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P2‐9: Soluble ST2 enhances IL‐33–induced neutrophilic airway inflammation: A potential mechanism of neutrophilic asthma

Background and Aims: The release of membrane vesicles can mediate intercellular communication. Exosomes contain nucleic acids including messenger RNA and small non-cording microRNA (miRNA). Lung fibroblasts are both targets and sources of inflammatory mediators. In this study, we examined whether miR-146a containing exosomes could modulate cyclooxygenase 2 (COX-2) gene expression in human lung fibroblasts. Methods: Human fetal lung (HFL) cells, 1 x 10 cells per plate, were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) with 10% fetal calf serum for 2 days. Culture media were then changed to DMEM without serum for 2 hours following which serum-free DMEM supplemented with and without IL-1β/TNF-α (1 ng/ml) was added. Media were harvested and cell debris was removed by centrifugation at 3000g; EVs were precipitated at 130000g. Size distribution of the particles was then evaluated by NanoSight LM10. Microarray and Realtime Polymerase Chain Reaction (RT-PCR) for COX-2 mRNA and miR-146a were performed with the purified exosomes. Results: We confirmed that HFL-1 cells produced particles in the range of exosomes; 30-100 nm. After 72 hours, the number of exosomes released by IL-1β/TNF-α stimulated cells was increased significantly. Using microarray analysis, 81 miRNAs were found to be differentially expressed including miR-146a after IL-1β/TNF-α stimulation. The differential expression of miR-146a was confirmed by RT-PCR. MiR-146a inhibitor blocked the role of miR-146a to suppress COX-2 mRNA expression in IL-1β/TNF-α treated condition significantly. Prostaglandin E2 levels determined by ELISA were consistent with these results. Conclusions: Exosomal miR-146a derived from human lung fibroblasts can modulate COX-2 gene expression.