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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Int Forum Allergy Rhinol. 2020 Jul 28;11(2):136–143. doi: 10.1002/alr.22656

Azithromycin and ciprofloxacin inhibit IL-8 secretion without disrupting human sinonasal epithelial integrity in vitro

Dong-Jin Lim 1, Harrison M Thompson 3, Christopher R Walz 3, Samrath Ayinala 4, Daniel Skinner 1, Shaoyan Zhang 1, Jessica W Grayson 1, Do-Yeon Cho 1,2,5,*, Bradford A Woodworth 1,2,*
PMCID: PMC7854841  NIHMSID: NIHMS1624815  PMID: 32725797

Abstract

BACKGROUND:

We recently developed a ciprofloxacin and azithromycin sinus stent (CASS) to target recalcitrant infections in chronic rhinosinusitis (CRS). The objective of this study is to evaluate the anti-inflammatory activity of azithromycin released from the CASS and assess the impact on the integrity and function of primary human sinonasal epithelial cells (HSNECs).

METHODS:

P. aeruginosa lipopolysaccharide (LPS)-stimulated HSNECs were treated with azithromycin and/or ciprofloxacin at concentrations attainable from CASS release. IL-8 secretion was quantified by enzyme-linked immunosorbent assay. Epithelial integrity (Transepithelial resistance (TEER), paracellular permeability (FITC-dextran), lactate dehydrogenase (LDH) assays) and function (cilia beat frequency (CBF)) were also evaluated.

RESULTS:

Azithromycin significantly reduced secreted IL-8 from P. aeruginosa LPS-stimulated HSNECs at all concentrations tested (control = 5.77±0.39 ng/ml, azithromycin (6 μg/ml) = 4.58±0.40 ng/ml, azithromycin (60 μg/ml) = 4.31±0.06, azithromycin (180 μg/ml) = 4.27±0.26 ng/ml, p<0.05). Co-incubation with azithromycin (6 μg/ml) and ciprofloxacin (2.4 μg/ml) in LPS-stimulated HSNECs also displayed a significant reduction in secreted IL-8 when compared to P. aeruginosa LPS alone (co-treatment = 4.61±0.29 ng/ml, P. aeruginosa LPS = 7.35±0.89 ng/ml, p<0.01). The drugs did not negatively impact TEER, paracellular permeability, LDH release, or CBF indicating retention of cell integrity and function.

CONCLUSION:

Azithromycin decreased P. aeruginosa LPS IL-8 production in HSNECs at drug concentrations attainable with sustained release of azithromycin from the CASS. In addition to antibacterial activity, anti-inflammatory properties of the CASS should provide further benefit for patients with recalcitrant CRS.

Keywords: Interleukin-8, Azithromycin, Ciprofloxacin, Sinus Stents, Chronic Rhinosinusitis, Sinusitis, Pseudomonas

INTRODUCTION

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease that turns recalcitrant when bacterial biofilms involving Pseudomonas aeruginosa reside on the sinonasal mucosal surfaces.1 Biofilms have significant immune triggering properties with several studies demonstrating a reduced treatment response to antibiotics and overall worse patient outcomes when biofilms are present in the sinuses.2–5 To minimize systemic side effects, we have previously evaluated topical antimicrobial delivery to the sinus cavities using ciprofloxacin in in vitro and in vivo animal models.6–9 However, the hydrophilic properties of ciprofloxacin contributed to release of high quantities of drug in an initial burst release when coated as poly D/L-lactide-co-glycolide nanoparticles on a poly-D/L-lactic acid sinus stent. To address this problem, we coated the single layer of ciprofloxacin with an outer layer of the hydrophobic antimicrobial, azithromycin (ciprofloxacin azithromycin coated sinus stent (CASS)).10,11 The CASS exhibited an extended release profile of both azithromycin and ciprofloxacin over 28 days with inhibition of new P. aeruginosa biofilms as well as robust treatment of preformed P. aeruginosa biofilms.10

Although azithromycin has been clinically used as an anti-inflammatory agent in respiratory diseases, the anti-inflammatory properties of azithromycin released from the CASS are currently unknown and require testing in relevant human in vitro models with and without ciprofloxacin.12,13 Interleukin-8 (IL-8) is chemoattractant cytokine that is upregulated in CRS with increasing levels correlated to higher disease severity (neutrophil infiltration).14 Previous studies have identified IL-8 inhibition as an off target effect of azithromycin and is considered one of the primary mechanisms of reduced airway inflammation in patients on chronic azithromycin therapy for airway disease.15,16 However, it is critical to assess the impact of azithromycin and ciprofloxacin on sinonasal epithelial integrity and toxicity for translation to future human clinical trials.

The objectives of the current study are to assess the anti-inflammatory activity of azithromycin with and without ciprofloxacin in vitro through inhibition of P. aeruginosa lipopolysaccharide (LPS)-induced IL-8 secretion, as well as determine the impact on the integrity and function of human sinonasal epithelial cells (HSNEC).

MATERIALS AND METHODS

Materials and Tested Concentrations

Azithromycin was obtained from TCI America (Portland, OR, USA), and ciprofloxacin HCl (99.5% purity) was purchased from GenHunter Corporation (Nashville, TN, USA). All other chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). The concentrations of ciprofloxacin and azithromycin were selected based on our previous in-vitro releasing studies from the CASS.10 The average dose of ciprofloxacin and azithromycin released daily were 2 μg and 120 μg, respectively. Therefore, the dosages of each drug were adjusted with incubation/exposure duration. For example, we used the 0.5 μg of ciprofloxacin and 30 μg of azithromycin as the duration of exposure was only 6 hours.

HSNEC cultured at an air-liquid interface (ALI)

Primary HSNECs were collected with prior approval of the Institutional Review Board at University of Alabama at Birmingham (UAB) (IRB-120305017). Written informed consent from each participant was collected and subjects were screened and negative for a mutation in the cystic fibrosis transmembrane conductance regulator gene. Primary HSNECs were derived from turbinate mucosa from 3 healthy donors undergoing surgery for non-inflammatory conditions and cultured on collagen-coated Costar 6.5-mm-diameter permeable filter supports (Corning, Lowell, MA) submerged in established culture media. Differentiation and ciliogenesis occurred in all cultures within 10 to 14 days.17–26 Matured HSNECs with resistance over 300 Ω/cm2 and 80% field ciliogenesis by inverted microscopy were subjected to experiments.

Interleukin-8 cytokine (IL-8) concentrations

IL-8 concentrations in the presence of azithromycin and/or ciprofloxacin were obtained using an enzyme linked immunosorbent assay (ELISA) kit for IL-8 (R&D systems, Minneapolis, MN). HSNECs were initially exposed to P. aeruginosa LPS for 2 hours, and then incubated up to 24 hours following treatment with azithromycin, ciprofloxacin, or a combination of these drugs. A dose escalation study of azithromycin (from 60 μg/ml to 180 μg/ml) based on our previous data (week 1 = 0.064 ± 0.061 mg/day, week 2 = 0.173 ± 0.026 mg/day, week 3 = 0.132 ± 0.012 mg/day and week 4= 0.063 ± 0.015 mg/day)10 was performed incubating HSNECs with LPS at the lowest dose 6 μg/ml (10 fold lower than 60 μg/ml).10 Based on these results, LPS treated HSNECs were incubated with ciprofloxacin and the lowest dose of azithromycin that inhibited IL-8 to evaluate synergy and additive effects. A 2.4 μg/ml dose of ciprofloxacin was used based on our in-vitro releasing studies (2.48 ± 0.46 μg/day on average).10 In each set of experiments, 100 μL of sterilized phosphate buffered saline (PBS) without P. aeruginosa LPS served as vehicle control. After the incubation period, the basal media (600 μL) were collected and measured for IL-8 concentration in each sample according to the manufacture’s protocol (R&D systems, Minneapolis, MN). The secreted IL-8 concentration in each sample was evaluated by measuring the absorbance at 450 nm using a microplate reader (Synergy HK, BIO-TEK Instruments, Winooski, VT). All samples were performed in triplicate.

Measurement of transepithelial electrical resistance (TEER)

Transepithelial electrical resistance (TEER) of HSNECs was measured using a EVOM2 epithelial volt-ohmmeter (World Precision Instruments, Sarasota, Florida, USA).27 The TEER is a summated value from both transcellular and paracellular resistances. The electrical impedance originating from the transcellular pathway represents the stability of the apical and basolateral plasma membranes, whereas the paracellular resistance is created when adequate cell-substrate or cell-cell contacts are formed in the HSNECs when cultured at an air liquid interface. 100μL of sterilized PBS or drug-containing PBS was dispensed into the apical chamber of the filter supports to produce an electrical circuit. After confirming the maturity of the cultured HSNECs by resistance and morphological observation under an inverted microscope, the planned experiments were conducted. The concentrations of azithromycin (30 μg/ml) and ciprofloxacin (0.5 μg/ml) and the period of incubation were selected to simulate clinically relevant conditions. For example, 30 μg/ml over the course of the six-hour incubation results in 120 μg/ml of azithromycin per day, a quantity similar to the 108 μg noted in our previous study.10 Azithromycin (30 μg/ml) or a mixture of azithromycin (30 μg/ml) and ciprofloxacin (0.5 μg/ml) were placed onto the apical surface of the cells. TEER was normalized against the PBS control and expressed as Ohms per square centimeter (Ω/cm2). These normalized values enable us to compare the difference of the epithelial membrane’s integrity between control and experimental groups. No disruption of the epithelial membrane in the presence of drugs can be observed as equal or higher than normalized TEER value 1.

In vitro diffusion-barrier function

To examine changes to the diffusion-barrier of HSNECs, paracellular permeability of model 10-kDa fluorescein isothiocyanate (FITC) labeled dextran was evaluated. The diffusion-barrier of HSNECs is determined by a tight epithelial barrier and tight junction formation and is impacted by a number of chemicals. By adding ciprofloxacin and azithromycin at given concentrations, the effects on altered permeability of 10-kDa FITC dextran can be measured. In brief, 0.5 mg/mL 10kDa FITC-dextran in 100 μL PBS was administered to the apical side of HSNECs for 6 hours in the presence or absence of ciprofloxacin (0.5 μg/ml) and azithromycin (6 μg/ml). The permeated concentrations of FITC dextran were quantified by a fluorescence microplate reader.

Ciliary Beat Frequency Analysis

To acquire ciliary beat frequency (CBF), the HSNECs were placed under a 20X objective on an inverted scope (Fisher Scientific, Pittsburgh, PA) at ambient temperature, and treated with azithromycin (30 μg/ml), ciprofloxacin (0.5 μg/ml), and azithromycin/ciprofloxacin (30 μg/ml and 0.5 μg/ml, respectively). After applying 100μL of sterilized PBS as control or PBS containing drug to the apical surface of the cells, the motion images were acquired for 30 minutes. Under each condition, image acquisition was performed using a Basler area scan high-speed monochromatic digital video camera (Basler AG, Ahrensburg, Germany) at a sampling rate of 100 frames per second. The Sisson-Ammons Video Analysis (SAVA) system was used to acquire CBF. Baseline CBF recording was conducted in the previously described manner, and each analysis of CBF was normalized to fold-change over baseline CBF Hz (treatment/baseline).28

HSNEC viability LDH assay

HSNEC cell viability was tested using a Lactate Dehydrogenase (LDH) assay as previously described.29 Relative cell viability in the presence of ciprofloxacin and azithromycin is reflective of concentrations of released LDH enzyme originating from damaged HSNECs. Concentration gradients generated from internal standards (with positive controls) provided by the manufacturer render a theoretical kit detection range in biological samples containing IL-8. In this study, the relative cell viability of HSNECs was measured after treatment with ciprofloxacin and azithromycin with concentrations similar to that of drug released from the CASS. Based on the in vitro drug release profile from our previous study, the average dose of ciprofloxacin released daily was 2 μg per day with maximum 2.48 ± 0.46 μg/day at week 3. In regard to azithromycin, an average of approximately 108 μg of azithromycin was released daily for 4 weeks. To reproduce in-vitro drug release conditions where ciprofloxacin/azithromycin remain on the epithelial surface for an entire day, the HSNECs cultured at ALI were treated up to 6 hours with 0.5 μg/ml of ciprofloxacin and/or 30 μg/ml of azithromycin at the apical surface. Released LDH at 3 and 6 hours after incubation is measured with a coupled enzymatic reaction that converts a tetrazolium salt (iodonitrotetrazolium (INT)) into the red color formazan (Cytoscan™ LDH cytotoxicity assay, G-biosciences, St. Louis, MO). Quantification of LDH was determined from the concentration of the converted formazan within the 20 minutes of incubation. The resulting formazan absorbs maximally at 492nm and can be measured quantitatively at 490nm. Total LDH in each sample was expressed as ng/ml.

Statistical Analysis

All experiments were performed at least in triplicate. Statistical analysis was performed with GraphPad Prism 6.0 (La Jolla, Ca) with significance set at p < 0.05. For assessing the anti-inflammatory activity of azithromycin and/or ciprofloxacin, a one-way ANOVA was performed with a Tukey’s multiple comparison test for all experiments except the TEER analysis. Normalized values for relative TEER were expressed as ± standard error of the mean.

RESULTS

Azithromycin inhibits production of IL-8

To evaluate the anti-inflammatory effects of azithromycin on HSNEC, cells were stimulated with 25 μg/ml of P. aeruginosa lipopolysaccharide (LPS) for 2 hours, and treated with incremental azithromycin concentrations (6, 60, and 180 μg/ml) (Figure 1). After a 24-hour incubation period, the basolateral media was harvested and secreted IL-8 was measured. Compared to the PBS treated group (2.656 +/− 0.150 ng/mL), a significant increase in IL-8 secretion was observed in the P. aeruginosa LPS treated group (5.770 +/− 0.395 ng/mL, p < 0.0001), indicating P. aeruginosa LPS stimulates the production of inflammatory cytokines including IL-8 from HSNECs. When treated with azithromycin (6, 60, and 180 μg/ml), there was a significant reduction in IL-8 production compared to those treated with LPS alone (4.579 +/− 0.399, 4.312 +/− 0.057 and 4.269 +/− 0.258 ng/mL, respectively, p < 0.05, n = 3 per condition). However, there was no statistically significant dose-dependent response among those cells treated with different concentrations of azithromycin.

Figure 1.

Figure 1.

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Comparison of LPS-induced Interleukin-8 (IL-8) levels at varying concentrations of azithromycin.

Reduced interleukin-8 expression by different azithromycin concentrations from P. aeruginosa lipopolysaccharide (LPS)-treated HSNEC (n=3). *, **, *** and ****: p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. HSNEC: Human sinonasal epithelial cell

Azithromycin-dependent reduction of IL-8 in the presence of ciprofloxacin

In our previous study, the CASS was able to simultaneously release both ciprofloxacin and azithromycin in a sustained manner.10 To study the anti-inflammatory effect of azithromycin in the presence of ciprofloxacin, HSNECs were treated with the lowest concentration of azithromycin (6 μg/ml) from our previous dose-response experiment, and co-incubated with ciprofloxacin at 2.4 μg/ml concentration - a concentration similar to the maximal release rate observed in our prior in vitro releasing study (2.48 ± 0.46 μg/day at week 3). As anticipated, secreted IL-8 following P. aeruginosa LPS exposure was significantly increased compared to the PBS control (7.35 +/− 0.89 ng/ml of IL-8 vs. 2.38 +/− 0.18 ng/ml of IL-8, p < 0.01, n = 3) (Figure 2). Co-treatment with azithromycin and ciprofloxacin significantly reduced the production of IL-8 (4.61 +/− 0.29 ng/ml, p < 0.01, n = 3). There was no meaningful reduction in secreted IL-8 in the presence of ciprofloxacin (2.4 μg/ml) alone with P. aeruginosa LPS exposure compared to LPS control (6.40 +/− 0.26 ng/mL vs. 7.35 +/− 0.89 ng/ml, p = 0.29, n = 3). This suggests that the anti-inflammatory activity is solely due to azithromycin.

Figure 2.

Figure 2.

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Comparison of LPS-induced Interleukin-8 (IL-8) levels in the presence or absence of the study drugs.

Reduced interleukin-8 expression by a combination of azithromycin and ciprofloxacin concentrations from P. aeruginosa lipopolysaccharide (LPS)-treated HSNEC (n = 3). **, *** and ****: p < 0.01, p < 0.001, and p < 0.0001, respectively. HSNEC: Human sinonasal epithelial cells

Azithromycin and ciprofloxacin do not affect HSNEC tight junction integrity

To study the integrity of HSNECs in the presence of azithromycin (30 μg/ml) and/or ciprofloxacin (0.5 μg/ml), the transepithelial electrical resistance (TEER) was assessed (Figure 3). The TEER was measured at predetermined time points up to 6 hours: 0, 30, 90, 180, 270, and 360 minutes. Each collected TEER values at different time point was normalized against the average values obtained from control groups (PBS). There was no significant reduction in normalized TEER over time. Compared to the azithromycin group (30 μg/ml), the azithromycin/ciprofloxacin (30 μg/ml and 0.5 μg/ml, respectively) group displayed similar or mildly greater resistance compared to control groups (PBS). For example, the normalized TEER values of the 30 μg/ml azithromycin group at 3 hours were 1.14 +/− 0.06 Ω/cm2, whereas the azithromycin/ciprofloxacin group was 1.43+/− 0.36 Ω/cm2. Similarly, it was found that the azithromycin/ciprofloxacin group displayed a TEER of 1.45+/− 0.31 Ω/cm2 after 6 hours incubation, which is higher than that of the azithromycin group (1.02+/− 0.37 Ω/cm2). There was no significant difference between groups. These findings confirm that the application of azithromycin and ciprofloxacin does not disrupt the integrity of the epithelial membrane, including tight junctions.

Figure 3.

Figure 3.

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Normalized transepithelial electrical resistance (TEER) in the presence of the study drugs

No significant reduction in normalized TEER over time in the presence of azithromycin (30 μg/ml) or azithromycin/ciprofloxacin (30 μg/ml and 0.5 μg/ml, respectively). TEER: Transepithelial electrical resistance

Paracellular permeability of 10kDa FITC dextran particles is unaffected by azithromycin and ciprofloxacin

Using 10kDa FITC dextran particles, the effect of azithromycin and ciprofloxacin on paracellular permeability of HSNECs was evaluated. (Figure 4). After 3 hours of incubation, average paracellular permeabilities (%) of 60kDa dextran particles were as follows: control = 0.32 +/− 0.45, azithromycin 30 μg/ml = 0.10 +/− 0.15, ciprofloxacin 0.5 μg/ml = 0.31 +/− 0.26, and azithromycin 30 μg/ml /ciprofloxacin 0.5 μg/ml = 0.37 +/− 0.31 (n = 3). Likewise, after 6 hours incubation the results were as follows: control = 3.70 +/− 0.46, azithromycin 30 μg/ml = 3.44 +/− 0.24, ciprofloxacin 0.5 μg/ml = 4.27 +/− 0.45, and azithromycin 30 μg/ml /ciprofloxacin 0.5 μg/ml = 4.72 +/− 0.48 (n = 3). As shown in Figure 4, there is no significant difference between the groups when compared to the control (PBS) group when analyzed at each time point. In each group, low percentages of loaded 10kDa FITC dextran particles were found on the basolateral surface of the HSNECs at each time point indicating retained integrity with exposure to azithromycin and ciprofloxacin.

Figure 4.

Figure 4.

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Paracellular permeability in the presence and absence of the study drugs

No alterations in average paracellular permeability of HSNECs over time in the presence of azithromycin and/or ciprofloxacin. Each value is the mean +/− standard deviation of 4 samples at each time point. HSNEC: Human sinonasal epithelial cell

Azithromycin and ciprofloxacin do not decrease CBF

By comparing CBF between groups, changes can reflect diminished function of the mucociliary clearance (MCC) apparatus caused by azithromycin and/or ciprofloxacin. Study drugs (azithromycin (30 μg/ml), ciprofloxacin (0.5 μg/ml), and azithromycin/ciprofloxacin (30 μg/ml and 0.5 μg/ml)) were administered to the apical surface of HSNECs and compared to controls (PBS) (Figure 5). There was no statistically significant difference in CBF between the groups. The fold changes were as follows: PBS, 1.28 +/− 0.28 CBF fold changes; azithromycin (30 μg/ml), 1.38 +/− 0.13 CBF fold changes (p = 0.8949); ciprofloxacin (0.5 μg/ml), 1.41 +/− 0.32 CBF fold changes (p = 0.8937); azithromycin/ciprofloxacin (30 μg/ml and 0.5 μg/ml, respectively), 1.39 +/− 0.25 CBF fold changes (p = 0.81).

Figure 5.

Figure 5.

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Ciliary Beat Frequency (CBF) in the presence and absence of the study drugs

No significant difference in ciliary beat frequency (CBF) was measured in the presence of azithromycin and/or ciprofloxacin. The CBF fold changes were described as mean +/− standard deviation of 4 samples at each time point.

Ciprofloxacin and azithromycin are not toxic to HSNEC

The basolateral media was also collected to measure LDH, which is a cytoplasmic enzyme that leaks extracellularly when the plasma membrane is damaged. As shown in Figure 6, there was no significant difference of LDH concentrations between groups. Compared to control (PBS) groups, all 3 study groups of HSNECs (azithromycin 30 μg/ml, ciprofloxacin 0.5 μg/ml, and azithromycin 30 μg/ml/ciprofloxacin 0.5 μg/ml) produced similar amounts of LDH at 3 and 6 hours after incubation. After 3 hours, LDH concentrations were as follows: control = 2.815 +/− 0.108, azithromycin 30 μg/ml = 2.614 +/− 0.122, ciprofloxacin 0.5 μg/ml = 2.729 +/− 0.177, and azithromycin 30 μg/ml /ciprofloxacin 0.5 μg/ml = 2.930 +/− 0.147 (n = 3 per condition). Similarly, LDH concentrations at 6 hours incubation were as follows: control = 5.314 +/− 0.122, azithromycin 30 μg/ml = 5.228 +/− 0.122, ciprofloxacin 0.5 μg/ml = 5.199 +/− 0.108, and azithromycin 30 μg/ml /ciprofloxacin 0.5 μg/ml = 5.486 +/− 0.254 (n = 3 per condition). Azithromycin and ciprofloxacin do not have a detrimental effect on the cellular viability of HSNECs when applied at concentrations attainable from the CASS.

Figure 6.

Figure 6.

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Lactate dehydrogenase (LDH) levels in the presence and absence of the study drugs

No detrimental effect on the cellular viability of HSNECs over time in the presence of azithromycin and/or ciprofloxacin compared to controls. The LDH values are presented as mean +/− standard deviation of 4 samples at each time point. HSNEC: Human sinonasal epithelial cell

DISCUSSION

Chronic bacterial infections with pathogenic organisms that form biofilms (i.e. Pseudomonas aeruginosa) and associated sinonasal inflammatory responses have been identified as common reasons for persistence of recalcitrant CRS. Pharmacological interventions for severe disease have been limited to surgical intervention (often repetitive) and systemic +/− topical antibiotic sinus irrigations in attempts to eliminate chronic infections due to these biofilm-forming organisms. Our innovative approach using ciprofloxacin and azithromycin in the form of a sustained release via an antibiotic-eluting sinus stent is an efficient treatment strategy with high translational potential to improve clinical outcomes in CRS. Although azithromycin’s antibacterial activity against organisms like Pseudomonas is weaker than drugs of first choice (e.g., ciprofloxacin), the anti-inflammatory effect of azithromycin is considered an important modulator of neutrophilic inflammation in chronic infections associated with bronchiectasis and CRS.30–32

Macrolide antibiotics have been shown to reduce bacterial virulence factors promoting biofilm formation in addition to reducing the IL-8 inflammatory response known to recruit neutrophils. For example, the las and rhl quorum sensing systems of P. aeruginosa are inhibited by azithromycin, leading to reduction of the autoinducer molecules, 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL) and C4-homoserine (C4-HSL).33 In a clinical trial, azithromycin improved the symptoms of CRS according to SNOT-22 (22-item Sino-Nasal Outcome Test) when using azithromycin 250 daily for 3 months compared to controls.34 However, in another trial using smaller accumulative doses (500 mg daily for 3 days, then 500 mg weekly for 11 weeks) there was no significant improvement in clinical outcomes.35 Because CRS is considered a mucosal disease, orally administrated azithromycin may be limited in achieving therapeutically effective doses with meaningful effects on neutrophilic inflammation as shown in the in vitro studies. The concentrations of azithromycin in sinus fluid and mucosal tissue were between 0.41 and 1.23 μg/ml, 48 hours after administration of a single dose,36 which is much lower than the average dosage (30 μg/ml) in the present study. In our current strategy, locally-delivered azithromycin to inflamed nasal and sinus mucosa could provide significant advantages in this regard.

The CASS was designed to release both azithromycin and ciprofloxacin from 2 distinctive layers containing an inner layer of hydrophilic ciprofloxacin and an outer layer of hydrophobic azithromycin. In our previous study, the CASS showed sustained delivery of both drugs for 28 days, which conferred anti-biofilm activity against P. aeruginosa PAO-1 strains. However, this previous study did not investigate the anti-inflammatory activity of the CASS or assess real world implications on human sinonasal epithelial cell function and integrity. In the current study, we successfully demonstrated that azithromycin exhibited significant anti-inflammatory activity at attainable concentrations released from the CASS. P. aeruginosa LPS-stimulated IL-8 secretion was significantly reduced without compromising the integrity or function of HSNECs. The decreased production of IL-8 from HSNECs treated with azithromycin corroborates other published studies showing the anti-inflammatory effect of macrolides including azithromycin in vitro and in vivo.37 Moreover, a recent study identified that sub-inhibitory azithromycin may also reduce S. aureus exoproteins that induce pro-inflammatory cytokines from HSNECs.38 In a study using primary nasal-polyps epithelial cells, several macrolides reduced the production of IL-8 under Escherichia Coli serotype 055:85 LPS-stimulation. CRS Patients with nasal polyps (CRSwNP) showed reduced polyp size and lower IL-8 levels in nasal lavage following treatment with macrolide antibiotics. The combination of azithromycin and ciprofloxacin did not affect TEER (maintenance of tight junctions), paracellular permeability (toxicity-induced leaking of substrate), LDH concentrations (measurement of cell toxicity and death), or CBF (cell function) indicating the CASS is highly promising from a safety standpoint as an antibiotic-based intervention for recalcitrant chronic infections in CRS. Advantages of the CASS include extended concurrent delivery of ciprofloxacin and azithromycin to reduce mucosal inflammation, eradication of pre-existing biofilms, and abrogation of new biofilms.

The concentration and type of antibiotic for creating a sinus stent has to be carefully selected due to emergent antibiotic-tolerant strains or antibiotic persisters, which are defined by a subpopulation of strains exhibiting tolerance to the antibiotic. With exposure to suboptimal concentrations, bacteria can easily adapt by altering their metabolism.39 In the case of biofilm-forming P. aeruginosa, reduced susceptibility of antibiotic agents directly results in clinical treatment failure of CRS.30 The average release of azithromycin from the CASS per day is in the range of the minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa PAO-1 (> 128 μg/ml).40 Without co-delivery of ciprofloxacin, (MIC <1 μg/ml), there is a possibility of azithromycin tolerance and persistence of P. aeruginosa in a biofilm. Based upon the concept of the mutant selection window (MSW), which is defined as a region between the MIC and the mutant prevention concentration (MPC), unfavorable combinations of antibiotics can result in reduced MSW, thereby increasing the probability of antibiotic-resistant strains.41–43 When the combination is antagonistic, wild-type bacteria can be alive in a multi-drug environment and thus may evolve into multi-drug resistant strains.43 Additionally, several studies indicate that the clinical isolates of P. aeruginosa in patients with recalcitrant CRS are capable of resistance against fluoroquinolones including ciprofloxacin.44–46 Taken together, further studies are required to optimize the final concentration of azithromycin and ciprofloxacin in order to create the ideally suited CASS for preclinical and human clinical trials to treat recalcitrant CRS infections.

There are several limitations to this study. The anti-inflammatory activity of azithromycin was only evaluated by measuring IL-8 proinflammatory cytokine in-vitro after P. aeruginosa LPS stimulation. To further evaluate the anti-inflammatory activity of the CASS, a more clinically relevant animal model of recalcitrant CRS such as the rabbit model of CRS is required.6,7,47 We can perform a more comprehensive and multi-dimensional analysis of proinflammatory cytokines in animal tissues. Preclinical studies can also evaluate changes to integrity and function of sinonasal epithelial cells in vivo in a physiologically relevant model to human CRS.

CONCLUSION

At releasing concentrations attainable from the CASS, azithromycin+/−ciprofloxacin decreases the LPS induced inflammatory response (IL-8) in HSNEC in the absence of toxicity and retains epithelial integrity and function in vitro. In addition to previously demonstrated anti-biofilm activity against P. aeruginosa, these findings provide support for transitioning the CASS to preclinical studies.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1 R01 HL133006-04) and National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482-05, CF Research Center Pilot Award) to B.A.W. and NIH/National Institutes of Allergy and Infectious disease (K08AI146220), John W. Kirklin Research and Education Foundation Fellowship Award, UAB Faculty Development Research Award, American Rhinologic Society New Investigator Award, and Cystic Fibrosis Foundation Research Development Pilot grant (ROWE15R0) to D.Y.C.

Funding Sources

This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (1 R01 HL133006-03) and National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK072482-05, CF Research Center Pilot Award) to B.A.W. and NIH/National Institutes of Allergy and Infectious disease (K08AI146220), John W. Kirklin Research and Education Foundation Fellowship Award, UAB Faculty Development Research Award, American Rhinologic Society New Investigator Award, and Cystic Fibrosis Foundation Research Development Pilot grant (ROWE15R0) to D.Y.C.

Footnotes

The manuscript was presented at the American Rhinologic Society Annual Meeting (Virtual) in September 2020.

REFERENCES

  • 1.Schleimer RP. Immunopathogenesis of Chronic Rhinosinusitis and Nasal Polyposis. Annual Review of Pathology: Mechanisms of Disease 2017; 12:331–357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bendouah Z, Barbeau J, Hamad WA, Desrosiers M. Biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa is associated with an unfavorable evolution after surgery for chronic sinusitis and nasal polyposis. Otolaryngology - Head and Neck Surgery 2006; 134:991–996. [DOI] [PubMed] [Google Scholar]
  • 3.Singhal D, Psaltis AJ, Foreman A, Wormald P-J. The Impact of Biofilms on Outcomes after Endoscopic Sinus Surgery. American Journal of Rhinology & Allergy 2010; 24:169–174. [DOI] [PubMed] [Google Scholar]
  • 4.Illing EA, Woodworth BA. Management of the upper airway in cystic fibrosis. Curr Opin Pulm Med 2014; 20:623–631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Fastenberg JH, Hsueh WD, Mustafa A, Akbar NA, Abuzeid WM. Biofilms in chronic rhinosinusitis: Pathophysiology and therapeutic strategies. World Journal of Otorhinolaryngology - Head and Neck Surgery 2016; 2:219–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Cho DY, Hoffman K, Skinner D et al. Tolerance and pharmacokinetics of a ciprofloxacin-coated sinus stent in a preclinical model. Int Forum Allergy Rhinol 2017; 7:352–358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Cho DY, Lim DJ, Mackey C et al. Preclinical therapeutic efficacy of the ciprofloxacin-eluting sinus stent for Pseudomonas aeruginosa sinusitis. Int Forum Allergy Rhinol 2018; 8:482–489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Cho DY, Lim DJ, Mackey C et al. Ivacaftor, a Cystic Fibrosis Transmembrane Conductance Regulator Potentiator, Enhances Ciprofloxacin Activity Against Pseudomonas aeruginosa. Am J Rhinol Allergy 2019; 33:129–136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Cho DY, Lim DJ, Mackey C et al. In-vitro evaluation of a ciprofloxacin- and ivacaftor-coated sinus stent against Pseudomonas aeruginosa biofilms. Int Forum Allergy Rhinol 2019; 9:486–492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Lim D-J, Skinner D, Mclemore J et al. In-vitro evaluation of a ciprofloxacin and azithromycin sinus stent for Pseudomonas aeruginosa biofilms. International Forum of Allergy & Rhinology 2020; 10:121–127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Aucamp M, Odendaal R, Liebenberg W, Hamman J. Amorphous azithromycin with improved aqueous solubility and intestinal membrane permeability. Drug Dev Ind Pharm 2015; 41:1100–1108. [DOI] [PubMed] [Google Scholar]
  • 12.Qiu S, Zhong X. Macrolides: a promising pharmacologic therapy for chronic obstructive pulmonary disease. Ther Adv Respir Dis 2017; 11:147–155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Blasi F, Cazzola M, Tarsia P et al. Azithromycin and lower respiratory tract infections. Expert Opinion on Pharmacotherapy 2005; 6:2335–2351. [DOI] [PubMed] [Google Scholar]
  • 14.Yoon BN, Choi NG, Lee HS, Cho KS, Roh HJ. Induction of interleukin-8 from nasal epithelial cells during bacterial infection: the role of IL-8 for neutrophil recruitment in chronic rhinosinusitis. Mediators Inflamm 2010; 2010:813610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cigana C, Nicolis E, Pasetto M, Assael BM, Melotti P. Anti-inflammatory effects of azithromycin in cystic fibrosis airway epithelial cells. Biochemical and Biophysical Research Communications 2006; 350:977–982. [DOI] [PubMed] [Google Scholar]
  • 16.Idris SF, Chilvers ER, Haworth C, McKeon D, Condliffe AM. Azithromycin therapy for neutrophilic airways disease: myth or magic? Thorax 2009; 64:186–189. [DOI] [PubMed] [Google Scholar]
  • 17.Zhang S, Fortenberry JA, Cohen NA, Sorscher EJ, Woodworth BA. Comparison of vectorial ion transport in primary murine airway and human sinonasal air-liquid interface cultures, models for studies of cystic fibrosis, and other airway diseases. Am J Rhinol Allergy 2009; 23:149–152. [DOI] [PubMed] [Google Scholar]
  • 18.Bhargave G, Woodworth BA, Xiong G, Wolfe SG, Antunes MB, Cohen NA. Transient receptor potential vanilloid type 4 channel expression in chronic rhinosinusitis. Am J Rhinol 2008; 22:7–12. [DOI] [PubMed] [Google Scholar]
  • 19.Woodworth BA, Antunes MB, Bhargave G, Palmer JN, Cohen NA. Murine tracheal and nasal septal epithelium for air-liquid interface cultures: a comparative study. Am J Rhinol 2007; 21:533–537. [DOI] [PubMed] [Google Scholar]
  • 20.Woodworth BA, Tamashiro E, Bhargave G, Cohen NA, Palmer JN. An in vitro model of Pseudomonas aeruginosa biofilms on viable airway epithelial cell monolayers. Am J Rhinol 2008; 22:235–238. [DOI] [PubMed] [Google Scholar]
  • 21.Antunes MB, Woodworth BA, Bhargave Get al. Murine nasal septa for respiratory epithelial air-liquid interface cultures. Biotechniques 2007; 43:195–196, 198, 200 passim. [DOI] [PubMed] [Google Scholar]
  • 22.Conger BT, Zhang S, Skinner D et al. Comparison of cystic fibrosis transmembrane conductance regulator (CFTR) and ciliary beat frequency activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in primary sinonasal epithelial cultures. JAMA Otolaryngol Head Neck Surg 2013; 139:822–827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhang S, Blount AC, McNicholas CM et al. Resveratrol enhances airway surface liquid depth in sinonasal epithelium by increasing cystic fibrosis transmembrane conductance regulator open probability. PLoS One 2013; 8:e81589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zhang S, Skinner D, Hicks SB et al. Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance. PLoS One 2014; 9:e104090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Woodworth BA. Resveratrol ameliorates abnormalities of fluid and electrolyte secretion in a hypoxia-Induced model of acquired CFTR deficiency. Laryngoscope 2015; 125 Suppl 7:S1–S13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Tipirneni KE, Grayson JW, Zhang S et al. Assessment of acquired mucociliary clearance defects using micro-optical coherence tomography. Int Forum Allergy Rhinol 2017; 7:920–925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER measurement techniques for in vitro barrier model systems. J Lab Autom 2015; 20:107–126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Cho DY, Skinner D, Zhang S et al. Cystic fibrosis transmembrane conductance regulator activation by the solvent ethanol: implications for topical drug delivery. Int Forum Allergy Rhinol 2016; 6:178–184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Kumar P, Nagarajan A, Uchil PD. Analysis of Cell Viability by the Lactate Dehydrogenase Assay. Cold Spring Harb Protoc 2018; 2018. [DOI] [PubMed] [Google Scholar]
  • 30.Barshak MB, Durand ML. The role of infection and antibiotics in chronic rhinosinusitis. Laryngoscope Investigative Otolaryngology 2017; 2:36–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Fastenberg JH, Hsueh WD, Mustafa A, Akbar NA, Abuzeid WM. Biofilms in chronic rhinosinusitis: Pathophysiology and therapeutic strategies. World J Otorhinolaryngol Head Neck Surg 2016; 2:219–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Foreman A, Jervis-Bardy J, Wormald PJ. Do biofilms contribute to the initiation and recalcitrance of chronic rhinosinusitis? Laryngoscope 2011; 121:1085–1091. [DOI] [PubMed] [Google Scholar]
  • 33.Tateda K, Comte R, Pechere J-C, Köhler T, Yamaguchi K, Van Delden C. Azithromycin Inhibits Quorum Sensing in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 2001; 45:1930–1933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Amali A, Saedi B, Rahavi-Ezabadi S, Ghazavi H, Hassanpoor N. Long-term postoperative azithromycin in patients with chronic rhinosinusitis: A randomized clinical trial. Am J Rhinol Allergy 2015; 29:421–424. [DOI] [PubMed] [Google Scholar]
  • 35.Videler WJ, Badia L, Harvey RJ et al. Lack of efficacy of long-term, low-dose azithromycin in chronic rhinosinusitis: a randomized controlled trial. Allergy 2011; 66:1457–1468. [DOI] [PubMed] [Google Scholar]
  • 36.Karma P, Pukander J, Penttila M. Azithromycin concentrations in sinus fluid and mucosa after oral administration. Eur J Clin Microbiol Infect Dis 1991; 10:856–859. [DOI] [PubMed] [Google Scholar]
  • 37.Yamada T, Fujieda S, Mori S, Yamamoto H, Saito H. Macrolide Treatment Decreased the Size of Nasal Polyps and IL-8 Levels in Nasal Lavage. American Journal of Rhinology 2000; 14:143–148. [DOI] [PubMed] [Google Scholar]
  • 38.Hu H, Ramezanpour M, Hayes AJ et al. Sub-Inhibitory Clindamycin and Azithromycin reduce S. aureus Exoprotein Induced Toxicity, Inflammation, Barrier Disruption and Invasion. J Clin Med 2019; 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Stokes JM, Lopatkin AJ, Lobritz MA, Collins JJ. Bacterial Metabolism and Antibiotic Efficacy. Cell Metab 2019; 30:251–259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Mustafa M-H, Khandekar S, Tunney MM et al. Acquired resistance to macrolides in Pseudomonas aeruginosa from cystic fibrosis patients. European Respiratory Journal 2017; 49:1601847. [DOI] [PubMed] [Google Scholar]
  • 41.Drlica K, Zhao X. Mutant selection window hypothesis updated. Clin Infect Dis 2007; 44:681–688. [DOI] [PubMed] [Google Scholar]
  • 42.Drlica K The mutant selection window and antimicrobial resistance. J Antimicrob Chemother 2003; 52:11–17. [DOI] [PubMed] [Google Scholar]
  • 43.Yeh PJ, Hegreness MJ, Aiden AP, Kishony R. Drug interactions and the evolution of antibiotic resistance. Nature Reviews Microbiology 2009; 7:460–466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Lister PD, Wolter DJ, Hanson ND. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 2009; 22:582–610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Guss J, Abuzeid WM, Doghramji L, Edelstein PH, Chiu AG. Fluoroquinolone-resistant Pseudomonas aeruginosa in chronic rhinosinusitis. ORL J Otorhinolaryngol Relat Spec 2009; 71:263–267. [DOI] [PubMed] [Google Scholar]
  • 46.Elies W [Current therapeutical management, new antibiotics and treatment of Pseudomonas aeruginosa in bacterial ENT-infections]. Laryngorhinootologie 2002; 81:40–45. [DOI] [PubMed] [Google Scholar]
  • 47.Lim DJ, McCormick J, Skinner D et al. Controlled delivery of ciprofloxacin and ivacaftor via sinus stent in a preclinical model of Pseudomonas sinusitis. Int Forum Allergy Rhinol 2020; 10:481–488. [DOI] [PMC free article] [PubMed] [Google Scholar]

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