Regulation of Macrophage Activation and Disease Pathogenesis following Ozone Exposure by Extracellular Vesicles and miRNA Cargo

Exposure to air pollutants like ozone contributes to the pathogenesis and severity of inflammatory diseases including asthma, emphysema, and ARDS. We previously demonstrated that lung injury following of ozone is due, not only to its direct effects on the lung, but indirectly to the actions of inflammatory mediators released by activated macrophages; moreover, macrophage activation is controlled, at least in part, by epigenetic regulators particularly, noncoding microRNAs (miRNA)s. Extracellular vesicles (EVs) are cell-derived particles that facilitate cell-cell communication by delivering cargo, including miRNAs, from donor to recipient cells. We found that miRNA cargo released from lung cell EVs after acute ozone exposure (0.8 ppm, 3 h) regulates macrophage-mediated inflammatory responses. Herein, we analyzed the EV- cargo miRNA profiles isolated from bronchoalveolar lavage fluid (BAL) after chronic ozone exposure. Mice (C57Bl6/J, 11-12 wk) were exposed to air or ozone (1.5 ppm, 2 h), 2x/wk for 6 wk. BAL was collected 24 h after the last exposure, EVs isolated and analyzed by flow cytometry to assess their origin. Ozone exposure resulted in histo-pathologic changes in the lung consistent with inflammation and chronic disease. In both control and ozone treated mice, BAL EVs originated mainly from CD45+ macrophages, with a smaller % from CD326+ epithelial cells and CD31+ endothelial cells. Ozone had no effect on the origin of the EVs, or absolute numbers of EVs that originated from these cells. Next, we assessed the effects of ozone on EV-cargo miRNA and their pre-miRNAs precursors by RNA sequencing. Among the 1,935 miRNAs detected in BAL EVs after ozone exposure, 8 miRNAs were found to be significantly upregulated (more than 2.85-fold change) relative to EVs from mice exposed to air control. We also identified 3 pre- miRNAs which were significantly altered (1.5 - 2 fold) after ozone exposure. All of these miRNAs and pre-miRNAs are newly identified, and their function and regulatory activity are unknown. These findings are important as they suggest novel pathways mediating macrophage activation, which can be targeted to reduce lung injury and chronic disease. Supported by NIH Grants ES004738, ES033698, and ES005022.