Neutrophil extracellular traps (NETs) are increased in the alveolar spaces of patients with ventilator-associated pneumonia

Abstract

Background: Neutrophils release neutrophil extracellular traps (NETs) in response to invading pathogens. Although NETs play an important role in host defense against microbial pathogens, they have also been shown to play a contributing mechanistic role in pathologic inflammation in the absence of infection. Although a role for NETs in bacterial pneumonia and acute respiratory distress syndrome (ARDS) is emerging, a comprehensive evaluation of NETs in the alveolar space of critically ill patients has yet to be reported. In this study, we evaluated whether markers of NET formation in mechanically ventilated patients are associated with ventilator-associated pneumonia (VAP).

Methods: We collected bronchoalveolar lavage fluid from 100 critically ill patients undergoing bronchoscopy for clinically suspected VAP. Subjects were categorized by the absence or presence of VAP and further stratified by ARDS status. NETs (myeloperoxidase (MPO)-DNA complexes) and the NET-associated markers peroxidase activity and cell-free DNA were analyzed by enzyme-linked immunosorbent assay and colorimetric assays, respectively. Quantitative polymerase chain reaction of nuclear and mitochondrial DNA was used to determine the origin of the extruded DNA. Interleukin (IL)-8 and calprotectin were assayed as measures of alveolar inflammation and neutrophil activation. Correlations between NETs and markers of neutrophil activation were determined using Spearman's correlation. We tested for associations with VAP and bacterial burden by logistic and linear regression, respectively, using log₁₀-transformed NETs.

Results: MPO-DNA concentrations were highly correlated with other measures of NET formation in the alveolar space, including cell-free DNA and peroxidase activity (r = 0.95 and r = 0.87, p < 0.0001, respectively). Alveolar concentrations of MPO-DNA were higher in subjects with VAP and ARDS compared with those with ARDS alone (p < 0.0001), and higher MPO-DNA was associated with increased odds of VAP (odds ratio 3.03, p < 0.0001). In addition, NET concentrations were associated with bacterial burden (p < 0.0001) and local alveolar inflammation as measured by IL-8 (r = 0.89, p < 0.0001).

Conclusions: Alveolar NETs measured by MPO-DNA complex are associated with VAP, and markers of NETosis are associated with local inflammation and bacterial burden in the lung. These results suggest that NETs contribute to inflammatory responses involved in the pathogenesis of VAP.

Keywords: Neutrophil extracellular traps (NETs); acute respiratory distress syndrome; alveoli; bronchoalveolar lavage; calprotectin; cell-free DNA; peroxidase; ventilator-associated pneumonia.

Fig. 1

NETs and NETosis markers by clinical group. a Myeloperoxidase (MPO)-DNA, b peroxidase activity, and c cell-free DNA (cf-DNA) are compared in ventilator associated pneumonia (VAP) with and without acute respiratory distress syndrome (ARDS). MPO-DNA is higher in ARDS patients with VAP. P values are for Kruskal-Wallis (nonparametric analysis of variance (ANOVA) with post-test Dunn’s to account for multiple pairwise tests). Error bars show the median and interquartile range. ***p < 0.0001, *p < 0.05

Fig. 2

NETs and neutrophil activation. NETs are associated with a–c the neutrophil cytosolic protein calprotectin and d–f the neutrophil chemoattractant interleukin-8 (IL-8). The correlation was a Spearman’s correlation for nonparametric data. Calprotectin (ug/mL) and IL-8 (pg/mL) were both measured by immunoassay. cf-DNA cell-free DNA, MPO-DNA myeloperoxidase DNA

Fig. 3

NETs and bacterial burden. Bacterial colony counts from quantitative bronchoalveolar lavage cultures are represented by quartile of increasing colony count versus measures of NETs and NETosis. Presented p values are from regression analysis with log-transformed a myeloperoxidase (MPO)-DNA, b peroxidase, or c cell-free DNA (cf-DNA) concentrations and quartile of colony count