Bronchial Epithelial Cell RNASeq Pilot

In this study, we tested two environmental exposure stimuli, bacterial lipopolysaccharide (LPS) and house dust mite (HDM), in an in vitro model of primary human bronchial epithelial cells (HBECs) from 6 individuals, to identify differentially expressed genes using RNAseq analysis in response to these exposures both individually and in combination. Our findings illustrate that exposure mixtures increase the strength and complexity of transcriptomic responses in HBECs as compared to individual stimuli alone, and that asthmatic's cells are more sensitive to these stimuli, eliciting a greater number of differentially expressed genes vs. controls. Our results provide a basis for future studies in this area, such as the expansion of this in vitro model to test multiple exposure combinations (i.e. the exposome) and their influence on molecular signaling in the bronchial epithelium. Overall design: Well-differentiated cells in ALI culture were stimulated with lipopolysaccharide (LPS) and/or house dust mite (HDM) extract. LPS was purchased from sigma (L2654) and dissolved in PBS. Low endotoxin HDM extract (product no. XPB70D3A2.5, lot no. 279020, 1590 endotoxin units/2.5 mL sized vial) were purchased from Greer Laboratories (Lenoir, NC) and dissolved in PBS. Prior to exposure to stimuli, cells were maintained for 20 hours in a minimal medium, which was depleted of hydrocortisone, epidermal growth factor, and bovine pituitary extract. Cells were apically stimulated with 250ul of minimal media containing either LPS (at 10 µg/ml), HDM (at 25 µg/ml), or the combination of LPS and HDM. As a vehicle treatment, minimal media containing PBS was used. After four hours, cells were used for total RNA isolation. For RNA sequencing analysis total RNA was isolated using miRNeasy Kit (Qiagen). From the validation experiment using additional donors, total RNA was isolated using RNeasy Mini Kit (Qiagen, Germantown, MD) for qPCR analysis.