The role of extracellular vesicle miRNA released in response to influenza A virus.

Abstract

Influenza A virus (IAV) is a common respiratory pathogen that has caused millions of deaths throughout history. Airway epithelial cells are the first line of defence and primary target against IAV. Recently, airway epithelial cells have been shown to release lipid bilayer particles, termed extracellular vesicles (EVs). EVs provide a stable capsule for the transfer of biological molecules such as microRNAs (miRNAs). MiRNAs are small, non-coding regulatory RNAs that have been shown to be altered in response to respiratory stimuli, including viral infections and have recently emerged as an EV cargo with the potential as novel biomarkers and therapeutics. This thesis investigates the hypothesis that the miRNA profile of EVs released from bronchial epithelial cells is altered in response to infection with IAV, promoting the anti-viral immune response. IAV infection of air-liquid interface (ALI) differentiated epithelial cells was characterised by analysing the expression of antiviral genes, cell barrier permeability and cell death. EVs were isolated by filtration and size exclusion chromatography from the apical surface wash of ALI cultured bronchial epithelial cells (BCi-NS1.1). The EV miRNA profile was then sequenced, and reads were mapped to miR Base. The sequencing results were validated by RT-qPCR using healthy and COPD primary epithelial cells. Functional analyses of the miRNAs was completed by in silico analyses and transfection of BCi with miRNA mimics and inhibitors. Infection of ALI cultured epithelial cells with IAV at 3.6 x 10⁶ IU/ml for 24 hours was determined to be a suitable condition for EV analyses due to the detection of significant upregulation of anti-viral genes without high levels of cell death or loss of barrier integrity. The presence of EVs released from ALI cultured bronchial epithelial cells was confirmed by visualisation using electron microscopy and detection of known EV markers such as CD9, CD63 and CD81 using western blot and the Exo View R100platform. Differential expression analyses identified 13 miRNAs that were differentially expressed between EVs released from IAV infected BCi compared to uninfected BCi (FDR <0.01). Of these 5 had a fold change of >1.5:miR-155-5p, miR-122-5p, miR-378a-3p, miR-7-5p and miR-146a-5p and 1 had a foldchange of <-1.5: miR-505-5p. Differences between EV, non-EV and cellular levels of these miRNA were detected. Furthermore, the EV miRNA response to IAV was reduced for PBECs obtained from COPD patients and was altered with participant age. miRNA target prediction and functional analysis was performed using miRNet to identify biological processes and pathways that were enriched for the target genes of these miRNAs. Target genes were found to be associated with immune and damage response further suggesting that these miRNAs are involved in immune response to IAV.
EV miRNA may be a key mechanism in modulating the response to IAV and therefore are potential targets for future therapies. However further work to determine transfer of EV miRNA and fully characterise the function of EV miRNA in response to IAV is required.

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