SARS-CoV-2 infection induces inflammatory bone loss in golden Syrian hamsters

  1. Wei Qiao
  2. Hui En Lau
  3. Huizhi Xie
  4. Vincent Kwok-Man Poon
  5. Chris Chung-Sing Chan
  6. Hin Chu
  7. Shuofeng Yuan
  8. Terrence Tsz-Tai Yuen
  9. Kenn Ka-Heng Chik
  10. Jessica Oi-Ling Tsang
  11. Chris Chun-Yiu Chan
  12. Jian-Piao Cai
  13. Cuiting Luo
  14. Kwok-Yung Yuen
  15. Kenneth Man-Chee Cheung
  16. Jasper Fuk-Woo Chan
  17. Kelvin Wai-Kwok Yeung

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Abstract

Extrapulmonary complications of different organ systems have been increasingly recognized in patients with severe or chronic Coronavirus Disease 2019 (COVID-19). However, limited information on the skeletal complications of COVID-19 is known, even though inflammatory diseases of the respiratory tract have been known to perturb bone metabolism and cause pathological bone loss. In this study, we characterize the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on bone metabolism in an established golden Syrian hamster model for COVID-19. SARS-CoV-2 causes significant multifocal loss of bone trabeculae in the long bones and lumbar vertebrae of all infected hamsters. Moreover, we show that the bone loss is associated with SARS-CoV-2-induced cytokine dysregulation, as the circulating pro-inflammatory cytokines not only upregulate osteoclastic differentiation in bone tissues, but also trigger an amplified pro-inflammatory cascade in the skeletal tissues to augment their pro-osteoclastogenesis effect. Our findings suggest that pathological bone loss may be a neglected complication which warrants more extensive investigations during the long-term follow-up of COVID-19 patients. The benefits of potential prophylactic and therapeutic interventions against pathological bone loss should be further evaluated.

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  1. Version published to 10.1038/s41467-022-30195-w
  2. ScreenIT

    SciScore for 10.1101/2021.10.08.463665: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variableBriefly, 6–10-week-old male golden Syrian hamsters (Mesocricetus auratus) were obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Centre for Comparative Medicine Research.
    Randomizationnot detected.
    Blindingnot detected.
    Power AnalysisThe sample size was decided based on preliminary data, as well as on observed effect sizes.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, after blocking with 10% goat serum, the sections were incubated with primary antibodies to CD68 (Abcam, ab31630/ab125212), RANK (Abcam, ab13918), TRAP (Abcam, ab216025), osteocalcin (TAKARA, M186), IL-1β (Abcam, ab9722), IL-1RA (Abcam, ab124962), TNF-α (Abcam, ab9635), IFN-γ (Abcam, ab9657), NF-κB (CST, #8242), anti-NFATc1 (CST, #8032) overnight at 4°C.
    CD68
    suggested: (Leica Biosystems Cat# NCL-CD68-KP1, RRID:AB_563621)
    IL-1RA
    suggested: (Abcam Cat# ab124962, RRID:AB_11130394)
    The primary antibodies used in this study included anti-CD68 (Abcam, ab31630, USA), anti-TRAP (Abcam, ab216025), antii-RANK (Abcam, ab13918), anti-IL-1β (Abcam, ab9722), anti-NP (ThermoFisher, USA), anti-ACE2 (ThermoFisher).
    anti-CD68 (Abcam,
    suggested: (Abcam Cat# ab31630, RRID:AB_1141557)
    antii-RANK
    suggested: None
    anti-NP
    suggested: None
    anti-ACE2
    suggested: None
    For the inhibition of IL-1β, 10 ng/mL IL-1b neutralizing antibody (Abcam, ab9722) was added to the cell culture.
    IL-1b neutralizing antibody (Abcam,
    suggested: None
    The primary antibodies used included mouse anti-NFATc1 (Santa Cruz, USA), mouse anti-TRAP (Abcam), rabbit anti-Cathepsin K (Abcam), mouse anti-RANK (Abcam), rabbit anti-NF-κB p65 (CST), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1RA (Abcam), rabbit anti-TNF-α (Abcam), rabbit anti-phospho-JNK (CST), rabbit anti-JNK (CST), rabbit anti-β-actin (Abcam), The protein bands were visualized by using HRP conjugated secondary antibodies and an enhanced chemiluminescence (ECL) substrate (Advansta, USA) and exposed under the Typhoon5 Biomolecular Imager 680 (GE Amersham, USA).
    anti-NFATc1
    suggested: None
    anti-TRAP
    suggested: None
    anti-Cathepsin K
    suggested: None
    anti-RANK
    suggested: None
    anti-NF-κB p65
    suggested: None
    anti-IL-1β
    suggested: None
    anti-TNF-α
    suggested: None
    anti-phospho-JNK (CST)
    suggested: None
    anti-JNK
    suggested: None
    anti-β-actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The plaque purified viral isolate was amplified by one additional passage in VeroE6 cells to make working stocks of the virus as described previously 58.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Cell culture: The mesenchymal stem cells (MSCs) and bone marrow macrophages (BMMs) were isolated from the long bones of 3-month-old or 6-month-old C57L6/J mice.
    C57L6/J
    suggested: RRID:MGI:5652281)
    Software and Algorithms
    SentencesResources
    The primers used in the RT-qPCR assay were synthesized using Integrated DNA Technologies (IDT, Singapore), based on sequences designed using Primer-BLAST (National Center for Biotechnology Information, NCBI, Table S1) or retrieved from the Primer Bank (http://pga.mgh.harvard.edu/primerbank/, Table S2).
    Primer-BLAST
    suggested: (Primer-BLAST, RRID:SCR_003095)
    http://pga.mgh.harvard.edu/primerbank/
    suggested: (PrimerBank, RRID:SCR_006898)
    Statistical analysis: All data analyses were performed and illustrated using the Prism software (version 7, GraphPad, USA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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.

  3. Version published to 10.21203/rs.3.rs-902819/v1 on Research Square
  4. Version published to 10.1101/2021.10.08.463665v1 on bioRxiv