Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

Effects of pH alteration on respiratory syncytial virus in human airway epithelial cells.

Background: Respiratory syncytial virus (RSV) is a leading cause of respiratory distress and hospitalisation in the paediatric population. Low airway surface pH impairs antimicrobial host defence and worsens airway inflammation. Inhaled Optate safely raises airway surface pH in humans and raises intracellular pH in primary human airway epithelial cells (HAECs) in vitro. We aimed to determine whether raising intracellular pH with Optate would decrease infection and replication of RSV in primary HAECs. Methods: We cultured HAECs from healthy subjects in both air-liquid interface and submerged conditions. We infected HAECs with green fluorescent protein-labelled RSV (GFP-RSV; multiplicity of infection=1) and treated them with Optate or PBS control. We collected supernatant after a 4-h incubation and then every 24 h. We used fluorescence intensity, fluorescent particle counts, plaque assays, Western blots and ELISA to quantitate infection. Results: In submerged culture, fluorescence intensity decreased in Optate-treated cells (48 h p=0.0174, 72 h p≤0.001). Similarly, Optate treatment resulted in decreased fluorescent particle count (48 h p=0.0178, 72 h p=0.0019) and plaque-forming units (48 h p=0.0011, 72 h p=0.0148) from cell culture supernatant. In differentiated HAECs cultured at ALI, Optate treatment decreased fluorescence intensity (p≤0.01), GFP via Western blot and ELISA (p<0.0001), and RSV-fusion protein via ELISA (p=0.001). Additionally, RSV infection decreased as Optate concentration increased in a dose-dependent manner (p<0.001). Conclusions: Optate inhibits RSV infection in primary HAECs in a dose-dependent manner. These findings suggest that Optate may have potential as an inhaled therapeutic for patients with RSV.