SARS-CoV-2 Infection of Microglia Elicits Pro-inflammatory Activation and Apoptotic Cell Death

  1. Gi Uk Jeong
  2. Jaemyun Lyu
  3. Kyun-Do Kim
  4. Young Cheul Chung
  5. Gun Young Yoon
  6. Sumin Lee
  7. Insu Hwang
  8. Won-Ho Shin
  9. Junsu Ko
  10. June-Yong Lee
  11. Young-Chan Kwon

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Abstract

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in coronavirus disease 2019 (COVID-19) patients. The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we report that SARS-CoV-2 can directly infect human microglia, eliciting M1-like pro-inflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA-seq analysis also revealed that ER stress and immune responses were induced in the early and apoptotic processes in the late phase of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumour necrosis factor α (TNF-α), but not the anti-inflammatory cytokine IL-10. After this pro-inflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of pro-inflammatory microglial IL-6 and TNF-α and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in COVID-19 patients.

IMPORTANCE

Recent studies reported neurological manifestations and complications in COVID-19 patients, which are associated with neuroinflammation. As microglia are the dominant immune cells in brains, it needs to be elucidate the relationship between neuroinflammation and host immune response of microglia to SARS-CoV-2. Here, we suggest that SARS-CoV-2 can directly infect human microglia with cytopathic effect (CPE) using human microglial clone 3 (HMC3). The infected microglia were promoted to pro-inflammatory activation following apoptotic cell death. This pro-inflammatory activation was accompanied by the high production of pro-inflammatory cytokines, and led to neurotoxic-M1 phenotype polarization. In vivo , murine microglia were infected and produced pro-inflammatory cytokines and provoked a chronic loss using K18-hACE2 mice. Thus, our data present that SARS-CoV-2-infected microglia are potential mediators of neurological problems in COVID-19 patients. In addition, HMC3 cells are susceptible to SARS-CoV-2 and exhibit the CPE, which can be further used to investigate cellular and molecular mechanisms of neuroinflammation reported in COVID-19 patients.

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    SciScore for 10.1101/2022.01.04.475015: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All protocols were approved by the Institutional Animal Care and Use Committee (Protocol ID 8A-M6, IACUC ID 2021-8A-02-01 & 2021-8A-03-03).
    Euthanasia Agents: Mock or SARS-CoV-2 infected mice were anesthetised by isoflurane, followed by perfusion with 10 or 20 ml of cold 1× DPBS (Gibco) into the left ventricle to remove blood from the tissues.
    Sex as a biological variableMice: Eight-week-old male B6.Cg-Tg(K18-hACE2)2Prlmn/J mice were purchased from the Jackson Laboratory and maintained in a biosafety level 2 (BSL-2) animal facility in the Korea Research Institute of Chemical Technology (KRICT).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For the virus neutralization, the inoculum was further incubated with CR3022 neutralizing antibody (ab273073, Abcam, Cambridge, UK) for 1 h.
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    anti-mouse CD45 Antibody (103114, BioLegend)
    CD45
    suggested: (BioLegend Cat# 103114, RRID:AB_312979)
    , APC anti-mouse TNF-α Antibody (506307, BioLegend)
    anti-mouse TNF-α Antibody ( 506307
    suggested: None
    FITC anti-mouse IL-6 Monoclonal Antibody (MP5-20F3) (11-7061-82, eBioscience, San Diego, CA, USA)
    anti-mouse IL-6
    suggested: None
    FITC anti-human ACE2 Antibody (NBP2-7211F
    anti-human ACE2
    suggested: None
    , Novus Biologicals, Centennial, CO, USA), and Alexa 647 anti-Iba1 Antibody (78060S, Cell signalling Technology, Danvers, MA, USA).
    anti-Iba1
    suggested: None
    Cells were stained with the anti-SARS-CoV-2 NP antibody (40143-R001, Sino biological, Beijing, China) and a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA).
    anti-SARS-CoV-2 NP
    suggested: None
    anti-rabbit IgG
    suggested: None
    The brain sections (30 μm thickness) were permeabilized with 0.2 % Triton X-100 in 1 % BSA/PBS for 30 min, washed in PBS, and blocked with 0.5 % BSA in PBS for 15 min, followed by incubation overnight at 4 °C with primary antibodies, namely, anti-SARS-CoV-2 S (40150-T62-COV2, Sino Biological), and anti-Iba1/AIF1 (MABN92, Merck Millipore, Burlington, MA, USA).
    anti-Iba1/AIF1
    suggested: (Millipore Cat# MABN92, RRID:AB_10917271)
    After washing twice, further incubation was carried out with Alexa Fluor 488-conjugated anti-rabbit antibody (A32731, Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 594-conjugated anti-mouse antibody (A32744, Thermo Fisher Scientific).
    A32731
    suggested: (Thermo Fisher Scientific Cat# A32731, RRID:AB_2633280)
    A32744
    suggested: (Thermo Fisher Scientific Cat# A32744, RRID:AB_2762826)
    Cells were immunostained overnight at room temperature with primary antibodies, namely, anti-dsRNA J2 (MABE1134, Sigma-Aldrich), anti-SARS-CoV-2 NP (40143-R019, Sino Biological), anti-SARS-CoV-2 S (40150-T62-COV2, Sino Biological), and anti-CD68 (sc-17832, Santa Cruz Biotechnology, Dallas, TX, USA).
    anti-dsRNA J2 ( MABE1134
    suggested: None
    anti-SARS-CoV-2 NP ( 40143-R019 , Sino Biological)
    suggested: None
    anti-CD68 ( sc-17832 , Santa Cruz Biotechnology , Dallas , TX , USA)
    suggested: None
    sc-17832
    suggested: None
    After washing thrice, further incubation was carried out with Alexa Fluor 488-conjugated anti-rabbit antibody (A32731, Thermo Fisher Scientific) and Alexa Fluor 594-conjugated anti-mouse antibody (A32744, Thermo Fisher Scientific).
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# A32731, RRID:AB_2633280)
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# A32744, RRID:AB_2762826)
    The membrane was incubated with 5 % skim milk (BD Biosciences) in Tris-buffered saline with 0.1 % Tween 20 (TBST) buffer and the primary antibodies, namely, anti-SARS-CoV-2 NP (40143-R001, Sino biological), anti-CD68 (sc-17832, Santa Cruz Biotechnology) anti-GSDMDC1 (sc-81868, Santa Cruz Biotechnology), anti-Actin (sc-47778, Santa Cruz Biotechnology), anti-Hsp70 (sc-24, Santa Cruz Biotechnology), anti-CX3CL1 (ab25088, Abcam), anti-CX3CR1 (ab8021, Abcam), anti-CD16 (80006S, Cell signalling Technology), anti-phospho-Stat1 (9167S, Cell signalling Technology), anti-Stat1 (14994S, Cell signalling Technology), anti-Fas (4233T
    anti-CD68
    suggested: (Santa Cruz Biotechnology Cat# sc-17832, RRID:AB_627157)
    anti-GSDMDC1
    suggested: (Santa Cruz Biotechnology Cat# sc-81868, RRID:AB_2263768)
    sc-81868 , Santa Cruz Biotechnology
    suggested: None
    anti-Actin ( sc-47778 , Santa Cruz Biotechnology)
    suggested: None
    anti-Hsp70
    suggested: None
    sc-24
    suggested: (Santa Cruz Biotechnology Cat# sc-24, RRID:AB_627760)
    anti-CX3CL1
    suggested: (Abcam Cat# ab25088, RRID:AB_448600)
    anti-CX3CR1
    suggested: (Abcam Cat# ab8021, RRID:AB_306203)
    anti-CD16
    suggested: None
    anti-phospho-Stat1
    suggested: None
    anti-Stat1
    suggested: (Cell Signaling Technology Cat# 14994, RRID:AB_2737027)
    anti-Fas
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    CRL-3304), Caco-2 (HTB-37), and Vero E6 (CRL-1586) cell lines were purchased from ATCC (Manassas, VA, USA).
    Caco-2
    suggested: None
    Vero E6
    suggested: None
    The SARS-CoV-2 Korean strain (GISAID Accession ID: EPI_ISL_407193), isolated from a patient in South Korea, was obtained from Korea Centres for Disease Control and Prevention (KCDC) and propagated in Vero cells (CCL-81, ATCC).
    Vero
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Eight-week-old male B6.Cg-Tg(K18-hACE2)2Prlmn/J mice were purchased from the Jackson Laboratory and maintained in a biosafety level 2 (BSL-2) animal facility in the Korea Research Institute of Chemical Technology (KRICT).
    B6.Cg-Tg(K18-hACE2)2Prlmn/J
    suggested: None
    Immunofluorescence assay: After transcardial perfusion with cold 4 % paraformaldehyde in PBS, brain tissues of SARS-CoV-2 infected K18-hACE2 mice were dissected and fixed by immersion in 4 % paraformaldehyde in PBS overnight at 4 °C.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Cells were then analysed by FACSAria III sorter (BD Biosciences, San Jose, CA, USA), and data was analysed by the FlowJo software (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    From the sequenced reads, adaptor sequences were removed using Cutadapt (version 3.1) (65) and aligned to the hybrid reference genomes of human (GRCh38.p13_ENS100) and SARS-CoV-2 (ASM985889_v3) with STAR aligner (version 2.7.6a) (66)
    Cutadapt
    suggested: (cutadapt, RRID:SCR_011841)
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Aligned reads were quantified in gene level by HTSeq (version 0.13.5) (67) with “intersection-nonempty” mode.
    HTSeq
    suggested: (HTSeq, RRID:SCR_005514)
    Differentially expressed gene analysis were processed with DESeq2 (version 1.30.1) (68) using abs (log2 fold change) > 1 and adjusted P-value (Benjamini-Hochberg) < 0.01 as cut-off
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Multidimensional scaling analysis were performed with clustermap function in python seaborn package (version 0.11.1) using genes with mean FPKM > 1 among the samples and transformed to log2(FPKM+1).
    python
    suggested: (IPython, RRID:SCR_001658)
    Over-representation analysis of the DEGs enriched to GO Biological Process 2018 with EnrichR (69) with adjusted P-value (Benjamini-Hochberg) < 0.05 cut-off.
    EnrichR
    suggested: (Enrichr, RRID:SCR_001575)
    Network analysis were presented by Metascape (70) using the GO Biological Process gene sets.
    Metascape
    suggested: (Metascape, RRID:SCR_016620)
    GO Biological
    suggested: None
    All data were analysed using the GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 10.1101/2022.01.04.475015v1 on bioRxiv