Review

How the airway smooth muscle in cystic fibrosis reacts in proinflammatory conditions: implications for airway hyper-responsiveness and asthma in cystic fibrosis Sarah McCuaig, James G Martin

Among patients with cystic fibrosis there is a high prevalence (40–70%) of asthma signs and symptoms such as cough and wheezing and airway hyper-responsiveness to inhaled histamine or methacholine. Whether these abnormal airway responses are due to a primary deficiency in the cystic fibrosis transmembrane conductance regulator (CFTR) or are secondary to the inflammatory environment in the cystic fibrosis lungs is not clear. A role for the CFTR in smooth muscle function is emerging, and alterations in contractile signalling have been reported in CFTR-deficient airway smooth muscle. Persistent bacterial infection, especially with Pseudomonas aeruginosa, stimulates interleukin-8 release from the airway epithelium, resulting in neutrophilic inflammation. Increased neutrophilia and skewing of CFTR-deficient T-helper cells to type 2 helper T cells creates an inflammatory environment characterised by high concentrations of tumour necrosis factor α, interleukin-8, and interleukin-13, which might all contribute to increased contractility of airway smooth muscle in cystic fibrosis. An emerging role of interleukin-17, which is raised in patients with cystic fibrosis, in airway smooth muscle proliferation and hyper-responsiveness is apparent. Increased understanding of the molecular mechanisms responsible for the altered smooth muscle physiology in patients with cystic fibrosis might provide insight into airway dysfunction in this disease.

Introduction Cystic fibrosis is a progressive multisystem disease that is caused by deficiency of a functional cystic fibrosis transmembrane conductance regulator (CFTR) and is a common inherited recessive disorder among white populations. Much of the research focus in this disease has been on CFTR dysfunction in the airway epithelium1 and its relation to the progressive airway obstruction and ultimately to the respiratory failure that ensues. However, the CFTR is expressed in various tissues and there is increasing evidence to suggest a functional role for this channel in the regulation of these non-epithelial tissues.2 Of these tissues, there is perhaps most evidence to suggest a potential contribution of CFTR-dependent alterations in properties of airway smooth muscle to the pathophysiology of cystic fibrosis. 40–70% of patients with cystic fibrosis have signs and symptoms of asthma such as cough and wheezing and airway hyper-responsiveness to inhaled histamine or methacholine.3,4 Furthermore, over 80% of patients have some degree of reversible airway obstruction after bronchodilator treatment, suggesting that many of these patients have asthma.5,6 The term cystic fibrosis asthma has been coined to describe patients with cystic fibrosis who have episodes of acute airway obstruction reversed by bronchodilators, a strong family history of asthma, or evidence of atopy.7,8 According to these criteria, about 20% of patients with cystic fibrosis have asthma (about two times the expected prevalence), and this proportion holds true across all ages.8 Also, there are reports that a higher than expected percentage of asthmatic patients are carriers of CFTR mutations:9 nine (45%) of 20 asthmatics were carriers of CFTR missense mutations expected to alter the aminoacid sequence of the CFTR protein and potentially modulate its function, whereas www.thelancet.com/respiratory Vol 1 April 2013

these mutations were present in only eight (15%) of 52 control individuals without airway disease. Airway hyper-responsiveness, a hallmark of asthma, affects 40–50% of patients with cystic fibrosis4,10 and, as in asthma, its cause has not been elucidated. There is evidence that in patients with cystic fibrosis with airway hyper-responsiveness to inhaled histamine and no symptoms of asthma, the hypersensitivity is neurally mediated because it can be abolished by ipratropium.3,4 However, airway hyper-responsiveness associated with asthma is not a neurally mediated phenomenon and in most patients must be a result of excessive shortening of airway smooth muscle cells. Increased smooth muscle sensitivity to cholinergic and α-adrenergic stimulation and a diminished response to β-adrenergic stimulation have also been reported in obligate heterozygotes for cystic fibrosis.11 Although in the short term an increase in forced expiratory volume in 1 s (FEV1) has been noted in patients with cystic fibrosis who were responsive to bronchodilator administration, findings from longitudinal studies have shown high variability and inconsistency of response.12,13 Differences in pulmonary function parameters between patients with cystic fibrosis with and without response to bronchodilators were not apparent and level of baseline lung function was not predictive of bronchodilator response.12 Findings from genetic studies support these data, showing that specific polymorphisms of the β2 receptor gene are associated with an increased severity of cystic fibrosis.14 Although airway smooth muscle is the principal effector of methacholine-induced and histamine-induced airway narrowing and hyper-responsiveness, potential abnormalities in airway smooth muscle cell contractility have not been investigated to any substantial extent in relation to cystic fibrosis. In addition to potential alterations in

Lancet Respir Med 2013; 1: 137–47 Published Online January 30, 2013 http://dx.doi.org/10.1016/ S2213-2600(12)70058-9 See Review pages 148, 158, and 164 Meakins Christie Laboratories, McGill University Health Centre, Montreal, Quebec Canada (S McCuaig, Prof J G Martin MD) Correspondence to: Prof James G Martin, Meakins Christie Laboratories, 3626 St Urbain, Montreal, QC, H2X 2P2, Canada [email protected]

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the properties of airway smooth muscle cells in CFTR deficiency, there is evidence of airway smooth muscle tissue remodelling in cystic fibrosis airways. The mass of airway smooth muscle is increased in patients with cystic fibrosis, suggesting that airway remodelling does occur in this disease, although perhaps not to the extent that it occurs in asthma.15,16 Whether abnormal airway responsiveness is attributable to primary abnormalities in cystic-fibrosisdeficient airway smooth muscle or altered properties secondary to the inflammatory environment of the airways in cystic fibrosis is not clear. In this Review, we will describe the evidence that one or both of these explanations might contribute to airway pathophysiology in cystic fibrosis.

The CFTR in smooth muscle The CFTR is a low-conductance, cyclic AMP (cAMP)regulated, ATP-gated chloride (Cl–) channel located in the apical membrane of epithelial cells lining the airways, intestines, ducts of the pancreas, and sweat glands.17 However, we now know that CFTR expression is not limited to epithelial cells. Over the past two decades, evidence of CFTR expression in lymphocytes,18 endothelial cells,19 hypothalamic neurons,20 cardiac muscle cells,21 tracheal smooth muscle cells,2 and rat22 and mouse23 aortic and ileal smooth muscle cells24 has emerged. A plausible hypothesis is that a defective CFTR in smooth muscle might affect its properties. Chloride transport is an important determinant of smooth muscle membrane potential and calcium signalling and the functional properties of contraction, relaxation, and proliferation. Previous studies have investigated the calcium-activated Cl– current (ICl,Ca), which contributes to agonist-induced vascular smooth muscle contraction25 and pulmonary vasoconstriction.26 A cAMP-dependent Cl– channel has also been studied in rat and bovine pulmonary arteries and has been implicated in pulmonary arterial relaxation,27 pulmonary arterial smooth muscle cell migration, and morphological changes.28 Although the molecular identity of this cAMP-dependent Cl– channel has not been characterised, it is possibly related to the CFTR. Chloride channels have also been implicated in the myogenic tone of cerebral vessels.29

CFTR in vascular smooth muscle cells The role of the CFTR in cAMP-dependent Cl– transport and smooth muscle function has been investigated. Primary cultures of aortic smooth muscle cells were examined by Robert and colleagues23 in 2005 in Cftr+/+ and Cftr–/– mice. Measurements of iodide efflux (¹²⁵I), a surrogate measure of chloride channel activity, revealed that when Cftr–/– mice were treated with isoproterenol, vasoactive intestinal peptide, or cAMP agonists there was no detectable stimulation of Cl– channels. Pharmacological activation of the CFTR with 6-hydroxy10-chlorobenzo[c]quinolizinium (MPB-07), 5-butyl-6-hydroxy138

10-chlorobenzo[c]quinolizinium chloride (MPB-91), and the isoflavone genistein stimulated iodide efflux from Cftr+/+ but not Cftr–/– mice. Furthermore, specific inhibition of the CFTR with thiazolidinone CFTRinh-172 prevented iodide efflux in Cftr+/+ mice but not Cftr–/– mice. Physiologically, the loss or dysfunction of the CFTR seems to impair the control of vascular tone in vascular smooth muscle cells. First, vascular tone is increased by nearly two times in aortic rings of Cftr–/– mice compared with wild-type animals in response to vasoactive drugs.23 Second, agonist-dependent vasorelaxation is impaired in pre-constricted Cftr–/– aortic rings after administration of vasoactive intestinal peptide and CFTR activators.23 In summary, mouse aortas without the CFTR are more constricted than control arteries, less sensitive to the relaxing action of vasoactive intestinal peptide, and fail to relax in the presence of CFTR activators. Although the biological significance of these findings for vascular regulation in patients with cystic fibrosis is unknown, the fact that abnormal arterial stiffness has been reported in the absence of significant hypertension is interesting.30 In a similar study, Robert and colleagues31 subsequently reported a functional role of the endogenously expressed CFTR in rat intrapulmonary arterial smooth muscle cells. Pulmonary vasorelaxation was induced in preconstricted rat intrapulmonary arterial rings treated with cAMP agonists. 4,4 -diisothiocyanato-stilbene-2,2 -disulphonic acid (DIDS), a calcium-activated chloride channel blocker, had no effect on cAMP-dependent Cl– transport through the CFTR. Furthermore, cAMP agonists still induced strong vasorelaxation in DIDS-treated rat intrapulmonary arterial rings. These findings suggest an important functional role of the CFTR channels in pulmonary vasorelaxation. Although CFTR-deficient pulmonary arterial muscle was not specifically studied, this evidence could potentially provide an additional explanation for the occurrence of pulmonary hypertension in patients with cystic fibrosis.

CFTR in ileal smooth muscle cells Patients with cystic fibrosis have gastrointestinal symptoms such as cramps, constipation, and intestinal obstruction or meconium ileus. Although there are several reasons why these symptoms and signs might occur, a possible abnormality of smooth muscle related to CFTR deficiency has not been extensively explored. Intestinal transit has been assessed in CFTR-deficient mice and is abnormally slow.32,33 Recently, Risse and colleagues24 compared the functional and morphological properties of ileal smooth muscle of Cftr–/– and Cftr+/+ C57BL/6 and BALB/cJ mice by investigating the response of the muscle to electric field stimulation, reactivity or sensitivity to muscarinic and β2-adrenergic receptor antagonists, velocity of shortening, and morphology of ileal smooth muscle strips. Resting tone was similar www.thelancet.com/respiratory Vol 1 April 2013

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across the two strains and both the wild-type and CFTRdeficient animals. Peak isometric force (Fmax) of ileal longitudinal smooth muscle contraction induced by electric field stimulation was increased in C57BL/6 Cftr–/– mice but was unchanged in BALB/cJ Cftr–/– mice. Enhanced smooth muscle relaxation was noted in the BALB/cJ Cftr–/– animals. This finding is contrary to the impaired vasorelaxation reported in CFTR-deficient mouse aortic rings.23 Treatment with the contractile agonist methacholine significantly increased ileal smooth muscle contraction in Cftr–/– C57BL/6 mice but not Cftr–/– BALB/cJ mice compared with wild-type mice. Thus, Cftr–/– C57BL/6 mice seem to have more reactive ileal smooth muscle compared with wild-type mice. Morphometric analysis of ileal smooth muscle mass revealed no significant differences in smooth muscle mass or cell density in the more reactive C57BL/6 Cftr–/– animals. However, significantly increased longitudinal and circular layers and a decreased density of cells in all ileal smooth muscle layers were reported in the BALB/cJ mice, suggesting hypertrophy of the muscle. An investigation of the relative expression of smooth muscle myosin heavy chain isoforms provided a possible explanation for the smooth muscle hyper-responsiveness in Cftr–/– C57BL/6 mice. Despite no significant difference in the fast(+) insert isoform, a lower total smooth muscle myosin heavy chain expression was noted in the Cftr–/– C57BL/6 mice. The proportional increase in fast(+) insert isoform (called SM-B) might have contributed to the increased Fmax maximum velocity of shortening (Vmax) reported in this CFTR-deficient strain. Thus, the structural and functional changes in CFTRdeficient ileal smooth muscle are strain dependent. C57BL/6 Cftr–/– mice have altered contractile properties and smooth muscle myosin heavy chain expression but unchanged morphology, whereas BALB/cJ mice have unaltered contractile properties but hypertrophy of the smooth muscle. The findings of alterations in ileal smooth muscle, contractile properties, and structural changes noted in CFTR-deficient animals suggests that the gastrointestinal pathology of cystic fibrosis might be related to abnormal smooth muscle function. However, in the aforementioned study24 the hyper-responsiveness to methacholine that occurred in the C57BL/6 Cftr–/– mice was not reproduced with chemical inhibition of CFTR in the Cftr+/+ mice with CFTRinh172. This important finding suggests that the function of CFTR in smooth muscle physiology might be independent of the functions targeted by this inhibitor. Changes in muscle function also probably result from chronic deficiency of CFTR, or perhaps an alteration in the smooth muscle environment might be necessary to elicit the reported changes in ileal smooth muscle. Abnormalities in intestinal transit time in Cftr–/– mice develop postnatally and are attributable to prostaglandin synthesis and to the intestinal microflora,33 suggesting that the www.thelancet.com/respiratory Vol 1 April 2013

consequences of CFTR deficiency create conditions that predispose to further abnormalities in smooth muscle function.

Airway smooth muscle in cystic fibrosis Expression of CFTR in sections of main or lobar bronchi of patients with cystic fibrosis has recently been shown by immunostaining with a monoclonal antibody to the CFTR carboxy-terminus.34 CFTR expression was visible in the airway smooth muscle and mucous glands. These findings of structural and functional consequences of CFTR deficiency in vascular and intestinal smooth muscle provide a basis for assessing the implications of CFTR-deficient airway smooth muscle. However, one must keep in mind that airway smooth muscle is phenotypically somewhere between a tonic muscle, such as aortic muscle, and phasic muscle, such as intestinal smooth muscle.35,36

Altered calcium responses in CFTR-deficient airway smooth muscle Calcium (Ca²+) is a crucial second messenger in smooth muscle contraction, activating myosin light chain (MLC) kinase through the formation of a Ca²+ and calmodulin complex. Thus, Ca²+ homoeostasis is essential for the maintenance of smooth muscle physiology. Histamine exerts its contractile properties by releasing Ca²+ from the sarcoplasmic reticulum. However, Ca²+ efflux increases the negative charge on the inner side of the sarcoplasmic reticulum, inhibiting further Ca²+ release. Leak of Cl– from the sarcoplasmic reticulum into the cytoplasm helps to neutralise this charge. However, cytosolic Cl– accumulation hinders the aforementioned processes.37,38 Janssen38 suggested that contractile agonists activate Cl– channels in the sarcolemma of airway smooth muscle. The activated channels allow for efflux of Cl–, which enables continued Cl– diffusion from the sarcoplasmic reticulum and thus enhances Ca²+ release and contractile response in the airway smooth muscle (figure 1). Janssen38 did not specifically implicate the CFTR in this phenomenon; however, consistent with this model Michoud and colleagues34 have reported that airway smooth muscle cells harvested from patients with cystic fibrosis show diminished Ca²+ release in response to histamine, as measured by the ratiometric Ca²+-sensitive dye, fura-2-acetoxymethyl ester. Administration of pharmacological CFTR-channel inhibitors, glibenclamide and N-phenylanthranilic acid, and anti-sense oligonucleotides to the CFTR transcript to non-cystic fibrosis airway smooth muscle cells also decreased the Ca²+ response to histamine. These results show an important role of the cAMP-dependent chloride channel in the contractile response to agonists but might have led one to expect a reduced contractile response to agonists. Further exploration of the regulation of airway smooth muscle contractile proteins is needed because this regulation could also account for altered properties of airway smooth 139

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Figure 1: A potential role of cystic fibrosis transmembrane conductance regulator in calcium signalling in airway smooth muscle (1) Cl– channels, potentially the CFTR, in the sarcolemma of airway smooth muscle might be activated by contractile agonists and allow for efflux of accumulating cytosolic Cl– (2), permitting continued release of Ca2+ from the sarcoplasmic reticulum (3).35 Decreased Ca2+ signalling in response to contractile agonists might occur in CFTR-deficient airway smooth muscle. However, cystic fibrosis airway smooth muscle cells express increased MLC20 and show increased and more rapid MLC20 phosphorylation, resulting in a hypercontractile phenotype. CFTR=cystic fibrosis transmembrane conductance regulator. DAG=diacyl glycerol. GPCR=G-proteincoupled receptor. IP3=inositol trisphosphate. IP3R=inositol trisphosphate receptor. MLC=myosin light chain. MLCK=myosin light chain kinase. MLCP=myosin light chain kinase phosphatase. P-MLC=phosphorylated myosin light chain. PLC=phospholipase C. RyR=ryanodine receptor.

muscle and might provide evidence to explain the asthma diathesis that occurs in patients with cystic fibrosis.

Airway smooth muscle abnormalities in cystic fibrosis neonatal pigs Evidence to support the finding of early-life abnormalities in the airway musculature in patients with cystic fibrosis has come from studies in CFTR–/– neonatal pigs. Using formalin-fixed trachea tissue cross-sections, Meyerholz and colleagues39 reported increased thickness of the mid-posterior inner tracheal wall, between the inner edge of the cartilage and the lumen, in CFTR–/– newborn pigs. This thickening corresponded to increased trachealis smooth muscle when normalised to inner wall area or luminal circumference. Morphometric analysis of tracheal sections revealed decreased tracheal diameter and luminal area in the CFTR–/– animals, which might be secondary to the increased trachealis muscle. Gene set enrichment analysis from a porcine trachea microarray dataset revealed increased transcript levels of airwaysmooth-muscle-related genes in the CFTR–/– pigs. This finding suggests that changes in expression of airway-smooth-muscle-related genes might mediate the increase in trachealis smooth muscle volume. Airway smooth muscle is active and produces peristalsis-like contractions in the human fetus.40 Dysregulated peristalsis might occur in CFTR–/– fetuses and contribute to the airway smooth muscle abnormalities noted in neonates. The porcine cystic fibrosis model might be useful for future studies investigating these changes in utero. 140

Altered airway smooth muscle mass in human cystic fibrosis airways The presence of bronchial hyper-responsiveness in cystic fibrosis has prompted studies of airway remodelling. Morphometric analysis of bronchial biopsy specimens from adult patients with cystic fibrosis by Hays and colleagues41 revealed increased airway smooth muscle content in patients with mild-to-moderate cystic fibrosis. The smooth muscle volume in the submucosa of airways of adult patients with cystic fibrosis was 1·63 times greater than in adults without cystic fibrosis when indexed to submucosal volume and 1·79 times greater when indexed to the basal lamina. The mean volume of individual smooth muscle cells in both cystic fibrosis and healthy control individuals was nearly identical. This finding suggests that the increased smooth muscle volume is due to hyperplasia without hypertrophy, implying that cell proliferation is the mechanism of airway smooth muscle remodelling in cystic fibrosis. In endobronchial biopsies of school-age children with cystic fibrosis compared with control individuals, Regamey and colleagues15 found airway smooth muscle was increased in magnitude to a similar extent to that reported by Hays and colleagues41 in their studies in adults. Therefore, airway remodelling associated with cystic fibrosis seems to occur early in life, as has been shown for asthma.42 The question of just how early airway remodelling occurs has not yet been answered, but the aforementioned porcine model might provide useful insights into this question. In Regamey and colleagues’15 study in school-age children, the volume fraction of airway smooth muscle www.thelancet.com/respiratory Vol 1 April 2013

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in subepithelial tissue was significantly increased in children with cystic fibrosis compared with control individuals and was related to myocyte number but not to myocyte size in a correlation analysis. Thus, one might conclude that, like in adults, hyperplasia rather than hypertrophy is the primary mechanism of increased smooth muscle mass in children with cystic fibrosis. However, unlike the findings in adults, mean myocyte size was significantly increased in children with cystic fibrosis, whereas the increases in myocyte number were not significant. The investigators attribute the absence of significance in increased myocyte number to a small sample size, leaving some uncertainty as to the interpretation of the results. Both hypertrophy and hyperplasia might plausibly play parts in the increase in airway smooth muscle mass in subepithelial tissue reported in patients with cystic fibrosis. Although defective ion transport in CFTR-deficient airway smooth muscle has been suggested to contribute directly to altered function, in Regamey and colleagues’15 study involving children with cystic fibrosis, an experimental group of children with bronchiectasis who did not have cystic fibrosis was included. Despite the presence of CFTR in bronchiectasis, the changes in airway smooth muscle mass were similar in magnitude to those in cystic fibrosis. A similarity between cystic fibrosis and bronchiectasis is that both are characterised by high levels of airway neutrophilia, which suggests that the inflammatory environment, rather than the deficiency of the CFTR, might be a key player in the reported changes in mass of airway smooth muscle in children (table).

Inflammation and airway smooth muscle function in cystic fibrosis Unlike in asthma, in which T lymphocytes, eosinophils, and mast cells play crucial parts in the pathogenesis of the disease, the inflammatory environment in cystic fibrosis is dominated by neutrophilia.43 Bacteria, characteristically Gram-negative Pseudomonas aeruginosa, stimulate macrophages to produce interleukin-1β and tumour necrosis factor α (TNFα). In consequence, airway epithelial cells release chemokines including interleukin-8, a potent neutrophil-chemoattractant. Neutrophil influx into the lungs further potentiates the inflammatory response by the release of the proinflammatory mediators TNFα and interleukin-8.44 As discussed later, these mediators might enhance contractile signalling in airway smooth muscle. The intrinsic deficiency of CFTR in neutrophils and macrophages has been linked to impaired bacterial killing.45 In particular, gene expression of toll-like receptor 4 (TLR4), which detects lipopolysaccharide from Gram-negative bacteria, is increased in cystic fibrosis neutrophils46 and in monocytes of children with cystic fibrosis who were uninfected at the time of study.47 The altered TLR expression in these CFTR-deficient cells prolongs the inflammatory response to lipopolysaccharide and reduces the effectiveness of pathogen clearing.45 Additionally, airway epithelium removed from patients with cystic fibrosis at transplantation displays increased interleukin-8, epidermal growth factor receptor (EGFR) and pro-transforming growth factor α (TGFα) immunohistochemical staining.48 Signalling through TLR4 activates downstream signalling leading ultimately to the activation of EGFR.49 Activation of EGFR increases transcription and release of interleukin-8 and increases

Tissue studied

Main findings

Robert et al (2005)23

Mouse aortic smooth muscle cells

Cftr–/– mice had increased vascular tone in aortic rings in response to vasoactive drugs compared with Cftr +/+ mice Impaired agonist-dependent vasorelaxation in preconstricted Cftr–/– rings after administration of vasoactive intestinal peptide and CFTR activators compared with Cftr +/+ mice

Hays et al (2005)41

Adult human airway smooth muscle tissue

Increased smooth muscle volume in submucosa of airways of patients with cystic fibrosis when indexed to submucosal volume and basal lamina Mean volume of airway smooth muscle cells was nearly identical in patients with cystic fibrosis and control individuals (hyperplasia vs hypertrophy)

Robert et al (2007)31

Rat intrapulmonary arterial DIDS specifically blocked Ca2+-activated Cl– channels and had no effect on cAMP-dependent transport through the CFTR smooth muscle cells cAMP agonists induced strong vasorelaxation in DIDS-treated rat intrapulmonary arterial rings, suggesting CFTR has an important functional role in vasorelaxation

Regamey et al (2008)15

Child human airway smooth muscle tissue

Michoud et al (2009)34

Human cystic fibrosis Diminished Ca2+ release in response to histamine in airway smooth muscle cells from patients with cystic fibrosis compared with those from control airway smooth muscle cells individuals

Meyerholz et al (2010)39

Neonatal pig tracheal sections

Decreased tracheal diameter, luminal area, and circularity of tracheal cross-sections in 4 × CFTR–/– vs 4 × CFTR+/+ neonatal pigs Increased thickness of trachealis muscle in 4 × CFTR–/– animals Increased gene transcript levels of genes related to airway smooth muscle in 4 × CFTR–/– animals

Risse et al (2012)24

Mouse C57BL/6 ileal smooth muscle cells

Increased smooth muscle contraction in response to methacholine in Cftr–/– vs Cftr+/+ mice No difference in smooth muscle mass or cell density in Cftr–/– vs Cftr+/+ mice Lower total smooth muscle myosin heavy chain expression and proportional increase in fast(+) insert isoform in Cftr–/– vs Cftr+/+ mice

Risse et al (2012)24

Mouse BALB/cJ ileal smooth muscle cells

No difference in smooth muscle contraction in response to methacholine in Cftr–/– vs Cftr+/+ mice Increased longitudinal and circular smooth muscle layers and decreased cell density in Cftr–/– vs Cftr+/+ mice, suggesting hypertrophy

Volume fraction of airway smooth muscle in subepithelial tissue was significantly increased in children with cystic fibrosis compared with control children without cystic fibrosis and was related to myocyte number but not myocyte size

cAMP=cyclic AMP. CFTR=cystic fibrosis transmembrane conductance regulator. DIDS=4,4 -diisothiocyanato-stilbene-2,2 -disulphonic acid.

Table: Cystic fibrosis transmembrane conductance regulator deficient smooth muscle in various tissues

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mucin production in the airways in response to cigarette smoke50,51 and might amplify the inflammatory response and possibly stimulate airway remodelling in patients with cystic fibrosis. In the past decade, accumulating evidence has suggested that TLR5 might also play an important part in the innate response to bacterial infection. The ligand of TLR5 is flagellin—the protein component of bacterial flagella characteristic of motile bacteria that infect the lungs in cystic fibrosis, especially P aeruginosa—which drives the production of proinflammatory mediators by the epithelium.52 CFTR-deficient helper T cells are skewed towards a type 2 helper T cell (Th2) phenotype.53 The resulting proallergic Th2-type response leads to increased interleukin-4 and interleukin-13 levels and stimulates immunoglobulin E (IgE) synthesis by B cells. IgE then activates mast cells, which release histamine, leucotrienes, prostaglandins, and heparin. CFTR-deficient mast cells release increased concentrations of the proinflammatory cytokine interleukin-6, which stimulates neutrophil production and inhibits activity of regulatory T cells.54 This proallergic Th2-type response is effective in combating parasitic infections but not in clearing the airways of pathogens such as P aeruginosa. Thus, the vicious cycle of bacterial infection and inflammation persists (figure 2).

The contribution of the inflammatory environment to airway hyper-responsiveness

Airway lumen

The pattern of inflammation in cystic fibrosis is mixed and is associated with the production of TNFα,

IL-8

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Figure 2: The inflammatory environment in cystic fibrosis The figure shows the neutrophil-dominated, Th2-skewed inflammatory environment in cystic fibrosis. Abundant IL-8 stimulates epithelial and possibly airway smooth muscle remodelling and induces greater contraction in cystic fibrosis airway smooth muscle than in non-cystic fibrosis airway smooth muscle resulting in airway hyper-responsiveness. EGFR=epidermal growth factor receptor. IgE=immunoglobulin E. IL=interleukin. TACE=TNFα-converting enzyme. TGF=transforming growth factor. Th2=type 2 helper T cell. TLR=toll-like receptor. TNF=tumour necrosis factor.

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interleukin-8, and interleukin-13. All three of these cytokines upregulate the contractile function of airway smooth muscle and are possibly involved in the cystic fibrosis asthma diathesis that occurs.

Interleukin-13 Allergic bronchopulmonary aspergillosis and Th2 inflammation are common in cystic fibrosis. The skewing of CFTR-deficient helper T cells toward a Th2 phenotype implies increased production and presence of interleukin-13 in the airway environment in patients with cystic fibrosis. Interleukin-13 is a Th2-type cytokine released by activated T cells, mast cells, and basophils that binds to the α-chain of the interleukin-4 receptor and the α-1 chain of the interleukin-13 receptor55 and is necessary and sufficient for the expression of allergic asthma.56 In murine models of allergic asthma, allergen exposure results in airway hyper-responsiveness and marked increases in airway eosinophilia, antigen-specific IgE, and airway epithelial mucous content.57 Airway hyper-responsiveness and goblet-cell hyperplasia are inter—leukin-13 dependent.56 Interleukin-13-mediated airway hyper-responsiveness effects are independent of eosinophils and IgE, and evidence points to direct effects on airway smooth muscle as a possible explanation for airway hyper-responsiveness. Risse and colleagues58 showed that interleukin-13 inhibits human airway smooth muscle cell proliferation and promotes the contractile responses to histamine. However, there was no alteration of expression levels of major contractile proteins, smooth muscle alpha actin, smooth muscle myosin heavy chain, calponin, or vinculin. Therefore, the increased contractility of airway smooth muscle in response to interleukin-13 stimulation seems to be independent of changes in the expression of phenotypic markers of smooth muscle and instead seems to be largely calcium dependent. Studies investigating the downstream signalling pathways through which interleukin-13 mediates the calcium response to histamine stimulation have revealed that interleukin-13 potentiates the activation of c-jun N-terminal kinase and extracellular signal-related kinase mitogen activated protein kinases (MAPKs).59 Interleukin-13 upregulates CD38, a bifunctional enzyme that synthesises cyclicadenine dinucleotide phosphate-ribose, which causes calcium release in response to agonist stimulation and seems to account for the enhanced calcium response to interleukin-13 (figure 3).60 CD38-deficient mice have an attenuated airway hyper-responsiveness when challenged with interleukin-13.61 To our knowledge, there have been no studies so far that examine the effect of interleukin-13 specifically on CFTR-deficient airway smooth muscle cells. If interleukin-13 does increase contractility in CFTR-deficient airway smooth muscle in response to contractile agonists, then interleukin-13 might be an important contributor to airway hyper-responsiveness in patients with cystic fibrosis. www.thelancet.com/respiratory Vol 1 April 2013

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Tumour necrosis factor α TNFα is an important cytokine involved in the development of airway hyper-responsiveness in some patients with asthma.62 TNFα activates the MAPK p38 via the TNFR1 receptor.63 The p38 intracellular signalling pathway modulates airway inflammation by regulating cytokine secretion by airway smooth muscle. TNFα also augments airway smooth muscle cell calcium responses in much the same manner as interleukin-13, increasing CD38 expression.64,65 Exaggerated p38 activation in response to TNFα has been reported in airway smooth muscle cells of patients with cystic fibrosis compared with control individuals (L Roussel, Meakins Christie Laboratories, Montreal, QC, Canada, unpublished). Whether altered contractile responses to TNFα occur in airway smooth muscle deficient in CFTR has not been explored (figure 3). Interleukin-8 Interleukin-8 is a chemokine that is also a potential contributor to altered airway smooth muscle function. It is a potent neutrophil chemoattractant that is released by neutrophils themselves, epithelia, and airway smooth muscle. CFTR-deficient airway neutrophils release high concentrations of interleukin-8 spontaneously.46 Under physiological conditions, interleukin-10, an antiinflammatory cytokine, is capable of inhibiting the production of interleukin-8 by neutrophils from healthy individuals66 yet is incapable of inhibiting interleukin-8 production by lipopolysaccharide-activated neutrophils from the blood of patients with cystic fibrosis.46 This insensitivity to the anti-inflammatory signalling of interleukin-10 did not seem to be mediated by reduced interleukin-10 receptor expression. The enhanced concentration of interleukin-8 might be in part attributable to this defect in regulatory processes. Govindaraju and colleagues67 reported that interleukin-8 induced greater contraction in CFTR-deficient airway smooth muscle cells despite lower interleukin-8-evoked calcium responses compared with airway smooth muscle cells from control individuals. Further investigation of MLC20 phosphorylation revealed significantly increased and more rapid MLC20 phosphorylation in cystic fibrosis airway smooth muscle cells compared with cells from control individuals. Additionally, MLC20 expression was increased in cystic fibrosis airway smooth muscle cells. The hypercontractile response to interleukin-8 suggests that the airway hyper-responsiveness reported in patients with cystic fibrosis might be in part mediated by the inflammatory environment in cystic fibrosis, which is characteristically abundant in interleukin-8.

Interleukin-17 In 2011, an important role of the proinflammatory cytokine interleukin-17 in mediating inflammation in cystic fibrosis emerged.68 Tan and colleagues68 detected increased interleukin-17 concentrations in the www.thelancet.com/respiratory Vol 1 April 2013

3 IL-13

1 TNFα

ADP ribosyl cyclase β-NAD

cADPR

cADPR hydrolase ADPR

Agonist (histamine) GPCR PLCβ

IL-13Rα1 IL-4Rα

↑CD38

?

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4

2

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+

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2+

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CD38 gene Nucleus

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cADPR ?

γ

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PIP2

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Ca2+

Ca

Ca2+ Ca2+ Ca2+

2+

Ca2+ Ca2+

Ca2+ Ca2+

Ca2+ Ca2+

Ca2+

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Ca2+

Figure 3: Augmentation of calcium responses in cystic fibrosis airway smooth muscle by interleukin-13 and tumour necrosis factor α IL-13 and TNFα bind their cognate receptors in the sarcolemma of airway smooth muscle (1) and upregulate CD38 expression (2). Ultimately cADPR, produced through the action of CD38, increases Ca2+ signalling (3), possibly resulting in a hypercontractile phenotype. IL-13 also promotes contractile responses to histamine. However, the mechanism through which histamine binding its GPCR causes activation of CD38 and cADPR production is not clear (4). β-NAD=β-nicotinamide adenine dinucleotide. cADPR=cyclic-adenine dinucleotide phosphate-ribose. CICR=calcium-induced calcium release. DAG=diacyl glycerol. GDP=guanosine diphosphate. GPCR=G-proteincoupled receptor. GTP=guanosine triphosphate. IL=interleukin. IP3=inositol trisphosphate. IP3R=inositol trisphosphate receptor. PIP2=phosphatidylinositol bisphosphate. PLCβ=phospholipase Cβ. RyR=ryanodine receptor. TNFα=tumour necrosis factor α. TNFR=TNF receptor.

bronchoalveolar lavage fluid of patients with established cystic fibrosis (n=33) and non-cystic fibrosis bronchiectasis (n=17) compared with newly diagnosed patients with cystic fibrosis (n=20) and healthy control individuals (n=13). They identified not only CD4+ T cells as sources of this cytokine, but additionally γδ T cells were found to be interleukin-17 positive in all disease groups studied, and interleukin-17-positive natural killer T cells were identified in patients with end-stage lung disease. P aeruginosa induces the release of interleukin-6 and interleukin-23 from macrophages and dendritic cells in the airways, which together with TGFβ induce Th17 cell differentiation and ultimately interleukin-17 production (figure 4).69 Interleukin-17 acts on a wide range of cell types; of interest in the context of airway inflammation in cystic fibrosis is its ability to promote neutrophil migration to the lungs.70 Concomitant with the studies showing raised interleukin-17 concentrations in patients with cystic fibrosis, recent investigation has revealed that interleukin-17 is a potential driving force of airway hyperresponsiveness in a mouse asthma model and in human airway smooth muscle contraction.71 Incubation of tracheal rings and agarose-filled lung slices from sensitised C57BL/6 mice for 12 h with interleukin-17A increased methacholine-induced and potassium chloride (KCl)-induced tracheal ring contraction and airway narrowing in lung slices in an epithelium-independent manner. Similarly, increased contraction of isolated human bronchi in response to methacholine and KCl was recorded after 12-h pre-incubation with interleukin-17A. 143

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Mechanistically, interleukin-17A increased MLC phosphorylation in mouse tracheal smooth muscle and was dependent upon the induced nuclear translocation of the transcription factor nuclear factor-κB (NF-κB). NF-κB upregulates the transcription of ROCK2, a negative regulator of myosin light chain phosphatase, thus preventing myosin dephosphorylation (figure 4). To our knowledge, the effects of interleukin-17 on airway hyperresponsiveness and airway smooth muscle contraction have yet to be explored in cystic fibrosis. This is a promising area of future study in view of the raised interleukin-17 concentrations reported in patients with cystic fibrosis and the apparent ability of interleukin-17 to increase airway hyper-responsiveness and airway smooth muscle contraction in allergic asthma.

The inflammatory environment and airway smooth muscle proliferation

Epithelium

Airway lumen

Until recently, the changes in airway smooth muscle in patients with cystic fibrosis elicited by the inflammatory environment seemed to be limited to alterations in contractility rather than in migration and proliferation of airway smooth muscle cells. Chemotaxis assays measuring the migratory response of airway smooth muscle to interleukin-8 reveal no difference in the amount of increase in migrated cells between cystic fibrosis airway smooth muscle cells and control airway smooth muscle cells.67 Furthermore, exposure of both cystic fibrosis and non-cystic fibrosis airway smooth muscle to interleukin-8 induced modest proliferation, but the proliferation was not significantly enhanced in the cystic fibrosis airway smooth muscle cells.67 Risse and colleagues58 investigated the effects of interleukin-13 on airway smooth muscle and found that interleukin-13, a prominent cytokine in the Pseudomonas aeruginosa 1

Neutrophils IL-6 IL-8 GM-CSF

TLR4 Macrophage

MLCK MLC active

MLC inactive 2

Submucosal compartment

proallergic inflammatory environment in cystic fibrosis, inhibits airway smooth muscle proliferation. However, in the past 2 years Th17 cytokines (interleukin-17A, interleukin-17F, and interleukin-22) have been shown to increase airway smooth muscle proliferation and reduce the rate of apoptosis in airway smooth muscle cells isolated from asthmatic and non-asthmatic individuals.72 Chang and colleagues72 showed that Th17 cytokinemediated airway smooth muscle proliferation is dependent upon Th17 cytokine receptors expressed in both asthmatic and non-asthmatic airway smooth muscle. A direct link between Th17 cytokines and airway smooth muscle proliferation has yet to be made in cystic fibrosis. However, the findings of Chang and colleagues72 in combination with the raised interleukin-17 levels reported in patients with cystic fibrosis68 suggest a possible link between interleukin-17 and the increased airway smooth muscle mass in endobronchial biopsies from patients with cystic fibrosis.15,41 Still, the exact molecular mechanisms that can account for airway smooth muscle hyperplasia in patients with cystic fibrosis remain to be elucidated and perhaps might be related to primary genetic defects in the CFTR or alterations in modifier genes. For example, screening of four large cohorts of patients with cystic fibrosis (the multicentre US cohort homozygous for the ∆F508 mutation [n=808], Seattle, WA, cohort [n=238], Ireland cohort [n=303], and Cleveland, OH, cohort [n=238]) revealed a significant correlation with polymorphisms in the endothelin receptor A gene, EDNRA.73 Endothelin is a proinflammatory peptide that acts as a smooth muscle agonist and is raised in patients with cystic fibrosis.74,75 When bound to its G-proteincoupled receptor, endothelin receptor A, it induces

IL-6 IL-23

NF-κB 3

IL-17

TGFβ

ROCK2 MLCP

Th 17 cell NK T cell γδ T cell

ROCK2 4

Airway smooth muscle cell

IL-17

Figure 4: Increased interleukin-17 production in cystic fibrosis and interleukin-17-mediated airway smooth muscle contraction (1) Pseudomonas aeruginosa induces macrophage release of IL-6 and IL-23 in the airways (2). Together with TGFβ, IL-6 and IL-23 induce Th17 cell differentiation and IL-17 production (3). IL-17 promotes neutrophil migration to the lungs (4) and increased airway smooth muscle contraction by increasing MLC phosphorylation in an NF-κB-dependent manner. GM-CSF=granulocyte-macrophage colony-stimulating factor. IL=interleukin. MLC=myosin light chain. MLCK=myosin light chain kinase. MLCP=myosin light chain kinase phosphatase. NF-κB=nuclear factor-κB. NK=natural killer. ROCK2=Rho-associated, coiled-coil containing protein kinase 2. TGFβ=transforming growth factor β. Th17=type 17 helper T cell.

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smooth muscle contraction and proliferation.76,77 Through genotypic analysis a single nucleotide polymorphism in the EDNRA gene, rs5335, was identified. Homozygosity for the C allele at rs5335 was associated with poorer lung function in all four cohorts studied (as measured by FEV1). Raised EDNRA mRNA concentrations were detected in primary smooth muscle cultures from non-cystic fibrosis trachealis smooth muscle isolated from individuals carrying the CC genotype at rs5335 compared with the heterozygous or homozygous GG genotype. This finding suggests a quantitative effect of the polymorphism on EDNRA expression. Furthermore, human tracheal airway smooth muscle of the CC genotype at rs5335 exhibited increased proliferation in culture after 72 h of growth. This effect was confirmed to be dependent on endothelin receptor A because the growth rate difference was attenuated with administration of the endothelin receptor A inhibitor, BQ-123. Thus, homozygosity for the EDNRA C allele at the rs5335 single nucleotide polymorphism, which is found in patients with cystic fibrosis with poor lung function, might increase expression of the endothelin receptor A and increase smooth muscle proliferation in these individuals. Understanding the interplay between primary genetic defects, modifier genes, and secondary inflammatory pathways is crucial for the development of treatments that can slow or inhibit this reported airway smooth muscle hyperplasia, which might contribute to asthmalike symptoms in patients with cystic fibrosis. In summary, persistent bacterial infection, especially with P aeruginosa, stimulates airway epithelial cells to secrete interleukin-8, resulting in neutrophil recruitment. Neutrophils potentiate the inflammatory response by further release of TNFα and interleukin-8. Airway smooth muscle releases interleukin-8 in response to TNFα. Thus, the environment of the cystic fibrosis airway contains an overabundance of interleukin-8, which induces greater contraction in cystic fibrosis than in non-cystic fibrosis airway smooth muscle cells because of increased MLC phosphorylation and expression in cystic fibrosis cells. Activation of EGFR through release of EGFR ligands mediated by TLR signalling might contribute to enhanced interleukin-8 and mucin production and possibly also to growth of the underlying airway smooth muscle. Furthermore, the T-helper cells of patients with cystic fibrosis seem to be skewed towards a Th2 proallergic response, characteristic of asthma. This Th2 response results in high interleukin-4 and interleukin-13 concentrations, which induce the production of IgE. Interleukin-13 induces airway hyperresponsiveness in murine models of allergic asthma. Recently, interleukin-17 was shown to be raised in patients with cystic fibrosis and to increase airway smooth muscle contraction and proliferation in murine and human samples and thus could be a key contributor in the cystic fibrosis asthma phenomenon. www.thelancet.com/respiratory Vol 1 April 2013

Th2 skewing of CFTR–/– helper T cells

Increased MLC20 expression and phosphorylation

Neutrophilic inflammation

IL-13, TNFα CD38

TLR4

cADPR Increased airway smooth muscle contractility

IL-17

IL-8 TLR5

? Airway smooth muscle proliferation

Increased airway smooth muscle mass

IL-6

EGFR

Absent CFTR

Cystic fibrosis asthma Cough, wheezing, acute airway hyper-responsiveness, and acute airway obstruction reversed by bronchodilators

Decreased Ca2+ responses to G-protein-coupled receptors?

Figure 5: Key contributing factors to cystic fibrosis asthma The figure shows a summary of our present understanding of the molecular mechanisms mediating the clinical display of cystic fibrosis asthma in 40–70% of patients with cystic fibrosis (dark blue box).3,4 Key alterations in airway smooth muscle physiology include increased mass, increased contractility, and decreased Ca2+ responses to histamine (light blue boxes). The effects of deficient ion transport through the CFTR, altered calcium signalling, and the neutrophil Th2-dominated inflammatory environment of the cystic fibrosis lungs all seem to play a part in the manifestation of asthma-like symptoms in patients with cystic fibrosis. cADPR=cyclic-adenine dinucleotide phosphate-ribose. CFTR=cystic fibrosis transmembrane conductance regulator. EGFR=epidermal growth factor receptor. IL=interleukin. MLC=myosin light chain. Th2=type 2 helper T cell. TLR=toll-like receptor. TNF=tumour necrosis factor.

Clinical implications There is substantial clinical evidence documenting physiological and phenotypic changes in the airway smooth muscle of patients with cystic fibrosis, which correlate with the reported airway hyper-responsiveness and asthma-like symptoms in 40–70% of patients with cystic fibrosis.3,4 In accordance with findings in CFTRdeficient vascular and ileal smooth muscle, CFTRdeficient airway smooth muscle cells have altered calcium responses to contractile agonists, which might contribute to the asthma diathesis of patients with cystic fibrosis.34 Deficient ion transport through the CFTR in patients with cystic fibrosis cannot be solely responsible for the altered airway smooth muscle physiology reported since increases in airway smooth muscle of similar magnitude occur in both cystic fibrosis and bronchiectasis (nonCFTR deficient) paediatric patients. However, both cystic fibrosis and bronchiectasis are diseases characterised by high levels of neutrophilia in the lungs, suggesting an important contribution of the inflammatory environment to alterations in airway smooth muscle (figure 5). Our understanding of the interplay between primary genetic and secondary inflammatory mechanisms in airway smooth muscle abnormalities in cystic fibrosis is complicated by the scarcity of clinical data from newborn babies or infants with cystic fibrosis who are free of bacterial infection. Despite the cross-sectional data available for children with cystic fibrosis who were uninfected at the time of study, the potential for a history of previous infection in these patients makes a definitive conclusion that altered airway smooth muscle function is 145

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Search strategy and selection criteria This Review was compiled using as primary sources the databases PubMed and HighWire (Stanford University, Stanford, CA, USA) with the keywords “cystic fibrosis”, “smooth muscle”, “airway smooth muscle”, “calcium signalling”, and “inflammation”. We focused on articles from 2003 to 2012 in addition to well cited review articles. The final search was undertaken in October, 2012.

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due to primary cystic fibrosis-related defects impossible. Therefore, although perhaps a difficult cohort to assess, data from uninfected newborn babies or infants will be necessary to answer the question of primary versus secondary mechanisms of airway dysfunction. Understanding the basis for airway hyper-responsiveness might provide insights into more appropriately targeted treatment in patients with cystic fibrosis. The interplay between the direct effects of CFTR deficiency and the neutrophil-dominated but also Th2-rich inflammatory environment in cystic fibrosis airways provides many potential targets for treatment. If airway remodelling in cystic fibrosis is primarily caused by the chronic inflammation characteristic of the cystic fibrosis lungs then new methods to combat inflammation early in life will help to diminish the pathogenesis of the disease. However, if airway remodelling and impaired smooth muscle physiology in cystic fibrosis is first and foremost independent of inflammation and mediated directly by CFTR deficiency, then gene therapy techniques or pharmacological activation of other endogenous chloride channels might be needed.

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Contributors SM researched, wrote, and edited the manuscript and drew the figures. JGM researched, wrote, and edited the manuscript.

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Conflicts of interest We declare that we have no conflicts of interest.

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