Neutrophil extracellular traps infiltrate the lung airway, interstitial, and vascular compartments in severe COVID-19

Abstract

Infection with SARS-CoV-2 is causing a deadly and pandemic disease called coronavirus disease-19 (COVID-19). While SARS-CoV-2-triggered hyperinflammatory tissue-damaging and immunothrombotic responses are thought to be major causes of respiratory failure and death, how they relate to lung immunopathological changes remains unclear. Neutrophil extracellular traps (NETs) can contribute to inflammation-associated lung damage, thrombosis, and fibrosis. However, whether NETs infiltrate particular compartments in severe COVID-19 lungs remains to be clarified. Here we analyzed postmortem lung specimens from four patients who succumbed to COVID-19 and four patients who died from a COVID-19-unrelated cause. We report the presence of NETs in the lungs of each COVID-19 patient. NETs were found in the airway compartment and neutrophil-rich inflammatory areas of the interstitium, while NET-prone primed neutrophils were present in arteriolar microthrombi. Our results support the hypothesis that NETs may represent drivers of severe pulmonary complications of COVID-19 and suggest that NET-targeting approaches could be considered for the treatment of uncontrolled tissue-damaging and thrombotic responses in COVID-19.

Figure 1.

DAPI⁺MPO⁺Cit-H3⁺ NETs are uniquely detected in lungs of COVID-19 patients. (A–D) Representative confocal microscopy pictures (magnification, 20×, maximal intensity projections of a Z-stack) of (A) DAPI (blue), (B) MPO (green), (C) Cit-H3 (red), and (D) merged stainings from non–COVID-19 and COVID-19 lungs. Control sections were incubated with rabbit and goat sera and with secondary antibodies (sec. Abs). Pictures are representative of one of four non–COVID-19 and one of four COVID-19 patients analyzed. Extracellular DAPI⁺MPO⁺Cit-H3⁺ NETs are indicated by large arrowheads, while one MPO⁺Cit-H3⁺ neutrophil is indicated by a thin arrow. (E–H) Representative high-resolution confocal microscopy pictures (magnification, 63×, maximal intensity projections of a Z-stack) of (E) DAPI (blue), (F) MPO (green), (G) Cit-H3 (red), and (H) merged stainings from COVID-19 lungs. DAPI⁺MPO⁺Cit-H3⁺ NETs are indicated by large arrowheads. (I) Quantification of NET volume from multiple fields (20×) of lung sections from the four non–COVID-19 and the four COVID-19 patients. Results show individual values and mean. P value compares non–COVID-19 and COVID-19 samples and was calculated using a nonparametric Mann–Whitney U test on mean values. *, P < 0.05. Scale bars, 20 µm.

Figure S1.

No evidence of NETs in postmortem lung biopsy specimens from non–COVID-19 patients. (A) Photographs of entire antibody-stained immunofluorescence lung sections from non–COVID-19 patients. (B) Magnification of NET-rich zones, indicated by white boxes in A. (C) Representative confocal microscopy pictures of immunofluorescence staining of lung sections from non–COVID-19 patients (MPO [red] and Cit-H3 [red] merged with DAPI [blue]). Scale bars, 1 mm (A); 100 µm (B); 20 µm (C).

Figure S2.

No evidence of NETs in postmortem biopsy specimens from other organs in COVID-19 patients. (A–D) Representative confocal microscopy pictures of immunofluorescence staining from (A) liver, (B) pancreas, (C) kidney, and (D) heart sections from two COVID-19 patients are shown (MPO [red] and Cit-H3 [red] merged with DAPI [blue]). Areas containing MPO⁺ neutrophils are shown. Scale bars, 20 µm.

Figure 2.

NETs are broadly distributed in postmortem lung specimens from COVID-19 patients. (A) Photographs of entire antibody-stained immunofluorescence lung sections from COVID-19 patients. (B) Magnification of NET-rich zones, indicated by white boxes in A. MPO⁺Cit-H3⁺ NETs are indicated by large arrowheads. (C) Photographs of entire H&E-stained lung sections from COVID-19 patients. NET-infiltrating areas are circled in black. Scale bars, 1 mm (A and C); 100 µm (B).

Figure 3.

NETs are found in lesional areas of the lung airway, interstitial, and vascular compartments. (A–D) Representative pictures of H&E staining from lung sections are shown on the left, and representative pictures of immunofluorescence staining from adjacent lung sections are shown on the right (MPO [red] and Cit-H3 [red] merged with DAPI [blue]). The black box on the left indicates the zone shown by immunofluorescence on the right. MPO⁺Cit-H3⁺ NETs are indicated by arrowheads. Examples of NETs found (A) in a terminal bronchiole and alveoli, (B) in small bronchi, (C) in the interstitial compartment, and (D) in a microthrombus within an arteriole are shown. Scale bars, 50 µm.