Background
Mechanical ventilation in critically ill adults can be complicated by ventilator-associated pneumonia (VAP). VAP is associated with mortality greater than 40% in some studies [
1,
2]. Mechanical ventilation may also contribute to the pathogenesis of acute respiratory distress syndrome (ARDS) [
3,
4]. In patients with ARDS, the development of VAP may be associated with higher risk of death [
5].
One of the main contributors to pathological inflammation in both ARDS and VAP are neutrophils. While neutrophils play a protective role against invading pathogens, unrestrained inflammation may lead to tissue injury in the lung [
6,
7]. One of the mechanisms by which neutrophils could promote both bacterial killing and contribute to tissue injury is through release of extracellular traps [
8‐
10] . These neutrophil extracellular traps (NETs) are characterized by extrusion of chromatin bound to cytosolic and granular contents of the cells. NETs are composed of DNA complexed with myeloperoxidase (MPO) or citrullinated histones [
9,
10]. Surrogate markers of NET formation include peroxidase activity and cell-free DNA (cf-DNA) levels [
11‐
13]. Although NET-derived DNA is primarily of nuclear origin, we recently demonstrated that mitochondrial extrusion could occur concomitantly with NETosis, with released oxidized mitochondrial DNA being highly inflammatory [
14,
15]. Thus, NET formation may be a key factor in balancing protective versus harmful inflammation.
In murine models of lung injury, NETs develop in response to a variety of infectious stimuli and contribute to lung injury [
16‐
19]. In humans, plasma NET levels were higher in patients with pneumonia-associated ARDS or transfusion-associated ARDS than in subjects without ARDS [
18,
19]. Extracellular histones, likely derived from dying cells, are elevated in bronchoalveolar lavage fluid (BALF) and the plasma of subjects with ARDS compared with healthy controls [
20]. Nevertheless, a thorough examination of NETs in the alveolar space collected by bronchoalveolar lavage in critically ill mechanically ventilated patients has not been reported to date.
In this study, we sought to evaluate the levels of traditional markers and novel markers of NETosis in BALF from mechanically ventilated ICU patients. We hypothesized that markers of NETosis would be associated with microbially confirmed VAP.
Discussion
Our study broadly assessed NETosis in the alveolar space of critically ill patients. We found that the most direct measure of NETs, MPO-DNA complexes, is higher in patients with microbially confirmed VAP. Increasing bacterial burden in the alveolar space was also associated with increased NETosis. This suggests that direct bacterial infection strongly drives NETosis and has implications for the future study of both ARDS and bacterial pneumonia. Extrapolation of our results would suggest that NETosis in the alveolar space of patients with direct ARDS due to pneumonia may differ from subjects with indirect ARDS. We could not directly address this in our study due to the limited number of patients with ARDS.
We also found that other markers of NETosis in the alveolar space correlate highly with MPO-DNA complexes. Cell-free DNA and peroxidase activity also differed by clinical condition and were highly correlated with MPO-DNA complexes suggesting they could serve as reasonable surrogates for NETosis. In particular, peroxidase activity was higher in patients with VAP alone compared with patients without VAP or ARDS. This again supports that bacterial infection is a strong driver of the process of NETosis. Consistent with this finding, we determined that MPO-DNA complexes, cell-free DNA, and peroxidase activity all increased with increasing bacterial burden measured by quantitative culture. Because this study measured alveolar concentrations rather than circulating concentrations of NETs, our findings suggest local, intra-alveolar NETosis in the presence of bacterial infection in the lung.
NETosis also correlated with alveolar inflammation as measured by IL-8. IL-8 is a neutrophil chemoattractant and has been shown to be associated with ARDS outcomes and VAP [
27‐
29].
Calprotectin is a dimer of calcium binding proteins S100A8 and S100A9 and is a measure of neutrophil degranulation [
30]. In our study, calprotectin also correlated with NETosis in the alveolar space. A previous proteomic study of BALF showed that S100A8 was differentially expressed in ARDS patients with VAP compared with those without VAP [
31]. Our study thus provides a strong link between local alveolar inflammation, neutrophil activation, and NETosis.
Our study has several limitations. First, we do not have corresponding plasma samples to correlate the degree of alveolar NETosis with circulating concentrations of NETs. Future studies will address whether circulating concentrations can serve as a surrogate for direct alveolar measurement. Second, subjects included in this study were ventilated for a variable number of days prior to alveolar sampling. This may in part explain why we found stronger associations for patients with VAP because clinical bronchoscopy was performed for this indication. Third, the use of samples obtained from clinically indicated bronchoscopy limits our ability to provide analyses for ARDS since patients had ARDS for a variable number of days prior to alveolar sampling. Finally, because all of our subjects were at risk for VAP with mechanical ventilation for > 48 h, the severity of critical illness is high for all subjects in this study including those without ARDS or VAP. We did measure MPO-DNA complexes from healthy subjects and the amounts were undetectable. This suggests that, at baseline, mechanical ventilation likely induces some degree of neutrophilic inflammation.
Whether NETs serve a protective role in the setting of bacterial infection with VAP and/or play a role in tissue damage remains unclear. Previous studies provide support for NETs playing a role in tissue injury in the lung [
16,
18,
32]. There are also dynamic factors that may influence NET formation in the alveolar space. For example, BALF from ARDS patients has recently been shown to promote NET production from neutrophils [
33]. In community-acquired pneumonia, higher circulating amounts of cell-free nucleosomes was associated with higher 30-day mortality, showing a link between a marker of NETosis and poor clinical outcome [
34]. These studies support our hypothesis that NETs serve as a marker of alveolar inflammation and may contribute to tissue injury and poor outcomes in critical illness.
Conclusions
Neutrophil-extracellular traps (NETs), measured as MPO-DNA complexes, are present in the alveolar space in critically ill patients. Higher MPO-DNA complexes are associated with VAP, higher bacterial burden, and with other markers of alveolar and neutrophilic inflammation. This study suggests a role for NETs in VAP pathogenesis, and NETs may be considered as a marker of neutrophilic inflammation in the alveolar space.
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