Neutrophil extracellular traps are increasingly recognized as pathogenic in acute injury, but their role in sepsis-related acute respiratory distress syndrome is unknown
In 35 patients with acute respiratory distress syndrome secondary to pneumonia, neutrophil extracellular traps were elevated in the blood and bronchoalveolar fluid, and associated with interleukin-8 and neutrophil levels; however, higher (vs. lower) levels of neutrophil extracellular traps were not associated with mortality or duration of mechanical ventilation
Neutrophil extracellular traps have been associated with tissue damage. Whether these are involved in the pathogenesis of human acute respiratory distress syndrome (ARDS) and could be a potential therapeutic target is unknown. The authors quantified bronchoalveolar and blood neutrophil extracellular traps in patients with pneumonia-related ARDS and assessed their relationship with ventilator-free days.
Immunocompetent patients with pneumonia and moderate or severe ARDS (n = 35) and controls (n = 4) were included in a prospective monocentric study. Neutrophil extracellular trap concentrations were quantified (as DNA–myeloperoxidase complexes) in bronchoalveolar lavage fluid and serum by enzyme-linked immunosorbent assay. The relationship between bronchoalveolar lavage neutrophil extracellular trap concentrations and the primary clinical endpoint (i.e., the number of live ventilator-free days at day 28) was assessed using linear regression analyses.
There was no significant relationship between bronchoalveolar lavage neutrophil extracellular trap concentrations and ventilator-free days by multiple regression analysis (β coefficient = 2.40; 95% CI, −2.13 to 6.92; P = 0.288). Neutrophil extracellular trap concentrations were significantly higher in bronchoalveolar lavage than in blood of ARDS patients (median [first to third quartiles]:154 [74 to 1,000] vs. 26 [4 to 68] arbitrary units, difference: −94; 95% CI, −341 to −57; P < 0.0001). Bronchoalveolar concentrations of patients were higher than those of controls (154 [74 to 1,000] vs. 4 [4 to 4] arbitrary units, difference: −150; 95% CI, −996 to −64; P < 0.001) and associated with bronchoalveolar interleukin-8 (Spearman’s ρ = 0.42; P = 0.012) and neutrophil concentrations (ρ = 0.57; P < 0.0001). Intensive care unit mortality (12%, n = 2 of 17 vs. 17%, n = 3 of 18; P > 0.99) and the number of ventilator-free days at day 28 (22 [14 to 25] vs. 14 [0 to 21] days; difference: −5; 95% CI, −15 to 0; P = 0.066) did not significantly differ between patients with higher (n = 17) versus lower (n = 18) bronchoalveolar neutrophil extracellular trap concentrations.
Bronchoalveolar neutrophil extracellular trap concentration was not significantly associated with mechanical ventilation duration in pneumonia-related ARDS.
The acute respiratory distress syndrome (ARDS) is the most severe form of acute respiratory failure, resulting from direct or indirect insults to the alveolo–capillary membrane. ARDS affects 10% of all intensive care unit patients and 23% of those receiving mechanical ventilation.1 Despite major improvements in mechanical ventilation strategies during the past decades,2 the hospital mortality of ARDS patients remains as high as 40%.1 In fact, none of the pharmacologic interventions tested in ARDS, including—but not limited to—steroids,3 β2-agonists,4 or statins,5 has shown significant survival benefit. The identification of biomarkers of disease severity that would be potential therapeutic targets is thus a key step for developing novel treatments for ARDS.
ARDS pathogenesis is characterized by the influx of protein-rich edema fluid into the air spaces as a consequence of increased permeability of the alveolo–capillary membrane. High alveolar concentrations of inflammatory mediators trigger lung neutrophil influx and activation, leading to the release of reactive oxygen species, proteases, and cytokines, all of which contribute to alveolar injury.6,7 The key role of neutrophils in the pathogenesis of ARDS was suggested by experimental models showing that their elimination decreased the severity of acute lung injury8 as well as by the clinical observation that neutropenia recovery in neutropenic patients with lung injury was associated with pulmonary function deterioration.9
Neutrophil extracellular traps are extracellular fibers composed of DNA, histones, and granule-derived proteins such as elastase or myeloperoxidase released by neutrophils during a process termed NETosis.10,11 Neutrophil extracellular traps can trap and kill extracellular pathogens and have been shown to be beneficial during infections: deficiency in neutrophil extracellular trap production12–14 or neutrophil extracellular trap scaffold destruction by bacterial DNases15,16 increased the susceptibility to infections. On the other hand, experimental data suggesting that neutrophil extracellular traps may also cause adverse tissue injury to the host have accumulated: extracellular histones (the major components of neutrophil extracellular traps) are highly toxic and induce respiratory failure when infused intravenously,17 and neutrophil extracellular traps have been detected in the lungs of mice subjected to endotoxemia18 or aggressive mechanical ventilation.19 Additionally, plasma20 and lung21 neutrophil extracellular trap concentrations have recently been correlated with the severity of human ARDS. A significant body of literature also illustrated the detrimental role of neutrophil extracellular traps in various clinical conditions, including autoimmune disorders, thrombosis, and cystic fibrosis, but lung and blood neutrophil extracellular trap production in human ARDS have not been comprehensively explored.
Whether neutrophil extracellular traps are involved in the pathogenesis of human ARDS and could be a potential therapeutic target is still unknown. In the current study, we hypothesized that (1) neutrophil extracellular traps are produced in the lungs of patients with pneumonia-related ARDS and associated with regional neutrophil infiltration, and that (2) bronchoalveolar neutrophil extracellular trap concentrations are associated with poorer outcome, as assessed by live ventilator-free days at day 28.
Materials and Methods
Methods are further described in the supplemental digital content (Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
This prospective, single-center, observational cohort study was approved by the institutional ethics committee (Comité de Protection des Personnes Ile-de-France V, Paris, France). Consecutive patients diagnosed with pneumonia-related ARDS22 admitted to the medical intensive care unit of Henri Mondor Hospital, Créteil, France, from January 2014 to May 2015 were eligible for inclusion in the study. Informed consent was obtained from all included patients or their relatives. No statistical power calculation was conducted before the study because of the lack of data available regarding lung neutrophil extracellular trap production during human ARDS at the time the study was designed. An 18-month inclusion period had a priori been defined.
Patients and Data Collection
All patients with moderate or severe pneumonia-related ARDS (Berlin definition22 ) were included consecutively with the following inclusion criteria: tracheal intubation and mechanical ventilation since less than 72 h; pulmonary infection diagnosed less than 7 days before; bilateral pulmonary infiltrates on chest x-ray; a PaO2/FiO2 ratio not greater than 200 mmHg with a positive end-expiratory pressure of at least 5 cm H2O. Noninclusion criteria were as follows: age younger than 18 yr, pregnancy, chronic respiratory failure requiring long-term oxygen therapy, Child-Pugh C liver cirrhosis, lung fibrosis, immunosuppression, SAPS II (Simplified Acute Physiology II score) greater than 90, irreversible neurologic disorders, or patients with withholding or withdrawing of life-sustaining therapies and profound hypoxemia (PaO2/FiO2 less than 75 mmHg).
Control patients (i.e., non–mechanically ventilated patients free of ARDS, any active infection, diffuse interstitial pneumonia, or immunosuppression; n = 4), undergoing a bronchoscopy with bronchoalveolar lavage and blood sampling as part of routine care, were also included (eTable 1, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
ARDS patients received mechanical ventilation using a standardized protective ventilation strategy.2,23 Other treatments, including neuromuscular blocking agents,24 nitric oxide inhalation,25 prone positioning,26 and venovenous extracorporeal membrane oxygenation, were administered depending on the severity of ARDS.27 The prevention of ventilator-associated pneumonia followed a multifaceted program28 ; Sedation and mechanical ventilation weaning followed standardized protocols.29
Demographics and clinical and laboratory variables were recorded upon intensive care unit admission, at sample collection time points, and during intensive care unit stay. Other recorded variables included the use of adjuvant therapies for ARDS, the need for hemodialysis or vasopressors, corticosteroids administration, the number of ventilator- and organ failure–free days at day 28, and the duration of mechanical ventilation and of intensive care unit stay in all patients and in survivors only and intensive care unit mortality. The primary clinical endpoint of the study was the number of live ventilator-free days at day 28.
Measurements of Bronchoalveolar Lavage Fluid and Blood Neutrophil Extracellular Trap and Cytokine Neutrophil Extracellular Trap Concentrations
Bronchoalveolar lavage fluid was collected from all ARDS patients during a bronchoscopy within 48 h of ARDS onset (day 1 to 2 sample) and, for patients who were alive and still had ARDS criteria, 5 to 7 days thereafter (day 5 to 7 sample). Bronchoalveolar lavage fluid samples were also collected from controls. A blood sample for the measurement of serum neutrophil extracellular trap and cytokine concentrations was obtained at the same time.
Neutrophil extracellular traps were quantified by measuring myeloperoxidase-DNA complexes in serum and bronchoalveolar lavage fluid samples using a previously described capture enzyme-linked immunosorbent assay.30 Optical density was measured at 405 nm. A standard curve was obtained by dilution of a strongly positive serum. Samples were interpolated from the standard curve using the sigmoidal dose–response equation and results were expressed in arbitrary units (detection range: 4–1,000 arbitrary units). Optical density values that were outside the detection range of arbitrary units (i.e., values less than 4 and greater than 1,000 arbitrary units) were replaced by the threshold value.
Serum and bronchoalveolar lavage fluid concentrations of the main pro- and antiinflammatory cytokines involved in neutrophil chemotaxis and activation (interleukin-6, interleukin-8, tumor necrosis factor–α31 ) or inhibition (interleukin-10), as well as lung epithelium injury markers (surfactant protein–D and receptor for advanced glycation end-products32 ) were measured using a Human Magnetic Luminex Assay (Biotechne – R&D Systems, UK) and expressed in ng/ml.
This is a primary analysis of this dataset. Continuous variables are reported as median [first–third quartiles] or mean ± SD, as appropriate, and compared using the unpaired Student’s t test or the Mann–Whitney test. For comparison of neutrophil extracellular trap bronchoalveolar lavage concentrations at different time points, the Wilcoxon matched-pairs signed rank test was used. The median differences and their 95% CI were computed using the Hodges–Lehman estimator. The normality of the data was tested using the Shapiro–Wilk normality test. Categorical variables are reported as number and percentages (95% CI) and compared using the chi-squared or Fisher test, as appropriate. When more than two groups were compared, comparisons were performed using the Kruskal–Wallis test and, when the global P value less than 0.05, post hoc comparisons were performed using Dunn’s multiple comparison test. Missing data were not replaced. No outlier value was excluded from the current dataset. Because no clinically relevant neutrophil extracellular trap concentration threshold value was previously reported in bronchoalveolar lavage fluid of ARDS patients, we had a priori planned to categorize patients according to median bronchoalveolar lavage neutrophil extracellular trap concentrations (i.e., patients with “higher” vs. patients with “lower” bronchoalveolar lavage neutrophil extracellular trap concentrations). The relationship between neutrophil extracellular trap concentrations in bronchoalveolar lavage (expressed as optical density) and live ventilator-free days at day 28 was further assessed using uni- and multiple linear regression analyses. Two covariates included in an exploratory multivariable analysis obtained on the day of bronchoalveolar lavage 1 collection were selected on their known prognosis value during ARDS. The relationship between neutrophil extracellular trap concentrations in bronchoalveolar lavage (expressed as optical density) and biologic (e.g., bronchoalveolar neutrophil and cytokines) variables was assessed using linear regressions or Spearman’s correlations. All statistical analyses were two-tailed, and a P value less than 0.05 was considered significant. Analyses were conducted using the SPSS Base 21.0 statistical software package (SPSS Inc., USA) and GraphPad Prism version 6.00 for Windows (GraphPad Software, USA).
Seventy-eight patients with moderate-to-severe pneumonia-related ARDS were admitted to the intensive care unit during the 17-month study period, of whom 43 had noninclusion criteria and 35 were included in the study (eFigure 1, Supplemental Digital Content 1, http://links.lww.com/ALN/B860). A microbiologic documentation was obtained in 83% (n = 29/35) of included ARDS patients (eTable 2, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
Production of Neutrophil Extracellular Traps in the Lungs of Patients with Pneumonia-related ARDS
The first bronchoalveolar lavage and blood samples were obtained after a median delay of 1 day after tracheal intubation (eTable 3, Supplemental Digital Content 1, http://links.lww.com/ALN/B860). Within 48 h of tracheal intubation (days 1 to 2 time point), patients with pneumonia-related ARDS exhibited significantly higher bronchoalveolar lavage fluid neutrophil extracellular trap concentrations than controls (n = 4) (median [1st to 3rd quartiles]: 154 [74 to 1,000] vs. 4 [4 to 4] arbitrary units, difference: −150; 95% CI, −996 to −64; P = 0.0002; fig. 1), whereas the difference of serum concentrations did not reach statistical significance (P = 0.072). Bronchoalveolar lavage fluid concentrations of neutrophil extracellular traps were also six times higher than serum concentrations (26 arbitrary units [4 to 68]; difference: −94; 95% CI, −341 to −57; P < 0.0001) in ARDS patients, suggesting a lung production of neutrophil extracellular traps during ARDS. Serum neutrophil extracellular trap concentrations of patients with higher versus lower bronchoalveolar lavage neutrophil extracellular traps did not differ significantly (eTable 4, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
Neutrophil extracellular trap kinetics, which could be studied in 18 patients also sampled at day 5 to 7, showed that median bronchoalveolar lavage fluid concentrations did not vary significantly over time (P = 0.854; fig. 2), consistent with the lack of significant changes in bronchoalveolar lavage neutrophils between the two time points (65% [31 to 75] vs. 23% [13 to 57], difference: −11; 95% CI, −38 to 5; P = 0.243 by Wilcoxon matched-pairs signed rank test). In contrast, serum neutrophil extracellular trap concentrations decreased significantly over time (from 47 [4 to 488] to 8 [4 to 57] arbitrary units, difference: −21; 95% CI, −80 to 0; P = 0.029 by Wilcoxon matched-pairs signed rank test; fig. 2).
Bronchoalveolar Lavage Cellularity, Cytokines, and Bronchoalveolar Injury Biomarker Concentrations
There was a strong correlation between bronchoalveolar neutrophils (in percentage of total bronchoalveolar lavage cell numbers: 65% [31 to 75] for the whole population) and neutrophil extracellular trap concentrations (Spearman’s ρ = 0.57; P < 0.0001; fig. 3), illustrating that more lung neutrophils were associated with higher bronchoalveolar neutrophil extracellular trap concentrations. The correlation between bronchoalveolar macrophages and neutrophil extracellular trap concentrations mirrored that of neutrophils (i.e., more bronchoalveolar macrophages were associated with less neutrophil extracellular trap concentrations; Spearman’s ρ = −0.52; P < 0.0001; eFigure 2, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
The quantification of the main pro- and anti-inflammatory cytokines (i.e., interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor–α) and of biomarkers reflecting bronchoalveolar epithelium injury (i.e., surfactant protein–D and receptor for advanced glycation end-products) showed significant differences between bronchoalveolar lavage fluid and serum concentrations of ARDS patients as compared with those of controls (fig. 4), consistent with the fact that all patients had pneumonia-related ARDS. Correlation matrixes were performed to further explore the relationship between bronchoalveolar neutrophil extracellular trap concentrations of ARDS patients and cytokine or alveolar epithelial injury biomarkers and displayed a significant correlation between bronchoalveolar neutrophil extracellular trap and interleukin-8 concentrations (Spearman’s ρ = 0.42; P = 0.012; table 1). In contrast, in sera of ARDS patients, none of the cytokines/alveolar epithelial injury biomarkers measured showed a significant correlation with neutrophil extracellular trap concentrations (eTable 5, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
Initial Presentation and Outcomes of Patients with Pneumonia-related ARDS According to Neutrophil Extracellular Trap Concentrations in Bronchoalveolar Lavage
Patients with higher (n = 17) versus lower (n = 18) neutrophil extracellular trap concentrations did not show clinically and statistically significant differences regarding age, comorbidities, and severity of illness scores at ARDS onset (eTable 3, Supplemental Digital Content 1, http://links.lww.com/ALN/B860). There was also no significant difference in terms of ARDS severity according to the Berlin definition. Although ARDS patients with higher bronchoalveolar lavage neutrophil extracellular trap concentrations did not show significantly different lung injury scores33 (P = 0.081), these were less frequently receiving neuromuscular blocking agents (P = 0.041) at ARDS onset than others. No other significant difference was observed between these two groups at ARDS onset, including for oxygenation and respiratory mechanics variables. At the time the first bronchoalveolar lavage was sampled (i.e., 24 h after ARDS onset in average), patients with higher bronchoalveolar neutrophil extracellular trap concentrations had higher PaO2/FiO2 ratios than others (142 [103 to 193] vs. 242 [149 to 350] mmHg, difference: 86 [20 to 167]; P = 0.006), while receiving lower tidal volumes (P = 0.021; eTable 3, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
The hospital and intensive care unit mortality of ARDS patients with higher versus lower bronchoalveolar neutrophil extracellular trap concentrations was not statistically different (table 2). Although patients with higher bronchoalveolar neutrophil extracellular trap concentrations trended to require less frequent extracorporeal membrane oxygenation support and showed shorter mechanical ventilation durations than their counterparts, there was no statistically significant difference between the former and the latter regarding the number of live ventilator-free days at day 28 (22 [14 to 25] vs. 14 [0 to 21], difference: −5; 95% CI, −15 to 0; P = 0.066). Furthermore, there was no statistically significant association between bronchoalveolar neutrophil extracellular trap concentrations, expressed as a continuous optical density variable, and the number of live ventilator-free days at day 28 (β coefficient = 3.70; 95% CI, −0.87 to 8.30; P = 0.084), even after entering potentially confounding variables (i.e., PaO2/FiO2 and tidal volume) in an exploratory multiple regression analysis (β coefficient = 2.40; 95% CI, −2.13 to 6.92; P = 0.288; eTable 6, Supplemental Digital Content 1, http://links.lww.com/ALN/B860).
The current study aimed at quantifying for the first time concomitant bronchoalveolar and circulating neutrophil extracellular trap production in immunocompetent patients with pneumonia-related ARDS. The main results of our study are as follows: (1) ARDS patients not only had six-times higher bronchoalveolar than circulating neutrophil extracellular trap concentrations, but also showed higher bronchoalveolar concentrations than controls (although serum concentrations did not significantly differ), suggesting a lung-borne production of neutrophil extracellular traps during ARDS; (2) bronchoalveolar but not serum neutrophil extracellular trap concentrations exhibited a stable kinetics over the first week of ARDS and were tightly correlated with lung neutrophil infiltration and bronchoalveolar interleukin-8 concentrations; and (3) bronchoalveolar neutrophil extracellular trap concentration was not significantly associated with mechanical ventilation duration, as reflected by live ventilator-free days at day 28.
Bronchoalveolar and circulating neutrophil extracellular trap concentrations were quantified in 35 patients with pneumonia-related ARDS. Of note, these patients were all immunocompetent, had a microbiologic documentation in more than 80% of cases, and had all been diagnosed with moderate-to-severe ARDS requiring mechanical ventilation since less than 24 h when included in the study. As such, neutrophil extracellular trap production was quantified in a homogeneous cohort of ARDS patients. In this well characterized group of patients, bronchoalveolar neutrophil extracellular trap concentrations were six times greater than circulating concentrations. Although bronchoalveolar lavage fluid dilution precludes any accurate comparison of neutrophil extracellular trap concentrations between bronchoalveolar lavage fluid and sera to be performed, such clinically significant concentrations differences between both compartments is suggestive of a lung-borne production of neutrophil extracellular traps during ARDS. Lung infiltration by activated neutrophils is a key feature of diffuse alveolar damage, the histopathologic hallmark of ARDS. Our finding of a lung-borne production of neutrophil extracellular traps during pneumonia-related ARDS is consistent with such lung neutrophil influx as well as with previous experimental studies.19,34,35 Indeed, several infectious and noninfectious stimuli typically associated with ARDS have been shown to trigger regional NETosis, including live bacteria, lipopolysaccharide, interleukin-8, or reactive oxygen species.12 In an experimental model of influenza pneumonia-related ARDS, Narasaraju et al.35 demonstrated that lung neutrophil extracellular trap production was induced by influenza virus and redox enzymes and correlated with the severity of lung injury. Recently, using a murine model of pneumococcal pneumonia, Moorthy et al.34 showed that neutrophil extracellular trap generation was associated with the pneumococcal capsule thickness, which determines the susceptibility of Streptococcus pneumoniae to neutrophil-mediated killing, and proportional to the disease severity. In contrast, in a two-hit lipopolysaccharide- or ventilator-induced lung injury mouse model leading to bronchoalveolar neutrophil extracellular trap production, DNase treatment did not significantly reduce lung injury, suggesting that the pathogenicity of neutrophil extracellular traps is equivocal and may depend on the cause of acute lung injury.19 The fact that more bronchoalveolar neutrophil extracellular traps were associated with more lung neutrophils (fig. 3) and higher bronchoalveolar interleukin-8 concentrations is consistent with regional NETosis, as interleukin-8 has been previously shown to be a potent inducer of neutrophil extracellular traps formation.36
Neutrophil extracellular traps kinetics were studied in 18 patients who were sampled both on days 1 to 2 and 5 to 7 after intubation. Interestingly, and although there were wide interindividual variations, the median bronchoalveolar lavage fluid concentrations did not vary significantly over time, consistent with the lack of significant changes in bronchoalveolar neutrophils. Such data suggest that lung neutrophil extracellular trap production is relatively stable during the first week of pneumonia-related ARDS. In contrast, serum neutrophil extracellular trap concentrations decreased significantly over time, illustrating that NETosis exhibited a different kinetics in blood, consistent with the fact that all included patients had a primary pulmonary—as opposed to extrapulmonary—infection, as a cause of ARDS. Circulating extracellular DNA kinetics have been reported in patients with trauma and were associated with organ failure time course.37 Stable extracellular DNA circulating concentrations have also been reported in ARDS patients.38 However, no previous study has described neutrophil extracellular trap kinetics in the bronchoalveolar compartment of ARDS patients. Our data show that neutrophil extracellular trap concentrations remained stable in the bronchoalveolar compartment over time, but the limited number of patients who had been sampled at the two time points precluded further exploring this aspect.
In our study, ARDS patients having higher bronchoalveolar neutrophil extracellular trap concentrations had the same severity of illness after intubation than others. Yet, they were less hypoxemic on day 1 (i.e., upon bronchoalveolar lavage fluid sampling) and eventually had a shorter duration of mechanical ventilation and of intensive care unit stay than others. However, no significant association was eventually demonstrated between bronchoalveolar neutrophil extracellular trap concentrations and the number of live ventilator-free days at day 28, even after accounting for clinically relevant potential confounders (i.e., PaO2/FiO2 ratio and tidal volume) in an exploratory multiple regression analysis. Importantly, our results do not support the hypothesis of a predominant detrimental role of neutrophil extracellular traps during ARDS, but would instead suggest that neutrophil extracellular traps might be beneficial during early pneumonia-associated ARDS. Such protective effect, if it were confirmed by further studies, could be consistent with the known antimicrobial effects of neutrophil extracellular traps, which have been previously shown to trap and kill a variety of pathogens including Salmonella typhimurium, Staphylococcus aureus, and Candida albicans.10,39 Conversely, patients experiencing chronic granulomatous disease, whose neutrophils fail to produce radical oxygen species–dependent neutrophil extracellular traps, are exposed to severe recurrent infections.12,13 Whether, during the acute phase of infectious ARDS, neutrophil extracellular traps have predominant antimicrobial protective effects will need to be confirmed by further studies.40 Yet, our results suggest that targeting neutrophil extracellular traps as an adjuvant therapy in ARDS might be at risk in this setting. On the other hand, one must certainly keep in mind that the excess or persistence of neutrophil extracellular traps release was previously shown to be potentially injurious to host organs and cells, especially in pulmonary diseases,18,35,41,42 suggesting that a potential detrimental role of neutrophil extracellular traps in these settings should not be ruled out. Our results, together with the existing body of literature on neutrophil extracellular traps, illustrate that the mechanisms underlying their production and the boundaries between their beneficial and detrimental effects during disease states in general, and during ARDS in particular, are still to be unveiled. The recent experimental study of Lefrançais et al.20 illustrated that a critical balance of neutrophil extracellular traps is required to prevent lung injury and maintain microbial control. Although complete peptidylarginine deiminase 4 deficiency in mice resulted in decreased bronchoalveolar and plasma neutrophil extracellular trap concentrations, together with lessened lung injury and increased bacterial load, mice with a partial peptidylarginine deiminase 4 deficiency showed lower lung injury scores and improved survival. Our study further illustrates in ARDS patients that the relationship between bronchoalveolar neutrophil extracellular traps and outcomes is equivocal. Further studies should be performed to explore the association between neutrophil extracellular traps kinetics and outcomes in infectious and noninfectious ARDS.
Our study certainly has a number of limitations: (1) This is a monocentric study including a homogeneous population of patients with pneumonia-related ARDS, thus limiting its external validity. (2) The number of patients in the cohort is relatively small and the study was not designed based on power calculations to observe differences between higher and lower bronchoalveolar lavage neutrophil extracellular traps groups, which certainly limited our ability to observe more outcome differences. We included a small number of control patients (n = 4) who underwent a bronchoscopy as part of routine care. However, the narrow distribution of bronchoalveolar lavage neutrophil extracellular traps and cytokines values obtained suggest this was a homogeneous group of patients with no obvious acute lung inflammation. (3) High concentrations of neutrophil extracellular traps that were above the detection range were replaced by the threshold value (i.e., values less than 4 and greater than 1,000 arbitrary units), limiting our ability to study patients with extreme neutrophil extracellular trap concentrations. And (4) although we hypothesize that the association between neutrophil extracellular trap bronchoalveolar concentrations and time to ARDS resolution is likely to result from their antimicrobial effects, our study does not allow for such a causal inference to be drawn. The fact that we could not obtain samples at both time points for all patients limited our ability to study neutrophil extracellular trap kinetics.
During early pneumonia-related ARDS, bronchoalveolar neutrophil extracellular traps are correlated with regional neutrophils and interleukin-8 concentrations. Bronchoalveolar neutrophil extracellular trap concentrations were not associated with the number of live ventilator-free days at day 28. Better understanding of the boundaries between the beneficial and detrimental effects of neutrophil extracellular traps during ARDS is crucial before embarking on trials aiming at assessing the effects of antineutrophil extracellular trap strategies.
The authors thank the nurses of the medical intensive care unit of Henri Mondor Hospital for their assistance in sampling patients, Aline Alves, Hôpital Henri Mondor, Créteil, France, for her help in processing bronchoalveolar lavage fluid samples, and Jeanne Tran Van Nhieu, M.D. Hôpital Henri Mondor, Créteil, France, for performing bronchoalveolar lavage fluid cytological analyses, Florence Canoui-Poitrine, Hôpital Henri Mondor, Créteil, France, for assistance in statistical analyses, and Dominique de Prost, M.D., Ph.D., Hôpital Louis Mourier, Colombes, France, for the constructive discussions on neutrophil extracellular traps.
Support for this study was provided by 2013 Clinical Research Grant and 2016 Master 2 grant of the French Intensive Care Society (Société de Réanimation de Langue Française), Paris, France.
The authors declare no competing interests.