ABCA3 mutations influence the stress level and immunological function of lung epithelial A549 cells

Abstract

Einleitung: ABCA3 is an ABC-transporter in the outer membrane of lamellar bodies (LBs) in alveolar epithelial type II cells (ATII) involved in the surfactant lipid transport. Mutations in the ABCA3 gene have been associated with genetic interstitial lung disease (ILD). Next to being essential for surfactant secretion, ATII are an active part of the lung immunological system and involved in lung repair. We investigate how ABCA3 mutations influence ATII cell homeostasis, primary the stress level and immunological function, and potential involvement of ATII cells in epithelial-mesenchymal transition (EMT). Methoden: A549 cells, a widely accepted ATII model, were stably transfected with vectors expressing HA-tagged WT ABCA3 protein and Q215K and E292V mutations. Initially, protein localization and processing features were analysed by immunofluorescence, immunoblotting and deglycosylation assay. Cellular stress was assayed by BiP immunoblotting and RT-PCR of XBP-1 mRNA splicing. To mimic viral and bacterial respiratory infections, cells were infected with respiratory syncitial virus (RSV) or treated with P. aeruginosa LPS. The immunological answer was measured by quantitative PCR of TLR3 and TLR4 receptors and associated cytokines (IL-6, IL-8, RANTES and GM-CSF). Ergebnisse: While WT and E292V proteins localized to LBs, Q215K protein was retained inside of the ER, indicating a misfolding defect. Also, ABCA3 mutations increased intracellular stress level. Compared to WT, mutants displayed a changed immunological status already in the absence of infectious agents with significantly lower gene expression of TLR3 and TLR4. After exposure to RSV and LPS, mutant lines were unable to upregulate TLR receptors, failing to become sensitized to further infections. Moreover, cytokine mRNA expression prior to infection and cytokine answer after exposure to infections were changed in mutants. Interestingly, RSV infection triggered a morphological transformation of Q215K and E292V cells from epithelial cells to fibroblasts. Diskussion: Besides elevating ER stress, we propose that non-functional ABCA3 protein can affect several important functions of ATII. Our initial data suggest that ABCA3 mutations can alter the immunological answer of ATII and make them more prone to EMT, leading to the speculation that stressed and non-functional ATII cells are a possible origin of fibrotic foci in genetic ILD. If all described is a secondary phenomenon to ABCA3, and if and to which extent raised ER stress level might be a connection between mutated ABCA3 and our immunology/EMT-related observations remains to be investigated. In addition, it would be necessary to confirm these data in patient samples and prove the significance on disease onset and progression.