Bile Acids Aspiration Reduces Survival in Lung Transplant Recipients with BOS Despite Azithromycin

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1 American Journal of Transplantation 2011; 11: Wiley Periodicals Inc. C 2011 The Authors Journal compilation C 2011 The American Society of Transplantation and the American Society of Transplant Surgeons doi: /j x Bile Acids Aspiration Reduces Survival in Lung Transplant Recipients with BOS Despite Azithromycin V. Mertens a, K. Blondeau a, L. Van Oudenhove a, B. Vanaudenaerde b,r.vos b,r.farre a, A. Pauwels a,g.verleden b,c, D. Van Raemdonck c, D. Sifrim a,d and L. J. Dupont b,c, a Center for Gastroenterological Research, KULeuven, Belgium b Laboratory of Pneumology, KULeuven, Belgium c Lung Transplantation Unit, University Hospital Gasthuisberg, KULeuven, Belgium d Barts and The London School of Medicine and Dentistry, University of London, UK Corresponding author: Lieven J. Dupont, lieven.dupont@uz.kuleuven.be Azithromycin (AZM) improved bronchiolitis obliterans syndrome (BOS) and reduced aspiration in lung transplant (LTx) recipients. We hypothesize that AZM could improve graft and overall survival more efficiently in LTx patients with BOS who have bile acid (BA) aspiration by protecting against the aspiration-induced progression of BOS. The goal was to compare FEV 1 (% baseline), BOS progression and overall survival in LTx recipients treated with AZM for BOS, both with versus without BA aspiration. Therefore, LTx recipients treated with AZM for BOS were recruited and bronchoalveolar lavage (BAL) samples were analyzed for the presence of BA and neutrophilia before the start of AZM treatment. Short-term effect of AZM on FEV 1 and BAL neutrophilia was assessed, progression of BOS and survival were followed-up for 3 years and results were compared between patients with/without BA aspiration. 19/37 LTx patients had BA in BAL. BA aspiration predisposed to a significantly worse outcome, in terms of decline in FEV 1, progression of BOS 1 and survival. AZM does not seem to protect against the long-term allograft dysfunction caused by gastroesophageal reflux (GER) and aspiration and an additional treatment targeting aspiration may be indicated in those LTx patients. Key words: Azithromycin, bile acids, chronic rejection, lung transplantation, respiratory aspiration Abbreviations: AR, acute rejection; AZM, azithromycin; BA, bile acids; BAL, broncho-alveolar lavage; BOS, bronchiolitis obliterans syndrome; BOSp, potential BOS; CF, cystic fibrosis; CMV, cytomegalovirus; COPD, chronic obstructive pulmonary disorder; FEV 1, forced expiratory volume in one second; GER, gastroesophageal reflux; GLM, general linear models; GNB, gram-negative bacteria; HLTx, heart lung transplantation; LTx, lung transplantation; PPI, proton pump inhibitor; SLTx, unilateral lung transplantation; SSLTx, bilateral lung transplantation; SP-A/D, surfactant protein-a/d. Received 15 April 2010, revised 12 October 2010 and accepted for publication 12 October 2010 At a Glance Commentary Scientific knowledge on the subject Aspiration of bile acids has been implicated in the pathogenesis of chronic rejection (BOS) after lung transplantation. Azithromycin (AZM), a macrolide with immunomodulatory properties has a beneficial effect in the treatment of BOS and may also reduce aspiration. It has not been shown whether AZM improves graft function more efficiently in LTx patients with evidence of bile acids aspiration. What this study adds to the field The present cohort study evaluated impact of aspiration on the short-term and long-term effects of treatment with AZM in LTx patients with BOS. Aspiration does not predict short-term improvement with AZM. The presence of bile acids in BAL was associated with progression to more severe BOS and reduced survival, despite continued treatment with AZM. AZM insufficiently protects against the long-term allograft dysfunction due to aspiration. Introduction Lung transplantation (LTx) has become an effective therapeutic option for several end stage pulmonary disorders (1). Studies showed a high prevalence of gastroesophageal reflux (GER) and aspiration of gastric components (pepsin and bile acids) after LTx (2 4) and both have been implicated as contributory nonalloimmune factors in the pathogenesis of bronchiolitis obliterans syndrome (BOS), which remains the major cause of late allograft failure and death in LTx recipients) (3,5). Aspiration of gastroesophageal refluxate, especially bile acids, may interact with the respiratory epithelium and induce neutrophil recruitment by promoting the expression of cytokines such as IL-8 by structural airway cells (6,7). 329

2 Mertens et al. Given observations from multiple studies that link reflux to the development of BOS (3;5;7;8), the most effective approach to prevent aspiration-related lung injury and improve long-term LTx outcomes remains to be determined (3;5;8;9). Limited data are available on the effect of GER treatment in preventing graft dysfunction after LTx. Gastric acid inhibitors (proton pump inhibitors (PPI) and H 2 antagonists) reduce gastric acid secretion, but do not eliminate reflux nor the risk for aspiration. LTx patients treated with PPI did not have a reduction in pepsin or bile acid concentration compared to patients not treated with PPI (9). Antireflux surgery has been proposed as an invasive but more efficient means to protect against the development of BOS (10 13). By reducing all types of reflux (acid and nonacid) fundoplication may significantly decrease aspiration of gastric contents and prevent the induction of airway inflammation leading to BOS. An alternative approach that might be advocated consists of treating the consequences of GER and aspiration rather than the cause itself. Azithromycin (AZM), a macrolide antibiotic, has a beneficial effect in the treatment of cystic fibrosis (CF) which has been attributed to its immunomodulatory effect (14;15). More recent (albeit smaller) studies have suggested a role for AZM in the treatment of BOS possibly through its ability to reduce airway neutrophilia (16). Mertens et al. recently showed that AZM is able to reduce GER severity and aspiration of bile acids after LTx, probably due to a prokinetic effect on esophageal and gastric motility (17). On the basis of these findings it could be argued that treatment with AZM in LTx patients with BOS might protect against the GER/aspiration-induced progression of BOS. We hypothesize that AZM could improve graft and overall survival more efficiently in LTx patients with BOS who have bile acid aspiration. The aim of this study was to compare FEV 1 (% baseline), BOS progression and survival, between AZM-treated LTx recipients with versus without bile acids aspiration, during a follow-up period of 3 years. Methods Study design and patient population This cohort study was performed in the LTx unit of the University Hospital Gasthuisberg, KULeuven. LTx recipients who were treated for BOS 0p with AZM were recruited during the period December 2002 December The severity stages of BOS were based on a decrement of FEV 1 and/or FEF 25 75% according to International Society of Heart and Lung Transplantation criteria. BOS stage 1 was defined as a FEV 1 between 66% and 80% of the baseline; BOS stage 2 as a FEV 1 between 51% and 65% of the baseline; and BOS stage 3 as a FEV 1 between 0% and 50% of the baseline. Potential BOS stage (BOS p ) was defined as a 10 19% decrease in FEV 1 or a 25% or more decrease in FEF 25 75% from the baseline. Patients undergoing retransplantation and patients who could not be classified according to the ISHLT BOS criteria were excluded. All LTx recipients received centerbased follow-up care scheduled throughout the postoperative period with a maximum interval between assessments of 3 months. Follow-up data were recorded for a period of at least 3 years after the initiation of AZM or until death/retransplantation. Spirometry was performed according to ATS/ERS guidelines. The study was approved by the local Ethical Committee. Immunosuppression Standard maintenance immunosuppression consisted of a triple drug regimen including a calcineurin inhibitor, a cell-cycle inhibitor and steroids (target trough levels for cyclosporin levels of ng/ml, for tacrolimus of 8 12 ng/ml and for mycophenolate of 2 4 mg/ml, azathioprine 1 2 mg/h/day adjusted to leukocyte count, methylprednisolone 2 8 mg/day). Prophylactic treatment with omeprazole 20 mg od was given since LTx. Standard of care for patients with BOS stage 0p in this study consisted of (AZM, 250 mg orally 3 times/week) for the whole duration of the study period while changing immunosuppression was avoided. BAL acquisition and analysis Bronchoscopy with broncho-alveolar lavage (BAL) was performed before the start of AZM therapy. BAL was performed by instilling two sequential 50 ml aliquots of isotonic saline, in the lingula or right middle lobe. Pooled samples of aspirated fluid were processed immediately for analysis. Bile acids determination was performed in BAL samples obtained prior to the start of treatment with AZM, by means of a commercially available (Bioquant, San Diego, CA, USA) enzymatic assay (lowest level of detection 0.2 lmol/l). Statistical analysis Statistical analyses were performed using Graphpad Prism 4.0 (San Diego, CA, USA) and SAS (with Enterprise Guide 4.0) statistical software. For all analyses, p values of <0.05 were considered statistically significant. Descriptive results are presented as median and interquartile range. Comparison between groups was done using Mann Whitney-U test and Fisher s exact test. In order to predict the effect of the presence of bile acids in BAL on survival and time to progression of BOS, Kaplan Meier survival analyses were performed. In addition, in order to be able to provide an estimate of the magnitude of the difference in survival or time to progression of BOS as well as to control for other potentially relevant variables, Cox proportional hazard regression models were estimated. Age at LTx, neutrophil levels before start of AZM, FEV 1 at the start of AZM and time of starting of AZM (days post-ltx) were entered as potential independent variables in a multiple regression with a forward selection procedure (criterion to be entered into the model: p<0.10); presence of bile acids was forced into the model. In addition, a general linear model analysis with change in FEV 1 3 months after AZM as the dependent variable was estimated, with the same potential independent variables and selection procedure. Indication for LTx was left out of the model because of multicollinearity with age at LTx. Results The presence of bile acids aspiration and patient characteristics A total of 37 LTx patients with BOS were recruited in this cohort study. Bile acids were detected in BAL, before the administration of AZM in 19 patients (51%). The median level of bile acids in the patients whose BAL tested positive was 1.2 lm ( ). The underlying diagnosis was COPD-emphysema (n = 15), CF (n = 6), pulmonary fibrosis (n = 5), primary pulmonary hypertension (n = 2), alpha American Journal of Transplantation 2011; 11:

3 Aspiration and Transplant Survival Table 1: Patient characteristics (expressed as median [25 75IQR] unless otherwise noted) of the lung transplant (LTx) recipients recruited into this study (n = 37), subdivided into two groups on the basis of the presence of bile acids in BAL No bile acids present Bile acids present in BAL (n = 18) in BAL (n = 19) p-value Baseline patient characteristics Gender (male/female) 9/11 11/ Underlying lung disease 0.57 COPD 7 8 Cystic fibrosis 3 3 Pulmonary fibrosis 3 2 Primary pulmonary hypertension 1 1 Alpha1 antitrypsin deficiency emphysema 1 2 Other 3 3 Type of LTx 0.57 Double LTx Single LTx 4 5 Heart-LungTx 0 1 Age at LTx (years) 55 (39 61) 54 (44 61) 0.78 Number of AR events (no. of events grade 1 AR) 2 ( ) 1 (0 2) 0.42 CMV status (D-R-/D+R-/DxR+) 8/3/7 7/4/ Time of diagnosis of BOS and start of AZM (days after LTx) 505 ( ) 408 ( ) 0.89 Number of patients in BOS stage 0p/1/>1 before start of AZM 7/8/3 6/11/ Median FEV 1 before AZM (% baseline) 78 (70 82) 77 (69 86) 0.72 BAL neutrophil count before AZM (% cell count) 16 (3 56) 34 (6 67) 0.43 BAL colonization status (colonized/not colonized with GNB) 5/13 5/ Short-term effects of AZM Improvement FEV 1 with AZM (% baseline) 7 ( 6 19) 5 ( 4 11) 0.79 Reduction in BAL neutrophil count with AZM (% cell count) 7 ( 33 4) 5 ( 39 6) 0.73 Other included bronchiectasis, lymphangioleiomyomatosis, obliterative bronchiolitis (other than BOS), Eisenmenger syndrome. antitrypsin deficiency emphysema (n = 3) and other (n = 6). Twenty-seven patients underwent bilateral lung transplantation (SSLTx), nine patients received unilateral lung transplantation (SLTx) and one patient a heart-lung transplantation (HLTx). On the basis of the presence of bile acids in BAL, patients were divided into two groups. There were no statistically significant differences in patient characteristics between both groups. The demographic and clinical characteristics of patients with and without bile acid aspiration arelistedintable1. The distribution of the BOS stages before the start of AZM is represented in Figure 1. When they started treatment with AZM, six patients with bile acids in BAL were diagnosed as having BOS stage 0p, 11 patients had BOS stage 1 and there were two patients with BOS stage 2. Patients without bile acids in BAL were similarly distributed between the different BOS severity stages at the start of AZM treatment: seven patients with BOS stage 0p, eight with BOS stage 1 and three patients had BOS stage 2. Short-term effect of AZM on FEV 1 and BAL neutrophilia Median FEV 1 before and 3 months after start of AZM was 78% of baseline FEV 1 and 84% of baseline FEV 1, respectively (p = 0.03). The overall increase in % baseline FEV 1 3 months after AZM treatment was 6% ( 5 16%). Twentythree LTx patients (62%) had an increase of 5% of baseline FEV 1 and 17 patients (46%) had 10% increase of baseline FEV 1 3 months post-azm treatment. Median BAL neutrophil count before and 3 months after start of AZM was 26% (3 66%) and 10% (4 32%), respectively (p = 0.05). The median decrease in % BAL neutrophilia 3 months after AZM treatment was 6% ( 39 6%). Effect of bile acids aspiration on improvement of FEV 1 and change in BAL neutrophilia 3 months after start of treatment with AZM The increase in % baseline FEV 1 3 months after AZM treatment was not significantly different between the group with bile acids and the patients who did not have bile acids in BAL (Table 1). Of the 23 LTx patients with an AZMinduced improvement 5% of baseline FEV 1,12hadbile acids in BAL (p = 1). Of the 16 LTx recipients with an AZMinduced improvement of 10% of baseline FEV 1, 7had bile acids in BAL (p = 0.51). The reduction in % neutrophil count in BAL 3 months after AZM treatment was not significantly different between the groups with bile acids versus the patients who did not have bile acids in BAL (Table 1). Neutrophil levels in BAL before start of AZM treatment were found to be the only significant independent predictor of change in FEV 1 3 months after start of AZM treatment American Journal of Transplantation 2011; 11:

4 Mertens et al. Figure 1: BOS grading of LTx patients before AZM treatment and 3, 12 and 36 months after initiation of treatment. in a GLM analysis, controlling for the presence of bile acids in BAL (Table 2). Effect of bile acids aspiration on progression of BOS BOS status was evaluated at different time points: before AZM treatment, 3 months after start AZM, 12 months after start AZM and 36 months after AZM. The distribution of the BOS stages at different time points is represented in Figure 1. The AZM-induced improvement in FEV 1 had as consequence that 3 months after start of AZM seven patients with bile acids in BAL and 10 patients without bile acids could no longer be labeled as having BOS (stage 0). The number of patients having BOS stage 0 decreased in both groups when evaluated at 12 months and at 36 months after the initiation of AZM while the number of patients with more severe BOS increased, especially at 36 months and more pronouncedly in the group of patients with bile acids in BAL. A significant difference was found in % freedom from BOS 1 in those patients with versus Table 2: Multiple regression models Hazard ratio Dependent variables Independent variables Beta±SE F/v 2 value [95% CI] p-value General linear model Change in FEV 1 3 months after AZM % BAL neutrophils before AZM 0.21 ± 0.07 F = 0.07 Not applicable versus prior to start of AZM Presence of bile acids in BAL 3.14 ± 4.48 F = 4.48 Not applicable 0.49 Cox proportional hazard models Time to onset to BOS (days) FEV 1 (% baseline) before AZM ± v 2 = [ ] 0.07 Presence of bile acids in BAL 0.96 ± 0.42 v 2 = [ ] Survival time (days) Start AZM (days post-ltx) ± v 2 = [ ] Presence of bile acids in BAL 2.41 ± 1.06 v 2 = [ ] American Journal of Transplantation 2011; 11:

5 Aspiration and Transplant Survival Figure 2: Kaplan Meier survival curve: effect of bile acids in BAL on freedom from BOS 1. The percentage of patients with freedom from BOS 1, either with (solid line) or without (dashed line) bile acids in BAL (p = 0.04). without bile acids in BAL (Kaplan Meier survival analysis p = 0.04) (Figure 2). Patients with bile acids in BALF had a significantly steeper decline in FEV 1 (ml/day) during the follow-up period of 3 years after the start of AZM compared to patients without bile acids in BALF ( 0.86 ml/day vs ml/day, p = 0.01) (Figure 2) (Figure 3). The presence of bile acids in BAL was found to be the only significant independent predictor of having BOS 1in Cox proportional hazard regression, with FEV 1 at the start of AZM showing a trend (p = 0.07) (Table 2). The hazard ratio for bile acids was 2.60, indicating that the hazard (risk) of progression to BOS 1 for patients with bile acids in BAL was 2.6 times higher compared to patients without bile acids in BAL. Effect of bile acids aspiration on 3 year survival Survival was observed up to 36 months after the start of AZM treatment. Three of 37 patients underwent a reltx for BOS and were censored at time of retransplant. All three patients had bile acids present in their BAL prior to AZM treatment was started. Seven of 37 patients died within 36 months after the start of AZM treatment. The cause Figure 3: Change in FEV 1 (ml/day) in LTx patients with or without bile acids in BAL (p < 0.05). Figure 4: Kaplan Meier survival curve: effect of bile acids in BAL on survival. The percentage of patients surviving without reltx, either with (solid line) or without (dashed line) bile acids in BAL (p = 0.03). of death was BOS (n = 6) and acute hemorrhage from a bronchovascular fistula (n = 1). In six of these seven patients BAL was positive for bile acids. Patients without bile acids in BAL were significantly more likely to be alive without retransplant 3 years after start of AZM compared to patients with bile acids in BAL [Kaplan Meier survival analysis, p = 0.03] (Figure 4). The presence of bile acids in BAL and time of starting AZM (days post-ltx) were found to be significant independent predictors of survival time in Cox proportional hazard regression (Table 2). The hazard ratio for bile acids was 11.16, indicating that the hazard (risk) of death for patients with bile acids in BAL was times higher compared to that for patients without bile acids in BAL; it should be noted, however, that the confidence interval for the hazard ratio is wide, due to the small sample size. Discussion GER and the presence of gastric components, like bile acids, have been associated with an increased risk of developing BOS after LTx (5,9,18,19). AZM was shown to have a beneficial effect in patients with BOS after LTx probably through modulation of systemic and pulmonary cytokine profiles and an inhibitory effect on the innate immune response (20). AZM is also able to reduce severity of GER and aspiration of bile acids in LTx patients (17). This suggests that AZM in LTx patients with BOS might protect against the GER/aspiration-induced airway inflammation and progression of BOS. The present cohort study evaluated the short-term and long-term effects of treatment with AZM in LTx patients with BOS and compared the effects between patients with or without bile acid aspiration. The main findings of this study were the following. (1) Approximately 46% of patients had an improvement of 10% of baseline FEV 1 3 months after starting AZM, 54% of patients had an improvement in BAL neutrophilia after 3 months treatment with AZM. (2). The presence of bile acids in BAL did not affect the short-term (3 months) change in FEV 1 and BAL neutrophilia. (3) The presence of bile acids in BAL was American Journal of Transplantation 2011; 11:

6 Mertens et al. associated with progression of BOS and reduced survival, despite AZM treatment. LTx patients transplanted at our center and diagnosed with BOS 0p, who were started on AZM, were included in this study. Several mostly uncontrolled studies have shown beneficial effects of AZM in BOS. Most LTx centers adopted a strategy of starting AZM in patients diagnosed with BOS. In the present study, initiation of AZM resulted in an important improvement in FEV 1 in more than 40% of the patient cohort and a significant overall increase in % baseline FEV 1 that coincided with an improvement of the distribution of the BOS stages in our population. This is in agreement with previous studies showing a positive effect of AZM in 30 40% of patients with BOS and an increase in FEV 1 ranging between 13% and 18% (14;21;22). We have also shown that AZM is able to significantly reduce the number of neutrophils in BAL, confirming data by Verleden et al. and Gotllieb et al. In these studies, high BAL neutrophilia predicted the FEV 1 response to AZM therapy in LTx recipients with BOS and AZM reduced IL-8 and neutrophilia in BAL (16,21). The short-term improvement in FEV 1 and BAL neutrophilia was not different between BOS patients with versus without bile acid aspiration. Bile acids in BAL have been associated with elevated levels of IL-8 and neutrophils in BAL and decreased BAL levels of pulmonary surfactant proteins SP-A and SP-D (7,23), which may predispose to the development of BOS. Although AZM is able to partially reduce the bile acid aspiration (17), it seems unlikely that the shortterm effect of AZM on FEV 1 and BAL neutrophilia could be attributed to an effect on bile acid aspiration. Other effects of AZM, possibly immunomodulatory (14,15), may be more important in accounting for the observed improvement 3 months after initiation. In the present study we found a significant link between the presence of bile acids in BAL and both the progression of BOS as well as reduced survival despite treatment with AZM, which was implemented in all patients throughout the whole study duration. Although this study was performed in a relatively small cohort and interpretation might be limited by risks for confounding and the lack of ability to adjust for all risk factors, a significantly steeper decline in FEV 1 (ml/day) was also noted in those patients with bile acids in BAL. These findings argue against the hypothesis that AZM might protect against the bile acid-induced inflammation and allograft dysfunction. A prospective study by D Ovidio et al. showed that bile acids in BAL as a marker of aspiration may predispose to BOS development which is in agreement with our data (5). In addition, our data also showed a significant difference in survival between patients with and without bile acid aspiration. These study data support the deleterious consequences of continuous aspiration of gastroduodenal contents in LTx recipients. Hartwig et al. and Melzer et al. documented in a rat model of LTx that chronic aspiration of acid gastric fluid accelerated the pulmonary allograft dysfunction (12,24). The injurious agents involved could be gastric acid or other components of the gastro-duodenal juices, such as pepsin, trypsin and bile acids. The ph of the bronchial epithelial lining fluid favors the activity of the duodenopancreatic agents rather than the gastric components. Bile acids carry a plausible biologic activity toward lung injury, and studies that associate their presence in BALF of patients with BOS support their role, whether primary or contributive to other agents, in the development of chronic lung allograft dysfunction (5,25). Recent in vitro evidence suggests that endotoxins present in the gastric juice as a result of bacterial overgrowth in the stomach (possibly secondary to PPI treatment) may also induce a proinflammatory effect (7). The aims of our study were not to evaluate the long-term effect on progression to BOS of AZM versus placebo, as this was considered unethical given the potential benefits of AZM in patients with BOS. As a result, our study does not preclude that giving AZM in patients with BOS may protect them from progression of BOS. However, the data from our study argue that AZM does not appear to protect against the specific contribution of GER/aspiration to chronic allograft dysfunction. Other treatment options such as antireflux therapy should probably be considered in these patients and it might be worthwhile to screen for aspiration in LTX patients before the initiation or perhaps even the onset of BOS. Preliminary data by Cantu et al. showed an improved survival and freedom from BOS in patients with early fundoplication (10 13). Fundoplication reduces all types of reflux (acid and nonacid) and aspiration of gastric components. Additional data on the long-term effect of fundoplication and a better insight into the optimal patient selection for antireflux surgery are needed. Other therapeutic options, like prokinetics, could be explored and the importance of conservative measures reducing the risk of aspiration in LTx patients should also be considered. In conclusion, aspiration of bile acids does not predict short-term improvement in lung function by AZM after start therapy. However, long-term freedom from progression of BOS and survival was significantly reduced in LTx patients with bile acids aspiration despite continuous AZM therapy. AZM does not seem to protect against the long-term allograft dysfunction due to GER and aspiration and an alternative treatment of GER/aspiration should be considered in those LTx patients. Disclosure The authors of this manuscript have no conflict of interest to disclose as described by the American Journal of Transplantation. 334 American Journal of Transplantation 2011; 11:

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