Abstract Description: Ozone is a major component of urban air pollution known to cause oxidative stress and lung injury. The alveolar epithelium is a main target of inhaled ozone; damage initiates an inflammatory response characterized by an accumulation of macrophages in the lungs. In response to mediators present in the tissue microenvironment (e.g., TLR4 agonists, TNFα, oxidized lipids, ROS), infiltrating macrophages become activated, releasing inflammatory mediators that contribute to acute lung injury. Research herein is focused on analyzing potential biochemical pathways regulating macrophage activation. The integrated stress response (ISR) is a signaling network involved in maintaining homeostasis in cells in the face of stressors; activation of the ISR causes upregulation of genes that either repair cellular damage or induce apoptosis. ISR activation is dependent on phosphorylation of eukaryotic initiation factor alpha (eIF2α). General control nonderepressible 2 (GCN2) is a key kinase that phosphorylates eIF2α; this is known to occur in response to oxidative stress. ISR signaling through GCN2 promotes both pro- and anti-inflammatory responses. We hypothesize that ISR signaling through GCN2 plays a role in regulating macrophage activation following ozone exposure. To test this hypothesis, we utilized GCN2 knock out mice (GCN2-/-) to investigate how dysfunctional ISR signaling affects lung injury and inflammation following ozone exposure. In both WT and GCN2-/- mice exposed to ozone, there was a significant increase in total protein compared to air-exposed mice, demonstrating alveolar epithelial injury. Cleaved caspase 3, a marker of apoptosis, was upregulated in bronchial and alveolar epithelia of WT mice; this was blunted in GCN2-/- mice, suggesting epithelial injury is not due to apoptosis. Histologic evaluation of lungs revealed significant neutrophilia in GCN2-/- mice, but not WT regardless of treatment. Flow cytometric analysis of isolated lung cells showed that ozone exposure resulted in an increase of neutrophils in WT mice, but not GCN2-/- mice. Numbers of infiltrating macrophages also increased in WT mice following ozone exposure; the majority of these cells displayed markers associated with a pro-resolution phenotype. In GCN2-/- mice, the effect of ozone on infiltrating macrophages was blunted; this indicates that GCN2, and thus the ISR, plays a role in regulating the macrophage response to ozone-induced lung injury. Future studies will investigate how loss of GCN2 controls macrophage function following ozone exposure and contribute to the growing knowledge of inflammatory mechanisms of ozone toxicity. Supported by ES004738, ES033698, ES005022, and ES007148, and the Air Pollution Educational and Research Grant Scholarship Program (APERG).
