Method development for flow-cytometric analysis of primary human airway epithelia infected with non-typeable Haemophilus influenzae

Horton, K.L., Cane, J.L., Jackson, C.L., Allan, R.N., Lucas, J.S. and Hinks, T.S.C. (2022) Method development for flow-cytometric analysis of primary human airway epithelia infected with non-typeable Haemophilus influenzae. European Respiratory Journal, 60 (66), [3926]. (doi:10.1183/13993003.congress-2022.3926).

Abstract

Introduction: non-typeable Haemophilus influenzae (NTHi) is a pathobiont which persists in the airways of patients with chronic obstructive pulmonary disease, cystic fibrosis and primary ciliary dyskinesia (PCD). NTHi airway colonisation is associated with chronic inflammation leading to bronchiectasis and disease exacerbation. It is not known if NTHi invades a specific subset of airway epithelial cells or if this differs by respiratory disease type.

Aim: develop a flow-cytometric method to identify major subsets of airway epithelial cells infected with NTHi.

Methods: primary human nasal (n=3) and bronchial (n=4) epithelial cells were air-liquid interface cultured for 4 weeks. Cultures were infected with a fluorescein-labelled NTHi PCD clinical isolate for 1-h and then treated with gentamicin (200 µg/mL for 1-h) to remove surface-attached bacteria. Fluorescein-labelled antibodies were used to identify cellular subsets by flow cytometry: Tubulin Tracker™ (ciliated), CD49f (basal), CD66c (secretory). Infected samples were compared to uninfected to confirm NTHi internalisation.

Results: secretory cells were the most abundant subset followed by ciliated then basal. Internalised NTHi (mean % infected per subtype) were detected within bronchial secretory (0.3%), ciliated (0.2%) and basal (0.2%) and nasal secretory (3.5%), ciliated (2.3%) and basal (2.7%), suggesting infection rate is higher in the upper airway compared to lower.

Conclusions: flow cytometry is a sensitive tool for detecting and quantifying NTHi internalisation in cultured airway epithelial cell subsets. This method could provide a readout analysing the therapeutic effects of drug treatments.

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Contributors

Author: C.L. Jackson

Author: J.S. Lucas

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