Articles

Neutrophil extracellular traps, disease severity, and

antibiotic response in bronchiectasis: an international,
observational, multicohort study
Holly R Keir, Amelia Shoemark, Alison J Dicker, Lidia Perea, Jennifer Pollock, Yan Hui Giam, Guillermo Suarez-Cuartin, Megan L Crichton,
Mike Lonergan, Martina Oriano, Erin Cant, Gisli G Einarsson, Elizabeth Furrie, J Stuart Elborn, Christopher J Fong, Simon Finch, Geraint B Rogers,
Francesco Blasi, Oriol Sibila, Stefano Aliberti, Jodie L Simpson, Jeffrey T J Huang, James D Chalmers

Summary
Background Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies Lancet Respir Med 2021;
that directly target neutrophilic inflammation because little is understood of the underlying mechanisms leading to 9: 873–84

severe disease. Neutrophil extracellular trap (NET) formation is a method of host defence that has been implicated in Published Online
February 17, 2021
multiple inflammatory diseases. We aimed to investigate the role of NETs in disease severity and treatment response
https://doi.org/10.1016/
in bronchiectasis. S2213-2600(20)30504-X
For the Spanish translation of the
Methods In this observational study, we did a series of UK and international studies to investigate the role of NETs in abstract see Online for
disease severity and treatment response in bronchiectasis. First, we used liquid chromatography-tandem mass appendix 1
spectrometry to identify proteomic biomarkers associated with disease severity, defined using the bronchiectasis For the Italian translation of the
severity index, in patients with bronchiectasis (n=40) in Dundee, UK. Second, we validated these biomarkers in abstract see Online for
appendix 2
two cohorts of patients with bronchiectasis, the first comprising 175 patients from the TAYBRIDGE study in the UK
Division of Molecular and
and the second comprising 275 patients from the BRIDGE cohort study from centres in Italy, Spain, and UK, using Clinical Medicine, University of
an immunoassay to measure NETs. Third, we investigated whether pathogenic bacteria had a role in NET concentrations Dundee, Ninewells Hospital
in patients with severe bronchiectasis. In a separate study, we enrolled patients with acute exacerbations of and Medical School, Dundee,
bronchiectasis (n=20) in Dundee, treated with intravenous antibiotics for 14 days and proteomics were used to identify UK (H R Keir BSc,
A Shoemark PhD, A J Dicker PhD,
proteins associated with treatment response. Findings from this cohort were validated in an independent cohort of J Pollock, Y H Giam BSc,
patients who were admitted to the same hospital (n=20). Fourth, to assess the potential use of macrolides to reduce M L Crichton MFM,
NETs in patients with bronchiectasis, we examined two studies of long-term macrolide treatment, one in patients M Lonergan PhD, E Cant BSc,
with bronchiectasis (n=52 from the UK) in which patients were given 250 mg of azithromycin three times a week for E Furrie PhD, C J Fong MBChB,
S Finch MBChB,
a year, and a post-hoc analysis of the Australian AMAZES trial in patients with asthma (n=47) who were given 500 mg Prof J D Chalmers PhD);
of azithromycin 3 times per week for a year. Respiratory Department,
Hospital Clinic, University of
Findings Sputum proteomics identified that NET-associated proteins were the most abundant and were the proteins Barcelona, IDIBAPS, CIBERES,
Barcelona, Spain (L Perea MSc,
most strongly associated with disease severity. This finding was validated in two observational cohorts, in which O Sibila MD); Respiratory
sputum NETs were associated with bronchiectasis severity index, quality of life, future risk of hospital admission, and Department, Hospital
mortality. In a subgroup of 20 patients with acute exacerbations, clinical response to intravenous antibiotic treatment Universitari de Bellvitge,
was associated with successfully reducing NETs in sputum. Patients with Pseudomonas aeruginosa infection had a IDIBELL, L’Hospitalet de
Llobregat, Spain
lessened proteomic and clinical response to intravenous antibiotic treatment compared with those without (G Suarez-Cuartin PhD);
Pseudomonas infections, but responded to macrolide therapy. Treatment with low dose azithromycin was associated Fondazione IRCCS Ca’ Granda
with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. Ospedale Maggiore Policlinico,
Respiratory Unit and Cystic
Fibrosis Adult Center, Milan,
Interpretation We identified NETs as a key marker of disease severity and treatment response in bronchiectasis. These Italy (M Oriano MSc,
data support the concept of targeting neutrophilic inflammation with existing and novel therapies. Prof F Blasi MD,
Prof S Aliberti MD); Department
of Molecular Medicine,
Funding Scottish Government, British Lung Foundation, and European Multicentre Bronchiectasis Audit and
University of Pavia, Pavia, Italy
Research Collaboration (EMBARC). (M Oriano); Department of
Pathophysiology and
Copyright: © 2021 The Author(s). Published by Elsevier Ltd. Transplantation, Università
degli Studi di Milano, Milan,
Italy (M Oriano, Prof F Blasi,
Introduction closely related to airway infection, tissue damage, and Prof S Aliberti); Centre for
Bronchiectasis is a chronic inflammatory lung disease mucociliary dysfunction.4,5 Each of these aspects of Experimental Medicine, School
defined by permanent bronchial dilatation.1 Patients have disease are poorly characterised and are likely to have of Medicine, Dentistry and
Biomedical Sciences, Queen’s
chronic cough, sputum production, and recurrent contributed to several unsuccessful clinical trials in the University Belfast, Belfast, UK
exacerbations, which are a major cause of morbidity and past 10 years.6 Guidelines for bronchiectasis recommend (G G Einarsson PhD,
mortality.2,3 The pathophysiological hallmark of this treatment with chest physiotherapy and antibiotics; Prof J S Elborn PhD);
disease is chronic neutrophilic inflammation, which is however, antibiotics are not effective in all patients Microbiome and Host Health,

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 Articles
South Australian Health and
Medical Research Institute,
Adelaide, SA, Australia
(Prof G B Rogers PhD); Priority
Research Centre for Healthy
Lungs, Faculty of Health and
Medicine, University of
Newcastle, Callaghan, NSW,
Australia (Prof J L Simpson PhD);
Division of Systems Medicine,
School of Medicine, University
of Dundee, Ninewells Hospital
and Medical School, Dundee,
UK (J T J Huang PhD)
Correspondence to:
Prof James D Chalmers, Division
of Molecular and Clinical
Medicine, University of Dundee,
Dundee, DD1 9SY, UK
jchalmers@dundee.ac.uk
874
and repeated antibiotic courses lead to anti­­ microbial
resistance.7,8 No treatments have been approved
for bronch­iectasis that directly target neutrophilic
inflammation. Drugs such as CXCR2 and leukotriene B4
antagonists decrease neutrophil recruitment to the
airway but also increase the rate of infections without
providing clinical benefits.9,10 There is a need to identify
mechanisms that can reduce neutrophil-mediated lung
damage without compromising bacterial killing.
Neutrophil extracellular trap (NET) formation is
emerging as a key mechanism in several inflammatory
conditions. NET formation is an active process in which
neutrophils extrude a meshwork of extra­ cellular fibres
composed of chromatin DNA, histones, and bactericidal
proteins to immobilise and disarm pathogens.11,12
NET-associated proteins include mediators that are
involved in the pathogenesis of bronchiectasis, including
neutrophil elastase, LL-37, and PR-3.4,5 Crucially, NET
formation can be inhibited without compromising
Research in context
Evidence before this study
Bronchiectasis has a diverse range of clinical phenotypes and its
pathophysiology is poorly understood. These factors have
contributed to the multiple failed clinical trials over the past
10 years, resulting in an absence of licensed therapies for
bronchiectasis. Understanding the biological mechanisms that
drive clinical phenotypes of bronchiectasis and treatment
responses might lead to more appropriate treatments for
patients. We searched PubMed for articles in English published
from database inception until July 30, 2020, using the terms
“bronchiectasis” AND “proteomics” OR “microbiome” OR
“endotype” OR “pathophysiology”. Although a small number of
biomarker and microbiome studies on bronchiectasis have
been published, we found no publications that integrated
multiple omic techniques to characterise the mechanisms of
neutrophilic inflammation. Individual neutrophil biomarkers
have been described but no studies have looked in detail at
neutrophil extracellular trap (NET) formation in bronchiectasis.
Added value of this study
Using proteomics, we identified a subgroup of patients with
severe disease characterised by increased neutrophil proteins
linked to NET formation. Patients with increased NET formation
had reduced microbial diversity and dominance of organisms
such as Pseudomonas. Patients with increased NET concentrations
had more exacerbations, shorter time to severe exacerbations,
and increased mortality. In response to antibiotic treatment,
innate immune and NET proteins were downregulated with an
increase in anti-protease defence. Patients with Pseudomonas
aeruginosa infection had a lessened response to antibiotic
treatment and worse outcomes. Macrolides were found to reduce
NETs in patients with bronchiectasis and P aeruginosa infection,
suggesting a novel non-antibiotic mechanism for their efficacy.
Validation in a cohort of patients with poorly controlled asthma
found that beneficial effects of macrolides on NETosis in vivo are
bacterial killing. For example, patients with the rare genetic
disease Papillon-Lefèvre syndrome do not have the enzyme
dipeptidyl peptidase-1 (DPP-1) that is required for activation
of neutrophil serine proteases. They are unable to form
NETs but can kill bacteria normally through phagocytosis.13
In a 2020 randomised, placebo-controlled, phase 2 trial of
patients with bronchiectasis, inhibition of DPP-1 was
found to extend the time to first exacerbation.14
In this study, we used a series of proteomic and
targeted approaches to investigate whether NETs are
associated with disease severity, bacterial infection, and
mortality in patients with bronchiectasis, and whether
NETs proteins can be reduced through treatment with
antibiotics and macrolide therapy.
Methods
Study design
In this multicohort observational study, we report results
from seven independent prospective cohort studies in
not limited to bronchiectasis. To our knowledge, this study is the
first to use sputum proteomics to characterise the impact of
antibiotics on airway inflammation in bronchiectasis. The study
design is unique in testing a series of hypotheses in linked cohort
studies using multiple techniques including proteomics,
microbiome characterisation, and targeted measurement of NETs
and other biomarkers.
Implications of all the available evidence
We used sputum proteomics as a powerful tool to assess the
host inflammatory response in patients with bronchiectasis.
Our results show that the presence of NETs is associated with
the burden of disease, and that treatment response is linked to
successful reduction of NET levels through intravenous
antibiotic or macrolide therapies. Intravenous antibiotics were
less effective at reducing inflammation in patients with
P aeruginosa infection but these patients had significantly
reduced concentrations of NETs after macrolide treatment,
which in turn was associated with reduced exacerbations in
patients with P aeruginosa infection. These observations
support a novel immunomodulatory effect of macrolides and
might justify consideration of initiating prophylactic macrolide
therapy for patients with bronchiectasis and P aeruginosa
infection, by contrast with current European Respiratory
Society guidelines, particularly in those who have responded
poorly to systemic antibiotics. A recent randomised study of
dipeptidyl peptidase 1 inhibition in bronchiectasis found for the
first time the benefit of directly targeting neutrophilic
inflammation. In our study we identified a mechanistic basis for
this therapy, which inhibits neutrophilic inflammation and has
now been shown elsewhere to extend time to first exacerbation
in patients with bronchiectasis. Recognition of NETosis as a
dominant mechanism of disease severity supports the further
development of a new generation of immunomodulatory
therapies for the treatment of bronchiectasis.
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the UK and in Spain, Italy, and Australia to test and
independently validate the role of NETs in bronchiectasis.
Each study was designed and run sequentially on the
basis of the results obtained. Each study generated new
hypotheses, which were then tested in further studies.
Our initial hypothesis was that there would be
differences in inflammation between severe and mild
bronchiectasis. We used sputum proteomics to
investigate this hypothesis. Based on the results of this
first study, we hypothesised that NETs would be
associated with disease severity, exacerbations, quality of
life, and mortality in patients with bronchiectasis. We
tested this hypothesis in a cohort of patients from the UK
and then validated our findings in a separate cohort. We
observed in these cohorts that bacteria were linked with
higher NET levels, and hypothesised that bacteria were
the main drivers of NETs. In a UK cohort of patients with
bronchiectasis, we investigated whether treatment with
antibiotics would result in beneficial changes to the lung
proteome, including reduced NETs. We found that
patients infected with Pseudomonas aeruginosa responded
poorly to this treatment, and therefore investigated a
further hypothesis that NET concentrations might be
reduced by macrolides. We investigated whether
macrolide treatment would reduce sputum NET
concentrations in 56 patients with bronchiectasis from
Dundee UK and 47 patients from the Australian
AMAZES trial in asthma.
Each study had independent patient eligibility criteria,
which are described in full in appendix 3 (pp 2–8) and
briefly summarised along with each of the predefined
procedures in each study in the following sections.
The studies were approved by local research ethics
committees and patients provided written informed
consent.
Proteomic analysis of mild and severe bronchiectasis
First, we aimed to identify sputum protein profiles
associated with severity of disease using the bronchiectasis
severity index.15 Patients with CT-confirmed idiopathic or
post-infective bronchiectasis attending a tertiary referral
clinic at Ninewells Hospital in Dundee, UK, were enrolled
and provided spontaneous sputum samples for proteomic
analysis. Eligible patients were clinically stable, defined by
stable symptoms and absence of antibiotic treatment for
4 weeks before enrolment (appendix 3 pp 2, 6). Prophyl­
actic antibiotic treatment (eg, macrolides and inhaled
antibiotics) were permitted. Samples from patients with
severe and mild disease were compared with each other.
Sputum protein profiling was done using a label-free
shotgun proteomic workflow with a nanoflow liquid
chromatography system (Agilent 1200, Agilent) linking to
an Orbitrap mass spectrometer (LTQ-Orbitrap, Thermo
Scientific; appendix 3 p 2).4 The findings were validated
using immunoassays for neutrophil elastase (ProAxsis,
Belfast, UK16), S100A12, and resistin (R+D systems,
Abingdon, UK).
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Articles
NETs, disease severity, and outcomes in bronchiectasis
We examined the association between NETs and disease
severity in two cohorts. The first observational cohort
study was the TAYBRIDGE cohort study, which was done
in two hospitals in the east of Scotland (2012–15;
appendix 3 pp 3, 6).16 Clinically stable patients with
bronchiectasis provided spontaneous sputum samples
for NET-associated proteomic biomarker quantification
using a validated immunoassay for histone-elastase
complexes, as previously described.17,18 Controls with
cystic fibrosis, chronic obstructive pulmonary disease
(COPD), or asthma were also recruited and healthy
individuals (health-care workers without lung disease;
full details on appendix 3 p 6). Participants who were able
to provide spontaneous sputum samples did so; however,
healthy controls and several patients with asthma
underwent sputum induction with 3% hypertonic saline.
Sputum NET concentration quantification was done
using a validated immunoassay for histone-elastase
complexes, as previously described.17,18 Results from
the histone-elastase complex assay correlated with
alternative methods to quantify NETs, including the
DNA-elastase immunoassay and microscopy methods
(appendix 3 p 17). For simplicity, subsequent results are
presented for the histone-elastase assay only.
Endpoints of interest in the context of the current study
were bronchiectasis severity index, exacerbations in the
12 months before the study, quality of life, severe
exacerbations requiring admission to hospital, and
mortality. Patients were followed-up until database lock on See Online for appendix 3
Jan 15, 2019. Quality of life was measured using the quality
of life bronchiectasis respiratory symptom score (QoL-B
RSS).19 For the purposes of the current study, these
endpoints of interest were used as measures of disease
severity and compared with patient sputum NET
concentration. Additionally, we measured the long-term
outcomes of mortality and admission to hospital in
patients with bronchiectasis by baseline NET concentration
tertile.
We characterised the microbiome of this UK cohort
using 16S rRNA gene sequencing (appendix 3 p 3).17
We prespecified to perform analyses at the phylum and
genus level. Differences between groups in microbiome
profiles were analysed by NET concentration tertile.
Extraction negative controls were done to account for
potential contamination.
We validated NETs as a biomarker for bronchiectasis in a
second independent study, the BRIDGE cohort study,
which was done in three European centres in 2018–19
(appendix 3 pp 4, 6–7).5 Patients with bronchiectasis were
enrolled at the Policlinico University Hospital (Milan, Italy),
Hospital de la Santa Creu I Sant Pau (Barcelona, Spain),
and Ninewells Hospital (Dundee, UK). Spontaneous
sputum and matched serum samples were obtained for
the measurement of 13 cytokines using a multiplex
immunoassay (Mesoscale diagnostics, Rockville, MD,
USA) plus neutrophil elastase activity, sputum pH, IL-8,
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IL-2, and IL-4 by ELISA (R+D systems, Abingdon, UK).
Endpoints for BRIDGE were bronchiectasis severity index,
forced expiratory volume in 1 s (FEV1), QoL-B RSS, and
severe exacerbations.
Antibiotics and the lung proteome in patients with
exacerbations of bronchiectasis
To test whether pathogenic bacteria had a role in the
concentrations of NETs observed in patients with severe
bronchiectasis, we enrolled patients who had been
admitted to hospital for treatment of exacerbations of
bronchiectasis (full study details are in appendix 3
[pp 4, 7]). Exacerbations were defined by the presence of
increased respiratory symptoms requiring antibiotic
treatment.20 Patients were treated with intravenous
antibiotics directed against the likely causative pathogen
(based on data from previous microbiological culture),
with antibiotic treatment subsequently modified on the
basis of results of cultures of spontaneous sputum
samples obtained on day 1 of treatment. For the purposes
of analysis, patients were classified as positive or negative
for P aeruginosa on the basis of the microbiological
culture sent on day 1. All patients were treated with
antibiotics for 14 days, and spontaneous sputum samples
were collected on day 14 (ie, at the end of treatment). We
first analysed samples in a discovery cohort, which we
used for profiling the proteins in the sputum, as
described for the first proteomics study. We then
validated our findings in a further cohort of patients, who
were recruited and treated in the same way as the
discovery cohort, and we used a targeted approach of
directly measuring the NET concentrations. We used the
Panther Classification System21 to analyse the reactome
pathway of upregulated and downregulated proteins.
This system classifies proteins according to a defined
ontological pathway and facilitates data interpretation in
proteomic and genomic studies. Detailed methods of
analysis are in appendix 3 (p 4).
Response to long-term macrolide therapy
To understand how macrolides, which do not have anti-
pseudomonal activity, might reduce exacerbations in
patients with chronic P aeruginosa infection, patients
with bronchiectasis were enrolled in a non-randomised
observational study at Ninewells Hospital, Dundee
(appendix 3 pp 4–5, 7–8).22 Patients with bronchiectasis
and P aeruginosa infection were treated with azithromycin
250 mg three times per week for 1 year, with sputum
samples taken at baseline and after 1 year for
measurement of NETs, and were compared with a
matched cohort of patients with bronchiectasis with
P aeruginosa infection who did not receive macrolide
therapy at any time during the previous 12-month period
from the same study site (appendix 3 pp 4–5, 7).
To validate the observation that macrolide therapy
reduces NETs, we investigated the use of macrolide
therapy in other chronic respiratory diseases beyond
876
bronchiectasis. We analysed sputum samples from
subset of patients with paired baseline and end of study
sputum samples23,24 who were treated with azithromycin
500 mg three times per week for 1 year or placebo in the
AMAZES study, a randomised controlled trial of long-
term azithromycin in patients with asthma in Australia.23
In the AMAZES study, patients were classified as
eosinophilic (>3% sputum eosinophils by cytospin) or
non-eosinophilic, as previously described.23
Statistical analysis
Sputum NET data were not normally distributed and so we
used the Mann Whitney U test for comparison of
two groups and the Kruskal-Wallis test for comparisons of
three or more groups. The Wilcoxon matched-pairs signed
rank test was used for comparisons of paired data. Data
were logarith­m­­ically transformed for easier visualisation.
We used Cox proportional hazards models incorp­­orating
age, sex, treatment (long-term macrolide, inhaled anti­
biotics, and inhaled corticosteroids), baseline exacer­bation
frequency, aetiology of bronchiectasis, and smoking
status to model time to admission to hospital due to
severe exacerbations, and mortality with the proportional
hazards assumption checked using log-minus-log plots.
We used receiver operator characteristic (ROC) curves to
assess the association between sputum NET concentration
and both mortality and admission to hospital due to severe
exacerbations. We used Youden’s index to calculate the
optimal cutoff of sputum NET concentration and we report
the sensitivity and specificity. For the microbiome
characterisation, we did beta-diversity analysis using
principal coordinates analysis with group compa­ risons
using permutational multivariate analysis of variance.
We compared the Shannon diversity and Berger Parker
indices between groups as measures of alpha diversity.
We analysed proteomic data using principal component
analysis (with the dataset logarithmically transformed,
mean centred, and unit variance scaled), partial least
square discriminant analysis, or Student’s t test. We
controlled the false discovery rate for the t test with
Storey’s q value, which we calculated using the qvalue
package in R (version 3.6.3) and a q value of less than or
equal to 0·05 is considered to be significant. For all other
analyses statistical significance was set at a p value of less
than 0·05, unless otherwise stated.
We did our statistical analyses using R (version 3.6.3),
SPSS (version 22), and GraphPad Prism (version 6.07).
Role of the funding source
The funders of the study had no role in study design,
data collection, data analysis, data interpretation, or
writing of the report.
Results
The cohorts contributing to this study, the hypotheses
being tested, and the summarised results are shown in
the table.
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Primary studies Validation studies Findings

Airway inflammation will be
different between severe and
mild bronchiectasis
NETs will be associated with
severe bronchiectasis and are
predictive of poor outcomes
Antibiotic treatment for 14 days
will result in beneficial changes
to the lung proteome including
reduced NETs
Macrolide treatment will reduce
sputum NETs
NA=not applicable. NETs=neutrophil extracellular traps. P aeruginosa=Pseudomonas aeruginosa.
Sputum proteomics study of patients NA
with 20 severe vs 20 mild disease
Observation cohort study of
175 patients from the UK
Sputum proteomics study of
20 patients admitted to hospital for
exacerbations of bronchiectasis
treated with intravenous antibiotics
in Dundee, UK
Cohort study of patients with
bronchiectasis P aeruginosa infection
treated with (n=26) or without
azithromycin (n=26)
Observation cohort study of
275 patients from Italy, Spain, and
the UK
Observational cohort study of
20 patients admitted to hospital for
exacerbations of bronchiectasis
treated with intravenous antibiotics
in Dundee, UK
Post-hoc analysis of randomised
controlled trial in poorly controlled
asthma in 47 patients (AMAZES)
A strong association was found between
multiple neutrophil proteins and disease
severity; proteins identified are suggestive
of NETs
NETs are associated with severe
bronchiectasis, frequent exacerbations,
symptoms, and future outcomes across
both cohorts
Antibiotic treatment reduces NETs;
patients with P aeruginosa infection have
higher baseline NETs and less of a
reduction in NETs, which correlates with a
worse clinical outcome
Macrolides reduce NETs in patients with
P aeruginosa infection, where antimicrobial
efficacy is not expected; NETs are also
reduced by azithromycin in neutrophilic
asthma

Table: Summary of the study hypotheses, study cohorts, and findings

To test our first hypothesis, we used an
extreme phenotype approach using quantitative label-
free proteomic analysis to compare the differences in
sputum proteome between patients with severe and mild
disease. We enrolled 40 patients from Ninewells Hospital
in Dundee, of whom 20 had severe disease and 20 had
mild disease. Patient characteristics are shown in
appendix 3 (p 9).
Using criteria of at least two peptides identified and a
false discovery rate of 1%, we identified 709 proteins from
the sputum samples of the 40 patients. Significant
differences were identified for 96 proteins differentially
expressed between the groups (figure 1). Analysis of the
most abundant and differentially expressed proteins
showed that the majority of proteins were associated with
neutrophilic inflammation, and particularly identified
proteins known to be components of NETs including
RETN (ADSF), S100-A9 (MRP-14) and S100-A8 (CFAG),
neutrophil elastase ELANE (HLE), AZU1 (azurocidin),
MPO (myeloperoxidase), and LCN2 (NGAL). We examined
the association between these proteins and individual
components of the bronchiectasis severity index and found
no significant differences by age and sex, whereas the
most consistent associations were found between
neutrophil proteins and frequent exacerbations, chronic
infection status, and radiological severity (appendix 3 p 15).
Selected proteins associated with severe disease were
validated by ELISA (appendix 3 p 16). The results from this
proteomic study suggest that bronchiectasis severity might
be associated with an increase in NETs.
The findings of the initial proteomic study suggested the
hypothesis that NETs were associated with disease severity
in bronchiectasis. To test this hypothesis, we compared
NET concentrations in the sputum of patients with
bronchiectasis (n=151) with the sputum from patients
with cystic fibrosis (n=9), COPD (n=66), asthma
(n=10), and induced sputum from healthy controls (n=13;
appendix 3 p 17). Sputum NET concentrations were similar
between those with bronchiectasis and cystic fibrosis but
much higher than in those COPD, asthma, and healthy
controls (p<0·0001; figure 2A). The association between
NETs and severity in bronchiectasis was analysed in
sputum samples from the 175 patients with bronchiectasis.
Increasing sputum NET concentration correlated with an
increase in disease severity using the bronchiectasis
severity index (p<0·0001; figure 2B). Additionally, sputum
NET concentrations were significantly higher in patients
with a history of severe exacerbations (p=0·0089; figure 2C)
and correlated with QoL-B RSS (r = –0·42, p<0·0001;
figure 2D). Median NET concentration in patients with
different aetiologies of bronchiectasis ranged 0·61–1·96
units per mL, and no difference was found between
the aetiological groups (Kruskal-Wallis test p=0·29).
We separated patients with bronchiectasis into NET
concentration tertiles (low: 0·0–6·2 units per mL, n=58;
intermediate: 6·6–24·4 units per mL, n=59; and high
25–289 units per mL, n=58). For long-term outcomes,
patients were followed-up for a median of 32 months (IQR
26–44; maximum 77 months). We found that NET tertiles
predicted time to first severe exacerbation (figure 2E) and
patients in the highest tertile had increased mortality
(figure 2F). NETs as a biomarker showed moderate
sensitivity, specificity, and discrimination to predict
admission to hospital due to severe exacer­ bations and
mortality in bronchiectasis (appendix 3 p 14). After
adjustment for confounding (age, sex, treatments,
exacerbation frequency, aetiology, and smoking), the
highest NET tertile was associated with increased mortality
(hazard ratio [HR] 2·88 [95% CI 1·08–7·65]; p=0·034)
compared with the lowest tertile. No significant increase in
risk of mortality was seen in the intermediate NET tertile
compared with the lowest tertile (HR 1·28 [0·41–3·97];

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A C
30 Protein Protein expression (log2) Diff q
Mild
Severe Severe Mild
20 MPO 31·5 28·2 3·3 0·0016
ADSF 29·4 26·2 3·2 0·0016
10
Integrin beta-2 27·1 24·3 2·8 0·0016
NGAL 30·9 28·2 2·7 0·0025
0
t[2]

HLE 30·5 28·0 2·5 0·0079

–10 Catalase 30·4 28·0 2·3 0·0079
OLM4 27·2 25·0 2·1 0·010
–20 PRIM2 26·5 24·5 2·0 0·016
PGRN 29·8 27·9 1·9 0·0076
–30 HP-3; HP-1 33·1 31·3 1·8 0·0047
MIG9 30·8 29·5 1·3 0·020
–40
–60 –40 –20 0 20 40 60 LEI 25·6 24·3 1·3 0·024
t[1] TCN-1 25·4 28·3 –2·9 0·0070
IBP-2 23·9 26·8 –3·0 0·0052
B HSP70-1 25·4 28·4 –3·1 0·016
Myosin-9
Uteroglobin 30·1 33·2 –3·1 0·0016
CFAG
MRP-14 T beta-4 PnSP-2 23·6 26·8 –3·2 0·014
0·1 MMP-9
Arginase-1 ZG16B Tlc 25·3 28·8 –3·5 0·0033
BASP1
Catalase ZG16B 27·0 30·6 –3·7 0·0062
0·05 ADSF TC-1
PRIM2 CA-VI 24·1 27·8 –3·7 0·0052
HLE SPLUNC1 B2M
NGAL Apo-AI 24·4 28·5 –4·1 0·0086
p[2]

0 Cystatin-D Apo-AI
Clusterin Clusterin 24·2 28·4 –4·2 0·0025
Integrin beta-2 PSP
–0·05 PGRN CA-VI PSP 23·7 28·0 –4·3 0·0047
MPO PnSP-2 B2M 24·7 29·2 –4·6 0·0025
TN-C
–0·1

Azurocidin
–0·15
–0·15 –0·1 –0·05 0 0·05 0·1 0·15
p[1]

Figure 1: Proteomics of patients with mild and severe bronchiectasis

(A) Scores plot of principal component analysis based on sputum protein profiles of patients with mild and severe bronchiectasis. t[1] and t[2] are the first two principal components. Each datapoint
summarises a patient’s proteomic profile and the ellipse shows Hotelling’s T2 (95% CI). (B) Loadings plot showing how strongly each protein variable influences a principal component in the scores
plot. p[1] and p[2] are the first two principal components. (C) Sputum proteins with the highest differential expression between patients with mild and severe diseases. Proteins known to be associated
with NETs are written in red. The expression levels and differences are expressed on a log2 scale, with blue indicating low, yellow medium, and red high expression (arbitrary unit).
B2M=beta-2-microglobulin. BASP1=brain acid soluble protein 1. NETs=neutrophil extracellular trap. PRIM2=DNA primase large subunit. ZG16B=zymogen granule protein 16 homolog B.

p=0·67). For the risk of severe exacerbations, after
adjustment for the same confounders, the highest NET
tertile was associated with increased admissions to hospital
due to severe exacerbations (HR 3·07 [1·51–6·23];
p=0·0020). The intermediate NET tertile was not associated
with increased risk of severe exacerbations (HR 1·66
[0·71–3·86]; p=0·24). In linear regression analyses,
adjusted for the above confounders, increasing NET
concentrations remained associated with worse quality of
life (p=0·0011). NETs were only moderately correlated with
neutrophil counts in sputum (appendix 3 p 17) and
neutrophil counts alone were not predictive of clinical
outcomes (data not shown). Analysis from this UK cohort
suggested that NETs are associated with disease severity in
bronchiectasis.
We examined associations between NETs and the
microbiome in the patients with bronchiectasis in this
cohort. Most sequences at the phylum level were
identified as either Proteobacteria or Firmicutes using
16S rRNA gene sequencing (negative control sequencing
data are shown on appendix 3 p 23). Individual
microbiome profiles at the phylum and genus level sorted
according to decreasing sputum NET concentration are
shown in appendix 3 (p 19–20). Decreasing alpha-
diversity, measured using the Shannon-Weiner diversity
index (p=0·012) and Berger-Parker dominance index
(p=0·011; appendix 3 p 21), was observed across the
three tertiles as NET concentration increased. Beta-
diversity analysis showed differences according to
NET tertiles that were significant using PERMANOVA
(p<0·0001). We found that microbiome profiles where the
most abundant taxa was Pseudomonas or Haemophilus
had higher sputum NET concentrations than those
dominated by Streptococcus or other organisms (p<0·0001;
appendix 3 p 21). NET concentra­tions increased with
higher relative abundance of Pseudomonas spp and with
an increasing bacterial load of either Pseudomonas or
Haemophilus by quantitative PCR (pPCR; appendix 3 p 21).
We validated our findings from the UK cohort in an
independent European cohort (BRIDGE; n=275; patient

878 www.thelancet.com/respiratory Vol 9 August 2021

 Articles

A B
p<0·0001*
3 400

p<0·0001*
Log histone-elastase complexes

Histone-elastase complexes
300

(units per mL)

1
(units/mL)

200
0

100
–1

–2 0

=5 te

=6 e
(n ild

(n ver
) s

=1 a

=1 y
) is

(n dera
(n OPD

9)
9)
51 asi

(n alth
(n thm
=9 os

Se
6)

7)
=4
3)
0)
=1 ct

(n fibr

o
C
=6

He
As
(n chie

M
c
sti
on

Cy

BSI score
Br

C D
400 100
p=0·0089†
Histone-elastase complexes (units per mL)

Quality of life bronchiectasis respiratory

80
300
symptom score

60

200
40

100 20

r =–0·42, p<0·0001
0 0
No (n=143) Yes (n=32) 0 1 2 3

A history of severe exacerbation Log histone-elastase complexes (units per mL)

E F
100 100
Proportion free from exacerbation (%)

80 80
Overall survival (%)

60 60

40 40

20 High 20 High
Intermediate Intermediate
Low p<0·0001 by log-rank test Low p=0·009 by log-rank test
0 0
0 12 24 36 48 60 72 84 0 12 24 36 48 60 72 84
Time from study start (months) Time from study start (months)
Number at risk
(number censored)
High 58 (0) 43 (0) 31 (4) 12 (18) 7 (20) 3 (23) 3 (23) 0 (24) 58 (0) 55 (0) 48 (24) 19 (26) 14 (34) 7 (37) 4 (40) 0 (40)
Intermediate 59 (0) 50 (3) 44 (6) 18 (29) 10 (36) 3 (43) 2 (44) 0 (45) 59 (0) 57 (0) 53 (4) 21 (33) 12 (41) 4 (49) 2 (51) 0 (52)
Low 58 (0) 53 (0) 48 (2) 25 (23) 14 (34) 3 (45) 1 (47) 0 (47) 58 (0) 58 (0) 53 (2) 27 (26) 14 (39) 3 (50) 1 (52) 0 (52)

Figure 2: Validating the association between NET concentrations and disease severity outcomes in bronchiectasis
(A) Sputum NET concentration according to disease. (B) Sputum NET concentrations against disease severity (n=175). (C) Sputum NET concentrations against a
history of severe exacerbations over the 12 months before the study (n=175). (D) Quality-of-life score against sputum NET concentrations (n=175;, with each
datapoint representing one patient. (E) Time to first exacerbation by NET concentration tertile (low: 0·0–6·2 units per mL, n=58; intermediate: 6·6–24·4 units per mL,
n=59; and high 25–289 units per mL, n=58). (F) Survival curve by NET tertile (n=175). For parts A, B, and C, each datapoint represents the values for an individual
patient and horizontal lines and whiskers show mean with SD. BSI=bronchiectasis severity index. COPD=chronic obstructive pulmonary disease. NET=neutrophil
extracellular trap. *Kruskal-Wallis test. †Mann Whitney U test.

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 Articles

A Downregulated Upregulated B C
2·5 on day 14 on day 14 Over-representative pathways (downregulated proteins on day 14) Downregulated proteins on day 14 related
Reactome pathways Number Fold False discovery to neutrophil degranulation
2·0 F5–20 ALP Uteroglobin detected enrichment rate
PZP MIG9 Cystatin–C
1·5 Catalase Innate immune system 15 11·79 1·52 × 10–10 Catalase, CD67 antigen, chitotriosidase-1,
Neutrophil degranulation 11 19·99 2·26 × 10–9 MPO, AGP 1, AGP7, ADSF, CFAG, MRP-14,
–Log q

Immune system 15 6·04 7·00 × 10–7 MIG9, PI-10

1·0
Regulation of TLR by endogenous ligand 4 > 100 6·69 × 10–6
0·5 Over-representative pathways (upregulated proteins on day 14) Upregulated proteins on day 14 related to
Neutrophil degranulation 6 17·45 1·40 × 10–3 neutrophil degranulation
0 Innate immune system 7 8·81 6·03 × 10–3
–3 –2 –1 0 1 2 3 4 PIgR, cystatin-C, lysozyme C, LTA-4 hydrolase,
ALP, hQSOX
Difference (log2)

D E
P aeruginosa (day 1) Protein expression (log2) + vs – (day 1) 300 Positive for P aeruginosa
Negative for P aeruginosa p<0·0001*
– +
Day 1 Day 14 Day 1 Day 14 Difference q
MPO 24·5 22·4 29·2 28·1 4·7 0·03 Histone-elastase complexes (units per mL)
IGHG1 19.1 20·1 22·5 21·9 3·4 0·03
HLE 21.8 20·5 25·0 24·3 3·2 0·04 200
Azurocidin 23·5 22·8 26·3 25·9 2·8 0·03
A1BG 19·2 18·4 21·3 19·6 2·1 0·04
Cystatin-C 21·0 23·8 17·6 19·9 –3·4 0·03
TIMP1 21·1 21·0 17·6 19·1 –3·4 0·01
SPLUNC1 21·8 24·4 18·2 19·8 –3·5 0·03
MLN 70 22·5 22·1 18·5 19·0 –3·9 0·01 100
T beta-4 22·7 23·1 18·6 19·0 –4·1 0·03
B2M 23·5 24·6 19·2 20·1 –4·3 0·00
Cystatin-SN 23·7 25·5 19·1 21·4 –4·7 0·01
Uteroglobin 25·2 27·7 19·8 23·1 –5·4 0·01
Cystatin-S 25·3 26·9 19·9 21·9 –5·4 0·01
BPI-like 1 24·8 26·6 19·3 22·0 –5·5 0·01 0
Actin 24·3 26·0 18·8 20·2 –5·5 0·01 Day 1 Day 14

F
100 Positive for P aeruginosa
Negative for P aeruginosa

75
Proportion of patients (%)

p=0·0050 by log-rank test

50

25

0
0 50 100 150 200 250
Time to next exacerbation (days)
Number at risk
Positive for P aeruginosa 11 6 2 0 0 0
Negative for P aeruginosa 9 9 6 4 1 0

Figure 3: Antibiotic responses and Pseudomonas aeruginosa status in patients with bronchiectasis exacerbations, discovery and validation cohorts
(A) Volcano plot showing differential expression of sputum proteins between day 1 of an exacerbation and day 14 after systemic antibiotic treatment (discovery cohort). Storey’s q value at the y axis
represents the p value adjusted for the false discovery rate. The proteins highlighted in red or blue are related to neutrophil degranulation. (B) Reactome pathway analysis of upregulated and
downregulated proteins by antibiotic treatment in this discovery cohort using the Panther classification system.21 The fold enrichment is in reference to the whole human proteome. (C) The
upregulated and downregulated proteins in part B that are associated with the neutrophil degranulation pathway. (D) Differentially expressed sputum proteins in patients with P aeruginosa infection
and those without on day 1 (discovery cohort). Only those with a q value of <0·05 are shown. Data are in arbitrary units on a log2 scale. (E) Change in sputum NET concentration from day 0 to day 14 in
patients with a positive P aeruginosa culture and those with a negative P aeruginosa culture (validation cohort). (F) Kaplain-Meier survival of analysis of time to exacerbation by P aeruginosa status at
baseline (validation cohort). A1BG=alpha-1B-glycoprotein. B2M=beta-2-microglobulin. IGHG1=immunoglobulin heavy constant gamma 1. PZP=pregnancy zone protein.
*Wilcoxon matched-pairs signed rank test.

880 www.thelancet.com/respiratory Vol 9 August 2021


characteristics are in appendix 3 [p 11]). In the
BRIDGE study, increases in sputum NET concentrations
correlated with an increase in disease severity
using the bronchiectasis severity index (p<0·0001),
FEV1 % predicted (r = –0·34, p<0·0001), QoL-B RSS
(r = –0·43, p<0·0001), and increased severe exacerbations
(p<0·0001; appendix 3 p 22). Sputum NET concentrations
were associated with increased sputum concentrations of
neutrophil elastase, IL-10, TNF-α, IL-1β, and IL-8
(appendix 3 p 22). Taken together, these findings from
two independent studies support that NETs are associated
with disease severity in bronchiectasis.
Our data from this investigation suggest that increased
relative abundance of pathogenic Proteobacteria are
associated with NET proteins that are upregulated in
severe bronchiectasis (figure 1; appendix 3 p 21). Hence,
we next hypothesised that antibiotic treatment could
reduce NETs in patients with exacerbations
of bronchiectasis. We investigated the protein profiles of
sputum samples from patients with exacerbations
of bronchiectasis in response to 14 days of systemic
antibiotic treatment. Sputum samples were collected
from 20 patients with bronchiectasis (the discovery
cohort; patient characteristics are shown in
appendix 3 [pp 11–12]) on day 1 of an exacerbation and
again on day 14, at the end of antibiotic treatment, to give
40 samples overall. Proteomic analysis showed
that the antibiotic treatment was associated with
altered expression of 39 proteins in the sputum
(23 downregulated and 16 upregulated; figure 3A).
Pathway analysis revealed that the upregulated
and downregulated proteins share the same
over-represented pathways of neutrophil degranulation
and innate immune system (figure 3B). Among the
11 downregulated proteins related to neutrophil
degranulation, ten of them were known NET-associated
proteins and the remaining one (CEACAM8
[CD67 antigen]) is known to be released upon activation
of neutrophils by extracellular chromatin (figure 3C).25
The upregulated proteins related to neutrophil
degranulation are predominantly protease inhibitors
(SLPI [ALP] and CST3 [cystatin-C]), PIgR, LTA4H
(LTA-4 hydrolase), lysozyme, and QSOX1 (hQSOX)
(figure 3C). With the exception of QSOX1, these proteins
are known to negatively regulate neutrophilic
inflammation. Interestingly, patients with a positive
P aeruginosa culture on day 1 were found to have higher
baseline levels of NET proteins (MPO, ELANE, and
AZU1) and lower levels of several proteins including
ACT1 (actin), BPIFB2 (BPI-like 1), and protease inhibitors
(CST1 [cystatin-SN], CST3 [cystatin-C], CST4 [cystatin-S],
B2M [beta-2-microglobulin], and TIMP1 [EPA]) than
patients without P aeruginosa infection (figure 3D).
Although this study was not sufficiently powered to show
differences between subgroups, we observed a trend
showing that patients with P aeruginosa by culture had a
lower overall magnitude of reduction in MPO, ELANE,
www.thelancet.com/respiratory Vol 9 August 2021
Articles
A
2·0 Macrolide group
Matched control
Log histone-elastase complexes
1·5
(units per mL)
1·0
0·5
0
Baseline Post-treatment
B
0·5
Log change in histone-elastase complexes
0
(units per mL)
–0·5
–1·0
–1·5
Overall Non-eosinophilic Eosinophilic
(n=47) (n=25) (n=22)
Figure 4: Changes in NET concentrations in response to long-term macrolide
therapy in bronchiectasis and asthma
(A) Effect of macrolide treatment (azithromycin 250 mg three times per week)
on sputum NET concentrations over 12 months in patients with bronchiectasis
(n=52). (B) Effect of macrolide treatment (azithromycin 500 mg three times per
week) on sputum NET concentrations over 12 months vs placebo in patients
with asthma from the AMAZES trial (n=47), by eosinophilic status. Datapoints
are the mean difference between azithromycin and placebo groups, with error
bars showing the 95% CI. Negative values indicate a greater reduction in the
macrolide group vs placebo group.
and AZU1 than did those without P aeruginosa infection
(figure 3D). To validate this observation, we carried out
an additional study of the same design in an independent
cohort (validation cohort; patient characteristics are
shown on appendix 3 pp 12–13). 20 additional patients
were enrolled, of whom 19 showed a reduction in sputum
NET concentration after 14 days of treatment with
antibiotics (p<0·0001; figure 3E). Patients with
P aeruginosa at baseline had a lesser reduction in
NET concentration with antibiotic treatment than those
without P aeruginosa infection (mean reduction 33·2%
[SD 21] vs 78·0% [22]; p=0·0015). This absence of
resolution of inflammation correlated with a shorter
time to next exacerbation (median time to exacerbation
49 days vs 108 days; p=0·0050; figure 3F).
Previously we did a meta-analysis of individual patient-
level data from three randomised controlled trials in
which macrolide treatment extended the time to first
exacerbation in patients with bronchiectasis versus
881
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placebo (n=31 macrolide therapy, n=30 placebo; HR 0·38
[95% CI 0·21–0·71]; p=0·0093; appendix 3 p 24).22 Long-
term macrolide therapy is regarded as not having
antimicrobial efficacy against P aeruginosa and did not
change the microbiome in patients infected with
Pseudomonas in a previous study but have been shown to
inhibit NET formation in vitro.26,27 Therefore, we
hypothesised that macrolides would be effective in
reducing NETs in patients with bronchiectasis with
P aeruginosa infection. In an observational cohort of
26 patients with bronchiectasis and P aeruginosa
(baseline characteristics are shown on appendix 3 pp 13–
14), azithromycin 250 mg three times per week for 1 year
significantly reduced the concentration of NETs
compared with a matched cohort (n=26) who did not
receive macrolides (log10 difference macrolide group
–0·45 [95% CI –0·80 to –0·09] vs control group 0·18
[–0·23 to 0·60; p=0·016; figure 4A).
Macrolides have established efficacy in COPD and
severe asthma where NETs are implicated.17 To validate
and extend our observation that macrolides reduce
sputum NET concentrations, we studied patients from
the AMAZES trial of long-term azithromycin in asthma.23
In this randomised controlled trial, in a subset of patients
with asthma (27 who had been treated with azithromycin
treated, and 20 who had been treated with placebo; of
whom 12 in the azithromycin group and ten in the
placebo group were eosinophilic) 12 months of treatment
with 500 mg azithromycin three times per week
significantly reduced sputum NET concentrations (log10
difference –0·50 [95% CI –0·77 to –0·22]; p<0·0001).
The effect was driven primarily by a reduction in
NET concentration in patients with non-eosinophilic
disease (n=25) at baseline (log10 difference macrolide
treatment vs placebo −0·68 [95% CI −0·99 to −0·37];
p<0·0001, with no significant effect in the eosinophilic
subgroup (n=22): log10 difference −0·22 [−0·62 to 0·18];
p=0·28, figure 4B).
Discussion
In this study, we found that sputum proteomics are a
powerful tool to assess host inflammatory response in
patients with bronchiectasis. By simultaneously studying
multiple markers of inflammation, we identified a key
role for NETs in disease severity and treatment response
in bronchiectasis.
We found that most of the differentially expressed
proteins between mild and severe disease were neutrophil-
derived and consistent with the published proteome of
NETs.18 NETs were abundant in the sputum of patients
with bronchiectasis and at levels similar to those seen in
the sputum of patients with cystic fibrosis, where NETs are
an accepted component of pathophysiology.28 Correlation
does not equal causation and showing that NETs are
higher in severe bronchiectasis does not prove that
reducing them would have clinical benefits. We therefore
extended our observations by showing that two therapies
882
known to reduce exacerbations and improve symptoms,
acute antibiotic therapy and macrolides, both reduce
sputum concentrations of NET in patients with
bronchiectasis. These findings suggest a link between
changes in NET concentration and clinical benefits.
A 2020 trial of the DPP-1 inhibitor brensocatib in
bronchiectasis14 strongly supports our interpretation.
Intercellular neutrophil elastase triggers formation of
NETs through the degradation of F-actin and histone H4
and is prevented through inhibition of DPP-1.13,29 Our
results might provide a potential mechanistic basis for the
beneficial effects of DPP-1 inhibition in bronchiectasis.
Identification of NETs as a dominant mechanism of
inflammation in severe bronchiectasis establishes a
potential opportunity to directly target inflammation with
a new generation of specific therapies for the first time.14
There is strong biological plausibility that NETs are
directly involved in bronchiectasis pathophysiology:
NETosis is an inefficient and ineffective form of bacterial
killing and key bronchiectasis pathogens, such as
P aeruginosa and Haemophilus influenzae, are resistant to
being killed by NETs and are capable of degrading NETs
to escape host defence.18,30 The findings of our microbiome
investigation support this theory. We found that higher
levels of NETs were associated with increasing load and
dominance of Pseudomonas and that microbial diversity
was reduced with increasing sputum NETs.
To our knowledge, this is the first study to examine
proteomic changes in response to antibiotic treatment in
bronchiectasis. We found that after 14 days of antibiotics
the most downregulated proteins were predominantly
NET-associated proteins, whereas the most upregulated
proteins were predominantly those known to negatively
regulate neutrophil degranulation. Patients who also had
P aeruginosa infection had a lesser response to antibiotics
in terms of proteomic changes. This observation is
consistent with the clinical observation that patients with
P aeruginosa have a worse outcome despite more frequent
antibiotic treatment.31
Macrolides are among the most effective therapies
available for bronchiectasis and have been shown to reduce
exacerbations by around 50%.22 The benefit in patients with
P aeruginosa infection appears to be greater even than the
effect in the overall bronchiectasis population.22 This
finding cannot be explained by antimicrobial effects
because macrolides are ineffective against P aeruginosa and
previous studies have shown that the microbiome was
unaffected by 12 months of macrolide therapy in patients
with a Pseudomonas-dominated microbiome.27 NETs are
reduced in the airways despite clear evidence that
neutrophil numbers are not reduced by macrolides in the
airway, neither in bronchiectasis nor in severe asthma.23,32
Macrolides, such as azithromycin, have been shown to
inhibit NETs ex vivo through reduced activation of the
oxidative burst and autophagy.26 To our knowledge, here we
show for the first time in vivo, in two different datasets, that
macrolides reduce NETs. Therefore, our study supports a
www.thelancet.com/respiratory Vol 9 August 2021

novel immunomodulatory effect of macrolides, particularly
in patients with bronchiectasis with P aeruginosa
infection. European Respiratory Society guidelines for
bronchiectasis recommend inhaled antibiotics rather than
macrolides as first-line therapy for patients with a
concomitant P aeruginosa infection who have frequent
exacerbations on the basis of the absence of activity of
macrolides against P aeruginosa.8 Our data, combined with
the known efficacy of macrolides in reducing exacerbations
in patients infected with P aeruginosa in our previous
individual patient-level data meta-analysis,22 would justify
revisiting these guideline recommendations.
Our study has important limitations. We used 16S rRNA
gene sequencing to characterise the sputum microbiome,
a method that has inherent limitations including
underestimation of some taxa and poor resolution to
species level. We used qPCR to overcome some of these
limitations, but metagenomic approaches should be
considered in future studies. We used qPCR that was
targeted to specific pathogens and did not assess total
bacterial burden in the large cohort studies. The use of
sputum samples is standard in bronchiectasis research
and sputum is the most acceptable and clinically useful
method of studying lung inflammation and the
microbiome. However, we acknowledge that sputum is
regarded as an intermediary between the upper and lower
airway microbiome.33 We have previously found a strong
correlation between NET concentrations in the sputum
and in bronchoalveolar lavage,17 which reduces concerns
that studies of NETs sputum would not be representative
of NET concentrations in the lung. We examined changes
in NET concentrations with macro­ lide treatment in
bronchiectasis via a non-randomised study, which is open
to bias by indication. All other studies of macrolides that
we identified at the time of study design that had a
randomised study design were done more than 10 years ago
and adequate contemporary samples were not available.22
Further prospective placebo-controlled studies would
arguably be unethical because guidelines recommend
macrolides for treatment on the basis of their established
efficacy.8 Nevertheless, we validated our findings using a
randomised controlled trial dataset of patients with severe
asthma (AMAZES), providing robust evidence that
macrolides reduce NETs in vivo. Likewise, our antibiotic
treatment study did not include a control group because
withholding antibiotic treatment from patients with acute
exacerbations of bronchiectasis would be unethical.
In summary, we found that proteins derived from NETs
are associated with severe disease and poorer outcomes
in patients with bronchiectasis. Furthermore, we found
that beneficial treatment responses are associated with a
reduction in NETs with both systemic antibiotics and
macrolides. These data provide a basis for future research
into the roles of NET proteins in bronchiectasis, and the
development of biomarkers of severity and treatment
response. Targeting NETosis could theoretically reduce a
broad range of neutrophil and downstream inflammatory
www.thelancet.com/respiratory Vol 9 August 2021
Articles
mediators without compromising bacterial clearance.
These data are important for understanding the potential
benefits of novel treatments, such as DPP-1 inhibition,
and targeting of macrolide treatment in those most likely
to benefit.
Contributors
HRK, AS, AJD, EF, OS, SA, JLS, JTJH, and JDC contributed to the study
design. HRK, AS, AJD, LP, JP, YHG, GS-C, MLC, MO, EC, CJF, SF,
GBR, OS, SA, and JDC contributed to data collection. HRK, AS, AJD,
ML, GGE, JLS, JTJH, and JDC contributed to data analysis. HRK, AS,
AJD, ML, GGE, JSE, FB, OS, SA, JLS, JTJH, and JDC contributed to data
interpretation. HRK, AS, JTJH, and JDC wrote the first draft of the
manuscript. All authors contributed to revising the manuscript for
important content and approval of final version. HRK, AS, AJD, ML, JP,
JLS, OS, SA, JTJH, and JDC had access to the raw data. JDC had final
responsibly to submit for publication. HRK and JDC accessed and
verified the underlying study data.
Declaration of interests
OS reports funding from Bayer, Grifols and Zambon. FB reports
grants and personal fees from AstraZeneca, Chiesi, GSK, Pfizer and
Menarini, grants from Bayer, personal fees from Grifols, Guidotti,
Insmed, Novartis, Zambon, and Vertex outside of the submitted work.
JSE reports grants from Novartis, and consultancy fees from Polyphor
and Bayer outside of the submitted work. ML reports personal fees
from AstraZeneca outside of the submitted work. SA reports grants
and personal fees from Bayer Healthcare, Aradigm Corporation,
Chiesi, Insmed, and Grifols and personal fees from AstraZeneca,
Basilea, Zambon, Novartis, Raptor, Actavis UK, and Horizon outside of
the submitted work. JDC reports grants and personal fees from
AstraZeneca, Boehringer Ingelheim, GSK, Insmed, and Zambon;
grants from Gilead; personal fees from Novartis, and Chiesi outside of
the submitted work. All other authors declared no competing interests.
Data sharing
Anonymised data from studies included in this manuscript can be made
available to researchers through an application process via the EMBARC For more on the application
website. process see www.bronchiectasis.
eu/dataaccess
Acknowledgments
This study was supported by the Scottish Government Chief Scientist
Office (Senior Clinical Fellowship to JDC), British Lung Foundation
through the British Lung Foundation Chair of Respiratory Research, and
European Bronchiectasis Network (EMBARC), a European Respiratory
Society Clinical Research Collaboration. EMBARC is supported by project
partners AstraZeneca, Chiesi, Grifols, Janssen, Insmed, Novartis, Zambon.
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