Ozone-induced changes in murine lung extracellular vesicle number and small RNA content

Inhalation exposure to ozone (O) causes adverse respiratory health effects that result from airway inflammation, a complex response mediated by changes to airway cellular transcriptional programs. These programs may be regulated in part by a subset of microRNAs transferred between cells (e.g. epithelial cells and macrophages) via extracellular vesicles (EV miRNA). To explore this, we exposed female C57BL/6J mice to filtered air (FA), 1, or 2 ppm O by inhalation and collected bronchoalveolar lavage fluid (BALF) 21 hours later for markers of airway inflammation, EVs, and EV miRNA. Both concentrations of O significantly increased markers of inflammation (neutrophils and total protein) and the number of EVs in the BALF. Using high-throughput small RNA sequencing, we identified several differentially expressed (DE) BALF EV miRNAs after 1 ppm (16 DE miRNAs) and 2 ppm (99 DE miRNAs) O versus FA exposure. O concentration response patterns in EV miRNA expression were apparent, particularly for the two most highly expressed (miR-2137 and miR-126-3p) and lowly expressed (miR-378-3p and miR-351-5p) miRNAs. Integrative analysis of EV miRNA expression and airway cellular mRNA expression identified EV miR-22-3p as a candidate regulator of transcriptomic responses to O in airway macrophages. In contrast, we did not identify candidate miRNA regulators of mRNA expression data from conducting airways (predominantly composed of epithelial cells). In summary, our data show that O exposure alters EV release and EV miRNA expression, suggesting that further investigation of EVs may provide insight into their effects on airway macrophage function and other mechanisms of O-induced respiratory inflammation.