Characterisation of a surrogate Type-II alveolar cell culture model for ATP binding cassette subfamily a member 3 (ABCA-3) deficiency

Introduction/Aim. ATP Binding Cassette Subfamily A Member 3 (ABCA-3) is a lipid transporter protein highly expressed in type-II alveolar (AT-II) cells. Mutations in ABCA3 can result in severe respiratory disease in infants and children. Cell cultures derived from patients with ABCA-3 deficiency that reflect the genetic and phenotypic features of ABCA-3 associated lung disease, would provide a means to study disease mechanisms and investigate therapeutics. Primary AT-II cells, while physiologically relevant to ABCA-3 deficiency, are not generally accessible. ABCA-3 is present in the nasopharynx, hence primary nasal epithelial cells could potentially provide a more accessible cell culture model. Our aim was to investigate the suitability of nasal epithelial cells to study ABCA-3 deficiency. Methods. Expression of ABCA3, and AT-II cell markers, SFTPB and SFTPC, in nasal epithelial cells and primary AT-II cells, were quantified by droplet digital PCR. Protein localisation of ABCA-3 was visualised by immunofluorescent microscopy. Functionality of the ABCA-3 protein in nasal epithelial cells derived from human subjects with or without ABCA-3 deficiency was assessed by the capacity of the cells to detoxify doxorubicin. Results. While ABCA-3 protein was localised in the cytoplasm of primary nasal epithelial cells, mRNA levels were 6.35x103-fold lower in nasal epithelial cells compared to primary AT-II cells. Gene expression of SFTPB and SFTPC was also significantly lower by 5.26 x 106- and 5.36 x 106-fold, respectively. Higher concentrations of doxorubicin reduced cell viability in ABCA-3 deficient nasal epithelial cells compared to controls in an assay-dependent manner when using an MTS assay but not with a lactate dehydrogenase assay or by calcein blue staining. Conclusion. There may be a role for nasal epithelial cell cultures to model ABCA-3 deficiency depending on endpoints of interest. However, alternative approaches that more accurately replicate the AT-II phenotype need to be explored.