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REVIEW

Early pulmonary inflammation and lung damage in children with cystic fibrosis ANDRÉ SCHULTZ1,2,3 AND STEPHEN STICK1,3 1

Department of Respiratory Medicine, Princess Margaret Hospital for Children, 2School of Paediatric and Child Health, University of Western Australia, and 3Telethon Kids Institute, Perth, Western Australia, Australia

ABSTRACT Individuals with cystic fibrosis (CF) suffer progressive airway inflammation, infection and lung damage. Airway inflammation and infection are present from early in life, often before children are symptomatic. CF gene mutations cause changes in the CF transmembrane regulator protein that result in an aberrant airway microenvironment including airway surface liquid (ASL) dehydration, reduced ASL acidity, altered airway mucin and a dysregulated inflammatory response. This review discusses how an altered microenvironment drives CF lung disease before overt airway infection, the response of the CF airway to early infection, and methods to prevent inflammation and early lung disease. Key words: biochemistry, cell biology, cystic fibrosis, paediatric lung disease, infection and inflammation. Abbreviations: 15-LO2, 15-lipoxygenase2; AEC, airway epithelial cells; ASL, airway surface liquid; BAL, bronchoalveolar lavage; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; CT, computed tomography; ENaC, epithelial sodium channel; ER, endoplasmic reticulum; IL, interleukin; LCI, lung clearance index; LT, leukotriene; NE, neutrophil elastase; LXA4, lipoxin A4; NF, nuclear factor; PPAR, peroxisome proliferator-activating receptor; PCL, periciliary layer; RV, rhinovirus; SPLUNC1, short palate lung and nasal epithelial clone 1; TLR, toll-like receptor.

INTRODUCTION Cystic fibrosis (CF) is the most common lifeshortening genetic disease affecting children of Caucasian descent, with an increasingly recognized prevalence in other populations. Individuals with CF suffer progressive airway inflammation, infection and lung damage. The mechanisms by which abnormal CF transmembrane regulator (CFTR) protein causes CF lung disease are not fully understood, but early Correspondence: André Schultz, Department of Respiratory Medicine, Princess Margaret Hospital for Children, GPO Box D184, Perth, WA 6840, Australia. Email: [email protected] Received 1 September 2014; invited to revise 22 September and 11 December 2014; revised 24 November 2014 and 30 January 2015; accepted 17 February 2015 (Associate Editor: Claire Wainwright). © 2015 Asian Pacific Society of Respirology

diagnosis through newborn screening has facilitated study of the earliest stages of CF lung disease.

Observations from surveillance of infants with CF diagnosed following newborn screening Building on the work of others,1,2 the Australian Respiratory Early Surveillance Team for CF programme provided some of the first snapshots of the earliest pathobiological changes in the lung associated with CF and the significant factors that contribute to structural lung disease, notably bronchiectasis.3–6 The initial cross-sectional analyses established that structural lung disease including bronchiectasis occurs soon after birth, is common in the first years of life and is associated with inflammation and infection.6,7 Bronchial dilatation, a precursor to bronchiectasis, was detected in nearly 20% of infants between 2 and 5 months of age, and overall nearly 80% of children at this age had evidence of pulmonary disease manifest by infection, inflammation or radiological changes.7 In children up to the age of 6 years, the overall prevalence of bronchiectasis was 22% and this increased with age and approached 70% by age 6 years.6 Factors in broncho-alveolar lavage fluid associated with bronchiectasis included: absolute neutrophil count, neutrophil elastase (NE) concentration, and Pseudomonas aeruginosa infection. Other studies have also demonstrated significant lung disease in early life using computed tomography (CT),8–10 with specific differences between studies largely due to methodological factors related to image acquisition and analysis.11 Early reports have suggested that there is a decline in lung function in the first 2 years of life.4 Infants with free NE detected in bronchoalveolar lavage (BAL) had lower forced vital capacities and forced expiratory flows than children in whom NE was undetectable. Significantly greater decline in forced expiratory flows occurs in infants and young children infected with Staphylococcus aureus or P. aeruginosa detected in BAL fluid compared with infants and young children who are uninfected. The impact of early infection has further been emphasized in a study by Mott et al.3 who reported an analysis of 301 paired, three-slice limited CT scans obtained 1 year apart in children whose first scan was at the age 1–3 years. The extent of Respirology (2015) doi: 10.1111/resp.12521

2 bronchiectasis increased over 1 year in 63% of scans and air trapping in 47%. Radiological progression of bronchiectasis and air trapping was associated with severe CFTR genotype, pulmonary infection and worsening neutrophilic inflammation. Thus, these studies implicate inflammation and infection in the earliest stages of lung damage, often before children are symptomatic. This review will focus on the basic mechanisms of CF lung disease before overt airway infection, the response of the CF airway to early infection, and briefly discuss methods to prevent inflammation and early lung disease in CF.

FACTORS RELATED TO CFTR GENE MUTATIONS THAT DRIVE EARLY LUNG DISEASE CF is caused by mutations of the genes that code for the CFTR protein. CFTR is a small linear cyclical membrane spanning protein located at the surface of airway epithelial and other cells. CFTR is an anion channel for chloride, bicarbonate, and the antioxidants thiocyanate and glutathione. CFTR is also a regulator of multiple transport proteins, including the epithelial sodium channel (ENaC), the outwardly rectifying anoctamin six chloride channel and the calcium-activated anoctamin one chloride channel (TMEM 16A). In addition, CFTR participates in many intracellular signalling pathways.12 With CFTR gene mutations, all the above functions can be disturbed. CFTR gene mutations can be classified into six classes based upon the nature of the underlying defect.13 A single mutation can belong to more than one class. Classes I–III are generally associated with lower chloride flux and more severe disease, although there is significant heterogeneity in the expression of disease. However, regardless of the nature of the mutation, the net effect is reduced chloride secretion into the airway lumen with associated sodium and water compartment shifts that tend to dehydrate the airway surface liquid (ASL) and contribute to an altered airway microenvironment. Other important factors that appear to contribute to dysregulation of the airway surface homeostasis include: decreased epithelial bicarbonate transport resulting in reduced ASL pH, abnormal airway mucin and a dysregulated inflammatory response.

FACTORS THAT CONTRIBUTE TO AN ABERRANT AIRWAY MICROENVIRONMENT ASL dehydration in CF The ASL layer is a thin fluid layer that that covers the airway epithelial cells (AEC). Impaired CFTR chloride ion channel function contributes to reduced ASL volume. Chloride transport to the ASL is further influenced through complex interaction between CFTR and anoctamins 1 and 6.12,14 However, sodium absorption through the ENaC, which is up regulated in CF, is the rate-limiting step in ASL volume regulation.15 ENaC is located in the apical membrane of AEC and is Respirology (2015)

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critical in the auto regulation of ASL volume through the active pumping of sodium from the ASL into AEC. In CF, CFTR mediated downregulation of ENaC is absent or diminished, resulting in excessive Na and water resorption contributing to ASL dehydration.16 Furthermore, because ENaC is activated by serine proteases, for instance NE,17 the increased levels of NE observed in CF4 might further enhance ENaC activity and promote ASL dehydration.

Reduced ASL pH in CF lung disease Reduced ASL pH occurs in CF bronchial epithelial cell cultures as well as in vivo in the lower airways of the CF pig.18,19 Impaired CFTR-related epithelial bicarbonate transport and a reduced ASL pH can result in ASL dehydration, abnormal mucins and impaired antimicrobial function. ASL pH and ASL dehydration The normal downregulation of ENaC activity is mediated through the action of short palate lung and nasal epithelial clone 1 (SPLUNC1) protein, a secreted protein that is abundant in ASL and is pH dependent. SPLUNC1 is unable to regulate ENaC function at low pH. Correction of ASL pH is associated with the normalization of ASL volume.18 The role of CFTR in maintaining pH homeostasis in the ASL appears to be related to SPLUNC1 function and regulation of ASL volume. Impaired CFTR bicarbonate transport and antimicrobial properties of ASL ASL pH further influences defence against pathogens through effects on proteins and peptides that are important for innate immune defences. Of particular importance for CF, the antimicrobial activity of SPLUNC1 has been shown to protect against P. aeruginosa infection20 and inhibits bacterial biofilm formation.21 Because SPLUNC1 activity is pH dependent,18 lower pH levels in CF ASL result in reduced SPLUNC1 mediated bacterial killing19 that is abrogated by normalizing ASL pH.19 Altered airway mucins in CF Airway hydration and mucin function Mucin glycoproteins (mucins), the major macromolecules in ASL, are concentrated in CF respiratory secretions.22 There are two main types of mucins on the airway surface: membrane mucins and gelforming mucins.23 Membrane mucins are attached to the surface of AEC and protrude up to 1.5 μm into the periciliary layer (PCL) of ASL. The high concentration of membrane mucins (including MUC1, MUC4, MUC16) in the PCL form a physical barrier protecting the airway surface24 and contribute to the osmotic modulus of the PCL. The predominant gel-forming mucins, MUC5B and MUC5AC, play a critical role in airway hydration.23 Under normal conditions, the osmotic modulus of the PCL is higher than the osmotic modulus of the gel layer, allowing the gel © 2015 Asian Pacific Society of Respirology

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3 unfolding of mucin molecules after exocytosis and by reduced release of tethered mucins from epithelial surfaces.

Figure 1 The gel on brush model of airway surface liquid dehydration in cystic fibrosis (CF). Diagramatic representation of the airway surface liquid (ASL) layer above an airway epithelial cell. (a) Healthy. (1) Gel forming mucin layer with lower osmotic modulus acts as a barrier to ASL dehydration. (2) Membrane mucins in periciliary layer. (b) Sequence of events leading to mucostasis in cystic fibrosis. (3) ASL dehydration crosses threshold as the osmotic modulus of the gel layer, now with concentrated mucins, exceeds the osmotic modulus of the periciliary layer. (4) Water movement from the periciliary layer to the gel layer. (5) Decreased ciliary beat function due to ciliary compression by the gel layer. (6) Mucus build-up in gel layer caused by mucostasis (based on Button et al.23).

layer to function as a buffer against changes in ASL hydration. When the ASL becomes dehydrated, the gel layer loses water to the PCL, where adequate hydration is essential for ciliary beat function. In CF, dehydration of the ASL passes a critical threshold causing the osmotic modulus of the gel layer to exceed the osmotic modulus of the PCL, resulting in water movement from the PCL to the gel layer and eventual PCL dehydration.23 If the airway surface is sufficiently dehydrated, the mucus layer compresses the ciliary brush causing the ciliary beat to slow, resulting in impaired mucociliary clearance (Fig. 1). Mucostasis per se can lead to airway inflammation by trapping non-infectious, noxious particles.25 A better known effect of mucostasis is the failure to clear pathogens, resulting in chronic airway infection and inflammation.

ASL pH and mucin function The bicarbonate pump function of CFTR is important for normal mucin function. Mucins are tightly packed in secretory granules in epithelial cells.26 On exocytosis, mucins unfold and expand in size up to 1000 times to form gel networks and strands; this process requires bicarbonate.27 Bicarbonate appears to facilitate the untethering of gel-forming mucins after exocytosis,27 allowing mucin molecules to be transported by ciliary activity. Recent evidence suggests that in CF, gel-forming mucins remain tethered to epithelial cell surfaces after exocytosis. Reduced ASL pH can therefore affect mucin function and reduce mucociliary clearance through impaired © 2015 Asian Pacific Society of Respirology

Pulmonary inflammation in CF Airway inflammation in CF starts early in life with markers of airway inflammation detected in BAL fluid of infants from as early as 4 weeks of age.2 Airway inflammation in CF is predominantly neutrophilic in nature, characterized in infants by elevated neutrophil counts, interleukin (IL)-8 levels and free NE. NE is a serine protease found mainly in the azurophil granules in neutrophil cytoplasm.28 NE is capable of degrading bacterial outer membrane proteins as well as proteins in the extracellular matrix. Antiproteases, including α1-antitrypsin, play an important role in the inactivation of free NE.28 When NE levels exceed the antiprotease activity of the lung, free NE activity can be detected in BAL fluid.29 Free NE hydrolyses proteins in the basal lamina and extracellular matrix causing damage to airways.4 Other proteases associated with inflammation and raised in the CF airway are also likely to contribute to lung damage. These include cathepsin S30 and metalloproteases.31 An important observation is that inflammation may be present before infection can be detected in airway secretions using standard culture techniques.2 In infants (median age 3.6 months) diagnosed with CF by newborn screening, IL-8 levels are detectable in 77.2%, and NE is detectable in 29.8%7 of BAL samples. However, bacterial pathogens are only detected in 21.1%. Possible explanations include: sterile inflammation (triggered by endogenous materials released during cell injury),32 failure of standard sampling and culture techniques to detect bacterial pathogens, non-bacterial pathogens, for example, viruses, or a protracted inflammatory response to a prior (viral) infection. Regardless of the underlying mechanisms leading to excessive airway inflammation in CF, viral and bacterial pathogens are important determinants of morbidity.4,33 Viral infection initiating a dysregulated inflammatory response Respiratory viruses, specifically rhinovirus (RV), are associated with a large proportion of CF exacerbations and hospitalizations in children. RV can be frequently detected in the lower airways of children with CF, during exacerbations and when clinically stable, with a higher viral load than in children with other respiratory diseases or control subjects.34 AEC provide a physical barrier between the lung and the airway surface and are actively involved in host defence.35 AEC function during viral infection includes the release of mediators that recruit and activate effector cells such as neutrophils, macrophages, dendritic cells and T cells.36 Evidence suggests that the inflammatory response to infection by AEC in young infants with CF is dysregulated.34,37 Paradoxically, the higher viral load seen in CF is associated with higher levels of pro-inflammatory mediators, but lower Respirology (2015)

4 levels of antiviral and anti-inflammatory mediators.34 In addition, CF AEC appear to have a dampened apoptotic response to viral infection.37 Apoptosis is an important mechanism by which cells limit viral replication and spread. Therefore, deficient antiviral control mechanisms and an ineffective, excessive inflammatory response to virus infection are likely to contribute to CF airway disease.

Bacterial infection and airway inflammation There are strong data to suggest that bacterial infection is an important driver of airway inflammation and progression of early CF lung disease.38 In particular, early infection may initiate a sustained effect on airway inflammation.38 With early surveillance programmes, pulmonary infection can be detected in CF from a very early age with BAL: approximately 20% of infants with CF at the age of three months,7 and in 44% of patients in the first 2 years of life show evidence of lower airway infection. The most commonly detected organisms in the lower airways of young infants are S. aureus and P. aeruginosa, followed by Aspergillus species, Haemophilus influenzae and Streptococcus pneumoniae. Infants with lower airway infection generally do not always have respiratory symptoms. During infancy, infection with P. aeruginosa, H. influenzae, S. pneumoniae and S. aureus is associated with higher levels of inflammation than with a range of other organisms.7,39 Furthermore, inflammation increases with organism density and the number of species isolated.40 Putative links among CFTR, mucosal immunity and inflammation Our understanding of the roles of abnormal CFTR in mucosal immunity is becoming clearer. Important

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recent observations include the links among CFTR and toll-like receptor (TLR) signalling, responses to endoplasmic reticulum (ER) stress and failure to resolve inflammatory responses.

TLR and the nuclear factor-κβ inflammatory signalling cascade. The TLR are a group of membrane spanning proteins on the cell surface that recognize pathogen associated molecular patterns and initiate a cellular response (Fig. 2). Upon stimulation TLR recruit myeloid differentiation primary response gene 88, an adaptor molecule, to start a process of intracellular signalling that results in the activation of nuclear factor (NF)-κβ and mitogen-activated protein kinase. The translocation of activated NF-κβ to the nucleus results in the upregulation of proinflammatory genes.41 CFTR normally regulates IL-8 activity via NF-κβ through the ΔNF-κβ-IL-8 promoter. In CF, there appears to be a disruption in the signalling link between CFTR and NF-κβ.42 TLR-5 appears to be implicated in the disrupted signalling: AEC rely almost exclusively on TLR-5 to recognize P. aeruginosa through its flagellin protein. The excessive cytokine production by CF airway cells and inflammatory cells after exposure to P. aeruginosa appears to be mediated by TLR and more specifically by TLR-5. Inhibition of TLR-5 abolishes the excessive immune response.43 ER stress, dysfunctional autophagosomes and inflammation. Another pathway through which inflammation could be accentuated in CF is dysfunctional autophagocytosis. Autophagy is a cellular process that maintains homeostasis by targeting abnormal proteins from the ER to autophagosomes that merge with lysosomes, causing degradation for

Figure 2 Drivers of inflammation in cystic fibrosis (CF). + = ACTIVATION; Θ = INHIBITION. Defective cystic fibrosis transmembrane regulator (CFTR) chloride transport and impaired inhibition of epithelial sodium channel (ENaC) results in airway surface liquid (ASL) dehydration and impaired mucociliary clearance. Defective CFTR bicarbonate transport accentuates ENaC hyperfunction and prevents bicarbonate mediated short palate lung and nasal epithelial clone 1 (SPLUNC1) antimicrobial activity in the ASL. CFTR-mediated downregulation of nuclear factor (NF)-κβ inhibited by flagellin-activated toll-like receptor (TLR)5, causing dysregulated inflammatory response and tissue damage. Defective CFTR accumulation (type 2 mutations) in endoplasmic reticulum (ER) accentuates NF-κβ inflammation. NE, neutrophil elastase. Respirology (2015)

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recycling of the abnormal proteins. In individuals with class II (the most common) CFTR mutations, misfolded CFTR accumulates in the ER, causing ER stress and autophagy.44 Activation of ER stress pathways correlates with inflammation in CF45 with direct and indirect interactions between ER stress pathway mediators and NF-κβ-dependent transcription of inflammatory mediators.46 Autophagocytosis can be impaired by TLR-5: ligation of TLR-5 by flagellin appears to impede autophagosome-lysosome fusion, resulting in the build up of autophagosomes containing abnormal CFTR in the ER, which in turn increases ER stress pathways that lead to increased inflammatory response via NF-κβ activation.47

Failure to resolve inflammation. Failure of pathways involved in the resolution of inflammation contribute to early CF disease. Leukotriene(LT)B4 and IL-8 are both powerful neutrophil chemoattractants. Lipoxin A4 (LXA4) counter-regulates the effects of LTB4, inhibiting LTB4-induced neutrophil transmigration and suppressing the production of IL-8 by epithelial cells and leukocytes. 15-Lipoxygenase2 plays a central role in LXA4 biosynthesis. Impoverished 15-lipoxygenase2 gene expression and a reduced LXA4/LTB4 ratio are found in the lower airways of children with CF.48

CONSEQUENCES OF AN ALTERED AIRWAY MICROENVIRONMENT Chronic inflammation in CF airways leads to progressive structural lung disease, even in infants with CF4 (Fig. 3). Structural lung disease is widely prevalent in young infants with CF and found in most children with CF by the age of 6 years. By 3 months of age 80% of infants diagnosed with CF by newborn screening will have abnormalities on chest CT imaging.7 Gas trapping, a marker of small airway disease is the most common finding (66.7%), followed by bronchial wall thickening (45%) and bronchial dilatation (18.6%).7 The majority of infants with abnormalities on CT scan do not have respiratory symptoms, but respiratory symptoms are more likely to be present in infants with bronchial dilatation.7 Bronchial dilatation is more extensive in the presence of infection and is associated with increased inflammation.7 Free NE activity in the lower airways appears to be the inflammatory marker most associated with the presence and progression of early structural CF lung disease5,7 and lung function decline.4 As airway inflammation, infection, reduced lung function and radiographic evidence of structural airway changes can be detected in CF shortly after diagnosis, early intervention strategies are needed to reduce the adverse clinical outcomes associated with early inflammation in CF airways.

EFFECT OF TREATMENT ON THE CF AIRWAY MICROENVIRONMENT Traditional methods for treating CF lung disease, still central in CF management, include airway clearance © 2015 Asian Pacific Society of Respirology

Figure 3 Early lung disease in cystic fibrosis (CF). (a) Computed tomography (CT) image of a 3-month-old child with CF showing air trapping. (b) CT image of a 3-month-old child with cystic fibrosis showing bronchiectasis.

and the aggressive use of antibiotics to combat infection. Improved understanding of CF pathogenesis has allowed for the development of novel therapies, some of which have resulted in remarkable results in clinical trials. The aim of this section is to link the preceding discussion about the pathophysiology of early inflammation in CF with treatment.

Drugs that correct the primary defect in CF CFTR potentiators and correctors One of the most exciting developments in CF treatment in recent years has been the development of CFTR potentiators and correctors. Ivacaftor, a CFTR potentiator, improves CFTR ion transport and reduce excessive sodium absorption from the ASL49 in individuals with CF with the class III mutation G551D50 or a number of other similar class III ‘gating’ mutations.13 Ivacaftor use results in marked improvements in lung function in adults and older children with class III mutations. CFTR correctors are designed to improve abnormal CFTR folding and trafficking as occurs in class 2 mutations. The CFTR corrector lumacaftor facilitates correct protein folding of ΔF508-CFTR during biogenesis and processing in the ER, allowing some mutant CFTR to exit the ER and traffic to the epithelial cell surface.51 The use of a CFTR potentiator (ivacaftor) in combination with a CFTR corrector (lumacaftor) has shown modest clinical effect in ΔF508-homozygous patients and very modest effect in ΔF508-heterozygous patients. A more detailed Respirology (2015)

6 discussion on CFTR repairing therapies can be found elsewhere.13 Of note, no potentiator or corrector studies in children under 6 years have been published. A safety study investigating the use of ivacaftor in 2–5-year-old children (NCT01705145) is currently underway.

Read through agents CF patients with CFTR non-sense mutations causing a premature termination signal (Class I mutations) may benefit from read through agents that prevent the aberrant truncation of protein during synthesis.52 The finding that the aminoglycoside antibiotic gentamycin has read through activity for these mutations53 resulted in the development of a novel small molecule read through agent ataluren. Ataluren use by children and adults with class I CFTR mutations results in the expression of full-length CFTR protein at the apical cell membrane and improved CFTR chloride channel function.54 Unfortunately, a recent phase 3 trial in patients with at least one non-sense CFTR mutation failed to show a significant difference in primary clinical end-points between the ataluren and placebo group. However, a subset of patients not treated with inhaled aminoglycoside antibiotics during the course of the trial did show a significant improvement in lung function.52 Another phase 3 trial in patients >6 years of age is currently underway (NCT02139306). Drugs that target airway inflammation As airway inflammation is central to the development and progression of CF lung disease, antiinflammatory medications have the potential to be beneficial in the treatment of CF lung disease. Antiinflammatory medications have to be used and developed with the greatest of caution as the suppression of the immune system can make an individual vulnerable to a wide range of complications and adverse effects, not least overwhelming infection. Glucocorticosteroids CF lung disease can be slowed by the use of systemic glucocorticosteroids,55 but their long-term use is precluded by unacceptable side-effects.55 Locally acting inhaled corticosteroids have been used widely to treat CF. Registry studies show a slower rate of lung function decline on patients who are prescribed inhaled corticosteroids.56 However, no clinical trial of inhaled corticosteroids has shown convincing evidence of reduced airway inflammation or improved lung function. In one study, the discontinuation of these drugs did not result in worsening of clinical outcomes,57 but patients were excluded from the study when the physician wished them to continue taking inhaled corticosteroids. Non-steroidal anti-inflammatory drugs The use of the non steroidal anti-inflammatory drug ibuprofen, which inhibits NF-κβ activation, has also been shown to result in decreased rates of lung function decline in children with CF.58 The use of ibuprofen as a long-term anti-inflammatory drug is comRespirology (2015)

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plicated by the need to establish the appropriate dose for individual patients with a 3-h pharmacokinetic test.59 Gastrointestinal bleeding, a rare but significant adverse effect, makes the benefit-risk ratio of longterm ibuprofen use debatable.

Macrolides Macrolides are commonly used antibiotics that have anti-inflammatory properties. Macrolides are known to inhibit NF-κβ and mitogen-activated protein kinase activation and to reduce IL-8 secretion.60 The long-term use of macrolides, like azithromycin, may be of some benefit in children with CF.61 The COMBAT CF trial where the use of long-term azithromycin is being tested in young infants from shortly after the diagnosis of CF is made is currently underway, and is one of the first clinical trials to study the prevention of early lung disease in young infants with CF. Similarly, the ongoing OPTIMIZing Treatment for Early Pseudomonas aeruginosa Infection in Cystic Fibrosis trial (NCT02054156) is evaluating the efficacy of chronic azithromycin initiated after early infection with P. aeruginosa on pulmonary exacerbation rates and microbiologic outcomes in patients aged 6 months to 18 years of age. Of note, azithromycin appears to reduce the efficacy of inhaled tobramycin when used concomitantly.62 A concern with the long-term use of macrolide antibiotics is the emergence of antimicrobial resistance. Synthetic macrolides with negligible antibacterial activity have been developed that appear to inhibit the proteolytic activation of ENaC channels by NE, thereby preventing NE induced ASL dehydration in human bronchial epithelial cell cultures.63 The clinical efficacy of non-antibiotic macrolides has yet to be examined. Inhibition of NF-κβ activity through the upregulation of peroxisome proliferator-activating receptor NF-κβ activity can be inhibited by peroxisome proliferator-activating receptor (PPAR). PPAR expression is downregulated in CF.64 The PPAR-γ agonists troglitazone and ciglitazone activate PPAR in primary CF airway epithelium and reduce airway inflammation in response to acute infection with P. aeruginosa in a CF mice model.65 A pilot study of pioglitazone in healthy individuals did not show a reduction in markers of airway inflammation after 28 days of use.66 The pilot study was limited by an inadequate dose on pioglitazone, a short study duration of 28 days, and a limited number of study participants (n = 20). Further evaluation of PPAR agonists is warranted. Inhibition of proteases The importance of proteases, particularly NE, in early CF airway inflammation is described above. α-1Antitrypsin is an antiprotease that can be derived from plasma. Inhaled α-1-antitrypsin has been shown to suppress markers of airway inflammation, including free NE and neutrophil levels.67 Other inhibitors of NE have been studied in CF with positive preliminary results.68 The protease cathepsin S also has potential © 2015 Asian Pacific Society of Respirology

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to be a future therapeutic target. Further study is required to determine if the use of protease inhibitors can prevent early inflammation in CF.

Antibiotic use as a strategy to target airway inflammation Antibiotic treatment is associated with a reduction in airway inflammation in CF.4 Antibiotics are routinely used to treat pulmonary exacerbations of CF disease. The knowledge that staphylococcal infection is associated with suboptimal clinical outcomes has prompted various CF centres around the world to treat infants with CF with prophylactic anti-staphylococcal antibiotics. Data on the benefits and risks of using prophylactic anti-staphylococcal antibiotics are conflicting. One study suggested that the use of prophylactic flucloxacillin in the first 2 years of life is associated with fewer chest symptoms and hospital admissions.69 Another study showed that the use of prophylactic amoxicillin clavulanic acid in the first 2 years of life was associated with improved nutritional parameters.70 In a slightly larger randomized controlled trial studying infants diagnosed by newborn screening for 5–7 years, the use of prophylactic cephalexin was not associated with improved clinical outcomes, but with increased colonization with P. aeruginosa.71 The knowledge that Pseudomonas in the lower airway is associated with a more rapid decline in lung function has lead to antibiotic-based Pseudomonas eradication regimens. Although P. aeruginosa, when detected in young infants and children with CF through early surveillance programmes, can be eradicated from the lower airways in 80% of children, eradication does not appear to influence airway inflammation38 or the deterioration in lung function associated with its detection.4 Although antibiotic use remains a cornerstone of CF management, it is clear that additional strategies are needed to alter the relentless course of CF lung disease. Antimicrobial strategies may include the use of antibodies or targeting different aspects of disease pathophysiology, for example, the development of synthetic human antibodies against alginate, an important pseudomonal virulence factor, holds promise for the more effective prevention of Pseudomonas infection in the future,72 whereas subjects with the G551D mutation treated with ivacaftor demonstrate reduced levels of Pseudomonas isolation.73 Drugs that target ASL hydration and/or mucus clearance Efforts to pharmacologically target ASL hydration through targets other than CFTR have included ENaC inhibitors, P2Y2 agonists and osmotic airway surface hydrators. ENaC inhibitors and purinergic receptor agonists ENaC inhibitors such as amiloride can reduce CF related ASL volume depletion and inhaled amiloride has been shown to improve mucociliary clearance in © 2015 Asian Pacific Society of Respirology

7 the short term.74 The failure of inhaled amiloride use in CF to result in clinically meaningful improvements in the longer term is thought to be due to the short half-life of amiloride in the airways limiting its efficacy.75,76 There may be rationale for testing the efficacy of amiloride to prevent early lung disease, as opposed to rescue therapy.77 A novel and more potent ENaC inhibitor with a longer half-life showed promising early results in preclinical trials78 but did not progress due to side-effects.78 Stimulation of the purinergic receptor P2Y2, abundant on the surface of airway epithelium, increases ASL volume by suppressing ENaC and stimulating chloride secretion.79 After preliminary success in the first clinical trail, the P2Y2 agonist denufosol did not result in clinical improvement over the use of placebo in further clinical trials.80 Non-CFTR ion channels still hold potential as therapeutic targets to compensate for impaired CFTR ion transport.13

Osmotic airway hydrators Osmotically active agents like hypertonic saline and mannitol, when delivered to the airway surface, draw up water into the airway lumen through the epithelial cell layer.81 Inhaled hypertonic saline has been shown to improve mucociliary clearance and reduce pulmonary exacerbations in patients with CF. However, in a large multicentre study evaluating the use of hypertonic saline in infants and young children (Infant Study of Inhaled Saline (ISIS) trial),82 there was no improvement in terms of primary outcome measures (exacerbations) in the treatment group. There were improvements in two physiologic end-points, FEV0.5 measured by infant lung function testing and the lung clearance index (LCI) measured by multiple breath washout,83 in two small substudies, suggesting that hypertonic saline may improve physiologic outcomes in this young age range. Larger trials to evaluate this hypothesis are planned. Dornase alfa Inhaled recombinant human DNAse (dornase alfa) acts by the enzymatic cleavage of viscous DNA that accumulates in CF airways as a result of neutrophilic inflammation and cell lysis. Dornase alfa use has also been shown to reduce the progression of neutrophillic airway inflammation over a 3-year study period in a cohort of patients with relatively mild CF lung disease.84 The beneficial effect of dornase alfa on lung function has been well demonstrated in adults and older children.85 Evidence in young children is limited to a pilot study (n = 12) where dornase alfa use resulted in improved CT outcomes in the treatment group after 100 days of therapy.86 Measuring the effect of treatment in young patients There is a clear need for more clinical trials in infants and children to determine the safety and efficacy of specific early treatment strategies to prevent structural lung disease. A challenge in setting up clinical trials in infants and young children has been the lack of standardized outcome measures for this age Respirology (2015)

8 group. The ISIS trial, described above, underlines the importance of using outcome measures in clinical trials that are sensitive to disease progression before symptoms become prominent.82 A number of imaging and lung function outcome measured are being studied, with CT chest and LCI currently the most developed and promising tools for monitoring lung disease in infants and young children. CT is the most sensitive marker of structural lung disease, but the use of ionizing radiation, ableit low dose, and the need for general anaesthetics limit CT to annual or biannual use. Infant and child lung function tests, with LCI currently the most promising, can be used more frequently. Changes in LCI correlate with CT changes in young children but not in infants.87 The advantages and disadvantages of CT chest and LCI as outcome measures in early disease has recently been reviewed,88 and both modalities are now developed well enough to use as primary outcome measures in clinical trials.

CONCLUSION AND FUTURE DIRECTIONS Airway inflammation in CF begins early in infancy and possibly before bacterial infection of the lung. CF lung disease may be present well before symptoms occur. Preventing early lung damage is preferable to having to treat established lung disease. Very few intervention studies have been published in infants and young children. Numerous exciting candidate drugs exist in various stages of development to target different aspects of the inflammatory response. The development of CFTR potentiators, correctors and read through agents hold particular promise for improving outcomes in CF. Once the safety and effectiveness of interventions, such as ivacaftor and dornase alfa, has been established in older patients, such medications need to be tested through robust clinical trials with appropriate outcome measures in infants and young children.

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