Towards the development of a tractable model to study innate immune responses of the respiratory tract to Staphylococcus aureus infection

The epithelium of the human airway provides critical protection to the respiratory tract because the cells are always exposed to inhaled microbes. The epithelial lining, therefore, acts as a physical shield against pathogens. This layer contains several cell types that are specialized to perform the functions of the innate defence system. Studies of interactions between the host and pathogens require the establishment of differentiated epithelial cell cultures that recapitulate the airway epithelium. Staphylococcus aureus is a bacterium found in 30% of the population in the upper tract of the respiratory system. The aim of this study was to develop in vitro models of the human airway epithelium that can be used to study the initial stages of infection of the respiratory tract. HBEC3-KT and BMI-1 cells (human immortalised bronchial epithelial cell lines) were differentiated at the air liquid interface (ALI) for three weeks. A direct comparison of these cells was performed with primary human bronchial epithelial cells (HBECs) cultured in the same manner. RT-PCR and qPCR was performed with several markers specific for different cell types (e.g., BPIFA1, BPIFB1, SCGB1A1, TEKT1) and this confirmed the differentiation of cells at the ALI. Expression of these markers were lower in the cell lines compared to the HBECs. Protein corresponding to some of these genes was examined under IF microscopy. An unbiased proteomic analysis was performed to directly compare the apical secretions of differentiated HBEC3-KT cells and differentiated primary HBECs. The proteomics data indicated that HBECs and differentiated HBEC3-KT cells appear phenotypically similar. Many proteins were shared between the two ALI cultures, but the production of primary mucus protein components and some host defence proteins was less in the HBEC3-KTs, which may limit the use of this cell line as a surrogate for the human airway epithelium. Effects of S. aureus infection were studied by using differentiated and undifferentiated cells with GFP-labelled S. aureus (SH1000) at different MOIs. Primary differentiated HBECs were also infected following treatment of the cells with IL-13. Bacterial infections were more marked in differentiated cells. qPCR and RT PCR was performed with several pro-inflammatory markers (e.g., CXCL10, CXCL1 and IL6). Inflammatory responses were also examined using IF microscopy and protein-based assays (cytokine arrays). It appeared that differentiated cells expressed higher levels of inflammatory mediators and induction was greater than in undifferentiated cells. This study established a number of in vitro models that can be used for the identification of pathogen and host factors that are necessary for the initial stages of S. aureus infection of the respiratory tract.