Validation of a bronchioepithelial cell culture model with primary cells derived from epithelial tissue to study the regulation of antimicrobial cholesteryl ester biosynthesis

Background: Respiratory tract infections are among the leading causes of death throughout the world. The bronchial epithelium plays a key role in the first line of lung defense. It senses microbial intruders via pattern recognition receptors (PRR) including toll like receptors (TLRs) that detect pathogen-associated molecular patterns (PAMPs). Upon binding, PRRs initiate the secretion of antimicrobial effector molecules such as antimicrobial peptides to directly kill the invading pathogens as well as antimicrobial cholesteryl esters (CE). Sterol O-acyltransferase 1 (SOAT1) is a key enzyme of cholesteryl ester biosynthesis. Preliminary studies with a human bronchial epithelial cell line suggested that SOAT1 expression is up-regulated by TLR ligands and proinflammatory cytokines. In this study, we wished to validate these findings with primary respiratory epithelial cells. Methods: Human sinus mucosa obtained as surgical excess material and pig trachea from a slaughterhouse were processed and cultured for epithelial cell outgrowth. Commercial human primary bronchial epithelial cells (EpiAirway, MatTek) were used in air liquid interface culture. Cells were stimulated with the TLR4 ligand lipopolysaccharide (LPS) and the pro-inflammatory cytokines IFNγ and TNFα for 6 or 24 h. For SOAT1 expression analysis, cells were extracted with Trizol. SOAT1 gene expression was quantified by quantitative RT-PCR employing SYBR Green and βactin as reference gene or by TaqMan methodology in duplex PCR and RPLOPO as a reference gene. SOAT1 protein expression was quantified by western immunoblotting. Results: The microbiota associated with human sinus mucosa and pig trachea routinely outgrew the tissue cultures. RT PCR analysis of one of the infected cultures suggested mRNA expression of SOAT1. In EpiAirway cultures SOAT1 gene expression was up-regulated 15.27 fold ?? 10.92 by 50 ng/mL IFNγ, and 8.34 fold ?? 0.53 by 100 ng/mL LPS after 6 h stimulation. In a second experiment, SOAT1 was upregulated when stimulated with LPS for 24h (3.65 fold ?? 0.83), but not after stimulation for 6h, and INFγ did not lead to an increase in SOAT1 expression. Variations in the culture medium and the time between EpiAirway arrival and stimulation may have contributed to this inconsistency. Faint SOAT1 protein signals were obtained by Western immunoblotting only in protein extracts from LPS treated EpiAirway cells further supporting the SOAT1 gene expression is upreguated by TLR ligands. Conclusion: The expression of SOAT1 in primary airway epithelial cells was appeared to be upregulated by the TLR ligand LPS and possibly the pro-inflammatory cytokine IFNγ. This data corroborates our preliminary data with a bronchial epithelial cell line and further supports the concept of a lipid mediated arm of mucosal innate immunity. Understanding not only the synthesis, but mechanisms and regulation of CEs and their relation to certain illnesses can possibly provide a way for early diagnosis and prevention of deadly infections