Bactchitis in Children

journal.publications.chestnet.org 1013

[ Original Research Signs and Symptoms of Chest Diseases ]

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 receptor-associated kinase; NF- k B 5 nuclear factor- k B; PBB 5 protracted bacte-rial 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), Th e 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), Th e University of Queensland, Brisbane, QLD; Qld Lung Transplant Service (Dr Yerkovich), Th e Prince Charles Hospital, Brisbane, QLD; Depart-ment of Respiratory Medicine (Drs Chang and Marchant), Queensland Children’s Medical Research Institute, Royal Children’s 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; Common-wealth of Australia) fellowships [Grants 569240, 511019, and 545216, respectively]. Dr Baines is supported by a research fellowship from Th e University of Newcastle. Th e 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: [email protected]

© 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

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

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

chronic wet cough in children in which the mechanisms are not understood. Th is study inves-

tigates the IL-1 pathway and a neutrophil gene expression signature in PBB.

METHODS: 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-1 b ,

IL-1 receptor antagonist (IL-1RA), and a -defensins 1-3 were assayed by enzyme-linked immu-

nosorbent assay, western blot, and quantitative real-time polymerase chain reaction, together

with selected IL-1 pathway members and neutrophil-related molecules.

RESULTS: In the experimental cohort, children with symptomatic PBB had signifi cantly higher

levels of IL-1 b and a -defensin gene and protein expression. Expression of the neutrophil che-

mokine receptor C-X-C motif receptor 2 was also higher in PBB. IL-1RA protein was higher,

however, the IL-1RA:IL-1 b ratio was lower in children with PBB than control subjects. In the

validation cohort, protein and gene expression of IL-1 b and a -defensins 1-3 were confi rmed

higher, as was gene expression of IL-1 pathway members and C-X-C motif receptor 2. IL-1 b

and a -defensin 1-3 levels lowered when PBB was treated and resolved. In children with recurrent

PBB, gene expression of the IL-1 b signaling molecules pellino-1 and IL-1 receptor-associated

kinase 2 was signifi cantly higher. IL-1 b protein levels correlated with BAL neutrophilia and

the duration and severity of cough symptoms. IL-1 b and a -defensin 1-3 levels were highly

correlated.

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

mediators were associated with BAL neutrophils, cough symptoms, and disease recurrence,

providing insight into PBB pathogenesis. CHEST 2014; 146(4):1013- 1020

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1014 Original Research [ 1 4 6 # 4 C H E S T O C TO B E R 2 0 1 4 ]

Protracted bacterial bronchitis (PBB) is an important

and common cause of chronic wet cough in children. 1

Once correctly diagnosed, the child’s cough resolves

with prolonged antibiotic therapy. 2 PBB is now interna-

tionally accepted as a diagnostic entity 3 and has been

incorporated into national 4 and international 5 pediatric

guidelines for cough management. However, despite

this increased clinical recognition, the underlying mech-

anisms of PBB remain to be elucidated.

Prior studies have shown that bacterial colonization and

airway neutrophilia are present in children with PBB. 2,6

Th is was associated with upregulation of the Toll-like

receptors (TLRs) TLR2 and TLR4 in the BAL of chil-

dren with PBB. 6 Th is implicates persistent neutrophilic

infl ammation in the pathogenesis of PBB and suggests

that neutrophil pathway mediators such as IL-1 b may

play an important role in pathogenesis. In adults with

neutrophilic asthma, using gene expression profi ling we

have implicated the IL-1 and tumor necrosis factor

(TNF)- a /nuclear factor- k B (NF- k B) pathways in

sputum 7 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.

Th erefore, this study evaluated these pathways and

mediators in two cohorts of PBB and control children.

We hypothesized that IL-1 b and the neutrophil gene

expression signature would be elevated in PBB and

related to symptoms and recurrence.

Materials and Methods Subject Recruitment and Sampling

Infl ammatory mediators were evaluated in two cohorts. Th e experi-

mental cohort (n 5 54) comprised subjects and BAL samples collected

in previous studies. 6,9 Th e validation cohort (n 5 47) was obtained

from a second cohort with purposive matching of control subjects. 2

Th e 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 card 10

used to document response to antibiotics, defi ned as absence of cough

or . 75% reduction in score (for � 3 days) within 2 weeks of antibi-

otic use (amoxycillin-clauvanate 45 mg/kg/d in two doses for 14 days 2 )

postbronchoscopy. In the validation cohort, children with PBB were

contacted at monthly intervals to document recurrence.

PBB was defi ned 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 defi ned as children with PBB who

were coughing when bronchoscopy was undertaken. Resolved PBB

was defi ned 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 defi ned

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.

Experimental cohort control was a convenient sample of children

undergoing gastroscopy, whereas in the validation cohort control sub-

jects 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 pre-

ceding 2 weeks. Informed consent was obtained, and the studies were

approved by the Ethics Committees of the Royal Children’s 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 identifi ed as

increased in sputum in neutrophilic asthma and include IL-1 b ( 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- k B

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

Protein Measurements

IL-1 b (undiluted) and IL-1 receptor antagonist (IL-1RA) (one-fi ft h

dilution) protein levels were measured in BAL supernatant using the

DuoSet enzyme-linked immunosorbent assay as per the manufacturer’s

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 manufacturer’s 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 .

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 signifi cant. Spearman rank

correlations were used to test relationships.

Results

Clinical Characteristics

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

( Table 1 ) and validation (n 5 36) ( Table 1 ) cohorts com-

prised mainly infants and young children with similar

profi les of moderate cough severity and a mean symp-

tom duration of . 20 weeks. Lung infl amma tion was

present with increased BAL cellularity including neutro-

phils. Th e validation cohort tended to have more males

and less intense airway neutrophilia than the experi-

mental cohort. Th e control subjects were older in the



TAB

LE 1

] C

linic

al a

nd B

AL

Dat

a fo

r th

e Chi

ldre

n in

the

Exp

erim

enta

l and

Val

idat

ion

Coh

orts

Cha

ract

eris

tics

Expe

rim

enta

l Coh

ort

Valid

atio

n Coh

ort

Con

trol

Sub

ject

sPB

B P

Valu

eCon

trol

Sub

ject

sPB

B P

Valu

e

No.

3321

1136

…

Clin

ical

det

ails

Age

, y

9.7

(5.5

, 12

.7)

2.3

(1.2

, 2.

8) ,

.001

0.7

(0.5

, 1.

9)2.

0 (1

.3,

4.1)

.015

G

irls

(bo

ys)

16 (

17)

11 (

10)

.780

6 (5

)9

(27)

.066

Cou

gh d

urat

ion,

wk

N/A

20 (

14,

80)

N/A

N/A

30 (

4, 5

6)N

/A

Cou

gh s

core

10

N/A

3 (2

, 3)

N/A

N/A

2 (1

, 3)

N/A

BAL

cell

coun

ts

To

tal c

ell c

ount

10 6

/L10

3 (7

1, 1

71)

365

(196

, 63

2) ,

.001

96 (

48,

110)

178

(100

, 34

0).0

04

N

eutr

ophi

ls,

%4

(2,

6)34

(10

, 44

) ,

.001

2 (1

, 4)

13 (

7, 5

7) ,

.001

M

acro

phag

es,

%90

(85

, 94

)52

(43

, 79

) ,

.001

92 (

90,

96)

67 (

30,

83)

.002

Eo

sino

phils

, %

0 (0

, 0)

0 (0

, 0)

.027

0 (0

, 0)

0 (0

, 0)

.174

Ly

mph

ocyt

es,

%3

(2,

6)12

(7,

22)

.001

5 (4

, 9)

9 (5

, 12

).1

32

BAL

cell

gene

exp

ress

ion

IL

-1 b

( IL1B )

0.94

(0.

72,

1.35

)2.

11 (

1.05

, 5.

24)

, .0

010.

84 (

0.58

, 1.

50)

4.44

(1.

31,

12.9

6).0

03

IL

-1 r

ecep

tor

2 ( IL1R2 )

0.87

(0.

69,

1.44

)1.

64 (

1.11

, 3.

65)

.002

1.08

(0.

42,

1.77

)2.

96 (

1.32

, 6.

73)

.014

IL

-1 r

ecep

tor

anta

goni

st (

IL1RN

)0.

74 (

0.59

, 1.

63)

0.47

(0.

37,

0.70

).0

010.

88 (

0.58

, 1.

35)

2.25

(1.

41,

6.27

).0

08

Pe

llino

-1 (

PELI1 )

1.01

(0.

74,

1.3)

0.72

(0.

66,

0.97

).0

321.

00 (

0.87

, 1.

30)

1.44

(0.

90,

5.95

).0

74

IL

-1 r

ecep

tor-

asso

ciat

ed k

inas

e ( IRAK2 )

0.91

(0.

74,

1.37

)2.

20 (

1.55

, 4.

37)

, .0

010.

92 (

0.64

, 1.

84)

1.26

(0.

93,

4.55

).0

77

a -

Def

ensi

ns 1

-3 (

DEFA1-3 )

1.21

(0.

67,

1.67

)1.

91 (

1.15

, 5.

03)

.004

0.90

(0.

68,

1.79

)3.

55 (

1.07

, 12

.42)

.017

C-

X-C m

otif

rece

ptor

2 (

CXCR2 )

1.12

(0.

73,

1.24

)2.

21 (

1.22

, 3.

57)

, .0

011.

38 (

0.28

, 3.

29)

4.43

(1.

34,

16.4

6).0

48

BAL

supe

rnat

ant

prot

ein

expr

essi

on

IL

-1 b ,

pg/

mL

5.01

(1.

30,

9.61

)12

.92

(7.7

9, 4

1.20

) ,

.001

0.70

(0.

35,

0.97

)3.

79 (

0.79

, 45

.3)

.014

I

L-1R

A,

pg/m

L1,

043

(627

, 1,

772)

2,42

4 (1

,557

, 5,

369)

.003

1,66

2 (1

,073

, 2,

135)

1,92

6 (6

06,

4,44

8).7

97

IL

-1RA t

o IL

-1 b

ratio

353

(126

, 72

1)14

0 (7

6, 2

88)

.008

2,63

1 (1

,997

, 2,

967)

357

(86,

796

) ,

.001

a -

Def

ensi

ns 1

-3,

pg/m

L0

(0,

305)

3,04

5 (1

,361

, 3,

805)

, .0

0116

3 (8

4, 2

73)

1,85

7 (4

89,

3,85

5).0

18

Dat

a ar

e gi

ven

as m

ean

(Q1,

Q3)

unl

ess

othe

rwis

e in

dica

ted.

IL-

1RA 5

IL-1

rec

epto

r an

tago

nist

; N/A

5 n

ot a

pplic

able

; PB

B 5

pro

trac

ted

bact

eria

l bro

nchi

tis;

Q 5

qua

rtile

.




experimental cohort, but ages were similar in the valida-

tion 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-1 b and IL-1RA protein were measured in 10 control

subjects and 29 PBB subjects, a -defensin 1-3 protein

was measured in fi ve control subjects and 24 subjects

with PBB, and gene expression was assessed in 10 con-

trol 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 insuffi cient remaining

samples.

Mediators in the Experimental Cohort

Th ere was altered expression of IL-1 pathway members

IL1B , IL1R2 , IL1RN , PELI1 , and IRAK2 and the neutro-

phil chemokine receptor CXCR2 in PBB ( Table 1 ) but

not TNF- a /NF- k B 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 sub-

jects: 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.

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

Table 1 ), IL-1 b ( Fig 1B , Table 1 ), and IL-1RA protein

levels ( Table 1 ) in PBB, and a lower IL-1RA to IL-1 b

ratio ( Fig 1C , Table 1 ). Western blot confi rmed the

presence of active IL-1 b ( Fig 1D ). Protein and gene

expression for IL-1 b were signifi cantly correlated in

PBB (Spearman r 5 0.65, P 5 .001).

Mediator in the Validation Cohort

Signifi cant diff erences in the experimental cohort were

evaluated in a clinical validation cohort to relate medi-

ator levels to symptom status and disease recurrence.

Th ere was signifi cantly higher gene expression of IL1B ,

IL1R2 , IL1RN , DEFA1-3 , and CXCR2 in PBB ( Table 1 ).

IL-1 b protein was also signifi cantly higher ( Table 1 ),

corresponding to a decreased IL-1RA to IL-1 b ratio in

PBB ( Table 1 ). However, gene expression of PELI1 and

IRAK2 did not reach signifi cance between patients with

PBB and control subjects. In PBB, the level of gene and

protein expression of a -defensins 1-3, IL-1 b , and IL-1RA

were signifi cantly correlated ( a -defensins 1-3: Spearman

r 5 0.46, P 5 .023; IL-1 b : Spearman r 5 0.68, P , .001;

IL-1RA: Spearman r 5 0.56, P 5 .002).

Clinical Signifi cance of Mediator Levels

Th e 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 investi-

gating 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-1 b protein ( Fig 2A ), and

a -defensin 1-3 protein ( Fig 2B ), but little diff erence in

gene expression of IL-1 pathway or neutrophil-related

mediators (data not shown).

When children with recurrent PBB were evaluated at

baseline and compared with those with nonrecurrent

PBB, there were no signifi cant diff erences in a -defensins

1-3, IL-1 b , IL-1RA, or CXCR2 expression. However,

downstream IL-1 pathway signaling molecules PELI1

( Fig 2C ) and IRAK2 ( Fig 2D ) were signifi cantly 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 partic-

ipants 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

Figure 1 – IL-1 b 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-1 b protein levels are higher in PBB, (C) the IL-1RA to IL-1 b 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-1 b (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.



Figure 3 – A-C, Levels of IL-1 b 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-1 b protein was signifi cantly correlated with a -defensin 1-3 protein in the BAL of children with PBB. See Figure 1 legend for expansion of abbreviations.

expression remained signifi cantly higher in symptom-

atic 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 recur-

rent PBB ( P 5 .09), however, we lost signifi cance with

the reduced sample size.

IL-1 b was signifi cantly associated with the BAL neutro-

philia ( r 5 0.75, P , .0001) (Fig 3A) and duration of

cough in weeks ( r 5 0.29, P 5 .046) (Fig 3B). IL-1 b levels

were signifi cantly higher in children with PBB and a

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

and a -defensin 1-3 levels were signifi cantly correlated

( Fig 3D ), and a -defensin 1-3 levels were also associated

with BAL neutrophils [Spearman r 5 0.48; P 5 .001].

Discussion

Th is study has identifi ed increased expression of

neutrophil-related mediators in PBB, including IL-1 path-

way members, neutrophil a -defensins, and the chemokine

receptor CXCR2. Elevated IL-1 b in PBB was con-

fi rmed in two clinical cohorts and was associated with

Figure 2 – A-D, In symptomatic PBB, the levels of (A) a -defensins 1-3 and (B) IL-1 b 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.




symptomatic PBB. Importantly, IL-1 b 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). Th e

expression of a -defensins 1-3 was increased in PBB and

was signifi cantly correlated with IL-1 b .

IL-1 b is an important mediator of the infl ammatory

response and host defense, however, dysregulated and

persistent IL-1 b 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 specifi c autophagocytic

conditions. 11 IL-1 b is secreted as the inactive pro mole-

cule, proIL-1 b , and is then processed enzymatically to

activated IL-1 b . Typically this occurs via caspase-1 and

infl ammasome activation. 12 However, activation of

IL-1 b when released from neutrophils is not exclusively

dependent on caspase-1, as enzymes including neutro-

phil elastase are able to cleave IL-1 b into its active

form. 13 Released and active IL-1 b binds to its receptor

IL-1R1 and initiates a signaling cascade through MyD88

and IRAK1/IRAK4, assisted by IRAK2 and PELI1.

Eff ects of IL-1 b are blocked through its decoy receptor

IL-1 receptor 2 and receptor antagonist IL-1RA. 14 Th is

study shows that IL-1 b is associated with increased BAL

neutrophilia, suggesting that either neutrophils are the

source of this cytokine production, or alternatively that

IL-1 b induces neutrophil infi ltration into the lung. Either

way, our previous reports 6 detail increases in neutrophil

proteases including matrix metalloproteinase-9, which

may lead to activation of IL-1 b -mediated neutrophil infl ux.

IL-1 b 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

fi brosis, 16 and non-cystic fi brosis bronchiectasis. 17 IL-1 b

is increased in stable COPD as well as acute exacerba-

tions, where it is associated with bacterial infection. 18

Bacterial infection is frequently detected in PBB and the

relationship between bacteria and IL-1 b activation in

PBB needs further research. In a number of models of

Pseudomonas pulmonary infection, IL-1 b production

occurs in response to bacterial infection 19 and is a deter-

minant of neutrophil infl ux likely through C-X-C motif

ligand (CXCL) 8. IL-1 plays a key role in coordinating

chemokine responses that lead to neutrophil infi ltration

in the lung. 20 Elevated IL-1 b in PBB is, therefore, consis-

tent with known host responses in neutrophilic airway

diseases, and suggests bacterial infection and neutrophil

infl ux are key features leading to ongoing IL-1 b release

and PBB symptoms.

Although PBB responds well to prolonged antibiotic

therapy, it can recur. In another infective disease, viral

encephalitis, levels of IL-1 b and IL-1RA were related to

prognosis. 21 We found that IL-1 b levels were highest in

symptomatic PBB and subjects with PBB who had

higher cough scores. IL-1 signaling molecules IRAK2

and PELI1 had signifi cantly increased gene expression

at baseline in children who went on to develop recur-

rent PBB. Th is suggests that IL-1 b pathway activation

may determine PBB recurrence. PELI1 is important in

regulating the innate immune response of the epithe-

lium to rhinovirus infection, including CXCL8 produc-

tion and neutrophil recruitment. 22 IRAK2 is critical in

sustaining late-phase infl ammatory responses aft er TLR

stimulation, which leads to increased cytokine produc-

tion. 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

Th is evidence collectively suggests that IRAK2 and

PELI1 promote neutrophilic airway infl ammation trig-

gered by infection and IL-1 b and that this response is

dysregulated in PBB and contributes to disease

recurrence.

Th is study also reports increased expression of the CXCL8

high-affi nity G-protein-coupled receptor CXCR2 and

the neutrophil a -defensins 1-3 in PBB. CXCR2 is

thought to be involved in uncontrolled neutrophil infl ux

into the airways in acute lung injury. 25 Defensins are

small arginine-rich cationic peptides that have antimi-

crobial activity against a broad range of pathogens and

exert their antimicrobial eff ect through membrane per-

meabilization. Th e level of neutrophil a -defensins 1-3

was higher in symptomatic PBB and was signifi cantly

correlated with IL-1 b , indicating that these molecules

may interact and infl uence PBB pathogenesis. Indeed,

recent evidence implicates a -defensins in the release of

IL-1 b from lipopolysaccharide-primed macrophages

through the P2X7 receptor. 26 Intratracheal instillation of

a -defensins in mice leads to acute lung infl ammation

and dysfunction involving neutrophil infl ux and elas-

tase release. 27 a -defensins have a cytotoxic eff ect,

induce IL-8 and IL-1 b gene expression, IL-8 protein

production, and NF- k B binding activity in human

bronchial epithelial cells. 28 Expression of TNF/NF- k B



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 interpreta-tion; K. J. B. wrote the fi rst draft of the manu-script 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/nonfi nancial disclosures: Th e authors have reported to CHEST the following confl icts: Dr Upham has been the recipient of peer-reviewed research fund-ing from 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, Th e Menarini Group, and Novartis Corp. Drs Baines, Yerkovich, Chang, Marchant, Simpson, and Gibson and Ms Carroll have reported that no potential confl icts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: Th e 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

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 tech-nical assistance with laboratory measurements.

Additional information: Th e e-Appendix can be found in the Supplemental Materials section of the online article.

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pathway members remained unchanged in PBB. Th is

suggests that while there are similarities between

neutrophil-related airway diseases in children and

adults, there are diff erences that indicate diff erent trig-

gers and underlying mechanisms.

While the sample size in this study was suffi cient to

establish a role for IL-1 b in PBB, it was nonetheless

insuffi cient to evaluate whether IL-1 b levels have prog-

nostic value or are related to specifi c subsets of PBB.

Further studies of IL-1 b in larger numbers of patients

with PBB are indicated. Th e role of IL-1 b in PBB could

be strengthened by showing changes in IL-1 b aft er

treatment, however, we could not justify a second

bronchoscopy for the children aft er PBB resolution.

Th e control groups in the two studies were dissimilar,

however, age diff erences were addressed in the valida-

tion cohort where purposive sampling led to matching

of age groups in the children with PBB and control

subjects.

In summary, we have identifi ed elevated IL-1 b and

implicated the IL-1 pathway in PBB. IL-1 b gene and

protein expression was increased, and the ratio of IL-1 b

to its antagonist was decreased in PBB. IL-1 b was asso-

ciated with symptomatic PBB compared with resolved

PBB, correlated with BAL neutrophilia, as well as dura-

tion 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 symp-

toms, and IL-1 b and a -defensin 1-3 protein levels were

signifi cantly 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.




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24 . Baines KJ , Hsu AC , Tooze M , Gunawardhana LP , Gibson PG , Wark PA . Novel immune genes associated with

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28 . Sakamoto N , Mukae H , Fujii T , et al . Diff erential eff ects of alpha- and beta-defensin on cytokine production by cultured human bronchial epithelial cells . Am J Physiol Lung Cell Mol Physiol . 2005 ; 288 ( 3 ): L508 - L513 .


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