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Macrolide Therapy Targets a Specific Phenotype in Respiratory Medicine: From Clinical Experience to Basic Science and Back
Bart M. Vanaudenaerde,1, Robin Vos,1,6, Isabelle Meyts3, Stephanie I. De Vleeschauwer1, Stijn E. Verleden1, Anna Widyastuti-Willems1, Wim A. Wuyts1,6, Dirk E. Van Raemdonck2,3,4,5, Peter H. Hoet1, Lieven J. Dupont1,5,6, Benoit Nemery1 and Geert M. Verleden*,1,5,6
Laboratory of Pneumology, 2Laboratory of Experimental Thoracic Surgery, Katholieke Universiteit Leuven, Leuven, Belgium
3 1

Department of Paediatrics, 4Department of Thoracic Surgery, 5Lung Transplantation Unit, 6Department of Respiratory Medicine, University Hospital Gasthuisberg, Leuven, Belgium
Abstract: For centuries a quest has been going on for the holy grail in respiratory medicine: a treatment for numerous devastating chronic lung disorders. Yet, it is only a decade ago that pharmacological interference with the activation of the innate immune system by a macrolide antibiotic silently moved into everyday clinical practice. Macrolides, with their unique molecular structure built around a lactone ring, are now known to target harmful exaggerated innate immune responses. However, not all chronic lung conditions benefit from macrolide therapy and interestingly, neither do all patients with an apparently identical chronic lung disease. A subgroup of responders seems to display a single specific phenotype that can be recognized in the various lung conditions and that seems to be related to inflammatory responses with a predominant innate immune system component. Recently we have contributed to the introduction of macrolide therapy in lung transplantation medicine. Also we attempted to analyse this phenotype by describing its clinical, immunological, histological and radiological characteristics. The aim of this manuscript is to review the use of macrolides in the respiratory field and to apply the macrolide-responsive phenotype beyond the setting of lung transplantation and other conditions in which macrolides have been successful. The description of this universal macrolide-responsive phenotype can both help rationalize macrolide therapy in respiratory disorders, in which its benefit is already well-known, as well as promote the use of this treatment in respiratory conditions of unknown etiology but with a macrolide responsive phenotype.

Keywords: Azithromycin, chronic respiratory disorders, inflammation, interleukin, macrolides, neutrophils. INTRODUCTION Macrolides are a group of antibiotics derived from Streptomyces species. They consist of a common macrolactone ring to which one or more sugars are attached. Macrolides are divided in groups according to number of elements and to the structure of the macrolactone ring. The first and oldest group of macrolides consists of the 14-membered lactone group, with a monobasic charge. The second group is the most recent one and consists out of ketolides. This group is derived from the 14 membered macrolide erythromycin, on which a 3-keto group is replaced by a L-cladinose moiety at position 3 of the macrolactone ring. The third group is the 15-membered lactone ring group, which includes a nitrogen element in the basic structure making it strictly spoken an azalide. This group has a bibasic charge. The last group is the 16-membered lactone group and has again a monobasic charge (Table 1) [1, 2]. The non-erythromycin macrolides as a group are also referred to as neo-macrolides. Macrolides are bacteriostatic (and only bactericidal at high concentrations) against Staphylococcus aureus,
*Address correspondence to this author at the Lung Transplantation Unit, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium; Tel: + 32-16346800; Fax: + 32-16346803; E-mail: geert.verleden@uz.kuleuven.ac.be

Table 1.
Macrolactone Ring Subgroups Name erythromycin*, roxithromycin*, clarithromycin*, troleandomycin, dirithromycin, flurithromycin telitromycin* azithromycin* spiramycin, josamycin, rokitamycin, miomycin, tylosin, rosarimicin, midecamycin
Classification of the different macrolide according to the macrolactone-ring structure. *Most commonly prescribed worldwide.

14-membered group

14-membered ketolide group 15-membered-ring 'azalide' group 16-membered group

Haemophilus influenzae and Streptococcus species, but they lack bacteriostatic or bactericidal activity against Pseudomonas aeruginosa. An important feature of macrolides is their ability to penetrate polymorphonuclear leukocytes thus making them more accessible at the site of inflammation. Especially neomacrolides accumulate intracellularly; azithromycin reaches up to 226 times the external concentration, leading to a concentration gradient (internal/external) that is 26 times higher than erythromycin in macrophages [3]. This may also ex 2008 Bentham Science Publishers Ltd.

These authors contributed equally to this review.

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plain the better delivery of azithromycin to the site of inflammation and its higher potency compared to erythromycin [3]. Independently of their antimicrobial activity, macrolides possess anti-inflammatory properties that may contribute to clinical benefits observed in patients with chronic airway inflammation [4]. Although this anti-inflammatory potential has been the subject of many reports and although it is now being accepted that macrolides interfere with innate immune responses, the specific presentation or phenotype of the patient likely to respond to macrolide therapy the topic of this review- has not received much attention. It is of the utmost importance that this macrolide responsive phenotype is specified as the potential side effects of long-term macrolide and especially azithromycin (with its long half-life) therapy are yet unknown and therefore preclude the generalized use of long term azithromycin treatment in any given lung condition. THE USE OF MACROLIDES IN MISCELLANEOUS LUNG DISORDERS Macrolide antibiotics possess anti-inflammatory properties that may contribute to clinical benefits observed in patients with miscellaneous chronic inflammatory lung disorders [4]. From a mechanistic point of view, exploring the lung conditions and the macrolide responsive patients phenotype in each one of these conditions can give us a clue towards a better understanding of the beneficial effect of macrolides and of a potential universal macrolide responsive phenotype. Macrolide therapy was initially described in diffuse panbronchiolitis (DPB), a condition common in Japan, which is characterized by a diffuse chronic neutrophilic airway inflammation restricted to the bronchiolar and centrilobular regions [5]. Clinical characteristics are productive cough, sputum production, wheezing, exercise dyspnea, Pseudomonas aeruginosa colonization and bronchiectasis. In these patients, long-term macrolide therapy had substantially reduced symptoms, especially in patients colonized with P. aeruginosa. In this specific subset of patients the ten year survival increased from 12% to more than 90% [6]. This survival benefit was accompanied by a decrease in airway neutrophilia [7-9] and Interleukin 8 (IL8, the main neutrophil chemo-attractant) in sputum and Broncho-Alveolar Lavage (BAL) [10]. Subsequently, interest arose in applying macrolide therapy in Cystic Fibrosis (CF) patients [4, 11]. Saiman et al, documented an improvement of Forced Expiratory Volume in one second (FEV1) between 3.6% and 7.2% in CF patients after 5 months of azithromycin treatment [12]. Prolonged use was well tolerated and the adverse effects (nausea, diarrhoea and vomiting) were generally mild to moderate and did not lead to discontinuation of the studied drug. Although resistance to macrolide antibiotics of S. pyogenes, S. pneumoniae, S. aureus and nontuberculous mycobacteria is a potential risk, this could not be clinically demonstrated [12]. Asthma, a condition of chronic lung inflammation, predominantly driven by TH2 lymphocytes, mast cells and eosinophils, can be treated effectively by inhaled corticosteroids. Yet, inhaled corticosteroids are much less effective in treating TH1- and neutrophilic-driven inflammation [13], associated with asthma exacerbations and severe asthma [14]. The

TELICAST study provides evidence of a therapeutic benefit of a ketolide antibiotic telithromycin (a new class of antibacterial agents related to macrolides) in asthma exacerbations [14]. Therapy with telithromycin reduced asthma symptoms and improved the lung function ten days after the exacerbation [14]. However, the presence of C. pneumoniae (38%) and M. pneumoniae (18%) was associated with more asthma exacerbations and was able to induce neutrophilic airway inflammation [15-18]. The TELICAST study indeed revealed that 61% of the patients showed evidence of colonization with these bacteria in blood or sputum, yet no clear indication was found for a greater treatment effect in these patients [14]. Although the pathophysiology of asthma is at first sight entirely different from CF and DPB, the innate immune response and ensuing neutrophilic inflammation plays a significant role during asthma exacerbations and in severe asthma. The inflammation associated with chronic obstructive pulmonary disease (COPD) typically includes macrophages, neutrophils and cytotoxic T-lymphocytes [19]. Macrolides have been approved for treatment of acute exacerbations of COPD, yet, rather on the basis of the antibacterial and not because of their anti-inflammatory activity [20, 21]. In 3 studies, the anti-inflammatory effect of macrolides was investigated in stable COPD patients, but failed to show a benefit either for health status, sputum bacterial colonies or frequency of exacerbations [19, 22-23]. No data are available on the benefit of macrolide treatment in COPD exacerbation. Our own experience with COPD patients leads us to think that patients with the specific macrolide responsive phenotype, which will be described in more detail further on, indeed improve after therapy (unpublished observation). Regarding lung transplantation (LTx), the use of azithromycin as additional treatment, on top of the usual immunosuppressive therapy, has been reported in 6 studies since 2003 and has revolutionized treatment in this field [2429], particularly for lung transplant recipients with chronic rejection or its clinical correlate Bronchiolitis Obliterans Syndrome (BOS). BOS is defined as an irreversible decline in FEV1 accompanied by a neutrophilic airway inflammation leading to a fibroproliferative process inducing a total obliteration of the airway [30]. Low-dose macrolide therapy was able to reverse the decline in lung function in some patients with BOS, questioning the current definition of BOS. Among the 116 patients investigated in these studies, 51 responded to the therapy with a mean FEV1 improvement of about 17%, which was maintained over time. However, again it is remarkable to observe that the treatment is only beneficial in a subgroup (about 44%) of the patients with BOS. A MACROLIDE RESPONSIVE PHENOTYPE COMMON TO VARIOUS LUNG DISORDERS Most of the aforementioned disorders allude on the same mechanisms of action for the macrolides. Already in DPB, not an anti-microbial action mechanism yet an antiinflammatory and immunomodulatory effect was assumed to be involved [5]. In CF, Saiman [31] outlined some more hypotheses: effects on biofilm production by P. aeruginosa (disrupting biofilm formation/quorum sensing molecules), a direct effect on cystic fibrosis transmembrane conductance

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regulator protein (CFTR), upregulation of the multi-drugresistant protein (MRD)(P-glycoprotein) and an effect on the epithelial adherence of P. aeruginosa due to decreased numbers of specific cell-surface receptors to which the organisms binds [32]. In the field of lung transplantation, more putative mechanisms as direct effect on innate immunity (neutrophils/IL8), gastro-oesophageal reflux disease (GERD), lymphocytic bronchitis/bronchiolitis and interaction with immunosuppressive trough levels were put forward [33]. In a recent pulmonary perspective we outlined a specific clinical phenotype of a group of patients with BOS after transplantation that benefits from macrolide therapy (Table 2) [34]. This phenotype can be clinically discriminated and diagnosed by neutrophilic airway inflammation (increase of neutrophils/IL8 in the BAL), which is clinically apparent by crackles on lung auscultation and increased sputum production. The evolution of this chronic inflammation seems to be a slow process, possibly due to repetitive chronic injury of the epithelium, leading to a slow obliterating process (Table 2) [34]. Histological examination of open lung biopsies during the inflammatory phase revealed inflammatory lesions characterized by a prominent peribronchiolar infiltrate of mononuclear cells (macrophages and lymphocytes), but not yet a pronounced tissue repair/remodeling and fibrosis. Radiological evaluation typically disclosed bronchiectasis, airway wall thickening and mucus plugging.
Table 2.
Macrolide Responsive Phenotype Inflammation Clinically Contributing cofactors Progression Histologically Radiologically Azithromycin neutrophilic airway inflammation coarse crackles, increased sputum production Pseudomonas aeruginosa colonization, gastro-oesophageal reflux slow progression (several years) initially inflammatory, ends up in pure fibrosis bronchiectasis, mucus plugging, airway wall thickening reversible (effective)

mostly with recurrent P. aeruginosa and Aspergillus infection, are found in over 65% of patients with BOS after lung transplantation and this more frequently in the later course of BOS. Nevertheless, in a case report of a 16-year-old lung transplant patient with BOS stage 3, azithromycin therapy resulted in a major improvement of the bronchiectases [37]. As in BOS, patients with DPB, CF and COPD go on to develop bronchiectases, mostly in a later stage of the disease together with colonization/infection with several bacteria such as P. aeruginosa, S. aureus, etc. Azithromycin appears to be able to break this chain of events leading to bronchiectases and this may be due to its antibacterial and antiinflammatory effects, but also to its property to increase the penetration and destruction of biofilm formation (Table 1). ANTI-BACTERIAL PROPERTIES OF MACROLIDES: P. AERUGINOSA IN THE HEAT OF THE FIGHT The anti-microbial properties of macrolide antibiotics have been extensively reviewed before and are beyond the scope of this review. We will however focus on the potential contribution of this anti-microbial effect to the benefit of macrolide treatment in the aforementioned lung disorders. Indeed, in these lung disorders, the macrolide responsive phenotype was associated with the presence of bacteria, particularly Pseudomonas (BOS, DPB and CF) [38, 39], Chlamydia (asthma and BOS) [40] and Mycoplasma (asthma). Yet none of the hitherto published studies was able to demonstrate a conclusive relationship. For instance, critical observation in CF and BOS demonstrated that patients not colonized with Pseudomonas also improved with azithromycin therapy. Likewise not all colonized CF patients responded to azithromycin [28, 41-42]. However the contribution of micro-organisms to the nefast progression of the various disorders cannot be over-estimated as persistent colonization with P. aeruginosa (non-mucoid and mucoid strains) occurs in about 70% of patients with DPB, 80% of patients with CF and 40% of the transplant patients with BOS. It has been suggested that the effect of macrolides on P. aeruginosa is anti-inflammatory rather than antibacterial, since these bacteria are inherently resistant to macrolides [43]. P. aeruginosa produces small (quorum-sensing) signal molecules (QsM), with a N-acetylhomoserine lactone structure, acting as auto-inducers. A large number of bacterial genes are regulated by quorum-sensing signals, many of which encode for putative virulence factors and alginate production. Formation of alginate by mucoid P. aeruginosa leads to biofilm formation, making individual bacteria unreceptive to therapeutic concentrations of antibiotics and host defences (such as ciliary clearance, opsonization, complement deposition, phagocyte engulfment and lymphocytemediated killing), impeding bacterial eradication [44]. It is also believed that the biofilm acts as an antigen and induces an antigen-antibody reaction on the surface of the airway leading to neutrophilia and lung damage [4, 44]. Furthermore, QsM have an intrinsic immune-modulatory capacity resulting in suppression of T-cell proliferation, direct induction of neutrophil chemotaxis, besides indirect neutrophil chemotaxis via chemokine (especially IL8) or cytokine release from human bronchial epithelial cells [45]. Low dose macrolides suppress protein synthesis, including quorumsensing molecules, virulence factors and alginate production

Characteristics of the responsive phenotype based on the experience in lung transplantation regarding treatment of chronic rejection.

A recurrent feature of the so-called responders over the different lung disorders is the presence of bronchiectasis, the key pathogenic factors of which is chronic bronchial infection/colonization and persistent airway inflammation. Initially, a genetic or environmental impairment of mucus clearance or host defence mechanisms fails to eliminate the micro-organisms, allowing them to persist in the bronchial tree, thereby inducing an inflammatory response by the production of toxins, which subsequently causes lung damage, eventually leading to a vicious circle of events. Long-term therapy with antibiotics may interrupt this vicious circle. Tsang et al. [35] indicated that macrolides are a potential therapeutic option. Yalcin et al. could associate the improvement of established bronchiectasis with a reduction of airway leukocytes, neutrophils and IL8 after three months of clarithromycin therapy [36]. Bronchiectases, which coincide

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in P. aeruginosa leading to a loss of viability with prolonged exposure. It has been suggested that treatment with azithromycin also impairs the transition from non-mucoid to mucoid phenotype and increases the penetration and destruction of the biofilm [46] (Fig. 1). There is emerging evidence that Pseudomonas colonization is linked to airway neutrophilia and BOS after transplantation [38, 47] and, as a consequence, it is often assumed that colonization with Pseudomonas is a prerequisite for the beneficial effect of azithromycin. Indeed in the Gerhardt study, 4 out of 6 patients with FEV1 improvement to azithromycin were colonized with Pseudomonas [24]. On the other hand, Verleden et al. reported that the effect of azithromycin on FEV1 did not differ between colonized and non-colonized patients [25]. Furthermore, Shitrit et al. found Pseudomonas in 9 out of 11 patients, but none of these responded to the therapy [27]. However, airway colonization is not always picked up by BAL culture, since there is also evidence of an increase in QsM in stable, non-colonized lung transplant patients [48-49]. Bacterial colonization, particularly by P. aeruginosa, is a potent inducer of neutrophilic airway inflammation in the different respiratory disorders, which needs to be monitored and reported more frequently and, if possible, eradicated to prevent further untoward airway damage. Despite its importance, however, (Pseudomonas) colonization is probably not the sole candidate to explain the presence of neutrophilic inflammation in various lung disorders. Therefore, it cannot be regarded as the sole discriminatory factor between responders and nonresponders in macrolide treatment.

ANTI-REFLUX PROPERTIES OF MACROLIDES The role of gastro-oesophageal reflux disease (GERD) with gastric aspiration, in the pathogenesis of miscellaneous respiratory disorders has been discussed for decades and is nowadays recognized as an important non-alloimmune mechanism associated with (difficult-to treat or severe) asthma and chronic cough [50-51]. Also in patients with established bronchiectasis, more extensive disease is observed in those patients with GERD [52]. In LTx, GERD is now recognized as a potential cause of reversible allograft dysfunction [33, 53] and, despite the fact that most LTx recipients are asymptomatic [54], GERD is common in these patients due to a number of risk factors such as post-operative iatrogenic vagal innervation, reduced gastric motility induced by immunosuppressive drugs as calcineurin inhibitors, impaired cough reflexes and mucociliary clearance [55]. This was also illustrated in a recent publication, describing that switching from a cyclosporine-A to a tacrolimusbased immunosuppressive treatment was able to improve gastric discomfort in a LTx recipient [56]. Gastric aspiration may be an ongoing source of respiratory epithelial injury as both pepsin and bile acids are potentially cytotoxic and have been shown to interact with key players of the pulmonary innate immune system, specifically with surfactant proteins and phospholipids [57], airway neutrophils and IL8-production [58-59] (Fig. 1). This ongoing epithelial injury caused by gastric aspiration may also play a crucial role in colonization of the respiratory tract, particu-

Fig. (1). Schematic overview of the chronic inflammatory process and the potential interactions of macrolides.

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larly by P. aeruginosa, as it facilitates adherence of bacterial adhesins to the epithelium by exposition of the underlying fimbrial receptors on the epithelial cell surface [60-61]. Particularly in gastroenterological literature, macrolides are well-known for their properties of enhanced gastric emptying, which is due to a motilin-like effect [62-63]. However, this prokinetic characteristic of macrolides might also be a possible explanation for the beneficial effect of azithromycin in respiratory disorders [64] as a recent observation in lung transplantation showed that BAL levels of pepsin were significantly lower in patients treated with azithromycin, compared to those who were not [65], suggesting an inhibitory effect on GERD. Despite its pathogenic importance in various respiratory disorders, however, it is unlikely that GERD is the sole discriminatory factor in the presence or absence of a clinical response to macrolide treatment. For instance, in the study of Verleden et al. none of the patients (all on a tacrolimusbased immunosuppressive treatment) had clinical symptoms of GERD, yet FEV1 improvement with azithromycin was seen in half of them. Shitrit et al. more closely investigated GERD as possible mechanism in their study by using 24hour pH-monitoring and gastroscopy, identifying only two patients with GERD, who had already been treated with a high-dose proton pump inhibitor. As a consequence, although GERD could to some extent be implicated in the action of macrolides, this does not explain the beneficial effect of azithromycin documented in these series of patients and it certainly does not discriminate between responders and nonresponders. THE ANTI-INFLAMMATORY PROPERTY OF MACROLIDES The actual mechanism of action of azithromycin remains obscure, as well as the reason why treatment is only effective in the specific phenotype of patients mentioned above. The beneficial effect of macrolides in different lung disorders is believed to be, at least partially, attributable to the antiinflammatory properties of the drugs [4] (Fig. 1). In most lung diseases this has been postulated, based on in vitro and in vivo animal studies [66-68], but it has not been investigated in depth. Additional in vitro and in vivo information on the anti-inflammatory effect of macrolides and their possible mechanisms of action in lung transplantation has recently been published by our group [28, 68] reporting on a 3 months follow-up study on azithromycin for lung transplant patients diagnosed with BOS [28]. The study went beyond the empiric evaluation of lung function parameters (FEV1/BOS) and was the first to investigate a potential antiinflammatory effect. Neutrophils [69] and the potent neutrophil chemoattractant IL8, were measured in the BAL. In the study population as a whole, azithromycin therapy resulted in a significant mean increase in FEV1 by 0.31L (or 13%) after 3 months of therapy. Equally important, this improvement was accompanied by a significant decrease of both BAL neutrophils and IL8. The effect of the aforementioned possible risk factors (GERD, bacterial growth of Mycoplasma pneumoniae, Chlamydia pneumoniae or P. aeruginosa and varying immunosuppressive trough levels) on the neutrophilic inflammation was confirmed as being cofactors,

yet not discriminators [38, 70]. The positive effect of azithromycin therapy was observed mainly in the small subset (phenotype) of patients characterized before. The significant discriminating variables in the dichotomy of responders and non-responders (FEV1 increase >10%) were the initial BAL neutrophilia and IL8. In fact, the results of this study suggested that neutrophils may be the prerequisite for a positive effect of azithromycin therapy on FEV1, as shown by a positive correlation (r=0.79) between initial BAL neutrophilia and changes in FEV1 over the study period. A second in vitro study in human airway smooth muscle cells (HASMC) further investigated the effect of macrolide therapy, adding further evidence to the plausibility that azithromycin acts via an anti-inflammatory mechanism in the specific phenotype in BOS after lung transplantation [55]. In this study we could indeed demonstrate that azithromycin inhibits the IL17-induced IL8 production in these HASMC, leading to inhibition of neutrophil activation and recruitment. Immunosuppressive agents classically used in the setting of lung transplantation even seem to further enhance the IL17-induced IL8 production, whereas steroids had no significant suppressive effect at all. The principal difference between immunosuppressive drugs/steroids and macrolides seemed to be the inhibition of the different mitogen-activated kinase (MAPK), p38-MAPK, extracellularregulated kinase (ERK) and Janus-N-terminal kinase (JNK), and the reduction of oxidative stress (as measured by 8isoprostane) by azithromycin, yet not by steroids and any of the tested immunosuppressive drugs [68]. Indirectly, these findings lead us to conclude that, in a simplified view, macrolides specifically reduce inflammation by inhibiting components of the innate immune system activation, whereas immunosuppressive agents suppress lymphocytes - key players in the adaptive immune system without any effect on the ongoing neutrophilic inflammation. By contrast, conventional immunosuppressive agents even seem to enhance chemokine production (and potentially neutrophil attraction). PULMONARY FIBROSIS: POTENTIAL TARGET FOR MACROLIDE THERAPY? However a major feature of many chronic lung disorders is fibrosis, a comparable dichotomy can be observed in interstitial lung disease, the prototype of a fibrosing pulmonary disorder. Despite its current anti-inflammatory or antifibrotic treatment is beyond the scope of this review, it has to be pointed out that histologically the pathogenic dichotomy regarding inflammation and fibroproliferation in OB parallels very much non-specific interstitial pneumonia (NSIP) on the one hand and usual interstitial pneumonia (UIP) on the other hand. In UIP, however, a restrictive lung function is confined to level of the lung interstitium, while the pulmonary function decline in BOS, is due to a disease along the entire bronchial tree [71]. NSIP may therefore resemble neutrophilic reversible allograft dysfunction (NRAD), where inflammation (lymphocytic or neutrophilic) leads to tissue damage, excessive repair and remodelling and eventually in fibrosis, whereas in UIP, as in fibroproliferative BOS (fBOS), fibrosis without cellular inflammation seems to be present and a key role for angiogenetic and remodelling factors can be put forward. UIP develops rapidly, while NSIP

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develops more gradually. Again, as in BOS, in both types the cause of the inflammation or direct fibrosis remain elusive, although one could also speculate about the severity and duration of airway epithelial damage in NSIP, as in NRAD, which could be triggered by repetitive, mild injury (infection/colonization, reflux, [38, 57, 70]). In spite of these resemblances, however, the potential therapeutic role of macrolides has thus far not been investigated in patients with interstitial lung disease, although recently a case report was published demonstrating a beneficial effect of clarithromycin in steroid resistant desquamative interstitial lung diseases [72]. RELEVANT DRUG-INTERACTIONS OF MACROLIDES THROUGH CYP-3A4 Macrolides are well-known for their prokinetic effect by stimulating motilin receptors, possibly altering absorption of other drugs, as well as their interaction with drugs like psychotropic agents, immunosuppressives, theophylline and non-sedating antihistamines [73], which can be potentially life-threatening. Furthermore, it is common knowledge that macrolides can have a proarrhythmic effect by retarding heart repolarization and prolongation the QT interval, with the risk of inducing potentially fatal ventricular arrythmias (i.e. Torsades de Pointes). Macrolides less likely to cause arrhythmias are azithromycin and roxythromycin as they seem to have a proarrhythmic potential at concentrations higher than those achieved with doses usually prescribed clinically, which may explain why this complication is hardly ever seen in our transplant population. Nevertheless, one needs to be very careful in the selection of a macrolide to address the proposed phenotype in patients already treated for concurrent comorbidities (e.g. CF, transplantation, COPD, cardiac diseases). Both monitoring of the serum concentrations of concurrently administered drugs and of the electrocardiogram should be considered when a macrolide is prescribed to patients at risk [74]. These aforementioned drug-interactions find their origin within the biotransformation of the drugs through their interaction with the hepatic cythochrome P-450. Macrolides differ in their abilities to bind to and inhibit the cytochrome P450 isoform CYP 3A4. These differences prompted von Rosenstiel et al. to reclassify macrolide antibiotics into three groups on the basis of in vitro experiments of the binding to and inhibition of CYP 3A4 [75]. The explanation for the success of the use of azithromycin in lung transplantation lies within its poor druginteraction. Erythromycin and clarithromycin, on the other hand, strongly inhibit P-glycoprotein (P-gp) and the hepatic or intestinal mixed-function oxidase system (CYP3A4, an isoform of cytochrome P-450) [75], which are known to take part in the absorption, distribution and elimination of immunosuppressive agents by the gastrointestinal epithelium [76]. Since tacrolimus and cyclosporine are also metabolized by the CYP3A4 and transported by P-gp in the intestine [73], adding erythromycin or clarithromycin will increase trough levels of tacrolimus/cyclosporine [77], causing more side effects. In lung transplantation it has even been postulated that the effect of azithromycin was due to its possible influence on the trough levels of the immunosuppressive drugs (tacrolimus/cyclosporine) [33], however, it poorly interferes

with CYP3A4 and P-gp and consequently does not affect trough levels of tacrolimus/cyclosporine [75]. CONCLUSIONS AND FUTURE PERSPECTIVES The introduction of azithromycin in lung transplantation is truly a milestone in the treatment of BOS. Earlier, this therapy had already proven to be superior to conventional treatment strategies in a subset of patients suffering from other pulmonary disorders like DPB, CF and asthma. In none of these therapeutical applications did one document the mechanism of action of macrolide treatment. Several potential mechanisms were put forward including an effect on colonization/infection (P. aeruginosa, Mycoplasma pneumoniae, Chlamydia pneumoniae), on gastroesophageal reflux disease (GERD), lymphocytic bronchitis/bronchiolitis, increase of immunosuppressive trough levels or an effect on innate immune responses (neutrophils/IL8). The most widely accepted hypothesis relates to an antiinflammatory effect of macrolide therapy involving neutrophils and some cytokines [4, 19, 41]. By [28, 68] a combination of an in vitro and in vivo clinical study, our group has strengthened the evidence for an anti-inflammatory mechanism of azithromycin in lung transplantation. The effect involves inhibition of IL8 production and inhibition of neutrophil activation and recruitment. The mechanistic difference between conventional immunosuppressives and macrolides is that the latter inhibit activation of the different MAPK (p38, ERK and JNK), and also reduce oxidative stress. From an immunological point of view, macrolides seem to specifically reduce the innate immune system activation and not the adaptive immune system activation. On the other hand, immunosuppressive agents are well known to non-specifically suppress adaptive immune responses (lymphocytes) while leaving the innate immune response unaffected (neutrophils). Our data even lead us to believe that the innate immune response is enhanced by conventional immunosuppressive therapy. Interestingly, this specific antiinflammatory effect of azithromycin is only translated into clinical improvement and thus efficacy in a distinct subset of patients over the various chronic inflammatory lung disorders. In lung transplantation, the documented response rate is hitherto 51 out of 116 published patients (or 44%), which is similar to other lung diseases [42]. In our clinical study [28] responders to azithromycin therapy were characterized by manifest neutrophilic inflammation, which had disappeared after three months of treatment. In the non-responders, neutrophilic inflammation was not present. As the pre-treatment airway neutrophilia correlates with the improvement of lung function after three months of therapy, this leads us to believe that we can predict whether a LTx recipient with BOS will yes or no benefit from azithromycin therapy, just by looking at the BAL neutrophilia. As a consequence, we defined two phenotypes of BOS: neutrophilic reversible allograft dysfunction (NRAD) and fibroproliferative BOS (fBOS) [34]. The first type (NRAD) is characterized by increased BAL neutrophilia and IL8, early onset after transplantation, slow FEV1 decline, coarse crackles on lung auscultation and responsiveness to azithromycin. The other type (fBOS) shows no increased BAL neutrophilia or IL8, no coarse crackles on lung auscultation, a rapid FEV1 decline and unresponsiveness to azithromycin therapy. If left untreated, both NRAD and fBOS will lead to inactive OB le-

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sions, yet the immunological mechanism underlying the fibroproliferative obliteration of the airways is entirely different: NRAD requires neutrophilic inflammation whereas fBOS does not. Exactly what triggers any of these two forms remains unknown, but one could speculate that the central element in the differentiation of both types of BOS is the severity and duration of airway epithelial damage. Subsequently, the proposed dichotomy could have major clinical consequences: patients developing BOS who are found to have substantial BAL neutrophilia have a rather good prognosis thanks to azithromycin treatment, whereas patients without BAL neutrophilia dont. In the latter case, retransplantation should be considered when the FEV1 further declines. From a semantic point of view, the acceptance of the NRAD phenotype, questions the current definition of BOS, which implies an irreversible loss of pulmonary function, characterized by a neutrophilic inflammation leading to fibroproliferation of the airways. Hence, a reformulation of BOS seems indispensable. NRAD should be seen as an exaggerated response of the innate immune system (which is by definition not specific) and it may even be a risk factor for the further development of fBOS [29]. Any patient suspected to have BOS should have BAL neutrophilia evaluated, as its presence seems to be the determining factor for an azithromycin responsive phenotype. By distinguishing NRAD from true BOS, the description of (f)BOS remains valid as an irreversible loss of pulmonary function, characterized by a fibroproliferative disorder of the small airways, after other possible concurrent post-transplant complications such as acute rejection, infection and GERD have been excluded. However, further progress in the treatment of BOS warrants a better understanding of its pathophysiology. Especially the pathogenesis of the NRAD phenotype as well as the non-antibiotic mechanism of action of macrolides need further unravelling. This implies an improved insight in the triggers of this type of airway inflammation: how does the intricate dialogue between innate and adaptive immune responses kindle the development of BOS? We have highlighted the involvement of IL17 as well as the importance of the TH17-IL17 pathway. How do the two important pathways involved in cell-mediated response, i.e. the TH1-IFN pathway and the TH17-IL17 pathway, fit into the scheme? Perhaps the answer can be found at the very first events of the immune response, in the dendritic cells, presenting the antigen to the other immune cells in the airway. A better understanding of the pathogenesis of BOS and thus of the macrolide-responsive phenotype will pave the way for the judicious application of this potential life-saving yet presumably not completely harmless treatment in other chronic inflammatory lung disorders ACKNOWLEDGEMENTS Research funds: GMV is holder of the GSK (Belgium) chair in respiratory pharmacology at the KULeuven, and is supported by the Research Foundation Flanders (FWO): G.0493.04, G.0518.06 and G.0643.08. BMV, LJD are senior research fellows and RV is a PhD fellow of the FWO.

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Received: July 16, 2008

Revised: August 20, 2008

Accepted: August 28, 2008