- Email: [email protected]
Mediators of Neutrophil Function in Children With Protracted Bacterial Bronchitis
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Original Research Signs and Symptoms of Chest Diseases
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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. This study investigates the IL-1 pathway and a neutrophil gene expression signature in PBB.
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:
CONCLUSIONS: 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
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 Children’s Medical Research Institute, Royal Children’s Hospital,
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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: [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
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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 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 ⱖ 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. 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.
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 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 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 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 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.
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 1014 Original Research
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12 (7, 22)
3 (2, 6)
0.91 (0.74, 1.37) 1.21 (0.67, 1.67) 1.12 (0.73, 1.24)
IL-1 receptor-associated kinase (IRAK2)
a-Defensins 1-3 (DEFA1-3)
0 (0, 305)
353 (126, 721)
1,043 (627, 1,772)
0.47 (0.37, 0.70)
3,045 (1,361, 3,805)
140 (76, 288)
2,424 (1,557, 5,369)
12.92 (7.79, 41.20)
2.20 (1.55, 4.37)
0.72 (0.66, 0.97)
, .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 5.01 (1.30, 9.61)
2.21 (1.22, 3.57)
1.01 (0.74, 1.3)
Pellino-1 (PELI1)
C-X-C motif receptor 2 (CXCR2)
1.91 (1.15, 5.03)
0.74 (0.59, 1.63)
IL-1 receptor antagonist (IL1RN)
2.11 (1.05, 5.24)
0.87 (0.69, 1.44)
1.64 (1.11, 3.65)
0.94 (0.72, 1.35)
IL-1b (IL1B)
IL-1 receptor 2 (IL1R2)
BAL cell gene expression
Lymphocytes, %
0 (0, 0)
0 (0, 0)
Eosinophils, %
52 (43, 79)
90 (85, 94)
Macrophages, %
34 (10, 44)
4 (2, 6)
365 (196, 632)
3 (2, 3)
20 (14, 80)
11 (10)
2.3 (1.2, 2.8)
21
PBB
Experimental Cohort
Neutrophils, %
Total cell count 106/L 103 (71, 171)
N/A
Cough score10
BAL cell counts
N/A
16 (17)
Girls (boys)
Cough duration, wk
9.7 (5.5, 12.7)
33
Age, y
Clinical details
No.
Control Subjects
] 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 Significance 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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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
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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 funding 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, 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
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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.
References 1. Chang AB, Landau LI, Van Asperen PP, et al; Thoracic Society of Australia and New Zealand. Cough in children: definitions and clinical evaluation. Med J Aust. 2006;184(8):398-403. 2. Marchant J, Masters IB, Champion A, Petsky H, Chang AB. Randomised controlled trial of amoxycillin clavulanate in children with chronic wet cough. Thorax. 2012;67(8):689-693. 3. Donnelly D, Critchlow A, Everard ML. Outcomes in children treated for persistent bacterial bronchitis. Thorax. 2007;62(1):80-84. 4. Gibson PG, Chang AB, Glasgow NJ, et al; CICADA. CICADA: Cough in children and adults: diagnosis and assessment. Australian cough guidelines summary statement. Med J Aust. 2010;192(5):265-271. 5. Shields MD, Bush A, Everard ML, McKenzie S, Primhak R; British Thoracic Society Cough Guideline Group. BTS guidelines: recommendations for the assessment and management of cough in children. Thorax. 2008;63(suppl 3): iii1-iii15. 6. Marchant JM, Gibson PG, Grissell TV, Timmins NL, Masters IB, Chang AB. Prospective assessment of protracted bacterial bronchitis: airway inflammation and innate immune activation. Pediatr Pulmonol. 2008;43(11):1092-1099.
7. Baines KJ, Simpson JL, Wood LG, Scott RJ, Gibson PG. Transcriptional phenotypes of asthma defined by gene expression profiling of induced sputum samples. J Allergy Clin Immunol. 2011;127(1):153-160. 8. Baines KJ, Simpson JL, Wood LG, Scott RJ, Gibson PG. Systemic upregulation of neutrophil a-defensins and serine proteases in neutrophilic asthma. Thorax. 2011;66(11):942-947. 9. Chang AB, Cox NC, Faoagali J, et al. Cough and reflux esophagitis in children: their co-existence and airway cellularity. BMC Pediatrics. 2006;6:4. 10. Chang AB, Newman RG, Carlin JB, Phelan PD, Robertson CF. Subjective scoring of cough in children: parentcompleted vs child-completed diary cards vs an objective method. Eur Respir J. 1998;11(2):462-466. 11. Dinarello CA, Simon A, van der Meer JW. Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nat Rev Drug Discov. 2012;11(8):633-652. 12. Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians of the body. Annu Rev Immunol. 2009;27: 229-265. 13. Guma M, Ronacher L, Liu-Bryan R, Takai S, Karin M, Corr M. Caspase 1independent activation of interleukin-1b in neutrophil-predominant inflammation. Arthritis Rheum. 2009;60(12):3642-3650. 14. Weber A, Wasiliew P, Kracht M. Interleukin-1 (IL-1) pathway. Sci Signal. 2010;3(105):cm1. 15. Pauwels NS, Bracke KR, Dupont LL, et al. Role of IL-1a and the Nlrp3/caspase-1/ IL-1b axis in cigarette smoke-induced pulmonary inflammation and COPD. Eur Respir J. 2011;38(5):1019-1028. 16. Eickmeier O, Huebner M, Herrmann E, et al. Sputum biomarker profiles in cystic
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fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function. Cytokine. 2010;50(2):152-157. Chalmers JD, Hill AT. Mechanisms of immune dysfunction and bacterial persistence in non-cystic fibrosis bronchiectasis. Mol Immunol. 2013;55(1): 27-34. Bafadhel M, McKenna S, Terry S, et al. Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers. Am J Respir Crit Care Med. 2011;184(6):662-671. Cai S, Batra S, Wakamatsu N, Pacher P, Jeyaseelan S. NLRC4 inflammasomemediated production of IL-1b modulates mucosal immunity in the lung against gram-negative bacterial infection. J Immunol. 2012;188(11):5623-5635. Marriott HM, Gascoyne KA, Gowda R, et al. Interleukin-1b regulates CXCL8 release and influences disease outcome
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in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages. Infect Immun. 2012;80(3):1140-1149. Griffiths MJ, Ooi MH, Wong SC, et al. In enterovirus 71 encephalitis with cardio-respiratory compromise, elevated interleukin 1b, interleukin 1 receptor antagonist, and granulocyte colony-stimulating factor levels are markers of poor prognosis. J Infect Dis. 2012;206(6):881-892. Bennett JA, Prince LR, Parker LC, et al. Pellino-1 selectively regulates epithelial cell responses to rhinovirus. J Virol. 2012;86(12):6595-6604. Kawagoe T, Sato S, Matsushita K, et al. Sequential control of Toll-like receptordependent responses by IRAK1 and IRAK2. Nat Immunol. 2008;9(6):684-691. Baines KJ, Hsu AC, Tooze M, Gunawardhana LP, Gibson PG, Wark PA. Novel immune genes associated with
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excessive inflammatory and antiviral responses to rhinovirus in COPD. Respir Res. 2013;14:15. Konrad FM, Reutershan J. CXCR2 in acute lung injury. Mediators Inflamm. 2012;2012:740987. Chen Q, Jin Y, Zhang K, et al. Alarmin HNP-1 promotes pyroptosis and IL-1b release through different roles of NLRP3 inflammasome via P2X7 in LPSprimed macrophages. Innate Immun. 2014;20(3):290-300. Zhang H, Porro G, Orzech N, Mullen B, Liu M, Slutsky AS. Neutrophil defensins mediate acute inflammatory response and lung dysfunction in dose-related fashion. Am J Physiol Lung Cell Mol Physiol. 2001;280(5):L947-L954. Sakamoto N, Mukae H, Fujii T, et al. Differential effects of alpha- and betadefensin 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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Original Research Signs and Symptoms of Chest Diseases
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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. This study investigates the IL-1 pathway and a neutrophil gene expression signature in PBB.
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:
CONCLUSIONS: 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
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 Children’s Medical Research Institute, Royal Children’s Hospital,
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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: [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
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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 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 ⱖ 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. 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.
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 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 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 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 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.
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 1014 Original Research
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12 (7, 22)
3 (2, 6)
0.91 (0.74, 1.37) 1.21 (0.67, 1.67) 1.12 (0.73, 1.24)
IL-1 receptor-associated kinase (IRAK2)
a-Defensins 1-3 (DEFA1-3)
0 (0, 305)
353 (126, 721)
1,043 (627, 1,772)
0.47 (0.37, 0.70)
3,045 (1,361, 3,805)
140 (76, 288)
2,424 (1,557, 5,369)
12.92 (7.79, 41.20)
2.20 (1.55, 4.37)
0.72 (0.66, 0.97)
, .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 5.01 (1.30, 9.61)
2.21 (1.22, 3.57)
1.01 (0.74, 1.3)
Pellino-1 (PELI1)
C-X-C motif receptor 2 (CXCR2)
1.91 (1.15, 5.03)
0.74 (0.59, 1.63)
IL-1 receptor antagonist (IL1RN)
2.11 (1.05, 5.24)
0.87 (0.69, 1.44)
1.64 (1.11, 3.65)
0.94 (0.72, 1.35)
IL-1b (IL1B)
IL-1 receptor 2 (IL1R2)
BAL cell gene expression
Lymphocytes, %
0 (0, 0)
0 (0, 0)
Eosinophils, %
52 (43, 79)
90 (85, 94)
Macrophages, %
34 (10, 44)
4 (2, 6)
365 (196, 632)
3 (2, 3)
20 (14, 80)
11 (10)
2.3 (1.2, 2.8)
21
PBB
Experimental Cohort
Neutrophils, %
Total cell count 106/L 103 (71, 171)
N/A
Cough score10
BAL cell counts
N/A
16 (17)
Girls (boys)
Cough duration, wk
9.7 (5.5, 12.7)
33
Age, y
Clinical details
No.
Control Subjects
] 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 Significance 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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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
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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 funding 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, 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
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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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