Influence of the Surfactant Tyloxapol on Mucociliary Clearance in Human Respiratory Cystic Fibrosis Cells

Mucociliary clearance is an important defence mechanism that protects deeper airways from inhaled particles like allergens, pathogens or other pollutants. The airway respiratory epithelium consists of hair-shaped structures called cilia, which are covered by a protective mucus layer, the periciliary liquid (PCL). Located on top of that is an airway surface liquid layer in which foreign particles and microorganisms get stuck [1,2]. In the tracheobronchial tree, mucus is always moved toward and through the larynx and then swallowed. PCL dehydration reduces mucociliary clearance, leading to airway obstruction [3]. Mucus hypersecretion with abnormal mucus consistency and reduced mucociliary clearance occurs in respiratory diseases like cystic fibrosis (CF), respiratory distress syndrome (RDS) of the newborn, chronic obstructive pulmonary disease, asthma and also in heavy smokers.

There are several categories of human disease models to study respiratory diseases and drug development: cell lines, primary cells, 3D cell models, cell co-cultures, explants and in silico models [4]. At present, the most popular models are cell lines, derived from human tissues, which are mentioned in this paper.

The influence of surfactants on depressed mucociliary clearance in different situations and with different methods has been demonstrated previously [5,6,7,8]. One model has used the sputum of 15 CF patients [8]. CF is a genetic disorder that affects the lungs, pancreas, kidneys, liver and intestines. In the airways, the disease causes difficulties in breathing and coughing up mucus. CF is caused by mutations of the gene for the CF transmembrane conductance regulator (CFTR) protein. When CFTR is not functional, secretions that are normally thin become thick and in the airways, mucociliary clearance is disturbed.

In this paper, the effect of the surfactant tyloxapol on mucociliary clearance was studied in tissue batches taken from CF donors and cultivated for 60 days.

MucilAir™-CF (Epithelix Sàrl, Geneva, Switzerland) is a fully differentiated and ready-to-use 3D model of human airway epithelium, constituted with primary epithelial cells isolated from bronchial biopsies. It is commercially available and ready to use [4]. The cells used for this experiment were taken from 3 CF patients and cultivated for 60 days. Tissues originate from patients belonging to the homozygons ρF508 mutation, where 70% of CF-patients belong to [3]. In this study, the effect of tyloxapol on mucociliary clearance was evaluated on 3 independent batches from these CF patients of MucilAir™-CF (CF_MD0220; CF_MD0437; CF_MD0485) and measurements of the movements of microbeads were performed in triplicates. The mucociliary clearance was monitored using a high-speed acquisition camera (Sony) connected to an Axiovert 200M microscope (Zeiss, Göttingen, BRD). Before exposure to tyloxapol, mucociliary clearance was quantified following the movement of plugs of mucus on the apical surface. Following the measurement of control clearance, 30 µl of a saline solution of tyloxapol containing also 30 µm polystyrene microbeads was applied on the apical surface of MucilAir™-CF. Then, 30 s movies showing the movement of the small beads were taken and analyzed using the imaging software Image Pro Plus (Mediacy, Media Cybernetics, Rockville, Md., USA). The first movies were acquired before the addition of the particles and then immediately after the addition of tyloxapol (1 min), then after 3, 6, 10, 20, 30, 60, 120 min, 24 and 48 h. The movement of the beads was tracked and the velocity of each particle was calculated in order to determine the mean speed of the particles as a measurement of mucociliary clearance. The tyloxapol solution was obtained from a 1:2,000 dilution of Tacholiquin® 1% Lösung, which contained 10 mg tyloxapol, 50 mg glycerol and 20 mg sodium bicarbonate. The final amount of tyloxapol in the 30 µl administered was 150 ng. Polystyrene microbeads are from Sigma-Aldrich Chemie GmbH, Buchs, Switzerland.

Mucociliary clearance was monitored on 3 independent batches from 3 different CF patients. All batches were covered by sticky mucus before the addition of vehicle or test solutions, and the mucociliary clearance of MucilAir™-CF was zero (fig. 1, before exposure). The stimulatory effect of tyloxapol was observed already during the first minute (fig. 1, 1 min) and increased with a maximal observed effect at 120 min, 24 h and even 48 h after administration the effect was still observable.

Due to the dehydration of the apical membrane of the CF airway epithelia [9,10,11], mucociliary clearance function is greatly impaired in CF patients. In this paper, an in vitro model of human airway epithelia, derived from CF patients and cultivated for 60 days, was used. According to the impaired mucociliary clearance function in CF-patients, mucociliary clearance in these MucilAir™-CF is zero. Surfactants are used for many years to restore mucociliary clearance in models of impairment of the respiratory tract in neonatal RDS [6], CF [8], the anaesthetized dog [7] and in a porcine-gastric-mucin-model [12]. In the present experiments, tyloxapol, a synthetic nonionic surfactant is able to restore mucociliary clearance of the CF epithelia. Immediately after the addition of tyloxapol, the mucus is transported because of increased fluidity and mucociliary clearance is restored. The lowest velocity measured of the mucociliary clearance after the addition of tyloxapol was 12 µm/s and the maximum velocity was about 22 µm/s. The result is consistent with the observation that tyloxapol reduced the viscosity of CF-sputum from 463 ± 133 to 128 ± 52 centipoise [13]. In conclusion, the surfactant tyloxapol is able to restore mucocilial clearance in the MucilAir™-CF model for at least 48 h, with a maximal observed effect after 120 min.

G.U. is an employee of bene Arzneimittel GmbH; E.B., R.F. and W.S. declare no conflict of interest.