Non-typeable Haemophilus influenzae biofilm formation on air-liquid interface cultured primary human airway epithelia

Introduction: defective motile cilia on primary ciliary dyskinesia (PCD) airway epithelia results in impaired mucociliary function and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways. It is unclear if biological pathways in response to NTHi infection differ in PCD, contributing to increased susceptibility to infection.Aim: to develop a biologically representative model of mature NTHi infection of airway epithelia to investigate PCD airway epithelia-NTHi interactions.Methods: air-liquid interface (ALI)-cultured primary nasal cells were used at 21-35 days post-ALI (n=30). Barrier properties were assessed by trans-epithelial electrical resistance (TEER), FITC-dextran passage and tight junctions staining. Cilia were assessed by high-speed video microscopy at 37˚C. NTHi PCD isolate was added to ALI cultures at multiplicity of infections (MOIs) 10, 50 or 100 for 1 hour and co-cultured for 3 days. TEER was measured pre- and post-infection. NTHi recoverability was assessed by colony forming units. Morphology was shown by scanning electron microscopy (SEM).Results: 100% of ALI cultures succeeded. Tight barriers were formed. Ciliary beat pattern and frequency (mean 14Hz) were normal. Mean cilia coverage at day 29 was 38%. Following infection, TEER increased relative to pre-infection at all MOIs but maximum NTHi recoverability was at MOI 50. SEM showed widespread ciliation and NTHi biofilm formation at MOI 50.Conclusions: a successful model of mature NTHi infection of human primary airway was established. The model will be used with PCD, and other airway disease, epithelia to investigate host-pathogen interactions