Visceral Fat Inflammation and Fat Embolism are associated with Lung’s Lipidic Hyaline Membranes in COVID-19 patients

  1. Georgia Colleluori
  2. Laura Graciotti
  3. Mauro Pesaresi
  4. Angelica Di Vincenzo
  5. Jessica Perugini
  6. Eleonora Di Mercurio
  7. Sara Caucci
  8. Patrizia Bagnarelli
  9. Cristina M. Zingaretti
  10. Enzo Nisoli
  11. Stefano Menzo
  12. Adriano Tagliabracci
  13. Annie Ladoux
  14. Christian Dani
  15. Antonio Giordano
  16. Saverio Cinti

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Abstract

Background

Visceral obesity is a critical determinant of severe coronavirus disease-2019 (COVID-19). Methods: In this study, we performed a comprehensive histomorphologic analysis of autoptic visceral adipose tissues (VAT), lungs and livers of 19 COVID-19 and 23 non-COVID-19 subjects.

Results

Although there were no between-groups differences in body-mass-index and adipocytes size, higher prevalence of CD68+ macrophages in COVID-19 subjects’ VAT was detected (p=0.005) and accompanied by crown-like structures presence, signs of adipocytes stress and death. Consistently, human adipocytes were successfully infected by SARS-CoV2 in vitro and displayed lower cell viability. Being VAT inflammation associated with lipids spill-over from dead adipocytes, we studied lipids distribution employing Oil-Red-O staining (ORO). Lipids were observed within lungs and livers interstitial spaces, macrophages, endothelial cells, and vessels’ lumen, features suggestive of fat embolism syndrome, more prevalent among COVID-19 individuals (p<0.001). Notably, signs of fat embolism were more prevalent among obese (p=0.03) independently of COVID-19 diagnosis, suggesting that such condition may be an obesity complication, exacerbated by SARS-CoV2 infection. Importantly, all infected subjects’ lungs presented lipids-rich (ORO+) hyaline membranes, formations associated with COVID-19-related pneumonia, present only in one control with non-COVID-19 pneumonia.

Conclusions

This study describes for the first time novel COVID-19-related features possibly underlying the unfavorable prognosis in obese SARS-CoV2-infected-subjects.

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    SciScore for 10.1101/2021.10.30.466586: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Given the observational (cross-sectional, case-control) nature of our study which was conducted on autoptic specimens and did not entail neither an intervention, nor the collection of subject’s sensitive information, we have not obtained an informed consent.
    Consent: Given the observational (cross-sectional, case-control) nature of our study which was conducted on autoptic specimens and did not entail neither an intervention, nor the collection of subject’s sensitive information, we have not obtained an informed consent.
    IRB: Therefore, our Institutional Review Board does not require an ethical approval for studies conducted on autoptic specimens and not collecting personal or sensitive data.
    Sex as a biological variablenot detected.
    RandomizationFive random fields were analyzed and at least 1700 lipid droplets were measured for each sample, and the difference between infected and non-infected cells was assessed by unpaired t-test.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: They were routinely tested for the absence of mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    To study SARS-CoV2 presence in VAT, we used the SARS-CoV2 nucleocapsid (Invitrogen #MA-17404) and spike protein (Sino Biological #40150-T62) antibodies at different dilutions.
    spike protein ( Sino Biological #40150-T62 ) antibodies at different dilutions
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Supernatants, collected as above, and cell pellets, collected at 96 hours post-infection, were analyzed using RT-qPCR as described in the VeroE6 cell section.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    For each subject the number of total macrophages and the density of CLS/104 adipocytes were counted with the ImageJ morphometric program (RRID:SCR_003070).
    ImageJ
    detected: ImageJ ( RRID:SCR_003070)
    Adipocytes’ area was measured in all patients by counting 100 adipocytes for each paraffin tissue section using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analyses were performed with Prism 6.0 (GraphPad Software Inc.,
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    La Jolla, CA) and IBM SPSS Statistics Data Editor (v.24).
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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 10.1101/2021.10.30.466586v1 on bioRxiv