Multiple-Organ Complement Deposition on Vascular Endothelium in COVID-19 Patients

  1. Paolo Macor
  2. Paolo Durigutto
  3. Alessandro Mangogna
  4. Rossana Bussani
  5. Luca De Maso
  6. Stefano D’Errico
  7. Martina Zanon
  8. Nicola Pozzi
  9. Pier Luigi Meroni
  10. Francesco Tedesco

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Abstract

Increased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at the tissue level. The mechanisms and pathways of local complement activation remain unclear. The aim of this study was to investigate the deposition of complement components in the lungs, kidneys, and liver in patients with COVID-19 patients and to determine the pathway/s of complement activation. We performed immunofluorescence analyses of autopsy specimens of lungs, kidney, and liver from 12 COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG, and spike protein of SARS-CoV-2. Lung deposits of C1q, C4, C3, and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. FB deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on the hepatic artery and portal vein of the liver. Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease and the contribution of the complement system to inflammation and tissue damage.

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  1. Version published to 10.3390/biomedicines9081003
  2. ScreenIT

    SciScore for 10.1101/2021.01.07.21249116: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This work was approved by the Ethical Committee of the Regione Friuli Venezia Giulia, Italy (Prot. N. 0025523 / P / GEN/ ARCS).
    RandomizationAt least three random sections of tissue samples collected from each case were examined independently by three observers.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableStudy group: The study group comprised 9 patients, 5 females and 4 males, aged 72 to 97 referred to the University Hospital in Trieste (Italy).

    Table 2: Resources

    Antibodies
    SentencesResources
    Tissue sections of seven µm were stained with the following primary antibodies (5μg/ml): goat anti-human IgG (Sigma-Aldrich, Milan, Italy), goat anti-C1q and C4 (The Binding Site, Birmingham, UK), goat anti-C3 (Quidel, San Diego, CA, USA), and anti-Factor B (Cytotech, San Diego, CA, USA); rabbit anti-MBL (Sigma-Aldrich) and anti-SARS-CoV-2 Spike S2 (Sino Biological, Beijing, China); murine monoclonal antibody against C9 neo-antigen (aE11, kindly provided by prof. T.E. Mollnes, Oslo, Norway).
    anti-human IgG
    suggested: None
    anti-C1q
    suggested: None
    C4
    suggested: None
    anti-C3
    suggested: None
    anti-Factor B
    suggested: None
    anti-MBL
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    C9
    suggested: (Thermo Fisher Scientific Cat# MA5-33373, RRID:AB_2815511)
    aE11
    suggested: None
    The following FITC-conjugated secondary antibodies were used to reveal bound antibodies: rabbit anti-goat IgG (Sigma-Aldrich), goat anti-rabbit IgG and anti-mouse IgG (Dako, Jena, Germany).
    anti-goat IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    anti-mouse IgG
    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: We detected the following sentences addressing limitations in the study:
    The pro-coagulant activity of MASPs may explain the beneficial effects observed in COVID-19 patients treated with Narsoplimab [25] which inhibits MASP-2, preventing C activation and thrombus formation, although the small number of treated patients and the concomitant use of other drugs represent important limitations, recognized by the authors of this study. The positive staining of the patients’ lung for C1q points to the classical pathway as an important route of C activation likely triggered by the IgG that are widely distributed in the lung of all patients examined. Unfortunately, the immunohistochemical analysis does not permit to define the antigenic specificity of these IgG. However, it is tempting to speculate that the spike protein of SARS-CoV-2 present in several tissue samples is a potential target, although we cannot exclude that other viral and tissue antigens may also be recognized by the antibodies. The latter possibility is suggested by the reports of sequence homology between SARS-CoV-2 and human proteins-derived peptides published by different groups [26-28] and is supported in this study by the finding of Spike protein-independent localization of IgG in the lung of COVID-19 patients. An important implication of these observations is that, because of cross-reactivity of human and viral proteins, the antibody response to SARS-CoV-2 may contribute to the pathogenesis of the disease, causing tissue damage possibly as a result of C activation. A similar conclusi...

    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.

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  3. Version published to 10.1101/2021.01.07.21249116v1 on medRxiv