COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds

  1. Beeke Tappe
  2. Chris D. Lauruschkat
  3. Lea Strobel
  4. Jezreel Pantaleón García
  5. Oliver Kurzai
  6. Silke Rebhan
  7. Sabrina Kraus
  8. Elena Pfeuffer-Jovic
  9. Lydia Bussemer
  10. Lotte Possler
  11. Matthias Held
  12. Kerstin Hünniger
  13. Olaf Kniemeyer
  14. Sascha Schäuble
  15. Axel A. Brakhage
  16. Gianni Panagiotou
  17. P. Lewis White
  18. Hermann Einsele
  19. Jürgen Löffler
  20. Sebastian Wurster

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Abstract

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus - and R. arrhizus -induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients.

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  1. Version published to 10.3389/fimmu.2022.954985
  2. ScreenIT

    SciScore for 10.1101/2022.04.21.22274082: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: This study was approved by the Ethics Committee of the University of Würzburg (protocol number 152/20).
    Consent: Informed written consent was obtained from all participants.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    , co-stimulatory antibodies (α-CD28 and α-CD49d, Miltenyi Biotec, Bergisch Gladbach, Germany), and RPMI medium, as summarized in Table S1.
    α-CD49d , Miltenyi Biotec , Bergisch Gladbach , Germany) ,
    suggested: None
    Antibodies for extracellular staining were added as detailed in Table S2, along with the fixable Viobility™ Live/Dead Dye (Miltenyi Biotec, Bergisch Gladbach, Germany) and dihydrorhodamine 123 (Sigma-Aldrich, St. Louis, MO, USA) for visualization of reactive oxygen species (ROS).
    ROS
    suggested: None
    Software and Algorithms
    SentencesResources
    Acquisition was performed using a Luminex detection system (Bio-Plex 200 system) and Bio-Plex Manager(tm
    Bio-Plex
    suggested: None
    Manager(tm
    suggested: None
    Downstream data analysis was performed with Kaluza v.
    Kaluza
    suggested: (Kaluza, RRID:SCR_016182)
    Statistics: Data compilation, statistical analyses, and visualization were performed using Microsoft Excel and GraphPad Prism v.9 (
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad Software, San Diego, CA, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Principal component analysis (PCA) of cytokine profiles was performed with ClustVis (https://biit.cs.ut.ee/clustvis/) [15] based on ln(x)+1-transformed data.
    ClustVis
    suggested: (ClustVis, RRID:SCR_017133)

    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:
    Due to the restrictive exclusion criteria, a major limitation of this study was the relatively small cohort size, especially for responses to R. arrhizus that were only tested during the Delta wave (after the emergence of CAM). The high consistency of our findings, among a heterogenous patient cohort, reduces the impact of this limitation on conclusions regarding global alterations of anti-mold immunity in COVID-19 patients, but downstream comparisons of immune impairment with specific clinical characteristics were severely underpowered and mostly relied on “soft statistics” such as correlation. This limitation is further compounded by the fact that some of the clinical variables were not entirely independent. For instance, due to the restriction of our study to patients with moderate disease severity (WHO scores 4 and 5) at the time of immune cell sampling, patients with higher initial COVID-19 severity tended to be enrolled at a later stage of their post-COVID-19 recovery (after release from the ICU). The size of our dataset precluded multivariate analysis to determine the independent impact of these and other clinical characteristics on immune readouts. Furthermore, the limited available blood volume precluded both a detailed wet-lab confirmation of the underlying immune pathways predicted by enrichment analysis and the assessment of immune responses to additional (i.e., non-fungal) antigens. Therefore, some of the observed immune alterations are likely not specific to fun...

    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.

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  3. Version published to 10.1101/2022.04.21.22274082 on medRxiv