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Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells
Pulmonary Pharmacology & Therapeutics xxx (2014) 1e6
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Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells Koichi Yamada a, b, Yoshitomo Morinaga a, Katsunori Yanagihara a, *, Norihito Kaku a, b, Yosuke Harada a, b, Naoki Uno a, Shigeki Nakamura b, Yoshifumi Imamura b, Hiroo Hasegawa a, Taiga Miyazaki b, Koichi Izumikawa b, Hiroshi Kakeya b, Hiroshige Mikamo c, Shigeru Kohno b, d a
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan b Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, Nagasaki, Japan c Department of Infection Control and Prevention, Aichi Medical University Graduate School of Medicine, Aichi, Japan d Global COE Program, Nagasaki University, Nagasaki, Japan
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Article history: Received 15 March 2014 Received in revised form 21 May 2014 Accepted 24 May 2014 Available online xxx
Acinetobacter baumannii is one of the main pathogens that cause ventilator-associated pneumonia (VAP). Hypersecretion of mucin in the airway is associated with the onset of VAP. Furthermore, macrolides are known to accelerate the resolution of VAP. However, this mechanism has not been elucidated. We examined whether macrolides inhibit MUC5AC production that is induced by multidrug-resistant A. baumannii (MDRAB). MUC5AC production in bronchial cells after MDRAB stimulation was analyzed by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction. For the inhibition study, cells were treated with azithromycin (AZM) or clarithromycin (CAM) simultaneously along with MDRAB stimulation. Western blotting was performed was performed to determine potential rules for signal modules. MDRAB induced MUC5AC production and gene expression. The EGFRERK/JNK-NF-kB pathway was involved in MDRAB-induced MUC5AC production. AZM but not CAM inhibited MUC5AC production. AZM suppressed the phosphorylation of ERK/JNK and the nuclear translocation of NF-kB. Our results suggest that the efficacy of macrolides against VAP may be due to the inhibition of mucin production. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Azithromycin Multidrug-resistant Acinetobacter baumannii MUC5AC Ventilator-associated pneumonia
1. Introduction Acinetobacter baumannii is a worldwide-known nosocomial pathogen that has become increasingly common over the past few decades and is associated with high rates of morbidity and mortality [1,2]. A. baumannii causes pulmonary, urinary tract, bloodstream, and surgical wound infections [2,3]. Ventilator-associated pneumonia (VAP) is caused by A. baumannii more often than by other pathogens [4]. Recently, an increasing number of drugresistant A. baumannii strains are being identified. The multidrugresistance rate of Acinetobacter is the highest among gramnegative pathogens [5], and the mortality rate of VAP caused by A. baumannii is the highest among all bacterial pathogens [4].
* Corresponding author. Tel.: þ81 95 819 7418; fax: þ81 95 819 7257. E-mail address: [email protected] (K. Yanagihara).
Furthermore, infections due to multidrug-resistant A. baumannii (MDRAB) strains are associated with worse prognosis than infections caused by non-MDRAB strains [6,7]. Therefore, it is very difficult to treat VAP caused by MDRAB. Airway mucus coats the surface of the respiratory tract, acts as a protective barrier against pathogens, and provides the setting for the innate immune system. However, excessive mucus production can be a risk factor for pathogenesis because mucus hyperproduction causes airway obstruction, impairment of gas exchange, and obstructive pneumonia. Mucus hypersecretion is observed not only for chronic airway diseases such as diffuse panbronchiolitis (DPB) and cystic fibrosis but also for VAP [8]. The control of mucus hypersecretion may thus contribute to the treatment of these diseases. More than 20 mucin genes have been identified in humans, and these genes are generally classified into 2 types : membraneassociated mucins and gel-forming mucins [9,10]. Gel-forming
http://dx.doi.org/10.1016/j.pupt.2014.05.006 1094-5539/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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K. Yamada et al. / Pulmonary Pharmacology & Therapeutics xxx (2014) 1e6
mucins are secreted mucins that provide the viscoelastic properties of the extracellular mucus layer. MUC2, MUC5AC, MUC5B, MUC6 and MUC19 belong to this family. MUC5AC is highly expressed in the lungs [11] and is up-regulated by various bacterial stimuli [12,13]. However, it remains unclear if A. baumannii can induce mucin production. It is gradually being accepted that macrolides not only possess antibacterial activity but also exert immunomodulatory effects. Macrolides have been shown to improve the survival rate of DPB patients [14]. Furthermore, clarithromycin (CAM) accelerates the resolution of VAP and weaning from mechanical ventilation [15]. The suppression of mucus hypersecretion has been observed with macrolides in vitro and in vivo [13,16]. In this study, we examined the effect of MDRAB on mucin production and the inhibition of MUC5AC production by macrolides. 2. Materials and methods 2.1. Materials CAM (Taishotoyama Pharmaceutical, Tokyo, Japan) and azithromycin (AZM; Pfizer, Groton, CT, USA) were generously provided by the respective companies; these drugs were dissolved in dimethyl sulfoxide. Mouse anti-MUC5AC monoclonal antibody (clone 45M1) was obtained from MONOSAN (Uden, the Netherlands). Goat anti-mouse horseradish peroxidase-conjugated secondary antibody was obtained from Bio-Rad (Hercules, CA). Anti-extracellular signal-regulated protein kinase (ERK), antiphospho ERK, and anti-c-Jun N-terminal kinase (JNK), antiphospho JNK, and anti-epithelial growth factor receptor (EGFR), anti-phospho EGFR antibodies were obtained from Cell Signaling Technology (Denvers, MA). Caffeic acid phenethyl ester (CAPE), PD98059, U0126, SB203580, SP600125, and AG1478 were obtained from Calbiochem (San Diego, CA). 2.2. Bacterial culturing and preparation of cell-free supernatant
2.4. Real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) Total RNA was extracted from NCI-H292 cells cultured in a 60mm dish using QuickGene-Mini80 and QuickGene RNA cultured cell kits (Fujifilm Co., Tokyo Japan) according to the manufacturer's instructions. Total RNA(1 mg) was reverse transcribed into cDNA using oligo (dT) primers and SuperScript III reverse transcriptase (Life Technologies) and then treated with RNaseH (Life Technologies). To quantify the expression of the MUC5AC gene, primers and Taqman probes were designed and used as previously reported (forward primer, 50 CAGCCACGTCCCCTTCAATA-30 ;reverse primer,50 ACCGCATTTGGGCATCC-30 ; Taqman probe, 50 -6-FAM-CCACCTCCGAGCCCCTCACTGAG-TAMRA-30 ) [19]. MUC5AC was amplified for 40 cycles (15 s at 95 C and 30 s at 60 C) using a LightCycler system (Roche Applied Science, Indianapolis, Indiana). MUC5AC expression was normalized to the expression of human porphobilinogen deaminase (hPBGD), which was measured using an hPBGD primer set (Roche Diagnostic GmbH, Mannheim, Germany) according to the manufacturer's instructions. 2.5. Enzyme-linked immunosorbent assay NCI-H292 cells were cultured in 12-well plates. MUC5AC protein was measured by an enzyme-linked immunosorbent assay (ELISA) [13]. After stimulation for 24 h, aliquots of the supernatant were incubated at 40 C in a 96-well plate until they had dried out. The plates were blocked with 2% bovine serum albumin for 1 h at room temperature and incubated with mouse anti-MUC5AC monoclonal antibody (MONOSAN, Uden, Netherlands) diluted in phosphatebuffered saline containing 0.05% Tween 20 for 1 h. Goat antimouse horseradish-peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, California) was dispensed into each well. After 1 h, immunoreactivity was colorimetrically detected with 3,30 ,5,50 tetramethylbenzidine-peroxidase solution. The reaction was stopped by adding 2 N H2SO4, and absorbance was measured at 450 nm.
In this study, we used the MDRAB strain AMU62852 (MIC: imipenem ¼ 16, amikacin S 32, ciprofloxacin S 4), which was kindly provided by the Aichi Medical University Graduate School of Medicine. This strain has the blaoxa-51-like gene, which is specific to A. baumannii [17].The bacteria were stored at 80 C in a Microbank system (Pro-Lab Diagnostics, Ontario, Canada) until use. Cell-free supernatant was prepared using a previously reported method [18].In brief, the A. baumannii strain was harvested from a MullerHinton II agar plate at 37 C for 18 h and grown in LuriaeBertani (LB) broth for 72 h at 37 C (to late log phase). Cell-free supernatant was obtained by centrifugation at 10,000 rpm for 50 min at 4 C and subsequent filtration through a 0.22-mg filter (Millipore Co. Billerica, USA). The supernatant was stored at 80 C until further use.
NF-kB/p65 in nuclear extracts from cells cultured in 6-cm dishes was measured using an NF-kB/p65ActivELISA (Imgenex, San Diego, CA) according to the manufacturer's instructions [20]. Briefly, 10 mg of nuclear samples from untreated or treated cells were added to a 96-well plate coated with the capture antibody. After incubation at 4 C overnight, the detecting antibody was added to the wells. The plate was incubated with an AKP-conjugated secondary antibody, after which pNPP substrate was added to the cells, and the absorbance of each sample was read at 405 nm by using a microplate reader.
2.3. Cell culture
2.7. Western blot analysis
NCI-H292 human airway epithelial cells were cultured in RPMI1640 medium (Life Technologies, Carlsbad, California) containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were grown at 37 C with 5% CO2 and were subcultured twice weekly. When the cells reached confluence, they were serum-starved for 24 h and then stimulated with the MDRAB culture supernatant. For the inhibition study, cells were simultaneously treated with the supernatant and macrolides. The cells were pretreated with signal transduction inhibitors for 30 min before stimulation. As a control, cells were incubated with RPMI medium and LB broth.
Cells were harvested, washed and homogenized at 4 C in a lysis buffer (0.1% sodium dodecyl sulfate, 1% Igepal CA-630, and 0.5% sodium deoxycholate). Cell lysates (20e50 mg) were resolved by electrophoresis on 12% polyacrylamide gel and transferred to a polyvinylidine difluoride membrane. After blocking the membrane in 10% PBS and 0.1% Tween 20 in Tris-buffered saline for 1 h at room temperature, the blots were hybridized overnight at 4 C with primary antibodies. After hybridization with secondary antibodies conjugated with horseradish peroxidase, immunocomplexes were visualized using an ECL Western Blotting Detection System (GE Healthcare, Chalfont St. Giles, United Kingdom).
2.6. Nuclear translocation of NF-kB/p65
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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2.8. Statistical analysis All data are expressed as means ± standard error of the mean. Differences were examined for statistical significance by using the one-way analysis of variance for comparisons involving more than 2 groups and the Student's t-test for comparison between 2 groups. P values lower than 0.05 were considered to indicate a statistically significant difference. 3. Results 3.1. AZM inhibits MUC5AC protein induced by MDRAB To determine the effect of the MDRAB supernatant concentration on MUC5AC protein expression, NCI-H292 cells were incubated with various concentrations of the MDRAB supernatant for 24 h and the levels of the MUC5AC protein secreted into the culture supernatant were evaluated by ELISA. The level of secreted MUC5AC increased with MDRAB addition in a dose-dependent manner except at a 1:5 dilution (Fig. 1A). On the basis of this result, a 1:40 dilution of the MDRAB supernatant was chosen for
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further studies. To determine the appropriate dosage of macrolides for the inhibition of MDRAB-induced MUC5AC protein secretion, we treated the cells with macrolides (CAM or AZM at 1e50 mg/ml). As shown in Fig. 1B, AZM (especially 50 mg/ml) reduced the level of secreted MUC5AC protein. On the other hand, CAM did not inhibit MUC5AC production. 3.2. AZM inhibits MDRAB-induced MUC5AC gene expression We performed an inhibition study of mRNA 6 h after stimulation, when the level of mRNA expression was maximum 6 h (data not shown). As shown in Fig. 2, AZM reduced MUC5AC expression at the mRNA level; however, CAM did not inhibit MUC5AC expression. 3.3. MDRAB-induced MUC5AC expression depends on ERK and JNK We evaluated the involvement of ERK and JNK in MDRABinduced production of MUC5AC by using selective inhibitors (Fig. 3A, B). Pretreatment with an ERK inhibitor, U0126, and a JNK inhibitor, SP600125, suppressed MDRAB-induced MUC5AC protein production. These results indicate that the MDRAB induction of MUC5AC production depends on the activation of ERK and JNK. 3.4. MDRAB-induced MUC5AC depends on EGFR and NF-kB We examined the involvement of EGFR and NF-kB in MDRABinduced MUC5AC production by using selective inhibitors (Fig. 4A, B). Pretreatment with an EGFR inhibitor, AG1478, and an NF-kB inhibitor, CAPE, suppressed MDRAB-induced MUC5AC protein production. These results indicate that the MDRAB induction of MUC5AC production depends on the activation of EGFR and NF-kB. 3.5. AZM inhibits the phosphorylation of ERK1/2 and JNK by MDRAB In order to investigate the effects of macrolides at the MAPK level, cells were simultaneously treated with the supernatant and macrolides for 60 min. After that, the phosphorylation of ERK1/2 and JNK was detected by western blotting. The phosphorylation of ERK1/2 and JNK was induced by MDRAB-treated cells and inhibited by AZM only (Fig. 5). 3.6. AZM inhibits nuclear translocation of the NF-kB p65 subunit In NF-kB, the p65 subunit plays an important role in MUC5AC gene expression. We evaluated the nuclear translocation of the NFkB p65 subunit by sandwich ELISA. As shown in Fig. 6A, MDRAB had a time-dependent effect on NF-kB p65 translocation. Furthermore, AZM, but not CAM inhibited the nuclear translocation significantly for 90 min of treatment (Fig. 6B). 4. Discussion
Fig. 1. Effect of multidrug-resistant Acinetobacter baumannii (MDRAB) on the MUC5AC protein. (A) MDRAB was grown in LB broth, and a cell-free supernatant was obtained. Confluent NCI-H292 cells were stimulated with various concentrations of the MDRAB supernatant (Sup) in RPMI medium for 24 h (n ¼ 3). Dilution ratio was defined as the ratio of volume of the supernatant to the medium. **indicates P < 0.01 compared with the control. þindicates P < 0.05 compared with a 1:80 MDRAB supernatant dilution. (B) For the inhibition study, cells were simultaneously treated with supernatant and macrolides. The MDRAB Sup dilution ratio was 1:40. Each bar shows the percentage value above control (LB broth and medium). *indicates P < 0.05 compared with MDRAB. **indicates P < 0.01 compared with MDRAB. These experiments were performed three times.
VAP is a leading cause of nosocomial infection-related mortality. VAP is difficult to treat because patients usually have serious concomitant diseases and sometimes cannot undergo an invasive examination. A. baumannii is the predominant pathogen in VAP. Carbapenems are recommended as empirical treatments for VAP associated with A. baumannii. However, carbapenem-resistant A. baumannii are becoming a serious problem [1,21]. Furthermore, A. baumannii easily acquires the resistance to antibiotics. Thus, treatment of VAP caused by A. baumannii is becoming more challenging. It was reported that ICU patients with VAP had high levels of mucins in their bronchoalveolar lavage fluid [8]. The presence of
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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Fig. 2. Effect of macrolides on MUC5AC gene expression. Cells were treated with each drug at 50 mg/ml for 6 h. After 6 h of treatment, gene levels were measured by quantitative real-time PCR and normalized against hPBGD (n ¼ 3). **indicates P < 0.01 compared with the control (LB broth and medium). þindicates P < 0.01 compared with MDRAB. These experiments were performed three times.
Fig. 3. Effect of MAP kinase inhibitors on MUC5AC production. Each bar shows the percentage value above the control (LB broth and medium). Cells were pretreated with each inhibitor for 30 min before stimulation. U0126 is an ERK inhibitor and SP600125 is a JNK inhibitor. **indicates P < 0.01 compared with MDRAB. These experiments were performed three times.
Fig. 4. Effect of EGFR and NF-kB inhibitors on MUC5AC production. Each bar shows the percentage above the control (LB broth and medium). Cells were pretreated with each inhibitor for 30 min before stimulation. AG1478 is an EGFR inhibitor and CAPE is an NFkB inhibitor. **indicates P < 0.01 compared with MDRAB. These experiments were performed three times.
high levels mucins prevents the efficacy of antibiotics in small airways and decreases lung function. Therefore, the control of mucus hypersecretion may contribute to the management of VAP. Giamarellos-Bourboulis et al. reported that CAM accelerates the resolution of VAP and weaning from mechanical ventilation [15]. In this study, A. baumannii was the most common pathogen that caused VAP. Because macrolides do not exhibit antibiotic effects against A. baumannii, it is thought that they act via mechanisms apart from the antibiotic effect. In the present study, we demonstrated that MDRAB could induce MUC5AC production. MUC5AC is up-regulated by various bacterial stimulants such as Pseudomonas aeruginosa, Haemophilus influenzae, and Chamydophila pneumoniae [13,22,23]. MUC5AC production reported in these reports was equal to those determined in our study. NF-kB is an important transcriptional factor that regulates MUC5AC gene expression. C. pneumoniae, Legionella pneumophila and H. influenza up-regulate NF-kB activation and increase MUC5AC production [22e24]. In this study, the NF-kB selective inhibitor CAPE significantly suppressed MDRAB-induced MUC5AC production. Thus, NF-kB plays a dominant role in the transcription of the MUC5AC gene through the stimulation of MDRAB. MAPKs are important signal transducers and are involved in many kinds of stress or inflammatory responses. The induction of MUC5AC production by L. pneumophila is dependent on ERK and JNK [24], whereas activation by H. influenzae is dependent on p38 [25]. Thus, the dominant MAPK involved in MUC5AC expression depends on the stimulant. Our data suggest that the ERK and JNK pathways could mediate MUC5AC production induced by MDRAB. However, p38 was not involved in MDRAB induced MUC5AC production (data not shown). EGFR is one of the important factors that regulate MUC5AC gene expression. P. aeruginosa and rhinovirus induce MUC5AC expression via the EGFR-ERK pathway [26,27]. In this study, the EGFR inhibitor, AG1478 significantly suppressed MUC5AC production by MDRAB. We suggest that MDRAB can induce MUC5AC production via the EGFR-ERK/JNK-NF-kB pathway. In this study, MDRAB-induced MUC5AC production was suppressed by AZM but not by CAM. Furthermore, AZM reduced the phosphorylation of ERK/JNK and the nuclear translocation of p65.
Fig. 5. Phosphorylation of ERK1/2 and JNK by MDRAB. Cells were simultaneously treated with the supernatant and macrolides for 60 min. Phosphorylation of ERK1/2 and JNK was induced by MDRAB treated cells and inhibited by AZM only.
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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increased effects against infection. We consider that macrolide therapy may contribute to controlling VAP by decreasing the amount of sputum, based on our study results. We need to establish a VAP model of MDRAB and evaluate whether macrolides can reduce mucin production. In conclusion, AZM inhibits the MUC5AC expression induced by MDRAB. AZM seems to reduce MUC5AC production by interfering with the phosphorylation of ERK/JNK and the activation of NF-kB. Our results provide a possible explanation for the clinical efficacy of macrolide therapy against VAP via resistant pathogens. Competing interests None declared. Ethical approval Not required. Acknowledgments This research was supported by Meiji Seika Pharma Co., Ltd., Tokyo, Japan, by a Grant-in-Aid for Scientific Research (no.21591294 to K.Y.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by a grant from the Global Centers of Excellence Program, Nagasaki University. References
Fig. 6. Nuclear translocation of the p65 subunit of NF-kB. The free NF-kB p65 in the nucleus was investigated by sandwich ELISA. (A) MDRAB had a time-dependent effect on NF-kB p65 translocation. Cells were treated with LB broth for 0min. *indicates P < 0.05 compared with 0 min. (B) Cells were treated with the supernatant and macrolides for 90 min. AZM significantly suppressed NF-kB p65 translocation. *indicates P < 0.05 compared with the MDRAB supernatant. These experiments were performed three times.
This indicates that AZM may suppress MDRAB-induced MUC5AC production through the inhibition of MAPK levels, especially ERK. H. influenzae -induced MUC5AC production was inhibited by only AZM, which was consistent with that reported in our study. However, AZM inhibited induced MUC5AC expression and secretion via the inhibition of activator protein-1 [22]. C. pneumoniae- induced MUC5AC production was inhibited by AZM, CAM, and telithromycin by the signal pathways between ERK and NF-kB [23]. CAM and AZM are known to have different effects on cytokine production and the inhibition in murine dendritic cells [28]. Previous studies suggest that the signal pathways by which mucus hypersecretion is modulated varies according to the type of macrolide or stimulant used. In this study, we used these antibiotics at a concentration of 50 mg/ml. The mean steady-state concentrations of these antibiotics in alveolar macrophages after oral administration were reported to reach over 300 mg/ml [29]. Furthermore, the concentration of AZM after intravenous administration was determined to be over 600 mg/ml [30]. Therefore, the concentration of antibiotics used in our study may be attainable in clinical cases. A reduction in sputum caused improved lung function, improved accessibility of antibiotics to the small airways, and
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Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt
Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells Koichi Yamada a, b, Yoshitomo Morinaga a, Katsunori Yanagihara a, *, Norihito Kaku a, b, Yosuke Harada a, b, Naoki Uno a, Shigeki Nakamura b, Yoshifumi Imamura b, Hiroo Hasegawa a, Taiga Miyazaki b, Koichi Izumikawa b, Hiroshi Kakeya b, Hiroshige Mikamo c, Shigeru Kohno b, d a
Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan b Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University Hospital, Nagasaki, Japan c Department of Infection Control and Prevention, Aichi Medical University Graduate School of Medicine, Aichi, Japan d Global COE Program, Nagasaki University, Nagasaki, Japan
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Article history: Received 15 March 2014 Received in revised form 21 May 2014 Accepted 24 May 2014 Available online xxx
Acinetobacter baumannii is one of the main pathogens that cause ventilator-associated pneumonia (VAP). Hypersecretion of mucin in the airway is associated with the onset of VAP. Furthermore, macrolides are known to accelerate the resolution of VAP. However, this mechanism has not been elucidated. We examined whether macrolides inhibit MUC5AC production that is induced by multidrug-resistant A. baumannii (MDRAB). MUC5AC production in bronchial cells after MDRAB stimulation was analyzed by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction. For the inhibition study, cells were treated with azithromycin (AZM) or clarithromycin (CAM) simultaneously along with MDRAB stimulation. Western blotting was performed was performed to determine potential rules for signal modules. MDRAB induced MUC5AC production and gene expression. The EGFRERK/JNK-NF-kB pathway was involved in MDRAB-induced MUC5AC production. AZM but not CAM inhibited MUC5AC production. AZM suppressed the phosphorylation of ERK/JNK and the nuclear translocation of NF-kB. Our results suggest that the efficacy of macrolides against VAP may be due to the inhibition of mucin production. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Azithromycin Multidrug-resistant Acinetobacter baumannii MUC5AC Ventilator-associated pneumonia
1. Introduction Acinetobacter baumannii is a worldwide-known nosocomial pathogen that has become increasingly common over the past few decades and is associated with high rates of morbidity and mortality [1,2]. A. baumannii causes pulmonary, urinary tract, bloodstream, and surgical wound infections [2,3]. Ventilator-associated pneumonia (VAP) is caused by A. baumannii more often than by other pathogens [4]. Recently, an increasing number of drugresistant A. baumannii strains are being identified. The multidrugresistance rate of Acinetobacter is the highest among gramnegative pathogens [5], and the mortality rate of VAP caused by A. baumannii is the highest among all bacterial pathogens [4].
* Corresponding author. Tel.: þ81 95 819 7418; fax: þ81 95 819 7257. E-mail address: [email protected] (K. Yanagihara).
Furthermore, infections due to multidrug-resistant A. baumannii (MDRAB) strains are associated with worse prognosis than infections caused by non-MDRAB strains [6,7]. Therefore, it is very difficult to treat VAP caused by MDRAB. Airway mucus coats the surface of the respiratory tract, acts as a protective barrier against pathogens, and provides the setting for the innate immune system. However, excessive mucus production can be a risk factor for pathogenesis because mucus hyperproduction causes airway obstruction, impairment of gas exchange, and obstructive pneumonia. Mucus hypersecretion is observed not only for chronic airway diseases such as diffuse panbronchiolitis (DPB) and cystic fibrosis but also for VAP [8]. The control of mucus hypersecretion may thus contribute to the treatment of these diseases. More than 20 mucin genes have been identified in humans, and these genes are generally classified into 2 types : membraneassociated mucins and gel-forming mucins [9,10]. Gel-forming
http://dx.doi.org/10.1016/j.pupt.2014.05.006 1094-5539/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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mucins are secreted mucins that provide the viscoelastic properties of the extracellular mucus layer. MUC2, MUC5AC, MUC5B, MUC6 and MUC19 belong to this family. MUC5AC is highly expressed in the lungs [11] and is up-regulated by various bacterial stimuli [12,13]. However, it remains unclear if A. baumannii can induce mucin production. It is gradually being accepted that macrolides not only possess antibacterial activity but also exert immunomodulatory effects. Macrolides have been shown to improve the survival rate of DPB patients [14]. Furthermore, clarithromycin (CAM) accelerates the resolution of VAP and weaning from mechanical ventilation [15]. The suppression of mucus hypersecretion has been observed with macrolides in vitro and in vivo [13,16]. In this study, we examined the effect of MDRAB on mucin production and the inhibition of MUC5AC production by macrolides. 2. Materials and methods 2.1. Materials CAM (Taishotoyama Pharmaceutical, Tokyo, Japan) and azithromycin (AZM; Pfizer, Groton, CT, USA) were generously provided by the respective companies; these drugs were dissolved in dimethyl sulfoxide. Mouse anti-MUC5AC monoclonal antibody (clone 45M1) was obtained from MONOSAN (Uden, the Netherlands). Goat anti-mouse horseradish peroxidase-conjugated secondary antibody was obtained from Bio-Rad (Hercules, CA). Anti-extracellular signal-regulated protein kinase (ERK), antiphospho ERK, and anti-c-Jun N-terminal kinase (JNK), antiphospho JNK, and anti-epithelial growth factor receptor (EGFR), anti-phospho EGFR antibodies were obtained from Cell Signaling Technology (Denvers, MA). Caffeic acid phenethyl ester (CAPE), PD98059, U0126, SB203580, SP600125, and AG1478 were obtained from Calbiochem (San Diego, CA). 2.2. Bacterial culturing and preparation of cell-free supernatant
2.4. Real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) Total RNA was extracted from NCI-H292 cells cultured in a 60mm dish using QuickGene-Mini80 and QuickGene RNA cultured cell kits (Fujifilm Co., Tokyo Japan) according to the manufacturer's instructions. Total RNA(1 mg) was reverse transcribed into cDNA using oligo (dT) primers and SuperScript III reverse transcriptase (Life Technologies) and then treated with RNaseH (Life Technologies). To quantify the expression of the MUC5AC gene, primers and Taqman probes were designed and used as previously reported (forward primer, 50 CAGCCACGTCCCCTTCAATA-30 ;reverse primer,50 ACCGCATTTGGGCATCC-30 ; Taqman probe, 50 -6-FAM-CCACCTCCGAGCCCCTCACTGAG-TAMRA-30 ) [19]. MUC5AC was amplified for 40 cycles (15 s at 95 C and 30 s at 60 C) using a LightCycler system (Roche Applied Science, Indianapolis, Indiana). MUC5AC expression was normalized to the expression of human porphobilinogen deaminase (hPBGD), which was measured using an hPBGD primer set (Roche Diagnostic GmbH, Mannheim, Germany) according to the manufacturer's instructions. 2.5. Enzyme-linked immunosorbent assay NCI-H292 cells were cultured in 12-well plates. MUC5AC protein was measured by an enzyme-linked immunosorbent assay (ELISA) [13]. After stimulation for 24 h, aliquots of the supernatant were incubated at 40 C in a 96-well plate until they had dried out. The plates were blocked with 2% bovine serum albumin for 1 h at room temperature and incubated with mouse anti-MUC5AC monoclonal antibody (MONOSAN, Uden, Netherlands) diluted in phosphatebuffered saline containing 0.05% Tween 20 for 1 h. Goat antimouse horseradish-peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, California) was dispensed into each well. After 1 h, immunoreactivity was colorimetrically detected with 3,30 ,5,50 tetramethylbenzidine-peroxidase solution. The reaction was stopped by adding 2 N H2SO4, and absorbance was measured at 450 nm.
In this study, we used the MDRAB strain AMU62852 (MIC: imipenem ¼ 16, amikacin S 32, ciprofloxacin S 4), which was kindly provided by the Aichi Medical University Graduate School of Medicine. This strain has the blaoxa-51-like gene, which is specific to A. baumannii [17].The bacteria were stored at 80 C in a Microbank system (Pro-Lab Diagnostics, Ontario, Canada) until use. Cell-free supernatant was prepared using a previously reported method [18].In brief, the A. baumannii strain was harvested from a MullerHinton II agar plate at 37 C for 18 h and grown in LuriaeBertani (LB) broth for 72 h at 37 C (to late log phase). Cell-free supernatant was obtained by centrifugation at 10,000 rpm for 50 min at 4 C and subsequent filtration through a 0.22-mg filter (Millipore Co. Billerica, USA). The supernatant was stored at 80 C until further use.
NF-kB/p65 in nuclear extracts from cells cultured in 6-cm dishes was measured using an NF-kB/p65ActivELISA (Imgenex, San Diego, CA) according to the manufacturer's instructions [20]. Briefly, 10 mg of nuclear samples from untreated or treated cells were added to a 96-well plate coated with the capture antibody. After incubation at 4 C overnight, the detecting antibody was added to the wells. The plate was incubated with an AKP-conjugated secondary antibody, after which pNPP substrate was added to the cells, and the absorbance of each sample was read at 405 nm by using a microplate reader.
2.3. Cell culture
2.7. Western blot analysis
NCI-H292 human airway epithelial cells were cultured in RPMI1640 medium (Life Technologies, Carlsbad, California) containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were grown at 37 C with 5% CO2 and were subcultured twice weekly. When the cells reached confluence, they were serum-starved for 24 h and then stimulated with the MDRAB culture supernatant. For the inhibition study, cells were simultaneously treated with the supernatant and macrolides. The cells were pretreated with signal transduction inhibitors for 30 min before stimulation. As a control, cells were incubated with RPMI medium and LB broth.
Cells were harvested, washed and homogenized at 4 C in a lysis buffer (0.1% sodium dodecyl sulfate, 1% Igepal CA-630, and 0.5% sodium deoxycholate). Cell lysates (20e50 mg) were resolved by electrophoresis on 12% polyacrylamide gel and transferred to a polyvinylidine difluoride membrane. After blocking the membrane in 10% PBS and 0.1% Tween 20 in Tris-buffered saline for 1 h at room temperature, the blots were hybridized overnight at 4 C with primary antibodies. After hybridization with secondary antibodies conjugated with horseradish peroxidase, immunocomplexes were visualized using an ECL Western Blotting Detection System (GE Healthcare, Chalfont St. Giles, United Kingdom).
2.6. Nuclear translocation of NF-kB/p65
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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2.8. Statistical analysis All data are expressed as means ± standard error of the mean. Differences were examined for statistical significance by using the one-way analysis of variance for comparisons involving more than 2 groups and the Student's t-test for comparison between 2 groups. P values lower than 0.05 were considered to indicate a statistically significant difference. 3. Results 3.1. AZM inhibits MUC5AC protein induced by MDRAB To determine the effect of the MDRAB supernatant concentration on MUC5AC protein expression, NCI-H292 cells were incubated with various concentrations of the MDRAB supernatant for 24 h and the levels of the MUC5AC protein secreted into the culture supernatant were evaluated by ELISA. The level of secreted MUC5AC increased with MDRAB addition in a dose-dependent manner except at a 1:5 dilution (Fig. 1A). On the basis of this result, a 1:40 dilution of the MDRAB supernatant was chosen for
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further studies. To determine the appropriate dosage of macrolides for the inhibition of MDRAB-induced MUC5AC protein secretion, we treated the cells with macrolides (CAM or AZM at 1e50 mg/ml). As shown in Fig. 1B, AZM (especially 50 mg/ml) reduced the level of secreted MUC5AC protein. On the other hand, CAM did not inhibit MUC5AC production. 3.2. AZM inhibits MDRAB-induced MUC5AC gene expression We performed an inhibition study of mRNA 6 h after stimulation, when the level of mRNA expression was maximum 6 h (data not shown). As shown in Fig. 2, AZM reduced MUC5AC expression at the mRNA level; however, CAM did not inhibit MUC5AC expression. 3.3. MDRAB-induced MUC5AC expression depends on ERK and JNK We evaluated the involvement of ERK and JNK in MDRABinduced production of MUC5AC by using selective inhibitors (Fig. 3A, B). Pretreatment with an ERK inhibitor, U0126, and a JNK inhibitor, SP600125, suppressed MDRAB-induced MUC5AC protein production. These results indicate that the MDRAB induction of MUC5AC production depends on the activation of ERK and JNK. 3.4. MDRAB-induced MUC5AC depends on EGFR and NF-kB We examined the involvement of EGFR and NF-kB in MDRABinduced MUC5AC production by using selective inhibitors (Fig. 4A, B). Pretreatment with an EGFR inhibitor, AG1478, and an NF-kB inhibitor, CAPE, suppressed MDRAB-induced MUC5AC protein production. These results indicate that the MDRAB induction of MUC5AC production depends on the activation of EGFR and NF-kB. 3.5. AZM inhibits the phosphorylation of ERK1/2 and JNK by MDRAB In order to investigate the effects of macrolides at the MAPK level, cells were simultaneously treated with the supernatant and macrolides for 60 min. After that, the phosphorylation of ERK1/2 and JNK was detected by western blotting. The phosphorylation of ERK1/2 and JNK was induced by MDRAB-treated cells and inhibited by AZM only (Fig. 5). 3.6. AZM inhibits nuclear translocation of the NF-kB p65 subunit In NF-kB, the p65 subunit plays an important role in MUC5AC gene expression. We evaluated the nuclear translocation of the NFkB p65 subunit by sandwich ELISA. As shown in Fig. 6A, MDRAB had a time-dependent effect on NF-kB p65 translocation. Furthermore, AZM, but not CAM inhibited the nuclear translocation significantly for 90 min of treatment (Fig. 6B). 4. Discussion
Fig. 1. Effect of multidrug-resistant Acinetobacter baumannii (MDRAB) on the MUC5AC protein. (A) MDRAB was grown in LB broth, and a cell-free supernatant was obtained. Confluent NCI-H292 cells were stimulated with various concentrations of the MDRAB supernatant (Sup) in RPMI medium for 24 h (n ¼ 3). Dilution ratio was defined as the ratio of volume of the supernatant to the medium. **indicates P < 0.01 compared with the control. þindicates P < 0.05 compared with a 1:80 MDRAB supernatant dilution. (B) For the inhibition study, cells were simultaneously treated with supernatant and macrolides. The MDRAB Sup dilution ratio was 1:40. Each bar shows the percentage value above control (LB broth and medium). *indicates P < 0.05 compared with MDRAB. **indicates P < 0.01 compared with MDRAB. These experiments were performed three times.
VAP is a leading cause of nosocomial infection-related mortality. VAP is difficult to treat because patients usually have serious concomitant diseases and sometimes cannot undergo an invasive examination. A. baumannii is the predominant pathogen in VAP. Carbapenems are recommended as empirical treatments for VAP associated with A. baumannii. However, carbapenem-resistant A. baumannii are becoming a serious problem [1,21]. Furthermore, A. baumannii easily acquires the resistance to antibiotics. Thus, treatment of VAP caused by A. baumannii is becoming more challenging. It was reported that ICU patients with VAP had high levels of mucins in their bronchoalveolar lavage fluid [8]. The presence of
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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Fig. 2. Effect of macrolides on MUC5AC gene expression. Cells were treated with each drug at 50 mg/ml for 6 h. After 6 h of treatment, gene levels were measured by quantitative real-time PCR and normalized against hPBGD (n ¼ 3). **indicates P < 0.01 compared with the control (LB broth and medium). þindicates P < 0.01 compared with MDRAB. These experiments were performed three times.
Fig. 3. Effect of MAP kinase inhibitors on MUC5AC production. Each bar shows the percentage value above the control (LB broth and medium). Cells were pretreated with each inhibitor for 30 min before stimulation. U0126 is an ERK inhibitor and SP600125 is a JNK inhibitor. **indicates P < 0.01 compared with MDRAB. These experiments were performed three times.
Fig. 4. Effect of EGFR and NF-kB inhibitors on MUC5AC production. Each bar shows the percentage above the control (LB broth and medium). Cells were pretreated with each inhibitor for 30 min before stimulation. AG1478 is an EGFR inhibitor and CAPE is an NFkB inhibitor. **indicates P < 0.01 compared with MDRAB. These experiments were performed three times.
high levels mucins prevents the efficacy of antibiotics in small airways and decreases lung function. Therefore, the control of mucus hypersecretion may contribute to the management of VAP. Giamarellos-Bourboulis et al. reported that CAM accelerates the resolution of VAP and weaning from mechanical ventilation [15]. In this study, A. baumannii was the most common pathogen that caused VAP. Because macrolides do not exhibit antibiotic effects against A. baumannii, it is thought that they act via mechanisms apart from the antibiotic effect. In the present study, we demonstrated that MDRAB could induce MUC5AC production. MUC5AC is up-regulated by various bacterial stimulants such as Pseudomonas aeruginosa, Haemophilus influenzae, and Chamydophila pneumoniae [13,22,23]. MUC5AC production reported in these reports was equal to those determined in our study. NF-kB is an important transcriptional factor that regulates MUC5AC gene expression. C. pneumoniae, Legionella pneumophila and H. influenza up-regulate NF-kB activation and increase MUC5AC production [22e24]. In this study, the NF-kB selective inhibitor CAPE significantly suppressed MDRAB-induced MUC5AC production. Thus, NF-kB plays a dominant role in the transcription of the MUC5AC gene through the stimulation of MDRAB. MAPKs are important signal transducers and are involved in many kinds of stress or inflammatory responses. The induction of MUC5AC production by L. pneumophila is dependent on ERK and JNK [24], whereas activation by H. influenzae is dependent on p38 [25]. Thus, the dominant MAPK involved in MUC5AC expression depends on the stimulant. Our data suggest that the ERK and JNK pathways could mediate MUC5AC production induced by MDRAB. However, p38 was not involved in MDRAB induced MUC5AC production (data not shown). EGFR is one of the important factors that regulate MUC5AC gene expression. P. aeruginosa and rhinovirus induce MUC5AC expression via the EGFR-ERK pathway [26,27]. In this study, the EGFR inhibitor, AG1478 significantly suppressed MUC5AC production by MDRAB. We suggest that MDRAB can induce MUC5AC production via the EGFR-ERK/JNK-NF-kB pathway. In this study, MDRAB-induced MUC5AC production was suppressed by AZM but not by CAM. Furthermore, AZM reduced the phosphorylation of ERK/JNK and the nuclear translocation of p65.
Fig. 5. Phosphorylation of ERK1/2 and JNK by MDRAB. Cells were simultaneously treated with the supernatant and macrolides for 60 min. Phosphorylation of ERK1/2 and JNK was induced by MDRAB treated cells and inhibited by AZM only.
Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006
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increased effects against infection. We consider that macrolide therapy may contribute to controlling VAP by decreasing the amount of sputum, based on our study results. We need to establish a VAP model of MDRAB and evaluate whether macrolides can reduce mucin production. In conclusion, AZM inhibits the MUC5AC expression induced by MDRAB. AZM seems to reduce MUC5AC production by interfering with the phosphorylation of ERK/JNK and the activation of NF-kB. Our results provide a possible explanation for the clinical efficacy of macrolide therapy against VAP via resistant pathogens. Competing interests None declared. Ethical approval Not required. Acknowledgments This research was supported by Meiji Seika Pharma Co., Ltd., Tokyo, Japan, by a Grant-in-Aid for Scientific Research (no.21591294 to K.Y.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by a grant from the Global Centers of Excellence Program, Nagasaki University. References
Fig. 6. Nuclear translocation of the p65 subunit of NF-kB. The free NF-kB p65 in the nucleus was investigated by sandwich ELISA. (A) MDRAB had a time-dependent effect on NF-kB p65 translocation. Cells were treated with LB broth for 0min. *indicates P < 0.05 compared with 0 min. (B) Cells were treated with the supernatant and macrolides for 90 min. AZM significantly suppressed NF-kB p65 translocation. *indicates P < 0.05 compared with the MDRAB supernatant. These experiments were performed three times.
This indicates that AZM may suppress MDRAB-induced MUC5AC production through the inhibition of MAPK levels, especially ERK. H. influenzae -induced MUC5AC production was inhibited by only AZM, which was consistent with that reported in our study. However, AZM inhibited induced MUC5AC expression and secretion via the inhibition of activator protein-1 [22]. C. pneumoniae- induced MUC5AC production was inhibited by AZM, CAM, and telithromycin by the signal pathways between ERK and NF-kB [23]. CAM and AZM are known to have different effects on cytokine production and the inhibition in murine dendritic cells [28]. Previous studies suggest that the signal pathways by which mucus hypersecretion is modulated varies according to the type of macrolide or stimulant used. In this study, we used these antibiotics at a concentration of 50 mg/ml. The mean steady-state concentrations of these antibiotics in alveolar macrophages after oral administration were reported to reach over 300 mg/ml [29]. Furthermore, the concentration of AZM after intravenous administration was determined to be over 600 mg/ml [30]. Therefore, the concentration of antibiotics used in our study may be attainable in clinical cases. A reduction in sputum caused improved lung function, improved accessibility of antibiotics to the small airways, and
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Please cite this article in press as: Yamada K, et al., Azithromycin inhibits MUC5AC induction via multidrug-resistant Acinetobacter baumannii in human airway epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.006