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A1334 - Transcriptomic Analysis of Early Airway Epithelial Inflammatory Responses
Author Block: R. J. Langley1, S. S. Hussain2, N. Baumlin3, M. Nair4, M. A. Salathe5, H. S. Chand6; 1University of South Alabama College of Medicine, Mobile, AL, United States, 2Immunology, Florida Internationa University, Miami, FL, United States, 3University of Miami, Miami, FL, United States, 4Florida Internationa University, Mobile, FL, United States, 5Univ of Miami Sch of Med, Miami, FL, United States, 6Immunology, Florida International University, MIAMI, FL, United States.
Rationale: Recurring inhalational exposures to microbial ligands have been shown to propagate a memory-based or ‘trained’ response in immune cells; however, contribution of the airway epithelial cells in this trained response is not known. Several cross-sectional and longitudinal studies suggest that exposure to endotoxin (LPS) affects the onset and progression of asthma, and therefore, we analyzed the immediate early response of differentiated human airway epithelial cells (HAECs) following the LPS exposure. Methods: Primary HAECs were differentiated at the air-liquid interface and were treated with LPS (10 µg/ml) for 2 h. Total RNA was isolated from cells and transcriptomic analysis was performed. Briefly, Illumina TruSeq Stranded Total RNAseq libraries with ribosomal depletion were prepared per manufacturer’s instructions. RNASeq data was aligned to the GRCh38 reference genome with Hisat2, and assembled into transcripts with Cufflinks2. DESeq2 was used to analyze the differential expression between treatments. Briefly, counts data from DESeq2 were normalized as read per million. Any gene that didn’t have at least one read in 50% of the samples was removed from the analysis leaving a total of 15,148 genes that were relatively expressed. Expression analysis was performed with JMP Genomics 8. Counts were log2(x+1) transformed and ANOVA with 5% FDR was performed. ToppFun was utilized for functional annotation pathway analysis. Results: 3,164 genes were determined to be significantly different following LPS treatment. Applying the filter of genes with at least 1.5-fold change, there were 192 genes showing upregulation compared to control, while 120 genes were downregulated. Pathway analysis revealed that Inflammatory and defense responses, cytokine signaling, and response to LPS were the primary upregulated pathways, while decreased genes related to leukotriene B4 receptor activity, epithelial development, cell fate and differentiation pathways. In addition, 10 miRNA’s were identified by the pathway analysis as potential response modulators including miR222, miR26a and miR26b that are implicated in cell proliferation and differentiation pathways. Conclusions: LPS exposure alters the airway epithelial cell fate by affecting several inflammatory and metabolic pathways to promote cell survival and differentiation Further studies are currently being followed to gain mechanistic insight into how these pathways shape a memory-dependent response in airway epithelial cells.