Immediate early transcriptional responses of airway epithelial cells

Airway epithelial cells are crucial for mucosal and adaptive immunity as they produce mucins that trap and clear inhaled particulates by mucociliary activity, and also secrete diverse inflammatory mediators. But whether these cells respond in a memory-dependent manner to a secondary exposure is poorly studied. Our recent studies have highlighted the role of 'memory-based' or 'trained' immune responses. In this proposal we would like to probe for the molecular basis underlying the memory-based response in AECs. We will first establish an in-vitro model of memory-based responses using differentiated AECs. Here we will delineate the kinetics of memory-based response in AECs using a microbial ligand, LPS or endotoxin. The cells will be grown in air-liquid interface to study the effect of memory response on mucous phenotype. The RNAseq analyses will be performed on AECs exposed to endotoxin and compared to that of non-exposed AECs to determine the changes in coding and noncoding RNA regions. Overall design: Air-liquid interface cultured 3D airway epithelial cells were treated with LPS (100 ng/ml) and cells analyzed at 2 h post treatment and compared to non-treated cells. The primary human airway epithelial cells (HAECs) were differentiated at the air-liquid interface for 14 days and were treated with LPS (100 ng/ml) for 2 h. Total RNA was isolated from cells treated with and without LPS using RNeasy isolation kit (Qiagen Inc.). RNA seq analysis was performed by HudsonAlpha Institute for Biotechnology, Huntsville, Alabama. Briefly, Illumina TruSeq Stranded Total RNAseq libraries with ribosomal depletion were prepared per manufacturer's instructions (Illumina, Inc, San Diego, CA).