Visualizing Cellular Adaptations during Ozone‐induced Lung Inflammation

Abstract

Ozone (O3) is a prevalent environmental pollutant with well‐documented acute and chronic health effects. Neutrophils are known as the prime orchestrators of lung immune surveillance. Although bacterial infections are known to elicit a robust lung neutrophil and alveolar macrophage response, it is not known why and how neutrophils are activated in response to sterile lung injury. Acute exposures to 2 ppm O3 induce lung macrophage and neutrophil extravasation and airway hyper‐reactivity at 24 h, however, these concentrations are manifold higher than ambient O3 concentrations, thus precluding any understanding of the initial events. Our pilot experiments established ozone's dose and time dependent killing of alveolar macrophages and concomitant recruitment of leukocytes. Our data indicated that 50 ppb of O3 induces cell death in half of the lung alveolar macrophage population. Thereafter, we exposed C57BL6 mice to either filtered air or 50 ppb of O3 for 2 h to understand the kinetics of murine ozone induced acute lung injury. We report acute vascular neutrophil concentration and activation at as early as 2 h post exposure which continues up to 24 h after ozone exposure. Immunofluorescent staining revealed CD11b positive lung alveolar macrophage and neutrophil recruitment. In addition to conventional bronchoalveolar lavage, lung vascular perfusate and peripheral blood analysis, we present novel application of simple, yet powerful, configuration of a fixed‐stage wide‐field upright fluorescent intravital microscopic set‐up to investigate ozone's oxidative lung damage. We observed extensive alveolar actin filament disorganization (Fig. 1), an initial drop (at 2 h post‐O3 exposure) followed by restoration (18 h post‐O3 exposure) of alveolar reactive oxygen species (ROS) (Fig. 2) as well as mitochondrial potential (Fig. 3) in live animals. Important findings from these preliminary experiments include CD11b predominant phenotypes of pulmonary leukocytes, alveolar necrosis, actin disorganization, acute alterations in alveolar ROS production as well as mitochondrial potential. Thus, we present evidence of systemic as well as lung toxicity at 40 fold lower O3 concentrations. The findings are important in establishing a sensitive model of ozone induced acute lung injury.Support or Funding InformationFedoruk Centre and Innovation Saskatchewan.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.