CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

  1. Pei-Shan Sung
  2. Shao-Ping Yang
  3. Yu-Chun Peng
  4. Cheng-Pu Sun
  5. Mi-Hwa Tao
  6. Shie-Liang Hsieh

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Abstract

Background

Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Methods

We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation.

Results

We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a −/− /tlr2 −/− mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a −/− /tlr2 −/− mice.

Conclusions

This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

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  1. Version published to 10.1186/s12929-022-00832-z
  2. ScreenIT

    SciScore for 10.1101/2022.02.01.478701: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: The protocol was approved by the Human Subject Research Ethics, Academia Sinica (AS-IRB-BM-20025)
    IACUC: All the animal experiments followed the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at AS core (protocol ID 20-10-1521).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies for immunofluorescence staining are as followings: rabbit anti-citrullinated histone H3 (#NB100-57135; Novus), goat anti-human/mouse myeloperoxidase polyclonal antibody (# AF3667, R&D system).
    anti-citrullinated histone H3
    suggested: None
    Antibodies for immunohistochemical (IHC) staining are: rabbit anti-citrullinated histone H3 (#NB100-57135; Novus), goat anti-human/mouse myeloperoxidase polyclonal antibody (#AF3667, R&D system)
    anti-human/mouse
    suggested: (R and D Systems Cat# AF3667, RRID:AB_2250866)
    , anti-CD11b antibody (#ab13357, Abcam), anti-CD64 antibody (#MA5-29704, Invitrogen)
    anti-CD11b
    suggested: None
    anti-CD64
    suggested: (Thermo Fisher Scientific Cat# MA5-29704, RRID:AB_2785528)
    , anti-Siglec-F antibody (#PA5-11675, Invitrogen), anti-F4/80 antibody (#ab74383, Abcam)
    anti-Siglec-F
    suggested: None
    anti-F4/80
    suggested: (Abcam Cat# ab74383, RRID:AB_1860121)
    , anti-CCR2 antibody (#NBP-35334, Novus Biologicals), anti-Ly6C antibody (#SC-23080, Santa Cruz).
    anti-CCR2
    suggested: None
    anti-Ly6C
    suggested: None
    Secondary antibodies: donkey anti-mouse IgG (H+L) Alexa 488-conjugated antibody (#715-545-151, Jackson ImmunoResearch),
    anti-mouse IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 715-545-151, RRID:AB_2341099)
    donkey anti-goat IgG (H+L) Alexa 647-conjugated antibody (#705-605-147, Jackson ImmunoResearch),
    anti-goat IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 705-605-147, RRID:AB_2340437)
    donkey anti-Human IgG (H+L) HRP-conjugated antibody (#709-035-149, Jackson ImmunoResearch),
    anti-Human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 709-035-149, RRID:AB_2340495)
    Visualization and quantification of NET structure: Cells were immersed in fixation buffer (containing 4 % paraformaldehyde) overnight, followed by permeabilization using 0.5 % Triton X100 in PBS, then incubated with anti-MPO antibody (1:100)
    anti-MPO
    suggested: None
    anti-citrullinated histone antibody (1:100), and Hoechst 33342 (1:100000).
    anti-citrullinated histone antibody ( 1:100)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 propagation: SARS-CoV-2 Taiwan/4/2020 was propagated in Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Viral titer was determined by observation of the cytopathic effect (CPE) in Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    In brief, HEK-293T cells were transiently transfected with pLAS2w.Fluc.
    HEK-293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mouse model for SARS-CoV-2 infection: Virus preparation and inoculation of SARS-CoV-2 into C57BL/6 and clec5a-/-tlr2-/- mice were as described [31].
    C57BL/6
    suggested: None
    clec5a-/-tlr2-/-
    suggested: None
    Recombinant DNA
    SentencesResources
    In brief, HEK-293T cells were transiently transfected with pLAS2w.Fluc.
    pLAS2w
    suggested: None
    Ppuro, pcDNA3.1-2019-nCoV-S, and pCMV-Δ R8.91 using TransITR-LT1 transfection reagent (Mirus)
    pcDNA3.1-2019-nCoV-S
    suggested: None
    pCMV-Δ R8.91
    suggested: None
    Software and Algorithms
    SentencesResources
    The level of NETs was calculated using the histone image captured by a Leica confocal microscope with white light laser system (TCS SP8 X-FALCON), and analyzed by MetaMorphTM software.
    MetaMorphTM
    suggested: None
    Data were further analyzed by the Ingenuity Pathways Analysis (IPA) software.
    Ingenuity Pathways Analysis
    suggested: None

    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

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  3. Version published to 10.1101/2022.02.01.478701v1 on bioRxiv