REVIEW URRENT C OPINION
The role of neutrophils in cystic fibrosis Alison M. Gifford a and James D. Chalmers b
Purpose of review Neutrophils are known to dominate the pulmonary inflammatory process observed in cystic fibrosis (CF). An enduring paradox is how these large numbers of neutrophils fail to eradicate colonizing bacteria. Major advances in our understanding of neutrophil dysfunction in CF and its effect on the innate immune system are leading to advances in our understanding of the pathophysiology and leading directly to new therapies. Recent findings New mechanisms of neutrophil dysfunction have been described in CF including disabled cystic fibrosis transmembrane conductance regulator recruitment to phagosomes and novel mechanisms of proteaseinduced neutrophil dysfunction. Neutrophil elastase has been shown to be present in the airway very early in life in CF patients, and appears a biomarker of disease progression, predicting lung function decline and bronchiectasis. Elastase has also been shown to induce a pro-inflammatory state of senescence in bronchial epithelial cells in vitro and potentially in vivo. Inhibitors of neutrophil elastase are now entering clinical trials with promising results. New avenues of CF therapeutics are being explored including novel macrolides, CXCR2 antagonists and exogenous opsonins. Summary This article reviews the past 12 months of research that contributes to our understanding of the role of neutrophils and immune dysfunction in CF. Keywords cystic fibrosis, cystic fibrosis transmembrane conductance regulator neutrophil elastase, neutrophils
INTRODUCTION Cystic fibrosis (CF) is the most common lethal inherited autosomal recessive disorder amongst Caucasians [1]. The clinical syndrome of CF results from an inherited defect in the cystic fibrosis transmembrane conductance regulator (CFTR) leading to airway obstruction by defective mucus clearance, infection and progressive bronchiectasis [2]. Failure of chloride secretion and sodium hyper-absorption at the apical airway surface leads to dehydration of airway surface liquid and impaired clearance of bacterial pathogens [3 ]. The resulting inflammatory process observed in CF is dominated by the presence of neutrophils, driving a vicious cycle of bacterial colonization, airway inflammation and structural damage that overwhelms normal resolution and repair pathways. These contribute to the irreversible lung damage, worsening of lung function and premature death of these patients [4]. Increasingly, therapies targeting the CF neutrophil are moving from experimental to translational and clinical studies, offering hope of a new avenue of therapy for this devastating disease. &
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This article reviews the past 12 months of research that contributes to our understanding of the role of neutrophils and immune dysfunction in CF.
NEW MECHANISMS OF NEUTROPHIL DYSFUNCTION IN CYSTIC FIBROSIS It is a paradox that the large numbers of neutrophils present in the CF airway fail to effectively kill colonizing bacteria. CFTR is expressed in leukocytes, including neutrophils, leading to the hypothesis that CFTR dysfunction may have effects on neutrophil function beyond those resulting from impaired mucociliary clearance. By contrasting neutrophil effector responses in healthy and CF neutrophils, a Department of Paediatrics, Ninewells Hospital and bTayside Respiratory Research Group, University of Dundee, Dundee, UK
Correspondence to Dr James D. Chalmers, Tayside Respiratory Research Group, University of Dundee, Dundee DD1 9SY, UK. Tel: +44 1382386339; e-mail: [email protected] Curr Opin Hematol 2014, 21:16–22 DOI:10.1097/MOH.0000000000000009 Volume 21 Number 1 January 2014
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The role of neutrophils in cystic fibrosis Gifford and Chalmers
KEY POINTS New mechanisms of neutrophil dysfunction have been found in CF including innate defects in CF neutrophils prior to migration to the airway and profound effects of neutrophilic inflammation on the airway epithelium. Neutrophil elastase is now known to be present very early in life in CF patients at higher levels, corresponding to earlier and more severe lung function decline and bronchiectasis. Neutrophil elastase inhibitors are now entering clinical trials. New avenues of CF therapeutics are being explored including novel macrolides and exogenous opsonization of bacteria.
previous studies have reported abnormalities in CF neutrophil function including priming, enhanced granule enzyme activity, increased reactive oxygen species (ROS) production [5], increased interleukin (IL)-8 secretion [6] and delayed apoptosis [7]. Zhou et al. [8 ] provide further evidence of the role of CFTR in intracellular bacterial killing by describing disabled recruitment of the CFTR protein to neutrophil phagosomes. CFTR is one of the chloride channels required for generation of hypochlorous acid (HOCl), a toxic agent involved in intracellular bacterial killing. It was previously demonstrated that CF neutrophils were defective in their ability to kill intracellular pathogens and that this could be recreated by CFTR knockdown using small interfering RNA in neutrophil cell lines [9]. In this study, the authors demonstrate that more than 95% of mature phagosomes (stained with the well recognized phagosome marker lysosomalassociated membrane protein 1; LAMP-1) also stained positive for CFTR in healthy peripheral blood neutrophils [8 ]. Using an enhanced green fluorescent protein (EGFP)–CFTR fusion protein, the authors demonstrated significantly less EGFP– DF508-CFTR localizing to phagosomes compared with EGFP–wild-type-CFTR, indicating a defect in the migration of the protein to the phagosomal membrane. This was improved by the pharmacological CFTR chaperone voltage regulator tester 325 (VRT-325). This provides evidence of the mechanism behind defective bacterial killing in CF neutrophils [8 ]. A recent small study investigated peripheral neutrophil inflammatory responses in the context of acute exacerbations of CF. Fourteen patients with CF and 10 healthy controls were studied. The authors reported ROS and IL-8 release were &
higher in CF than control at baseline (prior to antibiotic therapy) with a higher early apoptotic response [10]. When treated with antibiotics, there was an increase in IL-8 secretion by neutrophils, which the authors argue is a potential biomarker for response to therapy. The early apoptotic response opposes previous findings which have always suggested prolonged neutrophil survival in CF [7]. Stimulated peripheral blood neutrophils from patients with CF have been reported to release significantly more ROS than neutrophils from healthy controls [5]. A recent study examined ROS production in sputum neutrophils and found the converse, with reduced ROS production in sputum neutrophils as measured by flow cytometry [11 ]. These studies are complicated by the difficulties in isolating neutrophils from sputum and particularly in isolating viable cells from healthy donor airways for comparison. The authors speculate that there are subpopulations of neutrophils within the CF airway displaying what the authors call ‘functional exhaustion’ [11 ]. Metabolic adaptation of neutrophils on migration into the airway lumen with increased glucose and amino acid transporters is an example of changes in transcriptional profile as the neutrophil is primed, activated and transmigrates into the airway [12 ]. This is consistent with prior demonstration of the reprogramming of neutrophils, mostly by tumour necrosis factor (TNF)-a and IL-8 [13], that occurs when they arrive in the CF lung. A complete understanding of the diverse ways in which neutrophils adapt to the CF airway remains elusive (Fig. 1). &
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The neutrophil in cystic fibrosis
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Increased glucose and amino acid transporters
Reduced phagosomal CFTR
Altered apoptosis
Altered ROS
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FIGURE 1. Innnate defects in neutrophil function in cystic fibrosis. New data suggest reduced cystic fibrosis transmembrane conductance regulator (CFTR) localization to phagosomes. Previously reported data about alterations in apoptosis and reactive oxygen species (ROS) generation have been challenged.
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NEUTROPHIL ELASTASE
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Neutrophil elastase is a serine protease contained in azurophil (primary) granules designed to digest phagocytosed bacterial proteins. Neutrophil elastase has, however, been implicated in the pathology of CF and shown to activate the epithelial sodium channel (ENaC) by direct cleavage [14], thereby contributing to airway surface mucus accumulation [3 ]. The primary defences against neutrophil elastase are endogenous antiproteinases such as a1-antitrypsin, secretory leukocyte peptidase inhibitor (SLPI) and elafin [15]. In CF, the quantity of elastase released into the airway overwhelms the antiproteinase capacity leading to detectable elastase activity. A major contribution to understanding the role of neutrophil elastase in disease progression was made this year by Sly et al. [16 ] as part of the Australian Respiratory Early Surveillace Team for Cystic Fibrosis (AREST-CF) study. This study followed 127 infants diagnosed with CF by newborn screening and performed computed tomography (CT) and bronchoalveolar lavage (BAL) at 3 months, 1, 2 and 3 years of age. Free neutrophil elastase activity in BAL fluid at 3 months was associated with persistent bronchiectasis on two or more sequential CT scans [adjusted odds ratio (OR) 7.20 at 12 months and 4.21 for persistent bronchiectasis at 3 years of age]. As has been previously demonstrated in a number of contexts, elastase activity was associated with the presence of bacterial colonization, but the relationship persisted even after statistical adjustment for bacterial colonization [16 ]. This is a significant finding, which suggests that neutrophil elastase activity in BAL fluid in early life is associated with the early development of bronchiectasis in CF. Early identification of patients at risk of disease progression has therapeutic value, although detection of elastase activity in young children without the requirement for BAL remains an obstacle to clinical implementation. Surrogates such as urinary desmosine which measure systematic elastin breakdown are a possible solution but remain to be tested in this group [17 ]. Further evidence that neutrophil elastase may be a useful biomarker in CF came from a small study by Sagel et al. [18 ] of 35 school-aged children. Measuring longitudinal relationships over 3 years, those with higher sputum neutrophil elastase levels at first measurement had a significantly higher rate of decline in forced expiratory volume in 1 s (FEV1). Levels of neutrophil elastase also positively correlate with changes in total neutrophil counts, IL-8, IL-1ß and matrix metalloproteinase (MMP)-9 [18 ]. The authors evaluated multiple biomarkers but found neutrophil elastase was the superior marker &
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to predict disease progression [18 ]. The area under the receiver operator characteristic curve to predict rapid FEV1 decline was 0.68, but this was improved by including a panel of additional biomarkers. Thus, measurement of neutrophil elastase may provide a method of identifying patients at risk of rapid disease progression for early intervention. This requires validation in larger cohorts and identification of a clinically important ‘cut-off point’ for therapy, but nevertheless underlines the great importance of neutrophil elastase in CF pathogenesis. Advances have also been made in our mechanistic understanding of the effects of neutrophil elastase on the airway. The known effects of neutrophil elastase on bronchial epithelium, neutrophils and other airway leukocytes have been extensively reviewed [19]. New findings in the past year include that neutrophil elastase triggers the expression of senescence markers on bronchial epithelial cells. Senescence, which is a complete loss of replicative capacity, is pro-inflammatory as senescent cells secrete large amount of proinflammatory cytokines and MMPs [20]. Airway sections from patients with CF and controls were compared, with CF epithelium having increased expression of three senescence markers, p16, gH2A.X and phospho-Chk2 [21 ]. In-vitro neutrophil elastase exposure significantly increased p16 expression and decreased CKD4 activity in human bronchial epithelial cells, supporting the hypothesis that neutrophil elastase triggers senescence in CF airways [21 ]. Neutrophil elastase degrades many innate antimicrobial defences, and a recent study has identified that neutrophil elastase also cleaves the epithelial-derived antimicrobial midkine, demonstrating another mechanism by which neutrophil elastase indirectly promotes Pseudomonas aeruginosa infection by disabling the natural defence [22 ]. Another adverse effect of neutrophil elastase may be its ability to degrade the CFTR and disable its function. Neutrophil elastase is known to degrade a range of cell surface receptors including CD2, CD4 and CD8 on lymphocytes, phagocyte receptors on neutrophils such as complement receptor 1 and antigen presentation receptors on dendritic cells [22 ,23,24]. In a recent study, Le Gars et al. [25 ] demonstrate that neutrophil elastase degrades wild-type and DF508-epithelial CFTR in vitro and wild-type-CFTR in mice through an intracellular protease calpain pathway. This degradation has been shown to cause a loss of function in the CFTR by patch-clamping in vitro and by nasal potential difference recording in vivo. The authors went on to suggest that this mechanism occurred during P. aeruginosa infection and that &
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The role of neutrophils in cystic fibrosis Gifford and Chalmers
this was neutrophil elastase-dependent as CFTR activity was significantly protected in neutrophil elastase / mice [25 ]. This study has important implications not just in CF, as CFTR cleavage and dysfunction occurred in wild-type mice during infection. Neutrophil elastase cleavage of CFTR may therefore contribute to the pathogenesis of other inflammatory lung diseases such as chronic obstructive pulmonary disease and non-CF bronchiectasis where neutrophil elastase is also elevated [26]. Some of the known and recently described effects of neutrophil elastase are summarized in Fig. 2. &&
NEUTROPHIL EXTRACELLULAR TRAPS The novel form of neutrophil cell death with release of neutrophil extracellular traps (NETs) has recently been elegantly demonstrated with live in-vivo imaging of their formation [27]. NETs are composed of a DNA backbone with entangled histones and antimicrobial neutrophil granule components. Although used to ensnare and kill microbes, it is also hypothesized that the associated extracellular proteases and ROS may contribute to host tissue damage. Although research into the relevance of NETs in CF is still in its infancy, a recent study has shown that NETosis in peripheral blood neutrophils is dependent on ROS generation and particularly on the generation of HOCl [28 ]. Since &
generation of HOCl requires chloride channels including CFTR [28 ], this has potential implications for NET generation in CF. Given the importance of neutrophil proteases in CF pathogenesis, a fascinating recent finding was that DNA interaction with neutrophil elastase inhibits its activity [29 ]. DNase treatment dramatically increased elastase activity. These data suggest that NETs provide a kind of reservoir of inactive extracellular protease that is released upon NET degradation. Thus NETs may serve to protect the host from unregulated active protease. The implications of this for future antiproteinase and recombinant human DNase treatment are thus far unclear. There is increasing research into how bacteria evade NET-mediated killing or use NETs to promote airway inflammation. Recently, it was shown that pyocyanin, a P. aeruginosa virulence factor [30], enhances in-vitro NET formation in a time and dose-dependent manner [31 ], the first bacterial toxin found to do so. As with NET formation generally, this effect was NADPH oxidase-dependent [32]. Therefore, NET production induced by pyocyanin is a novel mechanism contributing to the immune dysfunction in CF. &
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THERAPIES TARGETING NEUTROPHILS The overwhelming evidence for two decades linking neutrophil elastase with disease progression in CF
Neutrophil
Neutrophil elastase (NE) Airway epithelium
p16
ENaC cleavage
CFTR disabling
Mucus production
Senescence
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FIGURE 2. Impact of neutrophil elastase on epithelial cells and neutrophils in cystic fibrosis. Elastase, represented by the scissors, has multiple reported effects including degradation of CFTR in addition to the epithelial sodium channel ENaC, thereby increasing mucus production. A new finding suggests neutrophil elastase may induce epithelial senescence. CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel. 1065-6251 ß 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
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Myeloid biology
has led to the development of neutrophil elastase inhibitors, with multiple compounds now entering clinical trials [33 ]. Elborn et al. [33 ] have published a phase IIa study in 56 patients of an oral neutrophil elastase inhibitor, designated AZD9668. Preclinical studies have shown AZD9668 to reversibly inhibit neutrophil elastase activity and to be highly potent and selective in vitro [34]. After 28 days of treatment, there was no effect on symptoms, lung function, sputum neutrophil counts, sputum weight or sputum neutrophil elastase activity compared with placebo. However, there was a significant reduction in sputum inflammatory markers IL-6 and RANTES (regulated on activation, normal T cell expressed and secreted), as well as a reduction in free and total urine desmosine [34]. AZD9886 was well tolerated and therefore is a promising subject of future research, although a larger sample size and &&
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longer duration of treatment may be required in order to see significant effects on clinical parameters. Neutrophil elastase inhibitors are not the only drugs that may target neutrophil-mediated inflammation. Macrolides are widely used in CF following randomized controlled trials showing improved clinical outcomes in patients with P. aeruginosa infection [35]. A large trial of the macrolide azithromycin in patients without P. aeruginosa did not find improvements in pulmonary function, but did show a reduction in exacerbations, leaving the role of macrolides in this group less certain [36]. In a recent sub-analysis of this study including 260 paediatric CF patients [37 ], absolute neutrophil count (ANC), the acute phase marker serum amyloid A (SAA), the neutrophil protein calprotectin, C-reactive protein &
ERK 1/2 p38 JNK
Doxycycline
Airway epithelium
IL-8, MMP-9
PTX3
SB-656933
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Pseudomonas
AZD9668 Neutrophil elastase (NE) GS-459755
ENaC Airway epithelium
FIGURE 3. Novel therapeutics targeting neutrophils and neutrophil dysfunction in cystic fibrosis. AZD9668 is an oral neutrophil elastase (NE) inhibitor recently reported in a phase IIa clinical trial. GS-459755 is a non-antibiotic macrolide which decreases NE-induced surface liquid volume in vitro. SB-656933 is a CXCR2 antagonist in early-phase clinical trials. Recombinant pentraxin-3 (PTX3) opsonizes Pseudomonas aeruginosa and results in a reduction in bacterial CFU in a murine model. Doxycycline is a tetracycline antibiotic that inhibits pro-inflammatory cytokine and matrix metalloproteinase-9 (MMP-9) release from epithelial cells. ENaC, epithelial sodium channel; ERK, extracellular signal-regulated kinase; IL-8, interleukin 8; JNK, c-Jun N-terminal kinase; MMP-9, matrix metalloproteinase 9. 20
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The role of neutrophils in cystic fibrosis Gifford and Chalmers
(CRP) and myeloperoxidase decreased significantly after 28 days of azithromycin treatment, with no changes seen with placebo. These changes positively correlated with improvement in lung function and weight, and at 168 days the treatment effect was sustained for ANC, SAA and calprotectin. The benefits are attributed to the anti-inflammatory properties of macrolides rather than their antimicrobial activity. Since antimicrobial macrolides may promote resistance, development of nonantibiotic macrolides would be desirable. Preclinical in-vitro testing of such a compound (GS-459755) has now been reported [38 ]. No effect was shown on neutrophil elastase activity in vitro, but pretreatment of human bronchial epithelial cells with GS-459755 was shown to reduce neutrophil elastaseinduced surface liquid depletion without affecting ciliary beat frequency. These agents are still some way from clinical use. Another antibiotic class with anti-inflammatory properties in the CF airway are the tetracyclines. Doxycycline has been shown to disrupt the TNFa-stimulated signalling pathways extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK) in CF bronchial epithelial cell lines [39 ]. At concentrations below the cytotoxic potential, this led to dose-dependent reductions in IL-8 and also inhibition of the activity and production of MMP-9. The role of tetracyclines in CF treatment algorithms is currently poorly defined, but they remain effective antimicrobials with potentially beneficial anti-inflammatory properties [39 ]. CXCR1 and CXCR2 are neutrophil cell surface receptors also found on subsets of T cells, macrophages, dendritic cells and mast cells. CXCR2 antagonists are hypothetically able to inhibit neutrophil recruitment but not microbial killing, which is performed through CXCR1 [40]. A recent clinical trial reported on a novel oral CXCR2 antagonist, SB-656933. The primary outcome of the study was safety over 28 days of treatment with 146 patients randomized to low-dose or high-dose active treatment or placebo. At high dose, the active drug reduced sputum neutrophils and elastase compared to baseline, but increased blood inflammatory markers CRP and IL-8 with no effect on lung function [41 ]. Determining whether these changes in airway inflammation will translate into clinical benefits will require larger and longer studies. Moving from clinical to experimental studies, enhancing opsonophagocytosis of bacteria by neutrophils represents a novel therapeutic strategy. Reduced levels of soluble pattern recognition receptors (PRRs) such as mannose-binding lectin [42], &
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ficolins [43 ] and the pentraxins [44] have been reported to be associated with disease severity in CF and other chronic lung diseases [45]. Replacement or augmentation of PRRs offers a new approach to therapy. In a recent study, CFTR null and wild-type mice were chronically infected with multi-drug resistant P. aeruginosa and subsequently treated with recombinant pentraxin-3 (PTX3) [46 ]. After treatment with PTX3, a significant reduction in colony forming units (CFU) was seen to a level even below that of the normal mice. Furthermore, in whole blood assay, pre-opsonization of P. aeruginosa with recombinant PTX3 significantly increased phagocytosis and killing by CF neutrophils. This new approach to CF therapeutics is worthy of further research. Some of the novel neutrophil-based approaches to CF therapy are summarized in Fig. 3. &&
CONCLUSION Neutrophils drive CF lung disease. The past 12 months have seen significant advances in our understanding of airway neutrophil dysfunction. These advances are leading directly to new therapies for the disease. Acknowledgements The authors thank Dr Hugh Gifford for assistance with the artwork. Conflicts of interest There are no conflicts of interest.
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Myeloid biology 8. Zhou Y, Song K, Painter RG, et al. Cystic fibrosis transmembrane & conductance regulator recruitment to phagosomes in neutrophils. J Innate Immun 2013; 5:219–230. Evidence of the mechanism behind defective bacterial killing in CF neutrophils. 9. Bonvillain RW, Painter RG, Adams DE, et al. RNA interference against CFTR affects HL60-derived neutrophil microbicidal function. Free Radic Biol Med 2010; 49:1872–1880. 10. Montemurro P, Mariggio` MA, Barbuti G, et al. Increase in interleukin-8 production from circulating neutrophils upon antibiotic therapy in cystic fibrosis patients. J Cyst Fibros 2012; 11:518–524. 11. Houston N, Stewart N, Smith DS, et al. Sputum neutrophils in cystic fibrosis & patients display a reduced respiratory burst. J Cyst Fibros 2013; 12:352– 362. Suggestion of different ‘sub-populations’ of neutrophils in the airway lumen and blood. 12. Laval J, Touhami J, Herzenberg LA, et al. Metabolic adaptation of neutrophils & in cystic fibrosis airways involves distinct shifts in nutrient transporter expression. J Immunol 2013; 190:6043–6050. A further advance to our understanding of how the neutrophil adapts to the unique inflammatory environment of the CF lung. 13. Taggart C, Coakley RJ, Greally P, et al. Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8. Am J Physiol Lung Cell Mol Physiol 2000; 278:L33–L41. 14. Caldwell RA, Boucher RC, Stutts MJ. Neutrophil elastase activates near-silent epithelial Naþ channels and increased airway epithelial Naþ transport. Am J Physiol Lung Cell Mol Physiol 2005; 288:L813–L819. 15. Stockley RA. Neutrophils and protease/antiprotease imbalance. Am J Respir Crit Care Med 1999; 160:S49–S52. 16. Sly PD, Gangell CL, Chen L, et al. Risk factors for bronchiectasis in children && with cystic fibrosis. N Engl J Med 2013; 368:1963–1970. A significant finding, which suggests that neutrophil elastase activity in BAL fluid in early life is associated with the development of bronchiectasis and lung function decline in CF. 17. Laguna TA, Wagner BD, Starcher B, et al. Urinary desmosine: a biomarker & of structural lung injury during CF pulmonary exacerbation. Pediatr Pulmonol 2012; 47:856–863. The development of urine desmosine as a marker of systemic elastin degradation promises to allow non-invasive assessment of neutrophil elastase activity. 18. Sagel SD, Wagner BD, Anthony MM, et al. Sputum biomarkers of inflam& mation and lung function decline in children with cystic fibrosis. Am J Respir Crit Care Med 2012; 186:857–865. Further evidence for neutrophil elastase as a biomarker in CF, predicting lung function decline. 19. Chalmers JD, Hill AT. Mechanisms of immune dysfunction and bacterial persistence in noncystic fibrosis bronchiectasis. Mol Immunol 2013; 55: 27–34. 20. Tsuji T, Aoshiba K, Nagai A. Alveolar cell senescence exacerbates pulmonary inflammation in patients with chronic obstructive pulmonary disease. Respiration 2010; 80:59–70. 21. Fischer BM, Wong JK, Degan S. Increased expression of senescence & markers in cystic fibrosis airways. Am J Physiol Lung Cell Mol Physiol 2013; 304:L394–L400. Evidence suggesting that neutrophil elastase can induce senescence in CF airway epithelial cells in vitro and perhaps in vivo. 22. Nordin SL, Jovic S, Kurut A, et al. High expression of midkine in the airways of & patients with cystic fibrosis. Am J Respir Cell Mol Biol 2013. [Epub ahead of print]. Describes an epithelial derived antimicrobial elevated in the CF lung that is cleaved and inactivated by neutrophil elastase. 23. Do¨ring G, Frank F, Boudier C, et al. Cleavage of lymphocyte surface antigens CD2, CD4, and CD8 by polymorphonuclear leukocyte elastase and cathepsin G in patients with cystic fibrosis. J Immunol 1995; 154:4842– 4850. 24. Roghanian A, Drost EM, MacNee W, et al. Inflammatory lung secretions inhibit dendritic cell maturation and function via neutrophil elastase. Am J Respir Crit Care Med 2006; 174:1189–1198. 25. Le Gars M, Descamps D, Roussel D, et al. Neutrophil elastase degrades && cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo. Am J Respir Crit Care Med 2013; 187:170–179. Evidence that neutrophil elastase degrades wild-type and DF508-epithelial CFTR in vitro that has implications for other chronic lung diseases. 26. Chalmers JD, Smith MP, McHugh B, et al. Short and long term antibiotic therapy reduces airway and systemic inflammation in non-CF bronchiectasis. Am J Respir Crit Care Med 2012; 186:657–665.
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27. Yipp BG, Petri B, Salina D, et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 2012; 18:1386– 1393. 28. Akong-Moore K, Chow OA, von Kockritz-Blickwede M, Nizet V. Influences of & chloride and hypochlorite on neutrophil extracellular trap formation. PloS One 2012; 7:e42984. Describes a potential link between CFTR and NET formation. 29. Dubois AV, Gauthier A, Bre´a D, et al. Influence of DNA on the activities and && inhibition of neutrophil serine proteases in cystic fibrosis sputum. Am J Respir Cell Mol Biol 2012; 47:80–86. An important finding that DNA reduces the activity of neutrophil serine proteases, suggesting NETs function to limit protease induced damage. 30. Lau GW, Ran H, Kong F, et al. Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice. Infect Immun 2004; 72:4275–4278. 31. Rada B, Jendrysik MA, Pang L, et al. Pyocyanin-enhanced neutrophil extra& cellular trap formation requires the NADPH oxidase. PLoS One 2013; 8:e54205. NET production induced by pyocyanin is a novel mechanism contributing to the immune dysfunction in CF. 32. Gray RD, Lucas CD, Mackellar A, et al. Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps. J Inflamm (Lond) 2013; 10:12. 33. Elborn JS, Perrett J, Forsman-Semb K, et al. Efficacy, safety and effect on && biomarkers of AZD9668 in cystic fibrosis. Eur Respir J 2012; 40:969–976. Phase IIa clinical study of a neutrophil elastase inhibitor in CF. 34. Stevens T, Ekholm K, Gra¨nse M, et al. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase. J Pharmacol Exp Ther 2011; 339:313–320. 35. Saiman L, Marshall BC, Mayer-Hamblett N, et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomised controlled trial. J Am Med Assoc 2003; 290:1749–1756. 36. Saiman L, Anstead M, Mayer-Hamblett N, et al. Effects of azithromycin on pulmonary function in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a randomized controlled trial. J Am Med Assoc 2010; 303:1707–1715. 37. Ratjen F, Saiman L, Mayer-Hamblett N, et al. Effect of azithromycin on & systemic markers of inflammation in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa. Chest 2012; 142:1259–1266. Recent trial that updates information on the use of azithromycin in CF without P. aeruginosa showing improvement in lung function and markers of infection. 38. Tarran R, Sabater JR, Clarke TC, et al. Nonantibiotic macrolides prevent & human neutrophil elastase-induced mucus stasis and airway surface liquid volume depletion. Am J Physiol Lung Cell Mol Physiol 2013; 304:L746– L756. Describes a novel macrolide anti-inflammatory with reduced antibacterial function. 39. Bensman TJ, Nguyen AN, Rao AP, Beringer PM. Doxycycline exhibits anti& inflammatory activity in CF bronchial epithelial cells. Pulm Pharmacol Ther 2012; 25:377–382. In-vitro demonstration of the anti-inflammatory properties of doxycycline, reducing IL-8 and MMPs. 40. Hartl D, Latzin P, Hordijk P, et al. Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat Med 2007; 13:1423–1430. 41. Moss RB, Mistry SJ, Konstan MW, et al. Safety and early treatment effects of & the CXCR2 antagonist SB-656933 in patients with cystic fibrosis. J Cyst Fibros 2013; 12:241–248. An early stage trial of a CXCR2 inhibitor in cystic fibrosis. 42. Chalmers JD, Fleming GB, Hill AT, Kilpatrick DC. Impact of mannose binding lectin (MBL) insufficiency on the course of cystic fibrosis: a review and metaanalysis. Glycobiology 2011; 21:271–282. 43. Haerynck F, Van Steen K, Cattaert T, et al. Polymorphisms in the lectin & pathway genes as a possible cause of early chronic Pseudomonas aeruginosa colonization in cystic fibrosis patients. Hum Immunol 2012; 73:1175–1183. Interesting small study relating genetic deficiency in the opsonins ficolin-1 and ficolin-2 with P. aeruginosa colonization in CF. 44. Chiarini M, Sabelli C, Melotti P, et al. PTX3 genetic variations affect the risk of Pseudomonas aeruginosa airway colonization in cystic fibrosis patients. Genes Immun 2010; 11:665–670. 45. Chalmers JD, McHugh BJ, Doherty CJ, et al. Mannose binding lectin deficiency and disease severity in non-CF bronchiectasis: a prospective study. Lancet Respir Med 2013; 1:175–274. 46. Paroni M, Moalli F, Nebuloni M, et al. Response of CFTR-deficient mice to && long-term chronic Pseudomonas aeruginosa infection and PTX3 therapy. J Infect Dis 2013; 208:130–138. New approach to CF therapeutics in experimental stages enhancing opsonophagocytosis of bacteria with the long pentraxin, pentraxin-3.
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The role of neutrophils in cystic fibrosis Alison M. Gifford a and James D. Chalmers b
Purpose of review Neutrophils are known to dominate the pulmonary inflammatory process observed in cystic fibrosis (CF). An enduring paradox is how these large numbers of neutrophils fail to eradicate colonizing bacteria. Major advances in our understanding of neutrophil dysfunction in CF and its effect on the innate immune system are leading to advances in our understanding of the pathophysiology and leading directly to new therapies. Recent findings New mechanisms of neutrophil dysfunction have been described in CF including disabled cystic fibrosis transmembrane conductance regulator recruitment to phagosomes and novel mechanisms of proteaseinduced neutrophil dysfunction. Neutrophil elastase has been shown to be present in the airway very early in life in CF patients, and appears a biomarker of disease progression, predicting lung function decline and bronchiectasis. Elastase has also been shown to induce a pro-inflammatory state of senescence in bronchial epithelial cells in vitro and potentially in vivo. Inhibitors of neutrophil elastase are now entering clinical trials with promising results. New avenues of CF therapeutics are being explored including novel macrolides, CXCR2 antagonists and exogenous opsonins. Summary This article reviews the past 12 months of research that contributes to our understanding of the role of neutrophils and immune dysfunction in CF. Keywords cystic fibrosis, cystic fibrosis transmembrane conductance regulator neutrophil elastase, neutrophils
INTRODUCTION Cystic fibrosis (CF) is the most common lethal inherited autosomal recessive disorder amongst Caucasians [1]. The clinical syndrome of CF results from an inherited defect in the cystic fibrosis transmembrane conductance regulator (CFTR) leading to airway obstruction by defective mucus clearance, infection and progressive bronchiectasis [2]. Failure of chloride secretion and sodium hyper-absorption at the apical airway surface leads to dehydration of airway surface liquid and impaired clearance of bacterial pathogens [3 ]. The resulting inflammatory process observed in CF is dominated by the presence of neutrophils, driving a vicious cycle of bacterial colonization, airway inflammation and structural damage that overwhelms normal resolution and repair pathways. These contribute to the irreversible lung damage, worsening of lung function and premature death of these patients [4]. Increasingly, therapies targeting the CF neutrophil are moving from experimental to translational and clinical studies, offering hope of a new avenue of therapy for this devastating disease. &
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This article reviews the past 12 months of research that contributes to our understanding of the role of neutrophils and immune dysfunction in CF.
NEW MECHANISMS OF NEUTROPHIL DYSFUNCTION IN CYSTIC FIBROSIS It is a paradox that the large numbers of neutrophils present in the CF airway fail to effectively kill colonizing bacteria. CFTR is expressed in leukocytes, including neutrophils, leading to the hypothesis that CFTR dysfunction may have effects on neutrophil function beyond those resulting from impaired mucociliary clearance. By contrasting neutrophil effector responses in healthy and CF neutrophils, a Department of Paediatrics, Ninewells Hospital and bTayside Respiratory Research Group, University of Dundee, Dundee, UK
Correspondence to Dr James D. Chalmers, Tayside Respiratory Research Group, University of Dundee, Dundee DD1 9SY, UK. Tel: +44 1382386339; e-mail: [email protected] Curr Opin Hematol 2014, 21:16–22 DOI:10.1097/MOH.0000000000000009 Volume 21 Number 1 January 2014
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The role of neutrophils in cystic fibrosis Gifford and Chalmers
KEY POINTS New mechanisms of neutrophil dysfunction have been found in CF including innate defects in CF neutrophils prior to migration to the airway and profound effects of neutrophilic inflammation on the airway epithelium. Neutrophil elastase is now known to be present very early in life in CF patients at higher levels, corresponding to earlier and more severe lung function decline and bronchiectasis. Neutrophil elastase inhibitors are now entering clinical trials. New avenues of CF therapeutics are being explored including novel macrolides and exogenous opsonization of bacteria.
previous studies have reported abnormalities in CF neutrophil function including priming, enhanced granule enzyme activity, increased reactive oxygen species (ROS) production [5], increased interleukin (IL)-8 secretion [6] and delayed apoptosis [7]. Zhou et al. [8 ] provide further evidence of the role of CFTR in intracellular bacterial killing by describing disabled recruitment of the CFTR protein to neutrophil phagosomes. CFTR is one of the chloride channels required for generation of hypochlorous acid (HOCl), a toxic agent involved in intracellular bacterial killing. It was previously demonstrated that CF neutrophils were defective in their ability to kill intracellular pathogens and that this could be recreated by CFTR knockdown using small interfering RNA in neutrophil cell lines [9]. In this study, the authors demonstrate that more than 95% of mature phagosomes (stained with the well recognized phagosome marker lysosomalassociated membrane protein 1; LAMP-1) also stained positive for CFTR in healthy peripheral blood neutrophils [8 ]. Using an enhanced green fluorescent protein (EGFP)–CFTR fusion protein, the authors demonstrated significantly less EGFP– DF508-CFTR localizing to phagosomes compared with EGFP–wild-type-CFTR, indicating a defect in the migration of the protein to the phagosomal membrane. This was improved by the pharmacological CFTR chaperone voltage regulator tester 325 (VRT-325). This provides evidence of the mechanism behind defective bacterial killing in CF neutrophils [8 ]. A recent small study investigated peripheral neutrophil inflammatory responses in the context of acute exacerbations of CF. Fourteen patients with CF and 10 healthy controls were studied. The authors reported ROS and IL-8 release were &
higher in CF than control at baseline (prior to antibiotic therapy) with a higher early apoptotic response [10]. When treated with antibiotics, there was an increase in IL-8 secretion by neutrophils, which the authors argue is a potential biomarker for response to therapy. The early apoptotic response opposes previous findings which have always suggested prolonged neutrophil survival in CF [7]. Stimulated peripheral blood neutrophils from patients with CF have been reported to release significantly more ROS than neutrophils from healthy controls [5]. A recent study examined ROS production in sputum neutrophils and found the converse, with reduced ROS production in sputum neutrophils as measured by flow cytometry [11 ]. These studies are complicated by the difficulties in isolating neutrophils from sputum and particularly in isolating viable cells from healthy donor airways for comparison. The authors speculate that there are subpopulations of neutrophils within the CF airway displaying what the authors call ‘functional exhaustion’ [11 ]. Metabolic adaptation of neutrophils on migration into the airway lumen with increased glucose and amino acid transporters is an example of changes in transcriptional profile as the neutrophil is primed, activated and transmigrates into the airway [12 ]. This is consistent with prior demonstration of the reprogramming of neutrophils, mostly by tumour necrosis factor (TNF)-a and IL-8 [13], that occurs when they arrive in the CF lung. A complete understanding of the diverse ways in which neutrophils adapt to the CF airway remains elusive (Fig. 1). &
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The neutrophil in cystic fibrosis
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Increased glucose and amino acid transporters
Reduced phagosomal CFTR
Altered apoptosis
Altered ROS
Increased IL-8
FIGURE 1. Innnate defects in neutrophil function in cystic fibrosis. New data suggest reduced cystic fibrosis transmembrane conductance regulator (CFTR) localization to phagosomes. Previously reported data about alterations in apoptosis and reactive oxygen species (ROS) generation have been challenged.
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NEUTROPHIL ELASTASE
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Neutrophil elastase is a serine protease contained in azurophil (primary) granules designed to digest phagocytosed bacterial proteins. Neutrophil elastase has, however, been implicated in the pathology of CF and shown to activate the epithelial sodium channel (ENaC) by direct cleavage [14], thereby contributing to airway surface mucus accumulation [3 ]. The primary defences against neutrophil elastase are endogenous antiproteinases such as a1-antitrypsin, secretory leukocyte peptidase inhibitor (SLPI) and elafin [15]. In CF, the quantity of elastase released into the airway overwhelms the antiproteinase capacity leading to detectable elastase activity. A major contribution to understanding the role of neutrophil elastase in disease progression was made this year by Sly et al. [16 ] as part of the Australian Respiratory Early Surveillace Team for Cystic Fibrosis (AREST-CF) study. This study followed 127 infants diagnosed with CF by newborn screening and performed computed tomography (CT) and bronchoalveolar lavage (BAL) at 3 months, 1, 2 and 3 years of age. Free neutrophil elastase activity in BAL fluid at 3 months was associated with persistent bronchiectasis on two or more sequential CT scans [adjusted odds ratio (OR) 7.20 at 12 months and 4.21 for persistent bronchiectasis at 3 years of age]. As has been previously demonstrated in a number of contexts, elastase activity was associated with the presence of bacterial colonization, but the relationship persisted even after statistical adjustment for bacterial colonization [16 ]. This is a significant finding, which suggests that neutrophil elastase activity in BAL fluid in early life is associated with the early development of bronchiectasis in CF. Early identification of patients at risk of disease progression has therapeutic value, although detection of elastase activity in young children without the requirement for BAL remains an obstacle to clinical implementation. Surrogates such as urinary desmosine which measure systematic elastin breakdown are a possible solution but remain to be tested in this group [17 ]. Further evidence that neutrophil elastase may be a useful biomarker in CF came from a small study by Sagel et al. [18 ] of 35 school-aged children. Measuring longitudinal relationships over 3 years, those with higher sputum neutrophil elastase levels at first measurement had a significantly higher rate of decline in forced expiratory volume in 1 s (FEV1). Levels of neutrophil elastase also positively correlate with changes in total neutrophil counts, IL-8, IL-1ß and matrix metalloproteinase (MMP)-9 [18 ]. The authors evaluated multiple biomarkers but found neutrophil elastase was the superior marker &
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to predict disease progression [18 ]. The area under the receiver operator characteristic curve to predict rapid FEV1 decline was 0.68, but this was improved by including a panel of additional biomarkers. Thus, measurement of neutrophil elastase may provide a method of identifying patients at risk of rapid disease progression for early intervention. This requires validation in larger cohorts and identification of a clinically important ‘cut-off point’ for therapy, but nevertheless underlines the great importance of neutrophil elastase in CF pathogenesis. Advances have also been made in our mechanistic understanding of the effects of neutrophil elastase on the airway. The known effects of neutrophil elastase on bronchial epithelium, neutrophils and other airway leukocytes have been extensively reviewed [19]. New findings in the past year include that neutrophil elastase triggers the expression of senescence markers on bronchial epithelial cells. Senescence, which is a complete loss of replicative capacity, is pro-inflammatory as senescent cells secrete large amount of proinflammatory cytokines and MMPs [20]. Airway sections from patients with CF and controls were compared, with CF epithelium having increased expression of three senescence markers, p16, gH2A.X and phospho-Chk2 [21 ]. In-vitro neutrophil elastase exposure significantly increased p16 expression and decreased CKD4 activity in human bronchial epithelial cells, supporting the hypothesis that neutrophil elastase triggers senescence in CF airways [21 ]. Neutrophil elastase degrades many innate antimicrobial defences, and a recent study has identified that neutrophil elastase also cleaves the epithelial-derived antimicrobial midkine, demonstrating another mechanism by which neutrophil elastase indirectly promotes Pseudomonas aeruginosa infection by disabling the natural defence [22 ]. Another adverse effect of neutrophil elastase may be its ability to degrade the CFTR and disable its function. Neutrophil elastase is known to degrade a range of cell surface receptors including CD2, CD4 and CD8 on lymphocytes, phagocyte receptors on neutrophils such as complement receptor 1 and antigen presentation receptors on dendritic cells [22 ,23,24]. In a recent study, Le Gars et al. [25 ] demonstrate that neutrophil elastase degrades wild-type and DF508-epithelial CFTR in vitro and wild-type-CFTR in mice through an intracellular protease calpain pathway. This degradation has been shown to cause a loss of function in the CFTR by patch-clamping in vitro and by nasal potential difference recording in vivo. The authors went on to suggest that this mechanism occurred during P. aeruginosa infection and that &
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The role of neutrophils in cystic fibrosis Gifford and Chalmers
this was neutrophil elastase-dependent as CFTR activity was significantly protected in neutrophil elastase / mice [25 ]. This study has important implications not just in CF, as CFTR cleavage and dysfunction occurred in wild-type mice during infection. Neutrophil elastase cleavage of CFTR may therefore contribute to the pathogenesis of other inflammatory lung diseases such as chronic obstructive pulmonary disease and non-CF bronchiectasis where neutrophil elastase is also elevated [26]. Some of the known and recently described effects of neutrophil elastase are summarized in Fig. 2. &&
NEUTROPHIL EXTRACELLULAR TRAPS The novel form of neutrophil cell death with release of neutrophil extracellular traps (NETs) has recently been elegantly demonstrated with live in-vivo imaging of their formation [27]. NETs are composed of a DNA backbone with entangled histones and antimicrobial neutrophil granule components. Although used to ensnare and kill microbes, it is also hypothesized that the associated extracellular proteases and ROS may contribute to host tissue damage. Although research into the relevance of NETs in CF is still in its infancy, a recent study has shown that NETosis in peripheral blood neutrophils is dependent on ROS generation and particularly on the generation of HOCl [28 ]. Since &
generation of HOCl requires chloride channels including CFTR [28 ], this has potential implications for NET generation in CF. Given the importance of neutrophil proteases in CF pathogenesis, a fascinating recent finding was that DNA interaction with neutrophil elastase inhibits its activity [29 ]. DNase treatment dramatically increased elastase activity. These data suggest that NETs provide a kind of reservoir of inactive extracellular protease that is released upon NET degradation. Thus NETs may serve to protect the host from unregulated active protease. The implications of this for future antiproteinase and recombinant human DNase treatment are thus far unclear. There is increasing research into how bacteria evade NET-mediated killing or use NETs to promote airway inflammation. Recently, it was shown that pyocyanin, a P. aeruginosa virulence factor [30], enhances in-vitro NET formation in a time and dose-dependent manner [31 ], the first bacterial toxin found to do so. As with NET formation generally, this effect was NADPH oxidase-dependent [32]. Therefore, NET production induced by pyocyanin is a novel mechanism contributing to the immune dysfunction in CF. &
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THERAPIES TARGETING NEUTROPHILS The overwhelming evidence for two decades linking neutrophil elastase with disease progression in CF
Neutrophil
Neutrophil elastase (NE) Airway epithelium
p16
ENaC cleavage
CFTR disabling
Mucus production
Senescence
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FIGURE 2. Impact of neutrophil elastase on epithelial cells and neutrophils in cystic fibrosis. Elastase, represented by the scissors, has multiple reported effects including degradation of CFTR in addition to the epithelial sodium channel ENaC, thereby increasing mucus production. A new finding suggests neutrophil elastase may induce epithelial senescence. CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel. 1065-6251 ß 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
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Myeloid biology
has led to the development of neutrophil elastase inhibitors, with multiple compounds now entering clinical trials [33 ]. Elborn et al. [33 ] have published a phase IIa study in 56 patients of an oral neutrophil elastase inhibitor, designated AZD9668. Preclinical studies have shown AZD9668 to reversibly inhibit neutrophil elastase activity and to be highly potent and selective in vitro [34]. After 28 days of treatment, there was no effect on symptoms, lung function, sputum neutrophil counts, sputum weight or sputum neutrophil elastase activity compared with placebo. However, there was a significant reduction in sputum inflammatory markers IL-6 and RANTES (regulated on activation, normal T cell expressed and secreted), as well as a reduction in free and total urine desmosine [34]. AZD9886 was well tolerated and therefore is a promising subject of future research, although a larger sample size and &&
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longer duration of treatment may be required in order to see significant effects on clinical parameters. Neutrophil elastase inhibitors are not the only drugs that may target neutrophil-mediated inflammation. Macrolides are widely used in CF following randomized controlled trials showing improved clinical outcomes in patients with P. aeruginosa infection [35]. A large trial of the macrolide azithromycin in patients without P. aeruginosa did not find improvements in pulmonary function, but did show a reduction in exacerbations, leaving the role of macrolides in this group less certain [36]. In a recent sub-analysis of this study including 260 paediatric CF patients [37 ], absolute neutrophil count (ANC), the acute phase marker serum amyloid A (SAA), the neutrophil protein calprotectin, C-reactive protein &
ERK 1/2 p38 JNK
Doxycycline
Airway epithelium
IL-8, MMP-9
PTX3
SB-656933
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Pseudomonas
AZD9668 Neutrophil elastase (NE) GS-459755
ENaC Airway epithelium
FIGURE 3. Novel therapeutics targeting neutrophils and neutrophil dysfunction in cystic fibrosis. AZD9668 is an oral neutrophil elastase (NE) inhibitor recently reported in a phase IIa clinical trial. GS-459755 is a non-antibiotic macrolide which decreases NE-induced surface liquid volume in vitro. SB-656933 is a CXCR2 antagonist in early-phase clinical trials. Recombinant pentraxin-3 (PTX3) opsonizes Pseudomonas aeruginosa and results in a reduction in bacterial CFU in a murine model. Doxycycline is a tetracycline antibiotic that inhibits pro-inflammatory cytokine and matrix metalloproteinase-9 (MMP-9) release from epithelial cells. ENaC, epithelial sodium channel; ERK, extracellular signal-regulated kinase; IL-8, interleukin 8; JNK, c-Jun N-terminal kinase; MMP-9, matrix metalloproteinase 9. 20
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(CRP) and myeloperoxidase decreased significantly after 28 days of azithromycin treatment, with no changes seen with placebo. These changes positively correlated with improvement in lung function and weight, and at 168 days the treatment effect was sustained for ANC, SAA and calprotectin. The benefits are attributed to the anti-inflammatory properties of macrolides rather than their antimicrobial activity. Since antimicrobial macrolides may promote resistance, development of nonantibiotic macrolides would be desirable. Preclinical in-vitro testing of such a compound (GS-459755) has now been reported [38 ]. No effect was shown on neutrophil elastase activity in vitro, but pretreatment of human bronchial epithelial cells with GS-459755 was shown to reduce neutrophil elastaseinduced surface liquid depletion without affecting ciliary beat frequency. These agents are still some way from clinical use. Another antibiotic class with anti-inflammatory properties in the CF airway are the tetracyclines. Doxycycline has been shown to disrupt the TNFa-stimulated signalling pathways extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK) in CF bronchial epithelial cell lines [39 ]. At concentrations below the cytotoxic potential, this led to dose-dependent reductions in IL-8 and also inhibition of the activity and production of MMP-9. The role of tetracyclines in CF treatment algorithms is currently poorly defined, but they remain effective antimicrobials with potentially beneficial anti-inflammatory properties [39 ]. CXCR1 and CXCR2 are neutrophil cell surface receptors also found on subsets of T cells, macrophages, dendritic cells and mast cells. CXCR2 antagonists are hypothetically able to inhibit neutrophil recruitment but not microbial killing, which is performed through CXCR1 [40]. A recent clinical trial reported on a novel oral CXCR2 antagonist, SB-656933. The primary outcome of the study was safety over 28 days of treatment with 146 patients randomized to low-dose or high-dose active treatment or placebo. At high dose, the active drug reduced sputum neutrophils and elastase compared to baseline, but increased blood inflammatory markers CRP and IL-8 with no effect on lung function [41 ]. Determining whether these changes in airway inflammation will translate into clinical benefits will require larger and longer studies. Moving from clinical to experimental studies, enhancing opsonophagocytosis of bacteria by neutrophils represents a novel therapeutic strategy. Reduced levels of soluble pattern recognition receptors (PRRs) such as mannose-binding lectin [42], &
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ficolins [43 ] and the pentraxins [44] have been reported to be associated with disease severity in CF and other chronic lung diseases [45]. Replacement or augmentation of PRRs offers a new approach to therapy. In a recent study, CFTR null and wild-type mice were chronically infected with multi-drug resistant P. aeruginosa and subsequently treated with recombinant pentraxin-3 (PTX3) [46 ]. After treatment with PTX3, a significant reduction in colony forming units (CFU) was seen to a level even below that of the normal mice. Furthermore, in whole blood assay, pre-opsonization of P. aeruginosa with recombinant PTX3 significantly increased phagocytosis and killing by CF neutrophils. This new approach to CF therapeutics is worthy of further research. Some of the novel neutrophil-based approaches to CF therapy are summarized in Fig. 3. &&
CONCLUSION Neutrophils drive CF lung disease. The past 12 months have seen significant advances in our understanding of airway neutrophil dysfunction. These advances are leading directly to new therapies for the disease. Acknowledgements The authors thank Dr Hugh Gifford for assistance with the artwork. Conflicts of interest There are no conflicts of interest.
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Myeloid biology 8. Zhou Y, Song K, Painter RG, et al. Cystic fibrosis transmembrane & conductance regulator recruitment to phagosomes in neutrophils. J Innate Immun 2013; 5:219–230. Evidence of the mechanism behind defective bacterial killing in CF neutrophils. 9. Bonvillain RW, Painter RG, Adams DE, et al. RNA interference against CFTR affects HL60-derived neutrophil microbicidal function. Free Radic Biol Med 2010; 49:1872–1880. 10. Montemurro P, Mariggio` MA, Barbuti G, et al. Increase in interleukin-8 production from circulating neutrophils upon antibiotic therapy in cystic fibrosis patients. J Cyst Fibros 2012; 11:518–524. 11. Houston N, Stewart N, Smith DS, et al. Sputum neutrophils in cystic fibrosis & patients display a reduced respiratory burst. J Cyst Fibros 2013; 12:352– 362. Suggestion of different ‘sub-populations’ of neutrophils in the airway lumen and blood. 12. Laval J, Touhami J, Herzenberg LA, et al. Metabolic adaptation of neutrophils & in cystic fibrosis airways involves distinct shifts in nutrient transporter expression. J Immunol 2013; 190:6043–6050. A further advance to our understanding of how the neutrophil adapts to the unique inflammatory environment of the CF lung. 13. Taggart C, Coakley RJ, Greally P, et al. Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8. Am J Physiol Lung Cell Mol Physiol 2000; 278:L33–L41. 14. Caldwell RA, Boucher RC, Stutts MJ. Neutrophil elastase activates near-silent epithelial Naþ channels and increased airway epithelial Naþ transport. Am J Physiol Lung Cell Mol Physiol 2005; 288:L813–L819. 15. Stockley RA. Neutrophils and protease/antiprotease imbalance. Am J Respir Crit Care Med 1999; 160:S49–S52. 16. Sly PD, Gangell CL, Chen L, et al. Risk factors for bronchiectasis in children && with cystic fibrosis. N Engl J Med 2013; 368:1963–1970. A significant finding, which suggests that neutrophil elastase activity in BAL fluid in early life is associated with the development of bronchiectasis and lung function decline in CF. 17. Laguna TA, Wagner BD, Starcher B, et al. Urinary desmosine: a biomarker & of structural lung injury during CF pulmonary exacerbation. Pediatr Pulmonol 2012; 47:856–863. The development of urine desmosine as a marker of systemic elastin degradation promises to allow non-invasive assessment of neutrophil elastase activity. 18. Sagel SD, Wagner BD, Anthony MM, et al. Sputum biomarkers of inflam& mation and lung function decline in children with cystic fibrosis. Am J Respir Crit Care Med 2012; 186:857–865. Further evidence for neutrophil elastase as a biomarker in CF, predicting lung function decline. 19. Chalmers JD, Hill AT. Mechanisms of immune dysfunction and bacterial persistence in noncystic fibrosis bronchiectasis. Mol Immunol 2013; 55: 27–34. 20. Tsuji T, Aoshiba K, Nagai A. Alveolar cell senescence exacerbates pulmonary inflammation in patients with chronic obstructive pulmonary disease. Respiration 2010; 80:59–70. 21. Fischer BM, Wong JK, Degan S. Increased expression of senescence & markers in cystic fibrosis airways. Am J Physiol Lung Cell Mol Physiol 2013; 304:L394–L400. Evidence suggesting that neutrophil elastase can induce senescence in CF airway epithelial cells in vitro and perhaps in vivo. 22. Nordin SL, Jovic S, Kurut A, et al. High expression of midkine in the airways of & patients with cystic fibrosis. Am J Respir Cell Mol Biol 2013. [Epub ahead of print]. Describes an epithelial derived antimicrobial elevated in the CF lung that is cleaved and inactivated by neutrophil elastase. 23. Do¨ring G, Frank F, Boudier C, et al. Cleavage of lymphocyte surface antigens CD2, CD4, and CD8 by polymorphonuclear leukocyte elastase and cathepsin G in patients with cystic fibrosis. J Immunol 1995; 154:4842– 4850. 24. Roghanian A, Drost EM, MacNee W, et al. Inflammatory lung secretions inhibit dendritic cell maturation and function via neutrophil elastase. Am J Respir Crit Care Med 2006; 174:1189–1198. 25. Le Gars M, Descamps D, Roussel D, et al. Neutrophil elastase degrades && cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo. Am J Respir Crit Care Med 2013; 187:170–179. Evidence that neutrophil elastase degrades wild-type and DF508-epithelial CFTR in vitro that has implications for other chronic lung diseases. 26. Chalmers JD, Smith MP, McHugh B, et al. Short and long term antibiotic therapy reduces airway and systemic inflammation in non-CF bronchiectasis. Am J Respir Crit Care Med 2012; 186:657–665.
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27. Yipp BG, Petri B, Salina D, et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 2012; 18:1386– 1393. 28. Akong-Moore K, Chow OA, von Kockritz-Blickwede M, Nizet V. Influences of & chloride and hypochlorite on neutrophil extracellular trap formation. PloS One 2012; 7:e42984. Describes a potential link between CFTR and NET formation. 29. Dubois AV, Gauthier A, Bre´a D, et al. Influence of DNA on the activities and && inhibition of neutrophil serine proteases in cystic fibrosis sputum. Am J Respir Cell Mol Biol 2012; 47:80–86. An important finding that DNA reduces the activity of neutrophil serine proteases, suggesting NETs function to limit protease induced damage. 30. Lau GW, Ran H, Kong F, et al. Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice. Infect Immun 2004; 72:4275–4278. 31. Rada B, Jendrysik MA, Pang L, et al. Pyocyanin-enhanced neutrophil extra& cellular trap formation requires the NADPH oxidase. PLoS One 2013; 8:e54205. NET production induced by pyocyanin is a novel mechanism contributing to the immune dysfunction in CF. 32. Gray RD, Lucas CD, Mackellar A, et al. Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps. J Inflamm (Lond) 2013; 10:12. 33. Elborn JS, Perrett J, Forsman-Semb K, et al. Efficacy, safety and effect on && biomarkers of AZD9668 in cystic fibrosis. Eur Respir J 2012; 40:969–976. Phase IIa clinical study of a neutrophil elastase inhibitor in CF. 34. Stevens T, Ekholm K, Gra¨nse M, et al. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase. J Pharmacol Exp Ther 2011; 339:313–320. 35. Saiman L, Marshall BC, Mayer-Hamblett N, et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomised controlled trial. J Am Med Assoc 2003; 290:1749–1756. 36. Saiman L, Anstead M, Mayer-Hamblett N, et al. Effects of azithromycin on pulmonary function in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a randomized controlled trial. J Am Med Assoc 2010; 303:1707–1715. 37. Ratjen F, Saiman L, Mayer-Hamblett N, et al. Effect of azithromycin on & systemic markers of inflammation in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa. Chest 2012; 142:1259–1266. Recent trial that updates information on the use of azithromycin in CF without P. aeruginosa showing improvement in lung function and markers of infection. 38. Tarran R, Sabater JR, Clarke TC, et al. Nonantibiotic macrolides prevent & human neutrophil elastase-induced mucus stasis and airway surface liquid volume depletion. Am J Physiol Lung Cell Mol Physiol 2013; 304:L746– L756. Describes a novel macrolide anti-inflammatory with reduced antibacterial function. 39. Bensman TJ, Nguyen AN, Rao AP, Beringer PM. Doxycycline exhibits anti& inflammatory activity in CF bronchial epithelial cells. Pulm Pharmacol Ther 2012; 25:377–382. In-vitro demonstration of the anti-inflammatory properties of doxycycline, reducing IL-8 and MMPs. 40. Hartl D, Latzin P, Hordijk P, et al. Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat Med 2007; 13:1423–1430. 41. Moss RB, Mistry SJ, Konstan MW, et al. Safety and early treatment effects of & the CXCR2 antagonist SB-656933 in patients with cystic fibrosis. J Cyst Fibros 2013; 12:241–248. An early stage trial of a CXCR2 inhibitor in cystic fibrosis. 42. Chalmers JD, Fleming GB, Hill AT, Kilpatrick DC. Impact of mannose binding lectin (MBL) insufficiency on the course of cystic fibrosis: a review and metaanalysis. Glycobiology 2011; 21:271–282. 43. Haerynck F, Van Steen K, Cattaert T, et al. Polymorphisms in the lectin & pathway genes as a possible cause of early chronic Pseudomonas aeruginosa colonization in cystic fibrosis patients. Hum Immunol 2012; 73:1175–1183. Interesting small study relating genetic deficiency in the opsonins ficolin-1 and ficolin-2 with P. aeruginosa colonization in CF. 44. Chiarini M, Sabelli C, Melotti P, et al. PTX3 genetic variations affect the risk of Pseudomonas aeruginosa airway colonization in cystic fibrosis patients. Genes Immun 2010; 11:665–670. 45. Chalmers JD, McHugh BJ, Doherty CJ, et al. Mannose binding lectin deficiency and disease severity in non-CF bronchiectasis: a prospective study. Lancet Respir Med 2013; 1:175–274. 46. Paroni M, Moalli F, Nebuloni M, et al. Response of CFTR-deficient mice to && long-term chronic Pseudomonas aeruginosa infection and PTX3 therapy. J Infect Dis 2013; 208:130–138. New approach to CF therapeutics in experimental stages enhancing opsonophagocytosis of bacteria with the long pentraxin, pentraxin-3.
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