[

Original Research Signs and Symptoms of Chest Diseases

Mediators of Neutrophil Function in Children With

Protracted Bacterial Bronchitis
Katherine J. Baines, PhD; John W. Upham, PhD; Stephanie T. Yerkovich, PhD; Anne B. Chang, PhD;
Julie M. Marchant, PhD; Melanie Carroll, BSc (Hons); Jodie L. Simpson, PhD; and Peter G. Gibson, MBBS

Protracted bacterial bronchitis (PBB) is a common and treatable cause of

chronic wet cough in children in which the mechanisms are not understood. This study investigates the IL-1 pathway and a neutrophil gene expression signature in PBB.

BACKGROUND:

BAL was collected from children in an experimental cohort (n 5 21, PBB; n 5 33,
control subjects), and a second validation cohort (n 5 36, PBB; n 5 11, control subjects). IL-1b,
IL-1 receptor antagonist (IL-1RA), and a-defensins 1-3 were assayed by enzyme-linked immunosorbent assay, western blot, and quantitative real-time polymerase chain reaction, together
with selected IL-1 pathway members and neutrophil-related molecules.

METHODS:

In the experimental cohort, children with symptomatic PBB had significantly higher
levels of IL-1b and a-defensin gene and protein expression. Expression of the neutrophil chemokine receptor C-X-C motif receptor 2 was also higher in PBB. IL-1RA protein was higher,
however, the IL-1RA:IL-1b ratio was lower in children with PBB than control subjects. In the
validation cohort, protein and gene expression of IL-1b and a-defensins 1-3 were confirmed
higher, as was gene expression of IL-1 pathway members and C-X-C motif receptor 2. IL-1b
and a-defensin 1-3 levels lowered when PBB was treated and resolved. In children with recurrent
PBB, gene expression of the IL-1b signaling molecules pellino-1 and IL-1 receptor-associated
kinase 2 was significantly higher. IL-1b protein levels correlated with BAL neutrophilia and
the duration and severity of cough symptoms. IL-1b and a-defensin 1-3 levels were highly
correlated.

RESULTS:

PBB is characterized by increased IL-1b pathway activation. IL-1b and related

mediators were associated with BAL neutrophils, cough symptoms, and disease recurrence,
providing insight into PBB pathogenesis.
CHEST 2014; 146(4):1013-1020

CONCLUSIONS:

Manuscript received January 15, 2014; revision accepted April 11, 2014;
originally published Online First May 29, 2014.
ABBREVIATIONS: CXCL 5 C-X-C motif ligand; CXCR 5 C-X-C motif
receptor; IL-1RA 5 IL-1 receptor antagonist; IRAK 5 IL-1 receptorassociated kinase; NF-kB 5 nuclear factor-kB; PBB 5 protracted bacterial bronchitis; PELI1 5 pellino-1; Q 5 quartile; TLR 5 Toll-like receptor;
TNF 5 tumor necrosis factor
AFFILIATIONS: From the Priority Research Centre for Asthma and
Respiratory Diseases (Drs Baines, Simpson, and Gibson), The University
of Newcastle, Callaghan, NSW; Department of Respiratory and Sleep
Medicine (Drs Baines, Simpson, and Gibson), Hunter Medical Research
Institute, John Hunter Hospital, New Lambton Heights, NSW; School of
Medicine (Drs Upham, Yerkovich, and Marchant and Ms Carroll), The
University of Queensland, Brisbane, QLD; Qld Lung Transplant Service
(Dr Yerkovich), The Prince Charles Hospital, Brisbane, QLD; Department of Respiratory Medicine (Drs Chang and Marchant), Queensland
Childrens Medical Research Institute, Royal Childrens Hospital,

Brisbane, QLD; and Child Health Division (Dr Chang), Menzies School
of Health Research, Darwin, NT, Australia.
FUNDING/SUPPORT: Drs Gibson, Upham, and Chang are supported by
National Health and Medical Research Council (NHMRC; Commonwealth of Australia) fellowships [Grants 569240, 511019, and 545216,
respectively]. Dr Baines is supported by a research fellowship from The
University of Newcastle. The study was funded by the Financial Markets
Foundation for Children [Grant 2010-005], NHMRC [Grant 1042601],
and NHMRC Centre of Research Excellence (CRE) in Lung Health of
Aboriginal and Torres Strait Islander Children [Grant 1040830].
CORRESPONDENCE TO: Katherine J. Baines, PhD, Level 2 West, HMRI
Bldg, Lot 1 Kookaburra Circuit, New Lambton Heights, NSW 2305,
Australia; e-mail: katherine.baines@newcastle.edu.au
2014 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of
this article is prohibited without written permission from the American
College of Chest Physicians. See online for more details.
DOI: 10.1378/chest.14-0131

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1013

Protracted bacterial bronchitis (PBB) is an important

and common cause of chronic wet cough in children.1
Once correctly diagnosed, the childs cough resolves
with prolonged antibiotic therapy.2 PBB is now internationally accepted as a diagnostic entity3 and has been
incorporated into national4 and international5 pediatric
guidelines for cough management. However, despite
this increased clinical recognition, the underlying mechanisms of PBB remain to be elucidated.
Prior studies have shown that bacterial colonization and
airway neutrophilia are present in children with PBB.2,6
This was associated with upregulation of the Toll-like
receptors (TLRs) TLR2 and TLR4 in the BAL of children with PBB.6 This implicates persistent neutrophilic

Materials and Methods

Subject Recruitment and Sampling
Inflammatory mediators were evaluated in two cohorts. The experimental cohort (n 5 54) comprised subjects and BAL samples collected
in previous studies.6,9 The validation cohort (n 5 47) was obtained
from a second cohort with purposive matching of control subjects.2
The selection of samples for analysis was based on the availability of
specimens and clinical diagnosis; details of enrollment of children is
described previously.2,6,9 A clinical history was obtained on the day of
the bronchoscopy, and parents were provided with a cough diary card10
used to document response to antibiotics, defined as absence of cough
or . 75% reduction in score (for 3 days) within 2 weeks of antibiotic use (amoxycillin-clauvanate 45 mg/kg/d in two doses for 14 days2)
postbronchoscopy. In the validation cohort, children with PBB were
contacted at monthly intervals to document recurrence.

inflammation in the pathogenesis of PBB and suggests

that neutrophil pathway mediators such as IL-1b may
play an important role in pathogenesis. In adults with
neutrophilic asthma, using gene expression profiling we
have implicated the IL-1 and tumor necrosis factor
(TNF)-a/nuclear factor-kB (NF-kB) pathways in
sputum7 and a blood gene expression signature
involving neutrophil defensins and proteases.8 Since
neutrophils play a role in both PBB and neutrophilic
asthma, there may be common mechanisms involved.
Therefore, this study evaluated these pathways and
mediators in two cohorts of PBB and control children.
We hypothesized that IL-1b and the neutrophil gene
expression signature would be elevated in PBB and
related to symptoms and recurrence.

approved by the Ethics Committees of the Royal Childrens Hospital

and University of Queensland (HREC/03/QRCH/17).
Target Selection and Gene Expression
Inflammatory gene expression was determined in RNA extracted
from BAL cell pellets using real-time quantitative polymerase chain
reaction and standardized TaqMan methods as described in detail
in e-Appendix 1. Genes tested include those previously identified as
increased in sputum in neutrophilic asthma and include IL-1b (IL1B),
IL-1 receptor 2 (IL1R2), IL-1 receptor antagonist (IL1RN), pellino-1
(PELI1), and IL-1 receptor-associated kinase 2 (IRAK2); TNF-a/NF-kB
pathway members TNF receptor superfamily member 1B (TNFRSF1B)
and NF-k light polypeptide gene enhancer in B cells 2 (NFKB2); and
the chemoattractant receptor C-X-C motif receptor 2 (CXCR2).7 Also
tested was a blood neutrophil gene expression signature including the
a-defensins (DEFA1-3 and DEFA4), protease elastase (ELANE), and
cathepsin G (CTSG).8

PBB was defined as the presence of a history of chronic (. 4 weeks)

wet cough and a response to antibiotic treatment with resolution of the
cough within 2 weeks in the absence of signs and symptoms of other
diseases. Symptomatic PBB was defined as children with PBB who
were coughing when bronchoscopy was undertaken. Resolved PBB
was defined as children who previously had a chronic wet cough that
responded to 2 weeks of antibiotics and who were cough-free at the
time when bronchoscopy was undertaken. Recurrent PBB was defined
prospectively as more than three episodes of wet cough responding to
antibiotic treatment within 12 months following the initial diagnosis,
and nonrecurrent PBB as those with fewer than three episodes in the
same timeframe.

Protein Measurements
IL-1b (undiluted) and IL-1 receptor antagonist (IL-1RA) (one-fifth
dilution) protein levels were measured in BAL supernatant using the
DuoSet enzyme-linked immunosorbent assay as per the manufacturers
instructions (R&D Systems, Inc). a-Defensins 1-3 (also known as the
human neutrophil peptides 1-3) were measured in BAL supernatant
(undiluted) using the Human HNP1-3 enzyme-linked immunosorbent
assay kit as per the manufacturers instructions (HK317; Hycult Biotech).
Western blot was performed on undiluted BAL from a subset of subjects
in the experimental cohort as described in e-Appendix 1.

Experimental cohort control was a convenient sample of children

undergoing gastroscopy, whereas in the validation cohort control subjects were age-matched and obtained opportunistically from children
undergoing bronchoscopy for assessment of the airways (eg, stridor)
with no history of chronic cough and no respiratory infection in the preceding 2 weeks. Informed consent was obtained, and the studies were

Statistical Analysis
Data were analyzed using Stata 11 (StataCorp LP) and reported as
median (quartile [Q]1, Q3). Statistical comparisons were performed
using the two-sample Wilcoxon rank sum (Mann-Whitney) test for
nonparametric data with P , .05 considered significant. Spearman rank
correlations were used to test relationships.

Results

profiles of moderate cough severity and a mean symptom duration of . 20 weeks. Lung inflammation was
present with increased BAL cellularity including neutrophils. The validation cohort tended to have more males
and less intense airway neutrophilia than the experimental cohort. The control subjects were older in the

Clinical Characteristics

The children with PBB in both the experimental (n 5 21)

(Table 1) and validation (n 5 36) (Table 1) cohorts comprised mainly infants and young children with similar
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1015

0 (0, 0)
3 (2, 6)

Eosinophils, %

Lymphocytes, %

0.47 (0.37, 0.70)

0.72 (0.66, 0.97)

0.74 (0.59, 1.63)

1.01 (0.74, 1.3)
0.91 (0.74, 1.37)
1.21 (0.67, 1.67)
1.12 (0.73, 1.24)

Pellino-1 (PELI1)

IL-1 receptor-associated kinase (IRAK2)

a-Defensins 1-3 (DEFA1-3)

C-X-C motif receptor 2 (CXCR2)

0 (0, 305)

3,045 (1,361, 3,805)

140 (76, 288)

2,424 (1,557, 5,369)

1,043 (627, 1,772)

353 (126, 721)

12.92 (7.79, 41.20)

5.01 (1.30, 9.61)

2.21 (1.22, 3.57)

1.91 (1.15, 5.03)

, .001

.008

.003

, .001

, .001

.004

, .001

.032

.001

.002

, .001

.001

.027

, .001

, .001

, .001

N/A

N/A

.780

, .001

P Value

163 (84, 273)

2,631 (1,997, 2,967)

1,662 (1,073, 2,135)

0.70 (0.35, 0.97)

1.38 (0.28, 3.29)

0.90 (0.68, 1.79)

0.92 (0.64, 1.84)

1.00 (0.87, 1.30)

0.88 (0.58, 1.35)

1.08 (0.42, 1.77)

0.84 (0.58, 1.50)

5 (4, 9)

0 (0, 0)

92 (90, 96)

2 (1, 4)

96 (48, 110)

N/A

N/A

6 (5)

0.7 (0.5, 1.9)

11

Control Subjects

1,857 (489, 3,855)

357 (86, 796)

1,926 (606, 4,448)

3.79 (0.79, 45.3)

4.43 (1.34, 16.46)

3.55 (1.07, 12.42)

1.26 (0.93, 4.55)

1.44 (0.90, 5.95)

2.25 (1.41, 6.27)

2.96 (1.32, 6.73)

4.44 (1.31, 12.96)

9 (5, 12)

0 (0, 0)

67 (30, 83)

13 (7, 57)

178 (100, 340)

2 (1, 3)

30 (4, 56)

9 (27)

2.0 (1.3, 4.1)

36

PBB

Validation Cohort

Data are given as mean (Q1, Q3) unless otherwise indicated. IL-1RA 5 IL-1 receptor antagonist; N/A 5 not applicable; PBB 5 protracted bacterial bronchitis; Q 5 quartile.

a-Defensins 1-3, pg/mL

IL-1RA to IL-1b ratio

IL-1RA, pg/mL

IL-1b, pg/mL

BAL supernatant protein expression

1.64 (1.11, 3.65)

0.87 (0.69, 1.44)

IL-1 receptor 2 (IL1R2)

IL-1 receptor antagonist (IL1RN)

2.20 (1.55, 4.37)

2.11 (1.05, 5.24)

12 (7, 22)

0 (0, 0)

52 (43, 79)

34 (10, 44)

365 (196, 632)

3 (2, 3)

20 (14, 80)

11 (10)

2.3 (1.2, 2.8)

21

PBB

0.94 (0.72, 1.35)

IL-1b (IL1B)

BAL cell gene expression

4 (2, 6)
90 (85, 94)

Macrophages, %

103 (71, 171)

Neutrophils, %

Total cell count 106/L

BAL cell counts

N/A
N/A

Cough score10

16 (17)

Cough duration, wk

9.7 (5.5, 12.7)

Girls (boys)

33

Age, y

Clinical details

No.

Control Subjects

Experimental Cohort

] Clinical and BAL Data for the Children in the Experimental and Validation Cohorts

Characteristics

TABLE 1

.018

, .001

.797

.014

.048

.017

.077

.074

.008

.014

.003

.132

.174

.002

, .001

.004

N/A

N/A

.066

.015

P Value

experimental cohort, but ages were similar in the validation cohort.

Gene expression was measured in the entire experimental
cohort, and protein was assessed in 30 control subjects
and 21 subjects with PBB. In the validation cohort,
IL-1b and IL-1RA protein were measured in 10 control
subjects and 29 PBB subjects, a-defensin 1-3 protein
was measured in five control subjects and 24 subjects
with PBB, and gene expression was assessed in 10 control subjects and 36 subjects with PBB, except for C-X-C
motif receptor (CXCR) 2 (eight control subjects and
25 subjects with PBB) due to insufficient remaining
samples.
Mediators in the Experimental Cohort

There was altered expression of IL-1 pathway members

IL1B, IL1R2, IL1RN, PELI1, and IRAK2 and the neutrophil chemokine receptor CXCR2 in PBB (Table 1) but
not TNF-a/NF-kB pathway members TNFRSF1B
[control subjects: 1.03 (0.77, 1.35); PBB: 1.09 (0.94, 1.49);
P 5 .183] and NFKB2 [control subjects: 1.06 (0.63, 1.53);
PBB: 1.16 (0.84, 1.48); P 5 .419]. Gene expression of
DEFA1-3 was higher in PBB, but ELANE [control subjects: 1.21 (0.49, 2.04); PBB: 1.45 (0.92, 2.06); P 5 .370]
and CTSG [control subjects: 0.91 (0.72, 1.29); PBB: 0.84
(0.57, 1.35); P 5 .299] were not changed, and DEFA4
was not detected.

Figure 1 IL-1b expression in BAL samples from the experimental

cohort including 30 control subjects and 21 subjects with PBB. A-D,
(A) a-defensins 1-3 and (B) IL-1b protein levels are higher in PBB,
(C) the IL-1RA to IL-1b ratio is decreased in PBB with results being
presented as the median and the error bar as the upper interquartile range,
and (D) Pro-IL-1b (17-kDa form) is present in the BAL supernatant of
subjects with PBB but not control subjects, demonstrated by western blot.
CTRL 5 control BAL sample; IL-1RA 5 IL-1 receptor antagonist;
PBB 5 protracted bacterial bronchitis BAL sample; STD 5 western
blot standard.

Clinical Signicance of Mediator Levels

Mediator in the Validation Cohort

The relationship between mediators and clinical features

was explored by comparing symptomatic PBB (n 5 26)
with treated and resolved PBB (n 5 10), recurrent
(n 5 29) with nonrecurrent PBB (n 5 6), and investigating correlations. Compared with symptomatic PBB,
resolved PBB showed much lower BAL neutrophils
[symptomatic PBB: 47% (12, 66); resolved PBB: 5% (2, 7)
neutrophils; P , .001], IL-1b protein (Fig 2A), and
a-defensin 1-3 protein (Fig 2B), but little difference in
gene expression of IL-1 pathway or neutrophil-related
mediators (data not shown).

Significant differences in the experimental cohort were

evaluated in a clinical validation cohort to relate mediator levels to symptom status and disease recurrence.
There was significantly higher gene expression of IL1B,
IL1R2, IL1RN, DEFA1-3, and CXCR2 in PBB (Table 1).
IL-1b protein was also significantly higher (Table 1),
corresponding to a decreased IL-1RA to IL-1b ratio in
PBB (Table 1). However, gene expression of PELI1 and
IRAK2 did not reach significance between patients with
PBB and control subjects. In PBB, the level of gene and
protein expression of a-defensins 1-3, IL-1b, and IL-1RA
were significantly correlated (a-defensins 1-3: Spearman
r 5 0.46, P 5 .023; IL-1b: Spearman r 5 0.68, P , .001;
IL-1RA: Spearman r 5 0.56, P 5 .002).

When children with recurrent PBB were evaluated at

baseline and compared with those with nonrecurrent
PBB, there were no significant differences in a-defensins
1-3, IL-1b, IL-1RA, or CXCR2 expression. However,
downstream IL-1 pathway signaling molecules PELI1
(Fig 2C) and IRAK2 (Fig 2D) were significantly higher
at baseline in those with recurrent vs nonrecurrent PBB,
suggesting that activation of IL-1 signaling predicts PBB
recurrence. Similar trends were seen whether the participants with recurrent and nonrecurrent PBB were
symptomatic or resolved at the time of sampling (for
resolved nonrecurrent PBB, n 5 3; resolved recurrent
PBB, n 5 7; and for symptomatic nonrecurrent PBB,
n 5 3; symptomatic recurrent PBB, n 5 22). PELI1 gene

There were higher levels of a-defensins 1-3 (Fig 1A,

Table 1), IL-1b (Fig 1B, Table 1), and IL-1RA protein
levels (Table 1) in PBB, and a lower IL-1RA to IL-1b
ratio (Fig 1C, Table 1). Western blot confirmed the
presence of active IL-1b (Fig 1D). Protein and gene
expression for IL-1b were significantly correlated in
PBB (Spearman r 5 0.65, P 5 .001).

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Figure 2 A-D, In symptomatic PBB, the levels of (A) a-defensins 1-3 and (B) IL-1b protein in BAL are higher. IL-1 pathway signaling is associated
with recurrence of PBB shown by elevated gene expression of (C) PELI1 and (D) IRAK2. Results are presented as median and the error bar as the upper
interquartile range. IRAK 5 IL-1 receptor-associated kinase; PELI1 5 pellino-1. See Figure 1 legend for expansion of other abbreviations.

expression remained significantly higher in symptomatic recurrent PBB (P 5 .03) or resolved recurrent PBB
(P 5 .04). IRAK2 gene expression was still higher in
symptomatic recurrent PBB (P 5 .13) or resolved recurrent PBB (P 5 .09), however, we lost significance with
the reduced sample size.
IL-1b was significantly associated with the BAL neutrophilia (r 5 0.75, P , .0001) (Fig 3A) and duration of
cough in weeks (r 5 0.29, P 5 .046) (Fig 3B). IL-1b levels
were significantly higher in children with PBB and a

cough score of three or more (P 5 .006) (Fig 3C). IL-1b

and a-defensin 1-3 levels were significantly correlated
(Fig 3D), and a-defensin 1-3 levels were also associated
with BAL neutrophils [Spearman r 5 0.48; P 5 .001].

Discussion
This study has identified increased expression of
neutrophil-related mediators in PBB, including IL-1 pathway members, neutrophil a-defensins, and the chemokine
receptor CXCR2. Elevated IL-1b in PBB was confirmed in two clinical cohorts and was associated with

Figure 3 A-C, Levels of IL-1b protein detected in the BAL of children with PBB are correlated with (A) BAL neutrophilia, (B) cough duration, and
(C) severity of cough symptoms (C, n 5 8 score of 1; n 5 10 score of 2; and n 5 26 score of 3 or more, results are presented as mean and the error bar as
SEM). **Dunn post hoc P , .01 vs children with PBB and a cough score of 1. D, IL-1b protein was significantly correlated with a-defensin 1-3 protein in
the BAL of children with PBB. See Figure 1 legend for expansion of abbreviations.

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1017

symptomatic PBB. Importantly, IL-1b protein levels

were correlated with BAL neutrophilia, as well as the
duration and severity of cough symptoms. Additionally,
baseline expression (time of bronchoscopy) of IL-1
pathway signaling members IL-1 receptor associated
kinase (IRAK) 2 and pellino-1 (PELI1) was higher in
those children who were more likely to experience
disease recurrence (more than three episodes of wet
cough in the year following baseline bronchoscopy). The
expression of a-defensins 1-3 was increased in PBB and
was significantly correlated with IL-1b.
IL-1b is an important mediator of the inflammatory
response and host defense, however, dysregulated and
persistent IL-1b release can harm the host and has been
linked to the pathogenesis of several diseases such as
rheumatoid arthritis, type 2 diabetes mellitus, and
atherosclerosis, as well as certain specific autophagocytic
conditions.11 IL-1b is secreted as the inactive pro molecule, proIL-1b, and is then processed enzymatically to
activated IL-1b. Typically this occurs via caspase-1 and
inflammasome activation.12 However, activation of
IL-1b when released from neutrophils is not exclusively
dependent on caspase-1, as enzymes including neutrophil elastase are able to cleave IL-1b into its active
form.13 Released and active IL-1b binds to its receptor
IL-1R1 and initiates a signaling cascade through MyD88
and IRAK1/IRAK4, assisted by IRAK2 and PELI1.
Effects of IL-1b are blocked through its decoy receptor
IL-1 receptor 2 and receptor antagonist IL-1RA.14 This
study shows that IL-1b is associated with increased BAL
neutrophilia, suggesting that either neutrophils are the
source of this cytokine production, or alternatively that
IL-1b induces neutrophil infiltration into the lung. Either
way, our previous reports6 detail increases in neutrophil
proteases including matrix metalloproteinase-9, which
may lead to activation of IL-1b-mediated neutrophil influx.
IL-1b has been observed to be elevated in other airway
diseases characterized by airway neutrophilia and/or
infection, such as neutrophilic asthma,7 COPD,15 cystic
fibrosis,16 and non-cystic fibrosis bronchiectasis.17 IL-1b
is increased in stable COPD as well as acute exacerbations, where it is associated with bacterial infection.18
Bacterial infection is frequently detected in PBB and the
relationship between bacteria and IL-1b activation in
PBB needs further research. In a number of models of
Pseudomonas pulmonary infection, IL-1b production
occurs in response to bacterial infection19 and is a determinant of neutrophil influx likely through C-X-C motif
ligand (CXCL) 8. IL-1 plays a key role in coordinating

1018 Original Research

chemokine responses that lead to neutrophil infiltration

in the lung.20 Elevated IL-1b in PBB is, therefore, consistent with known host responses in neutrophilic airway
diseases, and suggests bacterial infection and neutrophil
influx are key features leading to ongoing IL-1b release
and PBB symptoms.
Although PBB responds well to prolonged antibiotic
therapy, it can recur. In another infective disease, viral
encephalitis, levels of IL-1b and IL-1RA were related to
prognosis.21 We found that IL-1b levels were highest in
symptomatic PBB and subjects with PBB who had
higher cough scores. IL-1 signaling molecules IRAK2
and PELI1 had significantly increased gene expression
at baseline in children who went on to develop recurrent PBB. This suggests that IL-1b pathway activation
may determine PBB recurrence. PELI1 is important in
regulating the innate immune response of the epithelium to rhinovirus infection, including CXCL8 production and neutrophil recruitment.22 IRAK2 is critical in
sustaining late-phase inflammatory responses after TLR
stimulation, which leads to increased cytokine production.23 We have previously reported the upregulation of
PELI1 and IRAK2 in response to rhinovirus infection of
human primary bronchial epithelial cells in COPD.24
This evidence collectively suggests that IRAK2 and
PELI1 promote neutrophilic airway inflammation triggered by infection and IL-1b and that this response is
dysregulated in PBB and contributes to disease
recurrence.
This study also reports increased expression of the CXCL8
high-affinity G-protein-coupled receptor CXCR2 and
the neutrophil a-defensins 1-3 in PBB. CXCR2 is
thought to be involved in uncontrolled neutrophil influx
into the airways in acute lung injury.25 Defensins are
small arginine-rich cationic peptides that have antimicrobial activity against a broad range of pathogens and
exert their antimicrobial effect through membrane permeabilization. The level of neutrophil a-defensins 1-3
was higher in symptomatic PBB and was significantly
correlated with IL-1b, indicating that these molecules
may interact and influence PBB pathogenesis. Indeed,
recent evidence implicates a-defensins in the release of
IL-1b from lipopolysaccharide-primed macrophages
through the P2X7 receptor.26 Intratracheal instillation of
a-defensins in mice leads to acute lung inflammation
and dysfunction involving neutrophil influx and elastase release.27 a-defensins have a cytotoxic effect,
induce IL-8 and IL-1b gene expression, IL-8 protein
production, and NF-kB binding activity in human
bronchial epithelial cells.28 Expression of TNF/NF-kB

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pathway members remained unchanged in PBB. This

suggests that while there are similarities between
neutrophil-related airway diseases in children and
adults, there are differences that indicate different triggers and underlying mechanisms.
While the sample size in this study was sufficient to
establish a role for IL-1b in PBB, it was nonetheless
insufficient to evaluate whether IL-1b levels have prognostic value or are related to specific subsets of PBB.
Further studies of IL-1b in larger numbers of patients
with PBB are indicated. The role of IL-1b in PBB could
be strengthened by showing changes in IL-1b after
treatment, however, we could not justify a second
bronchoscopy for the children after PBB resolution.
The control groups in the two studies were dissimilar,
however, age differences were addressed in the validation cohort where purposive sampling led to matching

Acknowledgments
Author contributions: K. J. B., J. W. U., A. B. C.,
and P. G. G. are guarantors of this paper and
take responsibility for the integrity of the
work as a whole, from inception to published
article, and conceived and designed the
study. K. J. B., A. B. C., J. M. M., and P. G. G.
contributed to data collection and interpretation; K. J. B. wrote the first draft of the manuscript and performed data analysis; K. J. B.
and P. G. G. contributed to the writing of the
manuscript; and K. J. B., J. W. U., S. T. Y.,
A. B. C., J. M. M., M. C., J. L. S., and P. G. G.
contributed to the editing, revising, and
reviewing of the manuscript.
Financial/nonfinancial disclosures: The
authors have reported to CHEST the
following conflicts: Dr Upham has been the
recipient of peer-reviewed research fundingfrom the National Health and Medical
Research Council (Commonwealth of
Australia), received speaking fees from
AstraZeneca, Boehringer Ingelheim GmbH,
and Novartis Corp, and sits on the medical
advisory boards for Boehringer Ingelheim
GmbH, The Menarini Group, and Novartis
Corp. Drs Baines, Yerkovich, Chang, Marchant,
Simpson, and Gibson and Ms Carroll have
reported that no potential conflicts of interest
exist with any companies/organizations whose
products or services may be discussed in this
article.
Role of sponsors: The study sponsors had no
role in study design; the collection, analysis,
and interpretation of data; the writing of the
report; or the decision to submit the paper
for publication. No form of payment was
given to anyone to produce the manuscript.
Other contributions: We are grateful to all of
the parents and children who participated in
this study. We also thank Brent Masters, Helen
Buntain, Paul Francis, Nigel Dore, and Alan
Isles for allowing us to recruit their patients

of age groups in the children with PBB and control

subjects.
In summary, we have identified elevated IL-1b and
implicated the IL-1 pathway in PBB. IL-1b gene and
protein expression was increased, and the ratio of IL-1b
to its antagonist was decreased in PBB. IL-1b was associated with symptomatic PBB compared with resolved
PBB, correlated with BAL neutrophilia, as well as duration and severity of cough symptoms. IL-1 pathway
signaling was associated with PBB disease recurrence.
Expression of CXCR2 and a-defensins 1-3 was higher in
PBB, a-defensins 1-3 were associated with PBB symptoms, and IL-1b and a-defensin 1-3 protein levels were
significantly correlated. Further research into the role
of the IL-1 pathway and its relationship to a-defensins
1-3 in PBB is warranted. Future studies should also
examine a blood gene expression signature for PBB.

into the study; Carol Willis for maintaining

the database; and Sophie Anderson-James and
Helen Petsky for collecting the specimens and
clinical data. We also thank Naomi Fibbens,
Melinda Tooze, and Kellie Fakes for their technical assistance with laboratory measurements.
Additional information: The e-Appendix
can be found in the Supplemental Materials
section of the online article.

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