12. Lishomwa C Ndhlovu
  13. John R Greenland
  14. Satish K Pillai

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. In this study, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including immortalized AECs and primary AECs cultured at the air–liquid interface. Gal-9–glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an angiotensin-converting enzyme 2 (ACE2)-dependent manner, enhancing the binding of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induced the expression of key pro-inflammatory programs in AECs, including the IL-6, IL-8, IL-17, EIF2, and TNFα signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection.

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  1. Version published to 10.1093/jmcb/mjad030
  2. Version published to 10.1101/2022.03.18.484956v3 on bioRxiv
  3. Version published to 10.1101/2022.03.18.484956v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2022.03.18.484956: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: All the studies involving live viruses were conducted in the Vitalant Research Institute BSL-3 under approved safety protocols.
    Sex as a biological variablenot detected.
    RandomizationImage Analysis: To measure the frequency of infected cells, randomly-selected areas were imaged.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: All cells had been previously tested for mycoplasma contamination and incubated at 37°C in a humidified atmosphere with 5% CO2.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were then incubated with a primary antibody (monoclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (GeneTex, GTX135357)) in 1 X PBS (1:1,000) overnight at 4°C.
    anti-SARS-CoV-2
    suggested: None
    The following day, cells were washed three times with 1 X PBS and incubated with a secondary antibody (Goat anti-Rabbit IgG (H+L) secondary antibody, FITC (Thermo Fisher, 65-6111)) in 1 X PBS (1:200) for 1 h at 37°C.
    anti-Rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# 65-6111, RRID:AB_2533966)
    An unrelated anti-goat IgG antibody was used as a control.
    anti-goat IgG
    suggested: None
    The amount of captured ACE2, which is proportional to ACE2 binding activity, is then recognized by an ACE2 detection antibody and measured by reading the absorbance at a wavelength of 450 nm.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viruses were propagated in Vero E6-TMPRSS2 cells in DMEM with 2% FBS and 1% penicillin/streptomycin and viral stocks were stored at −80°C.
    Vero E6-TMPRSS2
    suggested: None
    Virus titer was measured in Vero E6 cells by TCID50 assay.
    Vero E6
    suggested: None
    In brief, Calu-3 cells cultured in 96-well cell culture plates were incubated with different concentrations of Gal-9 (0-5,000 nM).
    Calu-3
    suggested: None
    Pseudovirus production: VSVΔG-luciferase-based viruses, in which the glycoprotein (G) gene has been replaced with luciferase, were produced by transient transfection of viral glycoprotein expression plasmids (pCG SARS-CoV-2 Spike and pCAGGS VSV-G) or no glycoprotein control into HEK293T cells by TransIT-2020 as previously described (Ng et al., 2020).
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Pseudovirus production: VSVΔG-luciferase-based viruses, in which the glycoprotein (G) gene has been replaced with luciferase, were produced by transient transfection of viral glycoprotein expression plasmids (pCG SARS-CoV-2 Spike and pCAGGS VSV-G) or no glycoprotein control into HEK293T cells by TransIT-2020 as previously described (Ng et al., 2020).
    pCG
    suggested: RRID:Addgene_51476)
    pCAGGS VSV-G
    suggested: None
    Software and Algorithms
    SentencesResources
    The data from three independent experiments was used to calculate the CC50 by nonlinear regression using GraphPad Prism 8.0 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    RNA-seq and Ingenuity Pathway Analysis (IPA): RNA concentration and quality was measured using High Sensitivity RNA ScreenTape Analysis (Agilent, 5067-1500). cDNA libraries were prepared using the Illumina TruSeq Stranded total RNA library prep kit (Illumina, 20020597) and sequencing was performed on the Illumina Nextseq 550 platform generating 75 bp paired-end reads.
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    The quality of raw sequencing reads was assessed using FastQC.
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Differentially-expressed genes were identified by GSA or ANOVA in Partek® Flow® imported into the QIAGEN Ingenuity Pathway Analysis (IPA) software application.
    QIAGEN Ingenuity Pathway Analysis
    suggested: None
    Ingenuity
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    Data was analyzed using FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Data analyses were performed using Bio-Plex Manager™ 6.1.1 (Bio-Rad Laboratories, Hercules, CA, USA).
    Bio-Plex
    suggested: None
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Quantitative analysis of synergy: The combinatorial effects of Gal-9 treatment and SARS-CoV-2 infection on pro-inflammatory cytokine expression were analyzed using the SynergyFinder web application, implementing the Bliss Independence model.
    SynergyFinder
    suggested: (SynergyFinder, RRID:SCR_019318)

    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: We detected the following sentences addressing limitations in the study:
    Our study has limitations that must be considered. Firstly, we focused exclusively on the airway epithelium, and it is now established that SARS-CoV-2 is capable of infecting other cell lineages including monocytes, monocyte-derived macrophages, and microglia (Boumaza et al., 2021; Jeong et al.; Liu et al., 2021). It is possible that Gal-9 does not exert similar effects on viral replication or immune signaling in other target cell types. Secondly, our studies were all performed in vitro or in transplant tissue-derived primary epithelial cells ex vivo. As it is well-established that Gal-9 exerts conditional, pleiotropic immunomodulatory effects, the net effect of Gal-9 signaling on SARS-CoV-2 pathogenesis cannot be fully appreciated outside of an animal model with a functional immune system. Validation and extension of our observations in murine, hamster or nonhuman primate models of SARS-CoV-2 infection will help in evaluating the clinical relevance of our reported findings. In relevance to the implementation of animal models, our study did not elucidate the principal cell or tissue sources responsible for secreting Gal-9 in the setting of SARS-CoV-2 infection. Leveraging an animal model to identify these source compartments will be critical in developing interventions to manipulate Gal-9 signaling as a therapeutic approach. To our knowledge, the data presented here are the first to show that Gal-9 is directly involved in enhancing SARS-CoV-2 infection and virus-induced pro-i...

    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.

  5. Version published to 10.1101/2022.03.18.484956v1 on bioRxiv