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Interleukin-1 is associated with inflammation and structural lung disease in young children with cystic fibrosis

  • Author Footnotes
    1 S.T.M. and A.S.D. contributed equally to the manuscript.
    Samuel T. Montgomery
    Footnotes
    1 S.T.M. and A.S.D. contributed equally to the manuscript.
    Affiliations
    School of Paediatrics and Child Health, University of Western Australia, Nedlands 6009, Western Australia, Australia
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  • Author Footnotes
    1 S.T.M. and A.S.D. contributed equally to the manuscript.
    A. Susanne Dittrich
    Footnotes
    1 S.T.M. and A.S.D. contributed equally to the manuscript.
    Affiliations
    Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL),University of Heidelberg, Heidelberg, Germany

    Department of Pneumology and Critical Care Medicine, Thoraxklinik at the University Hospital Heidelberg, Heidelberg, Germany
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  • Luke W. Garratt
    Affiliations
    Telethon Kids Institute, University of Western Australia, Nedlands 6009, Western Australia, Australia
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  • Lidija Turkovic
    Affiliations
    Telethon Kids Institute, University of Western Australia, Nedlands 6009, Western Australia, Australia
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  • Dario L. Frey
    Affiliations
    Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL),University of Heidelberg, Heidelberg, Germany

    Department of Pneumology and Critical Care Medicine, Thoraxklinik at the University Hospital Heidelberg, Heidelberg, Germany
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  • Stephen M. Stick
    Affiliations
    School of Paediatrics and Child Health, University of Western Australia, Nedlands 6009, Western Australia, Australia

    Telethon Kids Institute, University of Western Australia, Nedlands 6009, Western Australia, Australia

    Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth 6001, Western Australia, Australia

    Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, University of Western Australia, Nedlands 6009,Western Australia, Australia
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  • Author Footnotes
    2 M.A.M. and A.K. contributed equally as senior authors.
    Marcus A. Mall
    Footnotes
    2 M.A.M. and A.K. contributed equally as senior authors.
    Affiliations
    Department of Translational Pulmonology, Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL),University of Heidelberg, Heidelberg, Germany

    Department of Pediatric Pulmonology and Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany

    Berlin Institute of Health (BIH), Berlin, Germany
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  • Author Footnotes
    2 M.A.M. and A.K. contributed equally as senior authors.
    Anthony Kicic
    Correspondence
    Corresponding author at: Telethon Kids Institute, Subiaco, Perth 6008, Western Australia, Australia.
    Footnotes
    2 M.A.M. and A.K. contributed equally as senior authors.
    Affiliations
    School of Paediatrics and Child Health, University of Western Australia, Nedlands 6009, Western Australia, Australia

    Telethon Kids Institute, University of Western Australia, Nedlands 6009, Western Australia, Australia

    Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth 6001, Western Australia, Australia

    Centre for Cell Therapy and Regenerative Medicine, School of Medicine and Pharmacology, University of Western Australia, Nedlands 6009,Western Australia, Australia

    School of Public Health, Curtin University, Bentley 6102, Western Australia, Australia
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  • Affiliations
    School of Paediatrics and Child Health, University of Western Australia, Nedlands 6009, Western Australia, Australia

    Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth 6001, Western Australia, Australia

    Murdoch Children's Research Institute, Parkville, 3052 Melbourne, Victoria, Australia

    Department of Paediatrics, University of Melbourne, Parkville, 3052 Melbourne, Victoria, Australia
  • Author Footnotes
    1 S.T.M. and A.S.D. contributed equally to the manuscript.
    2 M.A.M. and A.K. contributed equally as senior authors.
    3 The full membership of the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) is available at www.arestcf.org.
Open ArchivePublished:June 05, 2018DOI:https://doi.org/10.1016/j.jcf.2018.05.006

      Abstract

      Background

      Little is known about the role of interleukin (IL)-1 in the pathogenesis of cystic fibrosis (CF) lung disease. This study investigated the relationship between IL-1 signalling, neutrophilic inflammation and structural lung changes in children with CF.

      Methods

      Bronchoalveolar lavage fluid (BALf) from 102 children with CF were used to determine IL-1α, IL-1β, IL-8 levels and neutrophil elastase (NE) activity, which were then correlated to structural lung changes observed on chest computed tomography (CT) scans.

      Results

      IL-1α and IL-1β were detectable in BAL in absence of infection, increased in the presence of bacterial infection and correlated with IL-8 (p < 0.0001), neutrophils (p < 0.0001) and NE activity (p < 0.01 and p < 0.001). IL-1α had the strongest association with structural lung disease (p < 0.01) in the absence of infection (uninfected: p < 0.01 vs. infected: p = 0.122).

      Conclusion

      Our data associates IL-1α with early structural lung damage in CF and suggests this pathway as a novel anti-inflammatory target.

      Keywords

      1. Introduction

      Cystic fibrosis lung disease (CF) starts in the first months of life. The early lung disease observable on CT is characterized by mucus plugging and inflammation, often with no clinical symptoms and commonly without detectable respiratory infection [
      • Sly P.D.
      • Brennan S.
      • Gangell C.
      • et al.
      Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening.
      ,
      • Stick S.M.
      • Brennan S.
      • Murray C.
      • et al.
      Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.
      ]. Further, neutrophilic inflammation was identified as a key risk factor for progressive airway disease with structural airway remodelling resulting in bronchiectasis and decreased lung function [
      • Sly P.D.
      • Gangell C.L.
      • Chen L.
      • et al.
      Risk factors for bronchiectasis in children with cystic fibrosis.
      ,
      • Wielputz M.O.
      • Puderbach M.
      • Kopp-Schneider A.
      • et al.
      Magnetic resonance imaging detects changes in structure and perfusion, and response to therapy in early cystic fibrosis lung disease.
      ]. However, the nexus between airway mucus obstruction and neutrophilic inflammation in early CF lung disease has not been defined.
      Studies utilising βENaC-overexpressing mice with CF-like lung disease [
      • Mall M.
      • Grubb B.R.
      • Harkema J.R.
      • et al.
      Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice.
      ] have demonstrated the presence of mucus obstruction and airway neutrophilia in germ-free conditions [
      • Livraghi-Butrico A.
      • Kelly E.J.
      • Klem E.R.
      • et al.
      Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.
      ], suggesting that mucus plugging can trigger chronic airway inflammation even in the absence of bacterial infection [
      • Zhou-Suckow Z.
      • Duerr J.
      • Hagner M.
      • et al.
      Airway mucus, inflammation and remodeling: emerging links in the pathogenesis of chronic lung diseases.
      ]. Further research has suggested a link between airway mucus obstruction, hypoxic necrosis of airway epithelial cells (AEC) and sterile neutrophilic inflammation in CF via release of interleukin-1alpha (IL-1α) from dying cells triggering the interleukin-1 receptor (IL-1R) signalling pathway [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ]. This facilitates secondary activation and release of interleukin-1beta (IL-1β), which has been demonstrated to be elevated in the airways of children with CF [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ,
      • Armstrong D.S.
      • Hook S.M.
      • Jamsen K.M.
      • et al.
      Lower airway inflammation in infants with cystic fibrosis detected by newborn screening.
      ]. Secretion of IL-1α can maintain sterile inflammation in the absence of bacterial infection by binding to interleukin-1 receptor (IL-1R) [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ], while IL-1β in the CF airway is primarily increased in response to respiratory pathogens [
      • Armstrong D.S.
      • Hook S.M.
      • Jamsen K.M.
      • et al.
      Lower airway inflammation in infants with cystic fibrosis detected by newborn screening.
      ]. These findings suggest triggering IL-1R signalling via IL-1α may be an important mechanism in early CF inflammation, that is frequently observed in children with CF even in the absence of bacterial infection However, the role of the IL-1 receptor (IL-1R) pathway in the pathogenesis of early lung disease in children with CF and its relationship with early bacterial infection, neutrophilic inflammation and structural lung damage has not been studied.
      The aim was to investigate the relationship between the IL-1 signalling pathway, bacterial infection, airway inflammation and structural lung changes in a cohort of infants and young children with CF. To test the hypothesis that IL-1α is elevated in early CF lung disease and associated with inflammatory lung damage, we obtained bronchoalveolar lavage fluid (BALf) and computed tomography (CT) scans from 102 clinically stable children with CF. From BALf we determined infection status and measured differential cell counts and concentrations of free neutrophil elastase (NE), IL-1α, IL-1β, and interleukin-8 (IL-8) and compared them with structural lung changes observed on CT.

      2. Material and methods

      Please refer to the online supplement for full details.

      2.1 Study population

      This study was approved by the relevant institutional Human Ethics Committees. This cross-sectional study included samples from 102 (110 visits from 102 patients) clinically stable infants and children with CF (mean age 3.8, range 0.2–7.8; Table 1) participating in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) early surveillance program [
      • Stick S.M.
      • Brennan S.
      • Murray C.
      • et al.
      Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.
      ]. Samples were randomly selected from the AREST CF biobank. Data was obtained from children who underwent high-resolution CT scanning during the period of March 2007 to June 2015 including all children to the age of 6 years (110 visits from 102 patients; Table 1). Parents provided informed consent. Cystic fibrosis transmembrane conductance regulator (CFTR) genotype was determined as part of newborn screening (Supplementary Table 2).
      Table 1Demographics of study population and infection and inflammatory status.
      Demographics
      Subjects n (visits)102 (110 visits)
      Male n (%)52 (51%)
      Genotype homozygote p.Phe508del n (%)55 (54%)
      Any pathogen cultured n (%)51 (46.4%)
      Pancreatic Sufficiency n (%)19 (18.6%)
      BMI median z score (min-max)0.051 (−3.99–4.15)
      No respiratory infectionAny respiratory infection
      Median Age (IQR)4.1 (1.9–5.3)4.0 (1.8–5.4)
      Free neutrophil elastasedetected n (%)20 (33.9%)13 (25.5%)

      2.2 Chest CT, bronchoscopy, and bronchoalveolar lavage

      A chest CT followed by bronchoscopy was performed in children with CF, obtained under general anaesthesia as previously described [
      • Sly P.D.
      • Brennan S.
      • Gangell C.
      • et al.
      Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening.
      ,
      • Stick S.M.
      • Brennan S.
      • Murray C.
      • et al.
      Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.
      ]. During bronchoscopy, BALf was collected for the detection of pathogens and quantification of inflammation as described previously [
      • Brennan S.
      • Hall G.L.
      • Horak F.
      • et al.
      Correlation of forced oscillation technique in preschool children with cystic fibrosis with pulmonary inflammation.
      ]. Respiratory infection status was determined using microbiology [
      • Sly P.D.
      • Gangell C.L.
      • Chen L.
      • et al.
      Risk factors for bronchiectasis in children with cystic fibrosis.
      ], and was defined as colony counts for any organism (including mixed oral flora) >103 colony-forming units per milliliter BALf (Supplementary Table 1).

      2.3 Inflammation

      BALf was assessed for immune cell counts, IL-8 and NE activity as previously described [
      • Brennan S.
      • Hall G.L.
      • Horak F.
      • et al.
      Correlation of forced oscillation technique in preschool children with cystic fibrosis with pulmonary inflammation.
      ] and detailed in full in the online supplement. IL-1α and IL-1β concentrations were determined in cell-free BAL supernatants using a commercially available ALPHALisa kit (PerkinElmer, Waltham, MA). IL-1R antagonist (IL-1Ra) concentrations were determined in cell-free BAL supernatants using a commercially available Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). Samples below the detection range were arbitrarily reported as half the lower limit and included in the analysis with all other samples as previously described [
      • Garratt L.W.
      • Sutanto E.N.
      • Ling K.M.
      • et al.
      Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.
      ].

      2.4 PRAGMA-CF

      Chest CT scans were scored using the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis (PRAGMA-CF) method [
      • Rosenow T.
      • Oudraad M.C.
      • Murray C.P.
      • et al.
      PRAGMA-CF. a quantitative structural lung disease computed tomography outcome in young children with cystic fibrosis.
      ].

      2.5 Statistics

      Data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA) and Stata (StataCorp, College Station, TX). Data were natural log transformed to assume a normal distribution. Comparisons were performed using unpaired t-tests with Welch's correction presented as mean ± standard deviation. Associations between IL-1α, IL-1β, IL-8, neutrophil counts and neutrophil elastase activity were assessed using Pearson correlations. In addition, associations between IL-1α, IL-1β, IL-8, neutrophil counts, neutrophil elastase activity and extent of structural lung disease were evaluated using multiple linear regressions adjusted for age and sex. A two tailed p value < 0.05 was considered statistically significant.

      3. Results

      Demographic data for the study population are provided in Table 1. Of the 102 children included, 51 had pathogens identified in BALf (Supplementary Table 1).

      3.1 IL-1α correlates with IL-1β in early CF lung disease independent of airway infection

      To determine the role of IL-1α and IL-1β, and their relationship with airway infection in early CF lung disease, we measured levels of IL-1α, IL-1β and IL-1Ra in BALf collected from children with CF, with (n = 51) and without (n = 59) detectable bacterial airway infection. IL-1α and IL-1β were detected in BALf from most young children with CF (Fig. 1). Furthermore, IL-1α and IL-1β were significantly increased in BALf from children with CF with pulmonary infection when compared to those without (p < 0.05 and p < 0.01; Figs. 1A and B). IL-1α levels in BALf from children with CF strongly correlated with levels of IL-1β measured (r = 0.78, p < 0.0001; Fig. 1C), in line with the close interaction of these two mediators in inflammatory pathways. This relationship was independent from bacterial airway infection, as robust correlations between IL-1α and IL-1β were observed in BALf from both CF patients with and without respiratory infection (r = 0.83, p < 0.0001 and r = 0.62, p < 0.0001 respectively; Fig. 1C, Supplementary Fig. 1). IL-1Ra was detected in BALf from most young children with CF, and was significantly increased in in BALf from children with CF with pulmonary infection when compared to those without (p < 0.001, Supplementary Fig. 9). Correlations between IL-1Ra, IL-1α and IL-1β were also observed in BALf from both CF patients with and without respiratory infection (Supplementary Fig. 10).
      Fig. 1
      Fig. 1IL-1α and IL-1β are detectable in BAL fluid, are increased in airway infection and correlate in early CF lung disease. Data was stratified by respiratory infection status (A and B); closed circles: no infection (n = 59) and open circles: any infection (n = 51). IL-1α (A) and IL-1β (B) levels in BALf from children with CF with and without detectable respiratory infection. (C) Relationship between IL-1α and IL-1β in BALf from children with CF with and without detectable respiratory infection. *Indicates p < 0.05.

      3.2 IL-1α correlates with severity of neutrophilic inflammation in early CF lung disease independent of airway infection

      We next correlated levels of IL-1α and IL-1β with IL-8 concentrations, neutrophil counts and NE activity in BALf from children with CF, with and without detectable airway infection (Table 1). IL-1α and IL-1β were significantly correlated with levels of the neutrophil chemoattractant IL-8 (r = 0.64, p < 0.0001 and r = 0.64, p < 0.0001 respectively) which was independent of bacterial infection. BALf from children with CF with and without respiratory infection exhibited significant correlations between IL-1α and IL-8 (r = 0.74, p < 0.0001 and r = 0.44, p < 0.001 respectively; Fig. 2A , Supplementary Fig. 2A & B) as well as IL-1β and IL-8 (r = 0.65, p < 0.0001 and r = 0.59, p < 0.0001 respectively; Fig. 2B, Supplementary Fig. 2C & D). Next, we investigated the relationship between IL-1α, IL-1β and airway neutrophilia in early CF lung disease. IL-1α and IL-1β showed a strong correlation with the number of neutrophils per mL of BALf from children with CF (r = 0.71, p < 0.0001 and r = 0.67, p < 0.0001 respectively). This association was not affected by bacterial airway infection (Fig. 3A & B , Supplementary Fig. 3). Finally, we determined the relationship between levels of IL-1α and IL-1β, and levels of NE activity, a major product of activated neutrophils in CF airways and a key predictor of persistent bronchiectasis [
      • Sly P.D.
      • Gangell C.L.
      • Chen L.
      • et al.
      Risk factors for bronchiectasis in children with cystic fibrosis.
      ,
      • Gehrig S.
      • Duerr J.
      • Weitnauer M.
      • et al.
      Lack of neutrophil elastase reduces inflammation, mucus hypersecretion, and emphysema, but not mucus obstruction, in mice with cystic fibrosis-like lung disease.
      ]. Although we could detect free NE activity in only 34% of the uninfected and in 25% of the infected samples (Table 1), we found significant correlations of IL-1α and IL-1β with NE activity in BALf (r = 0.26, p < 0.01 and r = 0.32, p < 0.001 respectively) in our cohort of children with CF. When stratified according to respiratory infection status, IL-1α correlated significantly with NE activity in children with respiratory infection (r = 0.36, p < 0.01; Fig. 3C, Supplementary Fig. 4B), but not in those without respiratory infection (r = 0.19, p > 0.05; Fig. 3C, Supplementary Fig. 4A), where NE activity was only detectable in 13 out of 51 (25%) of children with CF in this subgroup. IL-1β correlated with NE activity in BALf from both uninfected and infected children with CF (r = 0.30, p < 0.05 and r = 0.42, p < 0.01 respectively; Fig. 3D, Supplementary Figs. 4C & 4D).
      Fig. 2
      Fig. 2IL-1α and IL-1β are associated with interleukin-8 in early CF lung disease independent of airway infection. Data was stratified by respiratory infection status; closed circles, dashed line: no infection (n = 59) and open circles, solid line: any infection (n = 51). Correlations between IL-1α and IL-1β with IL-8 (A and B) in BALf from children with CF with and without detectable respiratory infection.
      Fig. 3
      Fig. 3IL-1α and IL-1β are associated with neutrophilic inflammation in early CF lung disease independent of airway infection. Data was stratified by respiratory infection status; closed circles, dashed line: no infection (n = 59) and open circles, solid line: any infection (n = 51). Correlations between IL-1α and IL-1β with number of neutrophils per mL (A and B) and neutrophil elastase activity (C and D) in BALf from children with CF with and without detectable respiratory infection.

      3.3 IL-1α is associated with structural lung damage on CT in early CF lung disease

      We next examined the association between IL-1α, IL-1β, IL-8, neutrophil counts and NE activity in BALf and the extent of structural lung disease quantified by chest CT using the PRAGMA-CF scoring method [
      • Rosenow T.
      • Oudraad M.C.
      • Murray C.P.
      • et al.
      PRAGMA-CF. a quantitative structural lung disease computed tomography outcome in young children with cystic fibrosis.
      ] (Table 2). A significant association was observed between IL-1α and the extent of structural lung disease, with structural lung disease increasing 0.74 [0.23, 1.26] (95% CI) percent per loge pg/mL increase in IL-1α (p = 0.005). Similarly, there was a significant association between IL-1β in BALf and extent of structural lung disease, with structural lung disease increasing 0.30 [0.02, 0.59] percent per loge pg/mL increase in IL-1α (p = 0.037). No significant association was observed between neutrophil counts in BALf and extent of structural lung disease (p = 0.055), however significant associations were observed between both IL-8 and NE activity in BALf and extent of structural lung disease, with structural lung disease increasing 0.52 [0.22, 0.82] percent and 0.49 [0.12, 0.85] percent respectively per loge pg/mL increase in IL-1α (p = 0.001 and p = 0.01 respectively). Data was then stratified and reanalyzed according to the respiratory infection status of each child with CF at the time of bronchoscopy. Analysis showed no association between IL-1α and the extent of structural lung disease in children with respiratory infection (p = 0.12). However, there was a significant positive association between IL-1α and extent of structural lung disease in children without respiratory infection, with structural lung disease increasing 1.20 [0.33, 2.06] percent per loge pg/mL increase in IL-1α in BALf (p = 0.008). No association was observed between IL-1β in BALf in children with respiratory infection (p = 0.228), however there was a significant positive association between IL-1β in BALf and extent of structural lung disease in children without respiratory infection, with structural lung disease increasing 0.52 [0.01, 1.03] percent per loge pg/mL increase in IL-1β in BALf (p = 0.046). No association was observed between IL-8 or NE activity in BALf and extent of structural lung disease in children with respiratory infection (p = 0.052 and p = 0.139 respectively), while no association was observed between neutrophil count in BALf and extent of structural lung disease in children, irrespective of respiratory infection status.
      Table 2Associations of IL-1α, IL-1β, IL-8, Neutrophil count and NE activity with structural lung disease on CT in young children with CF.
      Extent of lung disease per IL-1α [95% Confidence Interval]0.74 [0.23, 1.26], p = 0.005
      Extent of lung disease per IL-1β [95% Confidence Interval]0.30 [0.02, 0.59], p = 0.037
      Extent of lung disease per IL-8 [95% Confidence Interval]0.52 [0.22, 0.82], p = 0.001
      Extent of lung disease per Neutrophil count [95% Confidence Interval]0.25 [−0.01, 0.51], p = 0.055
      Extent of lung disease per NE [95% Confidence Interval]0.49 [0.12, 0.85], p = 0.010
      Segregated by respiratory infection status
      No Respiratory InfectionAny respiratory infection
      Extent of lung disease per IL-1α [95% Confidence Interval]1.20 [0.33, 2.06], p = 0.0080.51 [−0.14, 1.17], p = 0.122
      Extent of lung disease per IL-1β [95% Confidence Interval]0.52 [0.01, 1.03], p = 0.0460.22 [−0.14, 0.58], p = 0.228
      Extent of lung disease per IL-8 [95% Confidence Interval]0.71 [0.22, 1.20], p = 0.0050.43 [−0.00, 0.87], p = 0.052
      Extent of lung disease per Neutrophil count [95% Confidence Interval]0.41 [−0.02, 0.85], p = 0.0620.17 [−0.19, 0.53], p = 0.355
      Extent of lung disease per NE [95% Confidence Interval]0.77 [0.22, 1.32], p = 0.0070.41 [−0.14, 0.97], p = 0.139

      4. Discussion

      This study provides a novel insight into the early process of CF lung disease, whereby IL-1 signalling drives neutrophilic inflammation and consequently structural lung changes. Our data show that both IL-1α and IL-1β were detectable in the airways in young children with CF and were associated with markers of neutrophilic inflammation in the presence and absence of bacterial infection. Additionally, we investigated the associations between IL-1α, IL-1β and structural lung changes detectable via CT and found a significant relationship between IL-1α and the extent of structural lung disease in children without detectable respiratory infection. Collectively, these results indicate that IL-1α is an important modulator of inflammation in early CF lung disease, even in absence of detectable microbial infection of the airways. Furthermore, our data support the hypothesis that IL-1α released from dying epithelial cells under hypoxic conditions in mucus-obstructed airways may induce secretion of IL-1β in the absence of bacterial infection and thus augment activation of the IL-1R signalling pathway [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ,
      • Chen C.J.
      • Kono H.
      • Golenbock D.
      • et al.
      Identification of a key pathway required for the sterile inflammatory response triggered by dying cells.
      ,
      • Montgomery S.T.
      • Mall M.A.
      • Kicic A.
      • et al.
      Hypoxia and sterile inflammation in cystic fibrosis airways: mechanisms and potential therapies.
      ]. These results suggest a significant role for IL-1α signalling in early neutrophilic inflammation and structural lung damage in young children with CF, specifically in the absence of respiratory infection.
      Neutrophilic airway inflammation is key in the onset and progression of lung disease in CF. Present in early CF lung disease, activated neutrophils release NE into the airway lumen where levels of NE activity are associated with structural lung damage detectable on CT in infants with CF as young as 3 months of age even in the absence of respiratory infection [
      • Sly P.D.
      • Brennan S.
      • Gangell C.
      • et al.
      Lung disease at diagnosis in infants with cystic fibrosis detected by newborn screening.
      ,
      • Stick S.M.
      • Brennan S.
      • Murray C.
      • et al.
      Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.
      ,
      • Sly P.D.
      • Gangell C.L.
      • Chen L.
      • et al.
      Risk factors for bronchiectasis in children with cystic fibrosis.
      ]. However, little is known regarding non-infective triggers of airway neutrophilia in CF. Recent studies utilising the βENaC-overexpressing mouse as a model of CF lung disease have linked mucus obstruction to airway inflammation, even in mice raised in germ-free environments [
      • Livraghi-Butrico A.
      • Kelly E.J.
      • Klem E.R.
      • et al.
      Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.
      ,
      • Zhou-Suckow Z.
      • Duerr J.
      • Hagner M.
      • et al.
      Airway mucus, inflammation and remodeling: emerging links in the pathogenesis of chronic lung diseases.
      ]. Mucus plugging early in life was associated with systemic and cellular hypoxia of epithelial cells resulting in necrosis of the airway epithelium, detected prior to the onset of airway neutrophilia [
      • Mall M.
      • Grubb B.R.
      • Harkema J.R.
      • et al.
      Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice.
      ,
      • Zhou Z.
      • Duerr J.
      • Johannesson B.
      • et al.
      The enac-overexpressing mouse as a model of cystic fibrosis lung disease.
      ,
      • Mall M.A.
      • Harkema J.R.
      • Trojanek J.B.
      • et al.
      Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.
      ]. These preclinical studies indicated that the number of necrotic AEC detected correlated with the severity of the mucus obstruction [
      • Johannesson B.
      • Hirtz S.
      • Schatterny J.
      • et al.
      CFTR regulates early pathogenesis of chronic obstructive lung disease in βENaC-overexpressing mice.
      ,
      • Zhou Z.
      • Treis D.
      • Schubert S.C.
      • et al.
      Preventive but not late amiloride therapy reduces morbidity and mortality of lung disease in betaenac-overexpressing mice.
      ]. Furthermore, AEC necrosis in mucus-obstructed airways has been associated with elevated levels of IL-1α that correlate with neutrophilic airway inflammation in βENaC-overexpressing mice, where genetic deletion of IL-1R was found to reduce airway neutrophilia and structural lung damage [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ]. Thus, we aimed to investigate the levels of IL-1α in BALf from young children with CF and determine its relevance in early lung disease.
      Since IL-1R signalling is also activated by IL-1β, we extended our studies to include this and found that levels of IL-1α and IL-1β in BALf correlated in children with CF. IL-1α and IL-1β exhibit highly similar structures, target the same receptor on the cell membrane (IL-1R), and reciprocally determine their secretion. Since IL-1α is constitutively active and does not contain a caspase-1 cleavage site, it can either be released into extracellular space directly from necrotic cells in the airway epithelium [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ] or indirectly from inflammatory cells after activation of the NLRP3 inflammasome [
      • Iyer S.S.
      • Pulskens W.P.
      • Sadler J.J.
      • et al.
      Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.
      ,
      • Fettelschoss A.
      • Kistowska M.
      • LeibundGut-Landmann S.
      • et al.
      Inflammasome activation and IL-1beta target IL-1alpha for secretion as opposed to surface expression.
      ]. Activation of NLRP3 and subsequent IL-1α secretion is potentially exacerbated in the CF airway by flagella from Pseudomonas aeruginosa [
      • Rimessi A.
      • Bezzerri V.
      • Patergnani S.
      • et al.
      Mitochondrial Ca2+-dependent Nlrp3 activation exacerbates the pseudomonas aeruginosa-driven inflammatory response in cystic fibrosis.
      ], reactive oxygen species generated during hypoxia [
      • Kim S.R.
      • Kim D.I.
      • Kim S.H.
      • et al.
      Nlrp3 inflammasome activation by mitochondrial ros in bronchial epithelial cells is required for allergic inflammation.
      ], and mitochondria released from necrotic AEC [
      • Iyer S.S.
      • Pulskens W.P.
      • Sadler J.J.
      • et al.
      Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome.
      ]. Release of IL-1β is predominately facilitated through toll-like receptor signalling from macrophages and the NLRP3 inflammasome [
      • Cullen S.P.
      • Kearney C.J.
      • Clancy D.M.
      • et al.
      Diverse activators of the Nlrp3 inflammasome promote IL-1beta secretion by triggering necrosis.
      ]. In βENaC-overexpressing mice, we previously found that increased IL-1α was associated with increased IL-1β in BALf [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ]. Genetic deletion of IL-1R had no measureable effect on the number of necrotic airway cells or levels of IL-1α but resulted in reduced levels of IL-1β, indicating that secretion of IL-1β occurred secondary to IL-1α-mediated activation of the IL-1R-MyD88 pathway, even in the absence of infection [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ]. We have now demonstrated in children with CF that the correlation of IL-1α and IL-1β is independent of detectable pulmonary infection; thus further supporting the hypothesis that IL-1α secretion plays an important role in triggering IL-1β release and sterile inflammation in early CF lung disease.
      Furthermore, we observed that IL-1α and IL-1β correlated with levels of IL-8 measured in BALf from young children with CF. Levels were associated with IL-8 independent from detectable respiratory infection, indicating the importance of IL-1R-MyD88 regulation for neutrophil recruitment in early CF lung disease. Based on these results, we investigated associations of IL-1α and IL-1β with airway neutrophilia and found IL-1α and IL-1β strongly correlated with neutrophil counts, independent from detectable respiratory infection. This suggests increased recruitment of neutrophils to the airway lumen occurs via activation of IL-1R-MyD88 signalling, NF-κB activation and subsequent release of IL-8, highlighting the therapeutic potential of IL-1R previously reported [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ,
      • Chen C.J.
      • Kono H.
      • Golenbock D.
      • et al.
      Identification of a key pathway required for the sterile inflammatory response triggered by dying cells.
      ]. When segregated by respiratory infection, we found a significant correlation between NE and IL-1α in children, but no correlation in those without infection. In contrast, we found significant correlations between NE and IL-1β in children with and without respiratory infection. This study has discovered associations between IL-1α and IL-1β with IL-8, the primary neutrophil chemoattractant, airway neutrophilia and increased levels of NE released from activated neutrophils in BALf in young children with CF. Taken together with data from preclinical studies in βENaC-transgenic mice [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ,
      • Mall M.A.
      • Harkema J.R.
      • Trojanek J.B.
      • et al.
      Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.
      ], our data suggest activation of the IL-1R-MyD88 pathway by elevated levels of IL-1α, may increase release of IL-8, subsequent neutrophil recruitment and activation, and a greater burden of neutrophil elastase activity in the pediatric CF airway.
      Neutrophil elastase activity detected in BALf is the major predictor for persistent bronchiectasis in CF [
      • Sly P.D.
      • Gangell C.L.
      • Chen L.
      • et al.
      Risk factors for bronchiectasis in children with cystic fibrosis.
      ], and structural lung changes are detectable on CT in children with CF without detectable respiratory infection [
      • Stick S.M.
      • Brennan S.
      • Murray C.
      • et al.
      Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening.
      ]. We therefore investigated associations between levels of IL-1α, IL-1β, IL-8, neutrophil count and NE activity in BALf from children with CF and structural lung changes detected on CT, and for the first time found significant associations between IL-1α, IL-1β and structural lung changes. When stratified by respiratory infection status, there were associations between IL-1α, IL-1β, IL-8, neutrophil count and NE activity and extent of structural lung disease on CT in children without respiratory infection, but no associations in children with respiratory infection. Although IL-1α, IL-1β, IL-8, neutrophil count and NE activity were all associated with extent of structural lung disease on CT, the association between IL-1α and extent of disease was the strongest association discovered. Our previous data implicated activation of the IL-1R-MyD88 pathway in structural lung damage in vivo via NE, which was reduced after genetic deletion of IL-1R [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ,
      • Gehrig S.
      • Duerr J.
      • Weitnauer M.
      • et al.
      Lack of neutrophil elastase reduces inflammation, mucus hypersecretion, and emphysema, but not mucus obstruction, in mice with cystic fibrosis-like lung disease.
      ]. Here, the extent of structural lung damage was associated with IL-1α, independent of pulmonary infection. This further strengthens the hypothesis that IL-1α is released prior to IL-1β from epithelial and potentially other cell types under hypoxic conditions is a key trigger of the IL-1R-MyD88 pathway, driving early structural lung damage in CF in the absence of infection.
      This study has a number of limitations; although sample size was large enough to segregate according to respiratory infection status, study subpopulations were not sufficient to segregate according to infection type. Larger groups of patients with known pathogenic bacteria including Ps. aeruginosa could potentially elucidate differences between patients with and without specific infections. While use of next-generation technologies such as 16S rRNA-sequencing and metagenomics analysis to understand the composition and dynamics of the lung microbiome in these patients may provide greater insight into the early inflammatory environment, the microbiology utilised in this study is the current clinical gold standard and advises patient treatment regimes in the clinic. Weak correlations between IL-1 cytokines and NE were observed in this study, however the minimum detection limit for NE was relatively high at 200 ng/mL when compared to IL-1α and IL-1β with detection limits of 1.3 pg/mL and 0.65 pg/mL respectively. As NE activity was detectable in 34% of uninfected and 25% of infected BALf, the relatively low sensitivity of the NE assay may have affected correlations observed with IL-1α and IL-1β. Although previous animal model work identified mucus obstruction as a cause of chronic airway inflammation [
      • Livraghi-Butrico A.
      • Kelly E.J.
      • Klem E.R.
      • et al.
      Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.
      ], this could not be measured directly in our study cohort. Encouragingly, studies have demonstrated that magnetic resonance imaging (MRI) of the lungs, in addition to detecting airway wall thickening and bronchiectasis, also has the sensitivity to detect airway mucus obstruction and deficits in lung perfusion in young children with CF [
      • Wielputz M.O.
      • Puderbach M.
      • Kopp-Schneider A.
      • et al.
      Magnetic resonance imaging detects changes in structure and perfusion, and response to therapy in early cystic fibrosis lung disease.
      ,
      • Stahl M.
      • Wielputz M.O.
      • Graeber S.Y.
      • et al.
      Comparison of lung clearance index and magnetic resonance imaging for assessment of lung disease in children with cystic fibrosis.
      ,
      • Mall M.A.
      • Stahl M.
      • Graeber S.Y.
      • et al.
      Early detection and sensitive monitoring of cf lung disease: prospects of improved and safer imaging.
      ]. Therefore, chest MRI in combination with BAL may be used in future studies to determine relationships between airway mucus obstruction, inflammation and infection in early CF lung disease.
      In summary, we have demonstrated IL-1α and IL-1β are present in BALf from young children with CF. Levels of these cytokines correlated with indicators of neutrophilic inflammation. Critically, the associations of IL-1α levels with inflammation and structural lung disease were observed in the absence of detectable airway infection. Collectively, these results suggest an important role of the IL-1R-MyD88 signalling pathway in early disease pathogenesis in CF, and potentially other muco-obstructive lung diseases characterized by airway mucus obstruction, localised hypoxia and neutrophilic inflammation. This study has demonstrated IL-1Ra is associated with IL-1α, IL-1β, and the presence of neutrophils in BALf from young children with CF. Previous studies utilising both human and animal models have highlighted the therapeutic potential of IL-1Ra with reduction of airway inflammation, inflammasome activation, airway neutrophilia and structural lung damage [
      • Fritzsching B.
      • Zhou-Suckow Z.
      • Trojanek J.B.
      • et al.
      Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease.
      ,
      • Mall M.A.
      • Harkema J.R.
      • Trojanek J.B.
      • et al.
      Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.
      ,
      • Iannitti R.G.
      • Napolioni V.
      • Oikonomou V.
      • et al.
      IL-1 receptor antagonist ameliorates inflammasome-dependent inflammation in murine and human cystic fibrosis.
      ]. With IL-1Ra already a routine treatment for rheumatoid arthritis and other autoinflammatory diseases [
      • Bresnihan B.
      • Alvaro-Gracia J.M.
      • Cobby M.
      • et al.
      Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist.
      ], there is potential for expedited translation of therapy into the CF setting.

      Acknowledgements

      The authors thank the subjects and families for their generous contributions to the AREST CF program. The authors also thank the respiratory fellows who performed bronchoscopies, and Mr. Luke Berry, Dr. Tim Rosenow and Ms. Clara Mok who all provided technical assistance.

      Funding

      This work was supported by; the US Cystic Fibrosis Foundation (STICK12A0), Cystic Fibrosis Australia (ACFRTMontgomery2015) and the German Federal Ministry of Education and Research (82DZL004A1). Stephen M. Stick is a NHMRC Practitioner Fellow.

      Appendix A. Supplementary data

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