Azithromycin Maintains Airway Epithelial Integrity ... - ATS Journals

Azithromycin Maintains Airway Epithelial Integrity during Pseudomonas aeruginosa Infection Skarphedinn Halldorsson1,2, Thorarinn Gudjonsson2,3,4, Magnus Gottfredsson5,6, Pradeep K. Singh7, Gudmundur Hrafn Gudmundsson1,2, and Olafur Baldursson2,8,9 1 Institute of Biology, 2Biomedical Center, 3Stem Cell Research Unit, Department of Anatomy, 4Department of Laboratory Hematology, 6Faculty of Medicine, and 8Faculty of Pharmacy, University of Iceland, Reykjavik, Iceland; 7Department of Microbiology, University of Washington, Seattle, Washington; and 5Department of Infectious Diseases and 9Department of Pulmonary Medicine, Landspitali University Hospital, Reykjavik, Iceland

Tight junctions (TJs) play a key role in maintaining bronchial epithelial integrity, including apical-basolateral polarity and paracellular trafficking. Patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) often suffer from chronic infections by the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which produces multiple virulence factors, including rhamnolipids. The macrolide antibiotic azithromycin (azm) has been shown to improve lung function in patients with CF without reducing the bacterial count within the lung. However, the mechanism of this effect is still debated. It has previously been shown that azm increased transepithelial electrical resistance (TER) in a bronchial epithelial cell line. In this study we used an air–liquid interface model of human airway epithelia and measured TER, changes in TJ expression and architecture after exposure to live P. aeruginosa PAO1, and PAO1-Drhl which is a PAO1 mutant lacking rhlA and rhlB, which encode key enzymes for rhamnolipid production. In addition, the cells were challenged with bacterial culture medium conditioned by these strains, purified rhamnolipids, or synthetic 3O-C12-HSL. Virulence factors secreted by P. aeruginosa reduced TER and caused TJ rearrangement in the bronchial epithelium, exposing the epithelium to further bacterial infiltration. Pretreatment of the bronchial epithelium with azm attenuated this effect and facilitated epithelial recovery. These data suggest that azm protects the bronchial epithelium during P. aeruginosa infection independent of antimicrobial activity, and could explain in part the beneficial results seen in clinical trials of patients with CF. Keywords: azithromycin; airway epithelia; tight junctions; Pseudomonas aeruginosa; rhamnolipids

The bronchial epithelium provides the first line of defense against infectious agents in the respiratory system (1). Not only does the bronchial epithelium provide a passive barrier between the respiratory lumen and the submucosal compartment, but it also recognizes and responds to potentially hazardous agents, secretes antimicrobial factors, and physically removes inhaled pathogens from the lung through mucociliary clearance (2). Maintaining bronchial epithelial integrity is therefore essential to keep the respiratory system infection-free. Bronchial epithelial integrity, including correct apical-basolateral polarity and paracellular trafficking, is largely maintained by tight junction (TJ) protein complexes that regulate ion transport across the epithelium and generate transepithelial electrical resistance (TER) (3). Dysfunction or disruption of TJs is evident in many disease conditions such as cancer and during (Received in original form September 15, 2008 and in final form April 9, 2009) Grant support was provided by the thematic program ‘‘Postgenomic medicine’’ from the Icelandic Centre for Research (RANNI´S) (Grant number 052010005), the University Hospital Research Fund, and the Eimskip University Fund. Correspondence and requests for reprints should be addressed to Olafur Baldursson, M.D., Ph.D., Landspitali, Eiriksgata 5, 101 Reykjavik, Iceland. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 42. pp 62–68, 2010 Originally Published in Press as DOI: 10.1165/rcmb.2008-0357OC on April 16, 2009 Internet address: www.atsjournals.org

bacterial infection (reviewed in Refs. 4 and 5). Indeed, many infectious agents are known to express virulence factors that specifically target TJs of the epithelium. For instance, Vibrio cholera produces Zonula occludens (ZO-1) toxin that causes delocalization of occludin and ZO-1 from the cell–cell boundaries and subsequent loss of intestinal epithelial integrity (6). Studies using respiratory epithelia suggest that TJ function is crucial for lung defense against infections (7, 8). In this regard, Pseudomonas aeruginosa is particularly interesting because well-differentiated and polarized respiratory epithelia are more resistant to P. aeruginosa internalization than epithelia with compromised integrity or junctional dysfunction (9, 10). P. aeruginosa’s extensive arsenal of virulence factors includes secreted exotoxins, elastase, proteases, and rhamnolipids (11), many of which have been reported to alter epithelial integrity (12–14). Recently, secreted rhamnolipids have been shown to promote infiltration of P. aeruginosa by altering tight junctions in the respiratory epithelium (14). N-(3-oxododecanoyl)–L– homoserine lactone (3O-C12-HSL), an autoinducer molecule in P. aeruginosa’s quorum sensing system, has also been reported to alter epithelial integrity, possibly via TJ modifications (13). These findings suggest the possibility that strategies aimed at strengthening respiratory epithelia, emphasizing TJ function, could be useful against P. aeruginosa airway infections. These infections are recognized as a significant clinical problem in patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (15). Unfortunately, the management of P. aeruginosa bronchitis under these circumstances is often characterized by a vicious cycle of antibiotic administration and antimicrobial resistance, allowing chronic inflammation and bronchial wall destruction (16). Better understanding of the complex interplay between airway epithelial defense mechanisms and bacterial virulence factors is therefore essential to allow the development of novel treatments. Our previous observations showed that the macrolide antibiotic azithromycin (azm) affected the expression of TJ proteins and increased TER in human airway epithelia in vitro (17). This result is interesting because data from clinical studies show that azm improves lung function and prognosis in patients with CF or diffuse panbronchiolitis (DPB), independent of its antibacterial effect (18, 19). This clinical effect is poorly understood, but earlier studies indicated that this may be explained by anti-inflammatory effects of azm or its ability to hinder bacterial biofilm formation (20, 21). In light of our findings, we hypothesized that azm could be used to augment TJ function during infection, thereby improving airway epithelial defense and resistance to P. aeruginosa infection. To test this possibility and to better understand the interaction between P. aeruginosa and the bronchial epithelium during airway infection, we challenged human airway epithelia with live P. aeruginosa bacteria, pathogen-free P. aeruginosa culture medium, purified rhamnolipids, and synthesized 3O-C12-HSL while measuring changes in TER and the expression of TJ proteins, in the presence and absence of azm.

Halldorsson, Gudjonsson, Gottfredsson, et al.: Azithromycin Maintains Airway Epithelial Integrity

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MATERIALS AND METHODS

Western Blotting

Cell Culture

Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany), homogenized, and cellular debris centrifuged and discarded. Protein concentration was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA) and equal amounts of protein were loaded and run on a 4–12% NuPage Bis-Tris gel (Invitrogen) and transferred to a PVDF membrane (Invitrogen). The membranes were blocked for 1 hour in 5% skimmed milk in 0.05% PBS-Tween and probed with primary antibody overnight and horseradish peroxidase– conjugated secondary antibody for 1 hour. Protein bands were visualized with ECL1 protein detection system and Typhoon imaging station (Amersham Biosciences, Little Chalfont, England).

A bronchial epithelial cell line (VA10), immortalized with E6/E7 viral oncogenes, was cultured as previously described (22). Briefly, the cells were maintained in bronchial epithelial growth medium (BEGM) with supplements (Lonza, Walkersville, MD) and penicillin/streptomycin (Gibco, Burlington, ON, Canada) at 378C in 5% CO2. Air–liquid interface cultures were set up on 12-well Transwell permeable filter supports with 0.4 mm pore size (Corning Costar Corporation, Acton, MA) and cultured in 50:50 Dulbecco’s modified Eagle medium/Ham’s F12 medium supplemented with 2% Ultroser G (Bioserpa, CergySaint-Christoph, France) and penicillin/streptomycin. Cells were seeded in BEGM. Two days after seeding, medium was changed to DMEM/F12 1 2% UG on both apical and basolateral sides. Two days later, medium was removed from the apical surface. Typically, cells were cultured at the air–liquid interface for 3 weeks before treatment, with medium changed every 2 days. For azm treatment, Zitromax (Pfizer, Cedex, France) was diluted in DMEM/F12 1 2% UG to a final concentration of 40 mg/ml and placed in the basal compartment.

Confocal Microscopy Immunofluorescence images were captured using a Zeiss LSM 5 Pascal Confocal Microscope system (Carl Zeiss AG, Munich, Germany) with Plan-Neofluar 40x and Plan-Apochromat 63x oil immersion objectives. Cell culture Transwell filters were mounted with coverslips and Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL). Z-scans were performed by taking a series of images at the same location with fixed focal intervals.

Immunofluorescent Staining Cells were fixed using either methanol at 2208C or 3.7% formaldehyde followed by 0.1% Triton X-100. The following primary antibodies were used: mouse anti–Claudin-1, rabbit anti-Occludin, rabbit anti–JAM-A, mouse anti–ZO-1, mouse anti-ezrin (all from Zymed, San Francisco, CA) and mouse anti-CFTR (R&D Systems, Minneapolis, MN). For F-actin staining, Alexa Fluor 488 phalloidin (Molecular Probes, Eugene, OR) was used. For immunofluorescent stainings, isotypespecific Alexa Fluor secondary antibody conjugates were used and TOPRO-3 nucleic acid stain, all from Molecular Probes.

Bacterial Strains and Preparations PAO1 is a wild-type (WT) P. aeruginosa strain. PAO1-Drhl is a PAO1 mutant lacking the rhlA and rhlB genes, key enzymes for rhamnolipid production. Before infections, the bacteria were cultured overnight in 50:50 DMEM/F12, conditions in which the strains grew at a similar rate. Bacterial culture supernatants were collected after 5 days in culture, centrifuged, and filtered through 0.22-mm pore-size filter (Whatman, Dassel, Germany). Rhamnolipids (JBR425) were purchased from Jeneil Biosurfactant Co. (Sauksville, WI). This is an aqueous solution of R1 and R2 rhamnolipids at 25% concentration, produced by P. aeruginosa. 3O-C12-HSL was a kind gift from Professor Paul Williams, University of Nottingham.

TER Measurements TER was measured with a Millicell-ERS volt-ohm meter (Millipore, Billerica, MA). All measurements were performed in triplicate, background readings of empty Transwell filters were subtracted.

RESULTS VA10 Cells Generate a Differentiated Bronchial Epithelium with High TER

Consistent with earlier data, VA10 cells formed a polarized and functionally intact stratified layer when cultured at the air–liquid interface for 2 to 3 weeks (22). The reconstructed human bronchial epithelia generated TER of 1,000 to 1,600 Vcm2 and expressed TJ complexes containing occludin, ZO-1 (Figure 1A), and JAM-A (not shown). Apical expression of ezrin and cystic fibrosis transmembrane conductance regulator (CFTR) was observed in some of the surface cells (Figure 1B), both of which are markers of ciliated epithelia (23, 24). In addition, Muc 5B expression was detected by RT-PCR in differentiated epithelia (data not shown). P. aeruginosa Infection Decreases TER and Affects the Expression of TJ Proteins and Associated Actin Cytoskeleton

Inoculation with live P. aeruginosa (PAO1, 20 cfu/cell) on the apical surface of the epithelia caused a gradual decline in TER, starting 5 hours after exposure and continued at a steady rate until reaching zero after 12 to 16 hours (Figure 2A). In contrast to apical actin, PAO1 infection affected the expression pattern of ZO-1 (Figure 2B) after 24 hours of exposure, suggesting that PAO1 attack on ZO-1 may explain the observed fall in TER. In addition, basal actin filaments (stress fibers) appeared to de-

Figure 1. Confocal images showing the apical expression of tight junctions (TJs), ezrin, and CFTR in airway epithelia. Cells were cultured at the air–liquid interface for 3 weeks. (A) Zonula occludens (ZO-1) (red) and occludin (green) surround each cell. Co-staining of these proteins appears yellow. The z-axis reveals that both proteins localize primarily to the sub-apical surface. (B) Ezrin (red) and CFTR (green) are also expressed apically. Nuclear stain is blue; bar 5 10 mm.

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expression of ZO-1, occludin, JAM-A, and claudin 4. Figure 3A shows that ZO-1 staining became more fragmented after 2 hours of exposure to the conditioned culture medium and was absent or disintegrated after 24 hours. Western blotting indicated that the expression of occludin and JAM-A decreased, while ZO-1 was processed to smaller protein products (Figure 3B). In contrast, claudin 4 expression was not affected. Together these data suggest that soluble PAO1 virulence factors specifically affect the expression and function of certain TJ proteins, leading to loss of TER and airway epithelial dysfunction. Rhamnolipids and 3O-C12-HSLs Cause Early TER Reduction in Respiratory Epithelia

Figure 2. Pseudomonas aeruginosa (PAO1) infection reduces transepithelial electrical resistance (TER) and affects localization of ZO-1 and F-actin in airway epithelia. (A) PAO1 growth on the apical surface (20 cfu/cell inoculum) gradually decreased TER, from 5 hours after infection, to baseline at 16 to 18 hours. Data are mean 6 SEM, n 5 3. (B) Confocal images showing that PAO1 infection disrupts the apical expression of ZO-1 (red) 24 hours after inoculation. In contrast, apical F-actin (green) expression is preserved, but basal F-actin expression appears decreased. Bar 5 10 mm. The figure represents one of three independent experiments with similar results.

crease after PAO1 infection (Figure 2B), without visible widespread cell death. To examine whether the disintegration of TJs was a direct effect of physical contact of the bacteria to the apical surface of the cells or due to soluble factors accumulating in the medium, we exposed the cells to filter-sterilized PAO1-derived culture medium. This produced a sharp fall in TER within the first hour of exposure, lasting for at least 24 hours, suggesting that soluble factors from PAO1 alone were sufficient to reduce TER. To determine which TJ proteins might be affected, we investigated the effect of PAO1 culture medium on the

A recent study indicated that secreted rhamnolipids play an important role in P. aeruginosa virulence, possibly through modifications of TJs (14). Therefore, we looked at rhamnolipids as potential virulence factors responsible for epithelial dysfunction in our model system. We exposed airway epithelia to conditioned culture medium from PAO1-Drhl, a P. aeruginosa strain that lacks two key enzymes in the rhamnolipid production pathway, rhlA and rhlB. In contrast to medium collected from WT-PAO1, filter-sterilized culture medium from PAO1-Drhl did not reduce TER over a period of 6 hours (Figure 4A). However, a significant decline in TER was seen after 24 hours in epithelia treated with either WT-PAO1 medium or PAO1-Drhl medium. To further test this possibility, we exposed airway epithelia directly to purified rhamnolipids and found a dose-dependent decline in TER (Figure 4B). In contrast to conditioned culture medium, a 50 mg/ml dose of purified rhamnolipids that caused a marked decrease in TER did not cause any visible degradation of TJ proteins (Figure 4C). 3O-C12-HSL is another P. aeruginosa–secreted inducer of virulence that has recently been reported to disrupt epithelial barrier integrity (13). To examine this effect in respiratory epithelia, we exposed our model to 100 mM synthesized 3O-C12HSL, a dose comparable to that of the purified rhamnolipids used earlier. Interestingly, 3O-C12-HSL had a TER-decreasing effect but, similar to the rhamnolipids, mediated little or no changes in TJ expression levels (data not shown). The data suggest that rhamnolipids and 3O-C12-HSL may be responsible for initial TER reduction during P. aeruginosa infection while other factors secreted by P.aeruginosa cause a slower, more permanent abolishment of TER and TJs. Azithromycin Prevents Decrease in TER Produced by P. aeruginosa Culture Medium or Rhamnolipids but Not from the Effects of 3O-C12-HSL

Our previous work showed that azm increased TER in human airway epithelia in vitro and affected the processing of TJ proteins (17). Therefore, we asked if azm could prevent TER reduction produced by conditioned medium, rhamnolipids or 3O-C12-HSL observed in the current experiments. Airway epithelia were incubated with 40 mg/ml azm for 4 days before exposure to sterile filtered bacterial culture medium, purified rhamnolipids, or 3O-C12-HSL. This azm concentration has been shown to be within the range found in sputum of patients receiving long-term daily azm treatment (25). Interestingly, azm-pretreated epithelia had a slower and less prominent fall in TER upon apical exposure of either conditioned culture medium (Figure 5A) or 50 mg/ml rhamnolipids (Figure 5B). In addition, pretreated epithelia had fully recovered 5 to 6 hours after initial exposure, while untreated epithelia showed little or no signs of recovery 24 hours after


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Figure 3. P. aeruginosa (PAO1)-derived culture medium affects the expression of TJ proteins in airway epithelia. (A) Confocal images showing gradual changes in ZO-1 expression (red) at 2 to 24 hours after apical exposure to PAO1 medium compared with control. Bar 5 20 mm. (B) WB analysis of ZO-1, occludin, JAM-A, and claudin 4 after exposure to PAO1 culture medium. ZO-1 and occludin appear to be down-regulated after 2 hours of exposure, an effect that is still apparent after 24 hours. The expression of JAM-A is also affected, but to a lesser extent. Claudin 4 expression remains unchanged throughout the experiment. The figure represents one of three independent experiments with similar results. L 5 protein standard.

exposure. In contrast, there were no signs of augmented TER drop or facilitated recovery in azm-pretreated cells exposed to 100 mM 3O-C12-HSL as compared with control cells (Figure 5C).

DISCUSSION In this study we show that P. aeruginosa decreases TER and affects the expression of TJ proteins in human airway epithelia in vitro. The data also show that direct bacterial invasion is not required to disrupt TJ function; soluble factors derived from P. aeruginosa are sufficient to produce this effect. Consistent with previous studies (13, 14), we found that rhamnolipids and 3OC12-HSL disrupt TJ function significantly and appeared to be important virulence factors without causing TJ degradation. Interestingly, epithelia pretreated with azm maintained TER after exposure to either P. aeruginosa culture medium or purified rhamnolipids. Maintenance of cell polarity in the bronchial epithelium plays a central role in host immunity. Not only does the epithelium constantly and in response to infection secrete antimicrobial factors and cytokines, but it also provides a mechanical barrier between the internal domain and the external environment. Receptor distribution on the apical surface versus the basolateral domain is well regulated and is of central importance for efficient epithelial response to external and endogenous factors (26). Epithelial integrity is therefore of key importance to withstanding infection in the respiratory tract. Tight junctions are central components of epithelial integrity due to their ability to maintain cell polarity and by regulating paracellular flux across epithelial tissue (7). Respiratory pathogens such as P. aeruginosa are known to disrupt epithelial integrity. Different P. aeruginosa virulence factors have been reported to decrease epithelial TER and the expression of TJ proteins, some produced by the type three secretion system (TTSS) (12) while others are under direct regulation of the P. aeruginosa quorum sensing (QS) system (13, 14). Among them are secreted rhamnolipids. Rhamnolipids contribute to P. aeruginosa function in several ways, such as biofilm de-

velopment, swarming motility and as antimicrobials (27). They have been shown to be virulent to mammalian cells in several ways, such as disruption of the polymorphonuclear leukocyte chemotactic responses, inhibition of normal macrophage function, stimulation of cytokine release from airway epithelial cells, and interference with normal ciliary function (27, 28). In addition, secreted rhamnolipids were recently implicated as early acting virulence factors by disrupting epithelial TJs (14). Our results confirm this finding by showing that unlike wild-type P. aeruginosa, bacterial culture supernatant from a P. aeruginosa strain lacking rhamnolipids failed to cause an immediate fall in TER. In addition, purified rhamnolipids at levels similar to those found in CF sputum (28) caused a sharp drop in TER. However, it should be kept in mind that stationary phase P. aeruginosa secrete a large amount of diverse virulence factors, many of which have been reported to affect cell–cell binding and epithelial integrity. One of the secreted factors that has been reported to affect epithelial integrity is the quorum sensing autoinducer molecule 3O-C12-HSL (13). Indeed, at concetrations of 100 mM, 3O-C12-HSL caused a sharp fall in TER in our model system. As with the rhamnolipids, this occurred without apparent reduction in TJ protein expression. However, although 3O-C12-HSL levels in laboratory cultured P. aeruginosa biofilms has been shown to be as high as 1 mM (29), reported concentrations of 3O-C12-HSL in CF sputum are in the lower nanomolar range (30). Our results provide further evidence that secreted rhamnolipids are an important factor in determining early disruption of epithelial integrity, most likely by inserting into the host membranes and altering TJ properties, without causing a reduction in TJ protein expression or degradation. The data also indicate that 3O-C12-HSL may contribute to an early fall in TER. Other factors are likely to follow the path created by the rhamnolipids and to cause a more permanent damage to TJ proteins. Due to their nature, TJs are becoming a potential drug target (31). Selective molecules that can open up TJs are considered promising tools for drug delivery in organs such as the respiratory system and the gastrointestinal tract. Interestingly,

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Figure 4. Rhamnolipids from P. aeruginosa (PAO1) decrease TER (Vcm2) in airway epithelia. (A) Culture medium from wild-type PAO1 decreased TER quickly after apical exposure (shaded bars), compared with control (open bars). In contrast, epithelia treated with PAO1-Drhl medium maintained TER 6 hours after exposure (solid bars). TER in all exposed epithelia had fallen after 24 hours of exposure. (B) Addition of purified rhamnolipids to the apical side of airway epithelia caused a dosedependent fall in TER. Data are mean 6 SEM, n 5 3. Figure represents data from three independent experiments showing similar results. (C ) WB analysis of TJ proteins of epithelia exposed to 50 mg/ml purified rhamnolipids.

Wong and Gumbiner (32) showed that a synthetic peptide, corresponding to the putative extracellular domain of occludin, modulated TJ permeability. In the same way, drugs that increase the function of TJs are candidates for cancer treatment and improved epithelial defenses against infectious agents. Disruption of TJs may be a key factor in P. aeruginosa virulence and host response. By opening the paracellular pathway followed by loss of epithelial integrity and polarity, bacteria are allowed to infiltrate the tissue resulting in receptor binding of bacterial-derived proteins, lipids and host cytokines to receptors otherwise located on the basolateral domain of the cell surface (33). This will in turn result in an enhanced immune response characterized by cytokine secretion, leukocyte migration, and

Figure 5. Azithromycin prevents decrease in TER produced by P. aeruginosa (PAO1) culture medium or rhamnolipids. Epithelia were pretreated with 40 mg/ml azm for 4 days before PAO1 culture medium, rhamnolipid, or 3O-C12-HSL exposure. (A) Compared with control, azm-pretreated epithelia had less TER reduction in the first 2 hours after exposure to PAO1 culture medium, began to recover earlier and, reached near-baseline TER in 6 hours. (B) In contrast to control, azmpretreated epithelia showed only slight TER reduction upon exposure to rhamnolipids and had fully recovered within 5 hours. (C ) Exposure of the apical surface to 3O-C12-HSL caused a sharp drop in airway epithelial TER. Azm pretreatment did not prevent this TER reduction. Data are mean 6 SEM, n 5 3. The figures represent one of three independent experiments with similar results.

inflammation. Chronic inflammation of the bronchial epithelium is present in diseases such as COPD, CF, and DPB. Incidentally, chronic colonization with P. aeruginosa is a major clinical problem in the management of these diseases (15, 34, 35). Azm has been used to treat patients with CF for a number of years after a Japanese study showed improved lung function in patients with DPB after administration of macrolide antibiotics (19). Interestingly, this improvement did not correlate with decreased bacterial counts in sputum samples from these


patients. Although long-term azm treatment of patients with CF is now widespread, its mechanism of action is still debated. The effects mediated by azm are likely to be synergistic, affecting both host and pathogen (36, 37). Previous studies indicate that azm treatment may attenuate inflammation (20, 38), inhibit alginate production and biofilm formation (39), and inhibit QS in P. aeruginosa (40). Interestingly, Tateda and coworkers have shown that azm decreases production of rhamnolipids in a P. aeruginosa strain without any effect on bacterial growth. However, it should be kept in mind that the beneficial effects of azm treatment are not limited to patients colonized by P. aeruginosa (41). Our previous results indicate that azm strengthens respiratory epithelial TER in vitro and affects TJ processing (17). In this study, we treated airway epithelia with azm and challenged them with conditioned culture medium from P. aeruginosa as well as purified rhamnolipids. Our results indicate that azm treatment can hinder epithelial TER reduction caused by secreted virulence factors. In addition, the dose of azm used in our experiments protected epithelial TER from the effects of purified rhamnolipids at concentrations that have been reported in P. aeruginosa biofilms and sputum from patients with CF (42, 43). The data indicate that azm has a secondary effect on the bronchial epithelium, strengthens epithelial integrity, and maintains polarity, thereby preventing infiltration of pathogens and subsequent inflammation. Interestingly, data from clinical trials in CF showing that azm decreases the rate and severity of pulmonary exacerbations are consistent with the notion that azm augments epithelial defenses (41). Our results suggest that a commonly prescribed antibiotic preserves TJ function in airway epithelia during infection, indicating that pharmacologic manipulation of TJ expression may be a plausible option to improve lung defense against infection. Such defense strategies could become clinically relevant in light of the impressive ability of microbes to evade and survive the constant crossfire of new antibiotics. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments: The authors thank Dr. Paul Williams at the University of Nottingham for providing the 3O-C12-HSL.

References 1. Whitsett JA. Intrinsic and innate defenses in the lung: intersection of pathways regulating lung morphogenesis, host defense, and repair. J Clin Invest 2002;109:565–569. 2. Bals R, Hiemstra PS. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur Respir J 2004;23:327–333. 3. Van Itallie CM, Anderson JM. The role of claudins in determining paracellular charge selectivity. Proc Am Thorac Soc 2004;1:38–41. 4. Cereijido M, Contreras RG, Flores-Benitez D, Flores-Maldonado C, Larre I, Ruiz A, Shoshani L. New diseases derived or associated with the tight junction. Arch Med Res 2007;38:465–478. 5. Landau D. Epithelial paracellular proteins in health and disease. Curr Opin Nephrol Hypertens 2006;15:425–429. 6. Schmidt E, Kelly SM, van der Walle CF. Tight junction modulation and biochemical characterisation of the zonula occludens toxin c-and ntermini. FEBS Lett 2007;581:2974–2980. 7. Shin K, Fogg VC, Margolis B. Tight junctions and cell polarity. Annu Rev Cell Dev Biol 2006;22:207–235. 8. Van Itallie CM, Anderson JM. Claudins and epithelial paracellular transport. Annu Rev Physiol 2006;68:403–429. 9. Plotkowski MC, de Bentzmann S, Pereira SH, Zahm JM, BajoletLaudinat O, Roger P, Puchelle E. Pseudomonas aeruginosa internalization by human epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity. Am J Respir Cell Mol Biol 1999;20:880–890. 10. Fleiszig SM, Evans DJ, Do N, Vallas V, Shin S, Mostov KE. Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity. Infect Immun 1997;65:2861–2867.

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11. Smith RS, Iglewski BHP. Aeruginosa quorum-sensing systems and virulence. Curr Opin Microbiol 2003;6:56–60. 12. Soong G, Parker D, Magargee M, Prince AS. The type III toxins of Pseudomonas aeruginosa disrupt epithelial barrier function. J Bacteriol 2008;190:2814–2821. 13. Vikstrom E, Tafazoli F, Magnusson KE. Pseudomonas aeruginosa quorum sensing molecule n-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of caco-2 cells. FEBS Lett 2006; 580:6921–6928. 14. Zulianello L, Canard C, Kohler T, Caille D, Lacroix JS, Meda P. Rhamnolipids are virulence factors that promote early infiltration of primary human airway epithelia by Pseudomonas aeruginosa. Infect Immun 2006;74:3134–3147. 15. Gomez MI, Prince A. Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis. Curr Opin Pharmacol 2007;7:244–251. 16. Rejman J, Di Gioia S, Bragonzi A, Conese M. Pseudomonas aeruginosa infection destroys the barrier function of lung epithelium and enhances polyplex-mediated transfection. Hum Gene Ther 2007;18:642–652. 17. Asgrimsson V, Gudjonsson T, Gudmundsson GH, Baldursson O. Novel effects of azithromycin on tight junction proteins in human airway epithelia. Antimicrob Agents Chemother 2006;50:1805–1812. 18. Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, Coquillette S, Fieberg AY, Accurso FJ, Campbell PW III. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2003;290:1749–1756. 19. Schultz MJ. Macrolide activities beyond their antimicrobial effects: macrolides in diffuse panbronchiolitis and cystic fibrosis. J Antimicrob Chemother 2004;54:21–28. 20. Cigana C, Nicolis E, Pasetto M, Assael BM, Melotti P. Anti-inflammatory effects of azithromycin in cystic fibrosis airway epithelial cells. Biochem Biophys Res Commun 2006;350:977–982. 21. Gillis RJ, Iglewski BH. Azithromycin retards Pseudomonas aeruginosa biofilm formation. J Clin Microbiol 2004;42:5842–5845. 22. Halldorsson S, Asgrimsson V, Axelsson I, Gudmundsson GH, Steinarsdottir M, Baldursson O, Gudjonsson T. Differentiation potential of a basal epithelial cell line established from human bronchial explant. In Vitro Cell Dev Biol 2007;43:283–289. 23. Huang T, You Y, Spoor MS, Richer EJ, Kudva VV, Paige RC, Seiler MP, Liebler JM, Zabner J, Plopper CG, et al. Foxj1 is required for apical localization of ezrin in airway epithelial cells. J Cell Sci 2003; 116:4935–4945. 24. Puchelle E, Gaillard D, Ploton D, Hinnrasky J, Fuchey C, Boutterin MC, Jacquot J, Dreyer D, Pavirani A, Dalemans W. Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium. Am J Respir Cell Mol Biol 1992;7:485–491. 25. Baumann U, King M, App EM, Tai S, Konig A, Fischer JJ, Zimmermann T, Sextro W, von der Hardt H. Long term azithromycin therapy in cystic fibrosis patients: a study on drug levels and sputum properties. Can Respir J 2004;11:151–155. 26. Vermeer PD, Einwalter LA, Moninger TO, Rokhlina T, Kern JA, Zabner J, Welsh MJ. Segregation of receptor and ligand regulates activation of epithelial growth factor receptor. Nature 2003;422:322– 326. 27. Soberon-Chavez G, Lepine F, Deziel E. Production of rhamnolipids by Pseudomonas aeruginosa. Appl Microbiol Biotechnol 2005;68:718–725. 28. Read RC, Roberts P, Munro N, Rutman A, Hastie A, Shryock T, Hall R, McDonald-Gibson W, Lund V, Taylor G, et al. Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating. J Appl Physiol 1992;72:2271–2277. 29. Charlton TS, de Nys R, Netting A, Kumar N, Hentzer M, Givskov M, Kjelleberg S. A novel and sensitive method for the quantification of n-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol 2000;2:530–541. 30. Erickson DL, Endersby R, Kirkham A, Stuber K, Vollman DD, Rabin HR, Mitchell I, Storey DG. Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis. Infect Immun 2002;70:1783–1790. 31. Kondoh M, Yoshida T, Kakutani H, Yagi K. Targeting tight junction proteins-significance for drug development. Drug Discov Today 2008; 13:180–186. 32. Wong V, Gumbiner BM. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J Cell Biol 1997;136:399–409.

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33. Humlicek AL, Manzel LJ, Chin CL, Shi L, Excoffon KJ, Winter MC, Shasby DM, Look DC. Paracellular permeability restricts airway epithelial responses to selectively allow activation by mediators at the basolateral surface. J Immunol 2007;178:6395– 6403. 34. Murphy TF, Brauer AL, Eschberger K, Lobbins P, Grove L, Cai X, Sethi S. Pseudomonas aeruginosa in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;177:853–860. 35. Poletti V, Casoni G, Chilosi M, Zompatori M. Diffuse panbronchiolitis. Eur Respir J 2006;28:862–871. 36. Giamarellos-Bourboulis EJ. Macrolides beyond the conventional antimicrobials: a class of potent immunomodulators. Int J Antimicrob Agents 2008;31:12–20. 37. Lopez-Boado YS, Rubin BK. Macrolides as immunomodulatory medications for the therapy of chronic lung diseases. Curr Opin Pharmacol 2008;8:286–291. 38. Cigana C, Assael BM, Melotti P. Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells. Antimicrob Agents Chemother 2007;51:975–981.

39. Hoffmann N, Lee B, Hentzer M, Rasmussen TB, Song Z, Johansen HK, Givskov M, Hoiby N. Azithromycin blocks quorum sensing and alginate polymer formation and increases the sensitivity to serum and stationary-growth-phase killing of Pseudomonas aeruginosa and attenuates chronic P. aeruginosa lung infection in cftr(2/2) mice. Antimicrob Agents Chemother 2007;51:3677–3687. 40. Tateda K, Comte R, Pechere JC, Kohler T, Yamaguchi K, Van Delden C. Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2001;45:1930–1933. 41. Clement A, Tamalet A, Leroux E, Ravilly S, Fauroux B, Jais JP. Long term effects of azithromycin in patients with cystic fibrosis: a double blind, placebo controlled trial. Thorax 2006;61:895–902. 42. Kownatzki R, Tummler B, Doring G. Rhamnolipid of Pseudomonas aeruginosa in sputum of cystic fibrosis patients. Lancet 1987;1:1026–1027. 43. Meers P, Neville M, Malinin V, Scotto AW, Sardaryan G, Kurumunda R, Mackinson C, James G, Fisher S, Perkins WR. Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections. J Antimicrob Chemother 2008;61:859–868.