American Journal of Respiratory and Critical Care Medicine

Rationale: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP.

Objectives: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth.

Methods: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses.

Measurements and Main Results: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air–liquid interface model of primary human subglottic epithelium.

Conclusions: Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.

Scientific Knowledge on the Subject

“Blind” subglottic secretion drainage significantly reduces ventilator-associated pneumonia in mechanically ventilated patients. However, remarkably little is known about the subglottic microenvironment in critically ill patients, and an improved understanding seems likely to suggest ways of improving the efficacy of subglottic secretion drainage.

What This Study Adds to the Field

The study shows for the first time that, in critically ill patients, prolonged mechanical ventilation is associated with mucin hypersecretion, mucus hyperviscosity, neutrophilic inflammation, reduced microbiological diversity, and enrichment of known respiratory pathogenic bacteria in subglottic secretions. Neutrophil proteases are secreted into the subglottic environment. We demonstrate that mucin from the subglottis of mechanically ventilated patients impairs the capacity of neutrophils to migrate, and to phagocytose and kill bacteria, and that the dysfunction is reversed on treatment with a mucolytic agent. The study also adds a novel air–liquid interface model using primary human subglottic epithelium, in which subglottic mucin promotes growth and invasion of bacterial pathogens. These data begin to explain the association between subglottic host defense dysregulation, microbiological changes, and ventilator-associated pneumonia, and importantly suggest novel preventive strategies.

Ventilator-associated pneumonia (VAP) is a common complication in the ICU, with 9–27% of intubated and mechanically ventilated patients developing the condition (13). VAP remains the most commonly fatal infection in critical illness, with an overall attributable mortality of 13% (4). VAP significantly increases the length and cost of ICU stay (5), accounts for one-half of all antibiotics given in critical care (6), and is commonly associated with antibiotic-resistant pathogens (79).

Introduction of the endotracheal tube (ETT) is a key contributing factor in VAP, providing a conduit for invasion of the airways with virulent microorganisms (10, 11). Bacteria cultured from the upper airway, above the ETT cuff before development of VAP, correlate strongly with organisms subsequently isolated from the lungs (1214). Microaspiration of infected secretions from the subglottis (the region located immediately below the vocal cords, and directly above the ETT cuff) into the lung is considered a key component in the pathogenesis of VAP. Indeed, subglottic secretion drainage (SSD) has consistently emerged as an effective strategy for prevention of VAP (1520), resulting in SSD being recommended in key guidelines and position statements (2125). Despite this, little is known about the subglottic microenvironment in critically ill patients. Identification of factors associated with bacterial colonization of the subglottis would likely suggest novel, logical targets for improved prevention of VAP.

The aims of this study were therefore to characterize subglottic host defenses in critically ill patients intubated in the ICU, through a systematic evaluation of subglottic microbes, neutrophils, mucus, and epithelium. Our data propose a significant role for mucins in neutrophil dysfunction and propagation of bacterial growth. Some of the results of these studies have been previously reported in the form of an abstract (26).

Patients and Volunteers

Patients who were intubated and mechanically ventilated for at least 4 days were recruited from two ICUs. Control subjects had elective laryngoscopy under general anesthesia for a range of upper airway symptoms of unidentified cause. Both groups had subglottic secretions aspirated under direct endoscopic vision. Control subjects also had subglottic brushings collected for epithelial culture, using a sheathed cytology brush (BC-202D-5010; Olympus). Healthy volunteers provided blood to generate neutrophils for in vitro experiments. Further details relating to subglottic sampling and blood sampling are to be found in the online supplement. Informed, witnessed assent (from a relative or main carer for patients in the ICU) or informed, written consent (from control subjects and healthy volunteers) was obtained. The relevant research ethics committees approved the study.

Processing of Subglottic Aspirates and Blood

Leukocytes were isolated from freshly aspirated mucus samples treated with 0.08% dithiothreitol (a mucolytic agent) (27) and from whole blood (28). Isolated cells were counted with a hemacytometer. Cytospins were prepared and cells were stained with Giemsa to allow identification by light microscopy. Details of processing are presented in the online supplement.

Bacterial and Fungal Identification in Subglottic Aspirates

Semiquantitative bacterial and fungal culture, in addition to 16S ribosomal RNA bacterial profiling, was performed on subglottic aspirates (details are presented in the online supplement) (29).

Quantification of Inflammatory Cytokines, Complement Factors, Mucins, DNA, and Proteases

Cytokines were assayed with V-PLEX and U-PLEX kits (Meso Scale Diagnostics). Human neutrophil elastase (HNE) was quantified by ELISA (Abcam). A cytometric bead array (BD Biosciences) was used to quantify C5a des-Arg (a stable form of activated complement factor 5). A Quant-iT fluorometric assay kit (Thermo Fisher Scientific) was used to quantify double-stranded DNA (dsDNA). Further details are provided in the online supplement.

Neutrophil Viability

Proportions of viable, apoptotic, or necrotic cells were estimated by flow cytometry. Neutrophils were stained with annexin V (AnnV, which binds to phosphatidylserine on the outer surface of apoptotic cells) and propidium iodide (PI, a vital dye that enters and stains only cells with a compromised cell wall, such as necrotic or late apoptotic cells). Samples were analyzed immediately with a FACSCanto II flow cytometer (BD Biosciences) and cell percentages were defined as AnnV/PI, AnnV+/PI, or PI+, corresponding to live, early apoptotic, or late apoptotic/necrotic cells, respectively (see the online supplement for greater detail and representative flow cytometry plots) (30).

Phagocytosis and Bacterial Killing by Neutrophils

Phagocytosis by mucus- or blood-derived neutrophils was estimated by flow cytometric quantification of pHrodo green Staphylococcus aureus Bioparticles (Thermo Fisher Scientific) taken up by neutrophils (see the online supplement for greater detail and representative flow cytometry plots) (31). Neutrophils were also incubated with live Pseudomonas aeruginosa and bacterial killing estimated as a percentage of P. aeruginosa colony-forming units, determined by serial dilution and incubation on Lennox B agar plates, compared with a no-neutrophil control (see the online supplement) (32).

Neutrophil Chemotaxis Assay

Chemotaxis was determined as the proportion of neutrophils migrating into the lower chamber of a Transwell permeability support (3.0-μm pore size; Corning) containing recombinant human IL-8 (Sigma-Aldrich; see the online supplement) (33).

Mucus Analysis and Mucin Purification

Shear viscosity of mucus was analyzed at 37°C with a rheometer (Kinexus Pro; Malvern Instruments) with 60-mm parallel plates, according to the manufacturer’s instructions. A table of shear rates was produced over the range of 0–100 s−1. Analysis was performed with rSpace software (version 1.6; Malvern Instruments) (34). The percentage dry weight of mucus was calculated with a microbalance to compare weight before and after drying the sample at 80°C overnight. The principal mucins in mucus are MUC5B and MUC5AC, and these were quantified by ELISA (Abcam).

Mucin was purified from subglottic aspirates as a single species by cesium chloride equilibrium density gradient ultracentrifugation and dialysis (see the online supplement) (35). The mucin applied to cells in experiments therefore contained “total mucin,” containing representative proportions of all constituent mucins. Total mucin (across a range of concentrations) was incubated with neutrophils (and the phagocytosis/bacterial killing assays repeated) and with epithelial cells (as described below).

Primary Subglottic Epithelial Cell Culture

Subglottic brushings were collected from newly intubated theater attender control subjects as described above. Epithelial cells were isolated and grown at an air–liquid interface (ALI) (see the online supplement) (36). Cultures were assessed 1) by light microscopy for the presence of apical cilia and 2) using an epithelial volt-ohmmeter (World Precision Instruments) to assess transepithelial electrical resistance. Separately, cells were stained with anti-cytokeratin antibody conjugated with Alexa Fluor 647 (BioLegend), counterstained with DAPI nuclear stain, and assessed by confocal microscopy (details are described in the online supplement).

Bacterial–Epithelial Coculture Experiments

Primary subglottic epithelial cells (PSECs), cultured at an ALI, had their apical mucus layer replaced with purified mucin and were cocultured with P. aeruginosa. At 7 hours the mucin layer was removed and the epithelial layer was homogenized. Bacterial growth was determined by serial dilution and incubation on LB agar plates (see the online supplement) (37).

Statistical Analysis

Analysis was conducted with Prism (GraphPad Software). Nonparametric data were analyzed by Mann-Whitney U test for two variables and by Kruskal-Wallis test for greater than two variables, using Dunn’s post hoc analysis test. Categorical data were assessed by Fisher’s exact test. Correlations were assessed by linear regression. A P value less than 0.05 was considered statistically significant.

Patients and Control Subjects

We enrolled 24 patients intubated and mechanically ventilated for at least 4 days and 31 newly (and transiently) intubated non-ICU control subjects. Of these, all the ICU patients and 27 control subjects had recoverable subglottic secretions and were included in the study. Clinical and demographic information for both cohorts are described in Table 1. Extended clinical details for the ICU cohort are described in the online supplement (Table E1). No oral antiseptics or ETTs coated with antimicrobial agents were used in our patients. Two of the 24 ICU patients had a clinical diagnosis of VAP made at the time of study or in the following 4 days.

Table 1. Demographic and Clinical Data for Patient and Control Groups

 ICU Patients (n = 24)Newly Intubated Control Subjects (n = 27)P Value
Age, yr, median (range)66 (20–79)58 (35–79)0.43*
Male patients, %6359>0.99
Time mechanically ventilated, median (IQR)5 (4–6) d9 (5–10) min 
ICU mortality, %42NA 
Receiving immunosuppressive drugs (including corticosteroids), %210 
Receiving systemic antimicrobial drugs, %920 

Definition of abbreviations: IQR = interquartile range; NA = not applicable.

* By Mann-Whitney U test.

By χ2 test.

At time of subglottic aspiration or during ICU admission before sampling.

Microbiological Analysis

The range of organisms identified in the subglottic aspirates of intubated and ventilated ICU patients was significantly lower than in newly intubated control subjects (Figure 1). More species recognized as potential respiratory pathogens complicating critical illness (e.g., Pseudomonas, Enterococcus) were isolated from patients in the ICU (Figures 1 and E1). Control samples tended to contain more commensal species, each in larger numbers, than did ICU patient samples (Figures 1 and E1, and Table E2).

Analysis of Mucins

To establish the constituents and physical properties of subglottic mucus aspirates we assessed viscosity and percentage dry weight (percentage of solids), and quantified dsDNA and the concentrations of the principal airway mucins MUC5B and MUC5AC (Figure 2). Mucus derived from patients in the ICU was significantly more viscous, and had a greater dry weight. In addition, significantly higher concentrations of dsDNA and mucin MUC5B (but not MUC5AC) were demonstrated, as compared with mucus from newly intubated control subjects. MUC5B was by far the most abundant mucin in mucus from ICU patients.

To investigate potential correlates between mucin concentrations in the subglottic aspirates and clinical status, total mucin concentrations in ICU subglottic aspirates were compared on the basis of various parameters. Subglottic mucin concentrations showed a weak, positive (but statistically significant) correlation with duration of intubation/mechanical ventilation and increasing age (r = 0.48, P < 0.05 for both; see Figure E2).

Analysis of Markers of Inflammation

Significantly higher concentrations of inflammatory cytokines, activated complement, and extracellular HNE were observed in subglottic aspirates from the ICU patients (Table 2).

Table 2. Concentrations of Cytokines and Other Mediators in Subglottic Aspirates

 ICU PatientsNewly Intubated Control PatientsP Value
MedianIQRnMedianIQRn
Proinflammatory cytokines 
 IL-8, pg/ml43,6858,285–106,242202,412819–4,41126<0.0001
 IL-6, pg/ml1,469514–3,80121124–2721<0.0001
 IL-1β, pg/ml3,686372–12,696238842–14522<0.0001
 IL-2, pg/ml4.42.0–112.5191.10.5–3.8130.0265
Th2 cytokine 
 IL-13, pg/ml226136–869214012–185140.0062
Proinflammatory, noncytokine mediators 
 C5a des-Arg, ng/ml46.810.9–68.8221.80.4–11.922<0.0001
 HNE, ng/ml13.05.0–15.4170.40.2–0.814<0.0001
Pleiotropic cytokines 
 IL-12, pg/ml20.911.0–52.9213.80.9–13.9170.0007
 IFN-γ, pg/ml14310–1,27121145–52170.0146
 GM-CSF, pg/ml1.60.9–2.5120.80.6–1.070.0378
Antiinflammatory cytokine       
 IL-10, pg/ml10.15.2–103.9202.60.6–14.2160.0056

Definition of abbreviations: C5a des-Arg = stable breakdown product of activated complement factor 5; GM-CSF = granulocyte–macrophage colony–stimulating factor; HNE = human neutrophil elastase; IQR = interquartile range; Th2 = T-helper cell type 2.

The Mann-Whitney U test was used for statistical comparisons.

Neutrophil concentrations were also significantly elevated in the subglottic aspirates taken from patients in the ICU compared with control subjects (Figure 3). There was no significant difference in macrophage concentration (Figure 3). No significant difference was demonstrable between the proportion of viable or apoptotic neutrophils in the two cohorts (Figure 3). A representative flow cytometry plot is shown in the online supplement, along with evidence that mucin itself did not compromise neutrophil viability (Figure E3). Given the large number of mucus-derived neutrophils we went on to investigate the phagocytic capacity of these cells on extraction from mucus samples. There was no demonstrable deficiency in phagocytic capacity of neutrophils extracted from the subglottic mucus of long-term intubated ICU patients, compared with newly intubated control subjects (Figure 3).

Mucin–Neutrophil Interactions

To assess whether the high mucin environment in the subglottis was responsible for neutrophil dysfunction, we incubated blood-derived neutrophils from healthy volunteers (who were not intubated at any point) with purified mucin, derived from subglottic secretions taken from patients in the ICU. Mucin (at a concentration of 20 mg/ml) induced a significant reduction in neutrophil chemotaxis compared with a mucin-free control (Figure 4A). This mucin concentration was selected as it approximated to the median concentration of mucin in ICU patients, and was more than twice the highest mucin concentration observed in any control. Neutrophil migration appeared to be paralyzed by high concentrations of mucin (see Video E1). In addition, mucin (20 mg/ml) significantly impaired the capacity of neutrophils to kill P. aeruginosa, one of the two most commonly isolated pathogens in VAP (Figure 4B) (1, 9). There was no significant reduction in either neutrophil chemotaxis or bacterial killing at the range of mucin concentrations found in subglottic aspirates from newly intubated control subjects. To corroborate these findings a further assay of phagocytosis by neutrophils from healthy volunteers was performed using S. aureus Bioparticles (S. aureus is the other of the two commonest pathogens causing VAP [1, 9]). Mucin at 20 mg/ml again impaired phagocytosis (Figure 4C). Dithiothreitol (a mucolytic agent) reversed the mucin-associated impairment of phagocytosis (Figure 4C).

Effects of Mucus on Bacterial Interaction with Subglottic Epithelium

To investigate potential interactions between infected mucus and the subglottic epithelium, we developed a novel in vitro ALI model of human PSECs and extensively characterized the model to determine how closely it recapitulated the in vivo environment (further detail and illustration are provided in Figures E4–E6). An epithelial phenotype was confirmed by expression of pan-cytokeratin, and at the ALI the cells demonstrated a highly differentiated pseudostratified respiratory epithelium with apical cilia (Figure E4). Transepithelial electrical resistance was demonstrated at the ALI and maintained in prolonged culture (Figures E4 and E5). Cultures also secreted appropriate airway mucins into an apical mucus layer (Figure E6).

The human PSEC model was used to investigate the interaction of the epithelium, P. aeruginosa, and purified mucin derived from patients in the ICU. Replacement of the PSEC apical mucus layer with purified ICU-derived mucin (at a concentration of 20 mg/ml), induced a significant increase in bacterial colony-forming units compared with a mucin-free control (Figure 5). This was evident in both the apical mucin layer and in the epithelial homogenate.

Our data suggest, for the first time, that intubation and mechanical ventilation in the ICU over four or more days is associated with neutrophilic inflammation and reduced microbiological diversity within the subglottic environment. Neutrophil function appears to be significantly impaired, at least in part due to an excess of subglottic mucin, which promotes colonization and growth of potential respiratory pathogens, and potentially invasion of the subglottic epithelium. These findings may explain the validated importance of SSD in the prevention of VAP, and suggest further preventive strategies. We also describe a novel in vitro model that reproduces several key features of the human subglottis.

Aspiration of infective subglottic secretions into the lung causes VAP in mechanically ventilated patients. We found that the microbiology of subglottic aspirates from intubated, critically ill patients shows reduced diversity and the presence of more pathogenic organisms associated with VAP, compared with control subjects.

It has previously been reported that even short-term tracheal intubation, for several hours, can cause significant mucosal inflammation around the tracheal cuff (38), and impede the function of the mucociliary escalator (39). This inflammation was characterized by a rapid accumulation of granulocytes and increased proinflammatory and chemotactic agents, such as IL-8, IL-6, IL-1β, and C5a (38). We also found elevated proinflammatory cytokines in subglottic aspirates from patients in the ICU, which may have contributed to the recruitment, and persistence, of unexpectedly high concentrations of viable neutrophils. The release of HNE, a serine protease with a broad range of substrates, is likely to have further precipitated the local inflammation, which may in turn contribute to a cycle of further neutrophil migration (40).

The persisting inflammation described above may have contributed to the differences in the mucus constituents and physical properties of ICU-derived mucus. In particular IL-8, IL-6, and IL-1β have been shown to upregulate MUC5B (4144), and in murine models of cystic fibrosis–like lung disease neutrophil elastase appears to drive mucus hypersecretion (45). In human studies, the inflammatory stimulus of e-cigarettes simultaneously produces airway neutrophilia and mucus hypersecretion (46).

Mucins are one of the main constituents of the mucus that covers epithelial surfaces, and are vital in determining mucus function as a lubricant and selective barrier. The predominant secreted airway mucins are MUC5B and MUC5AC (47). The proportions of these two airway mucins within the airway are not well characterized in humans. Both are secreted by goblet cells and seromucinous glands of the lamina propria at the apical epithelium (47). Consistent with our data, MUC5B appears to be the airway mucin most responsive to stimulation by inflammatory mediators. In keeping with this, MUC5B is the predominant airway mucin in chronic obstructive pulmonary disease (48).

Acute mucin hypersecretion is thought to aid innate immune defense by trapping particles (47). Nevertheless, prolonged, extensive, or inappropriate mucin hypersecretion has been implicated in airway obstruction and poor clearance of pathogens in numerous respiratory diseases (47). Our observation of high concentrations of mucin and dsDNA, as well as elevated mucus viscosity, suggests a potential failure of the mucus layer as an effective lubricant in the subglottis of critically ill patients. In parallel with the failure of the mucociliary escalator, demonstrated previously after intubation (39), this is likely to promote mucus stasis and a failure to clear pathogens.

The neutrophil is the key innate immune cell involved in clearance of bacteria and fungi (49). The inhibitory effect of mucin on phagocytosis by neutrophils is the most profound for any studied by our group to date. We have previously studied the effects of several cytokines and mediators (most of those described in Table 2) on neutrophil phagocytosis and found that only supraphysiological concentrations of C5a drive downregulation (32). In contrast, granulocyte-macrophage colony-stimulating factor and IFN-γ improve neutrophil phagocytosis. We cannot exclude the possibility that C5a contributed to impaired neutrophil phagocytosis in our patients, but the concentrations described here are associated with a small impairment of neutrophil phagocytosis in in vitro experiments (in contrast to the profound effect of mucin). On the basis of the available evidence we believe the most likely sequence is that, in the context of critical illness: intubation markedly upregulates local inflammation and cytokine production; this simultaneously recruits an excess of neutrophils and drives mucus hypersecretion; mucus (with a smaller potential contribution from C5a) profoundly impairs the ability of neutrophils to clear bacteria, while mucus itself provides a compatible environment for bacterial growth; and dysfunctional neutrophils release HNE, which may contribute further to local inflammation, potentially creating a vicious cycle.

We observed no demonstrable difference in phagocytic function between subglottic mucus–derived neutrophils from patients in the ICU or control subjects (Figure 3C). It should be emphasized that neutrophil functions were not measured in mucus aspirated fresh from patients (as neutrophils are “trapped” in the mucus, and the number of neutrophils and other leukocytes cannot be standardized), and that Figure 3C shows neutrophils liberated from mucus by a mucolytic (and studied at a known concentration).

Blood neutrophil phagocytic capacity was also similar in ICU patients and control subjects. This was unsurprising, as in our experience profound critical illness is required to impair blood neutrophil capacity (50) (our use of healthy volunteers’ blood neutrophils in Figure 3C could be criticized, but we were confident that control patients’ blood neutrophil phagocytosis would be normal).

We found that the phagocytic capacity of neutrophils in the subglottic aspirates from ICU patients and from control subjects was lower than that of blood neutrophils. This is consistent with our previous findings that extravascular neutrophils have lower phagocytic capacity than blood neutrophils from the same patient (Reference 32 and T. P. Hellyer, unpublished data). However, the phagocytic capacity of subglottic neutrophils from ICU patients remained equivalent to that from control subjects, and remained adequate for phagocytosis. While intubation and the inflammatory milieu may make a contribution to impaired neutrophil function, we believe these data suggest that neutrophils are functionally “trapped” by mucus. Importantly, however, they appear to retain function when removed from the mucus. We therefore went on to study the effect of purified mucin (from ICU patients) on healthy neutrophils. An in vitro cystic fibrosis model has suggested that high mucin concentrations may adversely affect the capacity of neutrophils for chemotaxis and bacterial killing (33).

Purified mucin (predominantly containing MUC5B and MUC5AC) at a concentration and ratio typically found in intubated, critically ill patients impaired the capacity of healthy neutrophils to migrate toward a chemoattractant and to phagocytose and kill bacteria. Importantly, a mucolytic agent capable of breaking down the polymeric structure of mucin, restored normal phagocytic function (Figure 4C) to levels observed in subglottic neutrophils from control patients (Figure 3C). This supports the earlier data suggesting that neutrophil function can be restored on liberation from mucus.

The novel human PSEC ALI model provides the potential to study subglottic diseases and potentially test therapeutic agents with a subsite-specific in vitro model. The model provided evidence that subglottic mucin promotes growth and invasion of bacterial pathogens. This was a preliminary exploration of this concept, and further validation is needed with other laboratory and clinical strains of VAP-producing pathogens, and the addition of neutrophils to the coculture in the future.

We believe that the identification of excess subglottic mucin as 1) a substrate for bacterial propagation and 2) a “disabling trap” for neutrophils, from which they can be functionally rescued, has some simple but potentially important clinical implications. In particular, more effective subglottic clearance by aspiration under direct vision and/or by physical or chemical disruption of subglottic mucus seems likely to decrease local infection and inflammation, while simultaneously creating an environment for more effective neutrophil function. Several of the ICU patients in this study were already undergoing SSD, yet subglottic mucus was freely aspirated from all patients under direct vision, suggesting SSD is suboptimally effective. We therefore believe the simple interventions of aspiration under direct vision and/or mucus disruption should be assessed in clinical trials with a view to establishing whether they can prevent VAP more effectively than SSD alone. Such studies will require careful design and substantial patient numbers to test whether VAP is effectively prevented.

In conclusion, intubated, critically ill patients have an inflammatory subglottic phenotype characterized by mucin MUC5B hypersecretion, emergence of potential respiratory pathogens, and release of neutrophil proteases such as HNE. The data suggest that more effective early removal and/or disruption of subglottic mucus represents an attractive strategy for improving prevention of VAP.

The authors thank Carmen Scott, Verity Calder, and Craig Samson (Newcastle Critical Care Research Team) for help with recruitment, and Jane White (surgical waiting list department) for help with recruitment.

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Correspondence and requests for reprints should be addressed to A. John Simpson, F.R.C.P.(Edin.), Ph.D., Institute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. E-mail: .

* These authors contributed equally to this work.

Supported by Wellcome Trust grant 108768; a Royal College of Surgeons of England Shears Northern Research Fellowship; a Royal Society of Medicine grant; Newcastle upon Tyne Hospitals NHS Charity; the Newcastle University Wellcome Trust Institutional Strategic Support Fund; UK Medical Research Foundation grant MRF-091-0001; and the Scottish Infection Research Network.

Author Contributions: J. Powell, J.D.P., S.E.W., J.A.W., J. Pearson, C.W., and A.J.S. designed the study. J. Powell, J.P.G., M.W.M., F.A.H.C., A.N., B.V., J.S., K.J., M.-H.R.-S., and S.P.C. performed experiments. J. Powell, C.W., and A.J.S. wrote the manuscript. All authors reviewed and approved the manuscript.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.201709-1819OC on February 9, 2018

Author disclosures are available with the text of this article at www.atsjournals.org.

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