Background: Impaired mucociliary clearance is a central feature of CF to which many factors contribute.Pseudomonas aeruginosa commonly infects the CF airway, and many strains harbor filamentous bacteriophage (Pf ) genomes within their bacterial chromosome.Our laboratories have shown that Pfs organize polymers of biofilms into higher-order liquid crystalline (LC) structures [1].Pf is present in the sputum of 40% to 60% of people with CF and P. aeruginosa infection and is associated with poor clinical outcomes [2], but the mechanisms by which Pf contributes to the pathogenesis of CF lung infections are unclear.We hypothesize that Pf interacts with the ciliated epithelia and disrupts effective mucociliary clearance (MCC).Methods: Explanted lungs from a person with CF infected with P. aeruginosa-harboring Pf and a person with CF infected with P. aeruginosa without Pf were harvested, sectioned, and evaluated for birefringent content within the airways to look for evidence of Pf-induced LC.To look for Pf impact on MCC, we generated air-liquid interface (ALI) cultures of nasal epithelial cells from a person with CF (F508del/F508del) and a healthy control (HC).ALI cultures were then treated overnight with Pf and other phage controls.Mucus was harvested from the apical surface to evaluate for birefringence.MCC in the ALI cultures was then assessed, first by measuring ciliary beat frequency and then adding fluorescent micro beads to the apical surface to track particle transport using high-speed video microscopy.Finally, inserts were fixed and imaged using scanning electron microscopy to evaluate the interaction between Pf and the epithelial surface.To further test the effects of Pf on an intact airway, MCC was assessed ex vivo in newborn piglet tracheas using video monitoring of the transport of mucus in the surface marked with ink particles.Results: We found that explanted CF lungs infected with P. aeruginosa harboring Pf have birefringent LC material embedded in mucus plugs within the airways.This was replicated in our ALI cell cultures treated with Pf.Although we found no effect on ciliary beat frequency among conditions tested ( p > 0.1), particle transport was significantly slower in HC and CF ALI cultures exposed to Pf ( p < 0.0001).Transport in untreated CF cells at ALI was rescued by elexecaftor-tezacaftor-ivacaftor (ETI) treatment to values comparable with those of HC cells ( p > 0.2).This rescue of CF cells by ETI was abrogated in the presence of Pf ( p < 0.0001).MCC in ex vivo newborn piglet tracheas likewise demonstrated significantly lower transport when Pf was added to the mucosal surface ( p = 0.04).Imaging of ALI filters using scanning electron microscopy demonstrated bundles of material likely representing mucus with Pf that appeared to entangle the ciliary brush.This was not present under control conditions.Conclusions: Pf induced formation of LC in the CF airway and impaired MCC in ALI cultures and in an ex vivo model.These data suggest that Pf contributes to the pathogenesis of P. aeruginosa-associated CF lung disease by impairing MCC.
原文链接
分享
引用
