Evaluating the role of alveolar macrophages in tolerance to ozone.

Acute exposure to ozone (O3) causes pulmonary inflammation and injury in humans and animal models. In rodents, acute O3-induced inflammation and injury can be mitigated by pre-exposure to relatively low concentrations of O3, a phenomenon referred to as tolerance. Although tolerance was first described long ago, the underlying mechanisms are not known. We hypothesized that alveolar macrophages (AMs) play a key role in tolerance to O3 based on prior studies with other exposures. To enable our studies, we first generated a mouse model in which female C57BL6/NJ mice were pre-exposed to filtered air or 0.8 ppm O3 for 4 days (4 h/day), then challenged with 2 ppm O3 (3 h) 2 days later, and phenotyped for airway inflammation and injury 6 or 24 h thereafter. As expected, pre-exposure to O3 resulted in significantly reduced airway inflammation and injury 24 h following O3 challenge. Tolerance was associated with regenerative hyperplasia in the terminal bronchioles and changes in the frequency of proliferating alveolar type 2 cells. O3 pre-exposure altered the expression of ∼1,500 genes in AMs, most notably downregulation of Toll-like receptor and proinflammatory cytokine signaling pathways, suggesting AMs had become hypo-responsive. Depletion of tolerized AMs prior to acute O3 challenge did not, however, alter inflammation and injury. Additionally, adoptive transfer of tolerized AM to naïve recipient mice failed to alter responses to acute O3 challenge. In total, our results argue against an important role for AMs in tolerance to ozone and suggest that other cell types are involved.