Neutrophil Extracellular Traps in SARS-CoV2 Related Pneumonia in ICU Patients: The NETCOV2 Study

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a poorly understood disease involving a high inflammatory status. Neutrophil extracellular traps (NETs) have been described as a new pathway to contain infectious diseases but can also participate in the imbalance of the inflammatory and the coagulation systems. NETs could be a therapeutic target in COVID-19 patients. Methods: Consecutive patients with SARS-CoV2 related pneumonia admitted to the intensive care unit were included in a prospective bicentric study. Neutrophil extracellular trap concentrations were quantified in whole blood samples at day-1 and day-3 by flow cytometry. The primary outcome was the association between the blood NET quantification at ICU admission and the number of days with refractory hypoxemia defined by a PaO₂/FIO₂ ratio ≤100 mmHg. Results: Among 181 patients admitted to the ICUs for acute respiratory failure related to SARS-CoV2 pneumonia, 58 were included in the analysis. Patients were 62 [54, 69] years old in median, mostly male (75.9%). The median number of days with severe hypoxemia was 4 [2, 6] days and day-28 mortality was 27.6% (n = 16). The blood level of NETs significantly decreased between day-1 and day-3 in patients who survived (59.5 [30.5, 116.6] to 47 [33.2, 62.4] p = 0.006; 8.6 [3.4, 18.0] to 4 [1.4, 10.7] p = 0.001 and 7.4 [4.0, 16.7] to 2.6 [1.0, 8.3] p = 0.001 for MPO+, Cit-H3+, and MPO+ Cit-H3+ NETs, respectively) while it remained stable in patients who died (38.4 [26.0, 54.8] to 44.5 [36.4, 77.7] p = 0.542; 4.9 [1.3, 13.0] to 5.5 [2.8, 6.9] p = 0.839 and 4 [1.3, 13.6] to 2.7 [1.4, 4.5] p = 0.421 for MPO+, Cit-H3+, and MPO+ Cit-H3+ NETs, respectively). In multivariable negative binomial regression, the blood level of MPO+ NETs was negatively associated with the number of days with severe hypoxemia within 7 days (0.84 [0.73, 0.97]), while neither Cit-H3+ NETs nor double-positive NETs were significantly associated with the primary outcome. Conclusion: The whole blood level of NETs at day-1 was negatively associated with the number of days with severe hypoxemia in patients admitted to the intensive care unit for SARS-CoV2 related pneumonia. The lack of decrease of the blood level of NETs between day-1 and day-3 discriminated patients who died within day-28.

Keywords: COVID19; SARS-CoV2; coronavirus; neutrophils extracellular traps neutrophils extracellular traps; pneumonia.

Figure 1

Typical staining of NETs in blood sample of a patient with SARS-COV2. After gating of nucleated cells (Leukocytes) with the DNA marker Hoechst 34580 (Plot A), SYTOX positive cells (ie cells with compromised plasma membranes) were isolated within the Leukocytes gate (Plot B). Single-positive (MPO+ or Cit-H3+) and double-positive (MPO+ Cit-H3+) NETs were then quantified in the SYTOX+ population (Plot C), respectively at 0.90, 0.03, and 0.52% for this patient.

Figure 2

Association between day-1 blood level of NETs and respiratory sequential organ failure assessment (SOFA) score at day-1, day-2, and day-3. The y-axis is shown using a logarithmic scale. The comparison is performed using a Jonckheere test to take into consideration the ordered respiratory SOFA score.

Figure 3

Evolution of blood levels of NETs between day-1 and day-3 according to the vital status at day-28.