Neutrophil extracellular traps in COVID-19

Abstract

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis - including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.

Keywords: Infectious disease; Inflammation; Neutrophils.

Figure 1. Detection of NETs in sera of COVID-19 patients.

Sera from COVID-19 patients (n = 50) and healthy controls (n = 30) were assessed for cell-free DNA (A), myeloperoxidase-DNA (MPO-DNA) complexes (B), or citrullinated histone H3 (Cit-H3) (C). COVID-19 samples were compared with controls by Mann-Whitney U test; ***P < 0.001, ****P < 0.0001. For the COVID-19 samples, correlation of cell-free DNA with MPO-DNA (D) and Cit-H3 (E) was assessed. Spearman’s correlation coefficients were calculated and are shown in the panels.

Figure 2. Association between NETs and clinical biomarkers in all available serum samples.

Cell-free DNA was compared with clinical laboratory results (when available on the same day), and correlation coefficients were calculated for C-reactive protein (A, n = 64), D-dimer (B, n = 56), lactate dehydrogenase (C, n = 55), and absolute neutrophil count (D, n = 69). In E (n = 69), MPO-DNA was compared with absolute neutrophils count, and in F (n = 81), Cit-H3 was compared with platelet count. The results of other relevant comparisons are discussed in the text. Pearson’s correlation coefficients were calculated and are shown in the panels.

Figure 3. Levels of NETs associate with mechanical ventilation in all available serum samples.

Serum samples were grouped by clinical status (room air versus mechanical ventilation) and analyzed for cell-free DNA (A, n = 51), MPO-DNA complexes (B, n = 51), Cit-H3 (C, n = 51), and absolute neutrophil count (D, n = 42). Groups were compared by Mann-Whitney U test; ****P < 0.0001, *P < 0.05. For D, the P value was 0.08.

Figure 4. COVID-19 sera trigger control neutrophils to release NETs.

COVID-19 samples (for which sufficient sera were available) were tested for their ability to trigger neutrophils isolated from healthy controls to undergo NETosis. (A) NETosis was quantified using the cell-impermeant dye SYTOX Green as described in Methods (n = 27 COVID-19 samples, and n = 20 controls). Fluorescence intensity (excitation/emission 504 nm/523 nm) is shown on the y axis. Bars demonstrate mean and standard deviation while each data point represents a unique patient/control; ***P < 0.001 by t test. (B) In an independent set of experiments, NETosis was quantified as nuclease-liberated MPO activity (n = 27 COVID-19 samples, and n = 17 controls). Absorbance at 450 nm is shown on the y axis after subtracting background from untreated cells. Bars demonstrate mean and standard deviation while each data point represents a unique patient/control; **P < 0.01 by t test. (C) Representative image of control neutrophils cultured with 10% heterologous control serum (upper) or COVID-19 serum (lower). Neutrophil elastase is stained green and DNA is stained blue. Scale bar: 100 μm. The yellow arrows highlight some examples of NET strands.