Broncho-alveolar inflammation in COVID-19 patients: a correlation with clinical outcome

  1. Laura Pandolfi
  2. Tommaso Fossali
  3. Vanessa Frangipane
  4. Sara Bozzini
  5. Monica Morosini
  6. Maura D’Amato
  7. Sara Lettieri
  8. Mario Urtis
  9. Alessandro Di Toro
  10. Laura Saracino
  11. Elena Percivalle
  12. Stefano Tomaselli
  13. Lorenzo Cavagna
  14. Emanuela Cova
  15. Francesco Mojoli
  16. Paola Bergomi
  17. Davide Ottolina
  18. Daniele Lilleri
  19. Angelo Guido Corsico
  20. Eloisa Arbustini
  21. Riccardo Colombo
  22. Federica Meloni

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Abstract

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19.

Methods

Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) ( n  = 28) and to the Intermediate Medicine Ward (IMW) ( n  = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels.

Results

ICU patients showed a marked increase in neutrophils (1.24 × 10 5  ml − 1 , 0.85–2.07), lower lymphocyte (0.97 × 10 5  ml − 1 , 0.024–0.34) and macrophages fractions (0.43 × 10 5  ml − 1 , 0.34–1.62) compared to IMW patients (0.095 × 10 5  ml − 1 , 0.05–0.73; 0.47 × 10 5  ml − 1 , 0.28–1.01 and 2.14 × 10 5  ml − 1 , 1.17–3.01, respectively) ( p  < 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p  < 0.01, IL8 p  < 0.0001), and also in patients who did not survive (IL6 p  < 0.05, IL8 p  = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab ( p  < 0.1) or antivirals ( p  < 0.05).

Conclusions

Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.17.20155978: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Research and data collection protocols were approved by the Institutional Review Boards (Comitato Etico di Area 1) (prot. 20100005334) and by IRCCS Policlinico San Matteo Foundation Hospital (prot. 20200046007), written informed consent was provided by patients when possible (IMW and conscious ICU patients) and was waived in all other cases.
    Consent: Research and data collection protocols were approved by the Institutional Review Boards (Comitato Etico di Area 1) (prot. 20100005334) and by IRCCS Policlinico San Matteo Foundation Hospital (prot. 20200046007), written informed consent was provided by patients when possible (IMW and conscious ICU patients) and was waived in all other cases.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The immunostaining of paraffin-embedded sections was done using SARS-CoV-2 (2019-nCoV) Nucleoprotein / NP Antibody, Rabbit MAb (Sino Biological, Catalog number: 40143-R019) – Dilution 1:1000 and SARS-CoV-2 (2019-nCoV) Spike Antibody, Rabbit MAb (Sino Biological, Catalog number: 40150-R007) - Dilution 1:400. ELISA Assays: To quantify IL8, IL10 and IL6, we used the SimpleStep ELISA® kit (Abcam, Cambridge, UK).
    SARS-CoV-2 (2019-nCoV) Nucleoprotein /
    suggested: None
    IL8
    suggested: None
    IL10
    suggested: None
    IL6
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 infected VERO E6 cells at 48 and 72 hours were used as positive controls.
    VERO E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were statistically analyzed with Graphpad Prism version 8.4.1.
    Graphpad Prism
    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: We detected the following sentences addressing limitations in the study:
    We are aware that this study has some limitations. We performed these preliminary analyses on limited sample size given the high risk of infection of the health care personnel. This limited our possibility to make further analyses, such as cell surface activation markers assessment by flow cytometry on macrophage or lymphocytes. Another limitation is the lack of paired assessment of cytokines in the peripheral blood due to the absence of stored serum samples from acute COVID19 patients in our institutions. Finally, we know that the infectious complications registered (Table 1) might have influenced cell differentials and cytokine levels however, the percentage of bacterial and fungal co-infection was very high in all patients in ICU without a significant difference between survivors and non-survivors. As a result of our study we wish to highlight the possible crucial role of IL8 in COVID-19 infection at the lung level. This cytokine showed higher values among ICU compared to IMW patients (Fig. 3a and b) and was associated to a negative outcome (Fig. 3d). It is known that IL8 acts as a chemoattractant and activating factor of neutrophils, in fact we assessed a direct correlation between IL8 and neutrophils percentage in BAL (Table 3 and Fig. S4b). Hence, we can infer that the release of IL8 represents a crucial step of SARS-CoV-2 pathogenesis and its pathway might represent a possible target of future intervention.

    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.

    About SciScore

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  2. Version published to 10.1186/s12890-020-01343-z
  3. Version published to 10.21203/rs.3.rs-49968/v5 on Research Square
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  5. Version published to 10.21203/rs.3.rs-49968/v3 on Research Square
  6. Version published to 10.21203/rs.3.rs-49968/v2 on Research Square
  7. Version published to 10.21203/rs.3.rs-49968/v1 on Research Square
  8. Version published to 10.1101/2020.07.17.20155978v1 on medRxiv