Tiotropium

Isoflurane was from Abbott Laboratories (UK). BD OptEIATM set for mouse immunoglobulin E was from BD Biosciences (US). Medical Oxygen was from BOC Industrial Gases (UK). FBS and Fixable near-IR dead cell stain kit for 633/635 nm excitation was from Invitrogen (UK). Collagenase and DNase was from Roche Diagnostics (Germany). Alum^TM was from ThermoFisher Scientific (UK). Glycopyrrolate, ethanol and 2 N H₂SO₄ was from VWR (UK). Tiotropium was a gift from Boehringer Ingelheim (Germany). Sterile saline was purchased from Fresenius Kabi Limited (UK) and pentobarbitone from National Veterinary Services Limited (UK). All other agents were purchased from Sigma-Aldrich (UK) unless otherwise described.

In experiments evaluating the effect of tiotropium (a gift from Boehringer Ingelheim, Ingelheim Germany) or fluticasone (Sigma-Aldrich, Zwijndrecht, The Netherlands) on HRV-induced mucin production, cells were pre-treated by incubation in HC-free complete medium containing tiotropium (10–1,000 nM) or fluticasone (10–1,000 nM) prior to infection with HRV. After infection, the basal medium was replaced by fresh free-HC medium containing tiotropium or fluticasone.
In experiments aimed to investigate the role of extracellular ATP release in virus-induced mucin production, suramin (a non-selective P2R antagonist, TOCRIS) was added to basal media at 10 μM for 30 min prior to HRV treatment. To investigate the ability of tiotropium and fluticasone to inhibit ATP- induced mucin expression, cells pre-treated with tiotropium/fluticasone were stimulated with 100 μM ATP at the basal side for 24 h.
In experiments with DAPT (γ-secretase inhibitor, TOCRIS), ALI-PBEC were incubated in complete medium supplemented with either 5 μM DAPT or solvent control (0.1% DMSO) during differentiation for 14 days. On day 14, cells were infected with HRV-A16 at MOI 1, and following infection the basal medium was replaced by fresh HC-free medium containing 5 μM DAPT or solvent control and cells were incubated for another 24 h.

Tiotropium treatment (nebulizer concentration 0.01–0.3 mM in saline; Boehringer Ingelheim Pharma GmbH) was administered via inhalation of aerosolized solutions for 3 min, as described above for ovalbumin challenges. Treatment was performed 1 h prior to each ovalbumin challenge. Ciclesonide treatment was not possible via nebulization because of the poor solubility, and was therefore administered via intranasal instillation (0.001–1 mg/kg in saline containing 0.2 % Tween 80; Bufa BV). Conscious guinea pigs were held in an upright position, while 200 μl ciclesonide was slowly instilled intranasally. After the instilled solution was aspirated, the animals were kept in the upright position for an additional 2 min to allow sufficient spreading of the fluid throughout the airways. Ciclesonide treatment was performed 24 h and 1 h prior to each ovalbumin challenge. Control animals were instilled with 200 μl sterile saline containing 0.2 % Tween 80.