Pro-viral miRNAs are detected in BALF extracellular vesicles of patients with influenza virus-induced ARDS

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EV) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs. In this study, we found significant changes in the miRNA composition of EVs in the broncho-alveolar lavage fluid (BALF) from patients with IAV-induced ARDS. Among the nine significantly deregulated microRNAs, miR-17-5p was upregulated in patients` BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication. A549 cells were routinely cultured in RPMI1640 with 10% FCS. Cells were infected with Influenza H1N1 (A/Memphis/14/96, A/WSN/33) for 12 hours with an MOI of 0.1 and 5. RNA was isolated from biological triplicates by phenol/chloroform extraction. miR-ath-159a was spiked in for later normalization. 400 ng RNA per sample were then reverse transcribed with the Life Technologies miR RT kit with megaplex primers. Reverse transcribed RNA was then loaded on the human Taqman Low Density Array (TLDA) Cards and run according to manufacturer´s recommendations Uninfected A549 cells were used as control to evaluate the impact of infection. microRNA expression was normalized to spiked-in miR-ath-159a