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

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 × 10⁵ ml^{- 1}, 0.85-2.07), lower lymphocyte (0.97 × 10⁵ ml^{- 1}, 0.024-0.34) and macrophages fractions (0.43 × 10⁵ ml^{- 1}, 0.34-1.62) compared to IMW patients (0.095 × 10⁵ ml^{- 1}, 0.05-0.73; 0.47 × 10⁵ ml^{- 1}, 0.28-1.01 and 2.14 × 10⁵ 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.

Keywords: Bronchoalveolar lavage; COVID-19; Cytokines; Lung; SARS-CoV-2.

Fig. 1

Cytological analysis of BAL collected. a Histograms of total cells ml^− 1 counted in IMW and ICU samples showed as median (IQR). ***, p < 0.001 vs. ICU b Cell counts are converted in percentage of leucocytes comparing IMW with ICU samples. Data are shown as median (IQR). ***, p < 0.001 vs. ICU. b-d Percentage of each leucocyte quantified in all analyzed BALs dividing them into survivors and non-survivors. Data are shown as median (IQR). *, p < 0.05

Fig. 2

a and b Electron micrographs showing both a cytopathic cells and still morphologically preserved mononuclear cells and b extensive cytopathy involving all cells. c the SARS-CoV-2–anti-spike antibodies immunoreactive multinucleated cells in the BAL sample of one of the ICU patients, 40x; d flaps of epithelial cells and inflammatory cells immunoreacting with anti-spike antibodies, 10x. PMN = polymorphonuclear leukocyte. Scale bar = 5 μm

Fig. 3

a Cytokines quantified in BAL of ICU patients and IMW patients (repeated BAL samples in the same patients have been excluded) has been transformed in Log10(pg ml^− 1) and represented as median (IQR). **p < 0.01 vs. ICU; ***p < 0.001 vs. ICU. b-d Quantified cytokines divided in survivors and non-survivors. *p ≤ 0.05 vs. survivors. Data has been transformed as Log10(pg ml^− 1) and represented as median (IQR)

Fig. 4

Differences between cytokines after tocilizumab, steroids, and anti-viral drugs treatment. Hydroxychloroquine has been added in the anti-viral group. Data are represented as median (IQR). **p < 0.05 vs. steroids

Fig. 5

Correlation cytokine/BAL cell differentials: a macrophages percentage vs. IL6/IL8 and b neutrophil percentage vs. IL6/IL8