Pulmonary tolerance to ozone is associated with changes in the alveolar macrophage transcriptome

Tolerance to the air pollutant ozone refers to the phenomenon by which pre-exposure to a relatively low concentration confers protection against from lung injury and inflammation caused by a subsequent challenge with high concentration ozone. We investigated whether alveolar macrophages play a central role in this process. First, we established a model of ozone tolerance in which inbred C57BL/6NJ mice were pre-exposed to 0.8 ppm ozone for 4 days then challenged with 2 ppm ozone on day 7, followed by sacrifice and phenotyping on Day 8. As expected, pre-exposure significantly reduced ozone-induced airway injury and inflammation on day 8. To investigate the role of alveolar macrophages, we treated mice with clodronate on day 5, which reduced macrophage number by ~5-fold. Following ozone challenge on day 7, clodronate treated mice exhibited increased injury compared to vehicle treated controls. Cytokines involved in neutrophil chemotaxis such as IL-6, MIP-1b, and KC, were also significantly elevated in clodronate treated mice. Collectively, these data indicate an important role of AMs in this model of tolerance. To identify differences in the phenotype of AMs caused by pre-exposure to ozone, we isolated AMs from ozone pre-exposed mice vs. filtered air controls using flow sorting and performed RNA-seq analysis of these samples. We found that a number of genes and pathways were altered by ozone pre-exposure, most importantly downregulation of pathways involved in toll-like receptor cell signaling, proinflammatory gene expression, and NF-kB mediated gene regulation.