Abstract
Ozone (O3) is a criteria air pollutant that causes lung injury, oxidative stress, impaired lung function, and exacerbation of pre-existing pulmonary diseases including COPD and asthma. The alveolar epithelium is a major target for O3. Damage leads to an inflammatory response characterized by an accumulation of macrophages in the lung. Our laboratory has previously demonstrated that these cells become activated and release pro-inflammatory mediators and cytotoxic oxidants that contribute to tissue injury. Biochemical pathways that regulate macrophage activation following O3 exposure are still poorly understood, and this represents the focus of my research. The integrated stress response (ISR) is a signaling pathway activated by cellular stress; it is known to downregulate protein synthesis and modulate gene transcription, resulting in restoration of cellular homeostasis or induction of apoptotic cell death. The ISR has been shown to regulate inflammatory signaling by inhibiting inflammasome activation, downregulating pro-inflammatory mediator production, and inducing macrophage phenotypic switching. The goal of my studies is to investigate the role of the ISR in macrophage proinflammatory activation and O3 toxicity. Two specific aims are proposed to test my hypothesis that ISR signaling plays a key role in regulating macrophage activation during the inflammatory response to O3. Aim 1 will determine if the ISR is activated in macrophages following O3 exposure and Aim 2 will evaluate whether ISR signaling plays a role in macrophage activation and O3 toxicity. For these studies GCN2 knockout mice (GCN2-/-) will be used. These mice have a reduced ability to handle stress due to targeted disruption of GCN2, a key kinase in the ISR pathway. C57BL/6 wild-type (WT) and GCN2-/- mice will be exposed to filtered air (FA) or O3 (0.8 ppm) in a whole-body Plexiglas chamber for 3 hr. At 24, 48 and 72 hr post-exposure, lung macrophages will be isolated and analyzed by flow cytometry to assess their phenotype; ISR proteins and gene targets will be assessed by western blotting and qPCR. Support for our hypothesis would come from findings that ISR signaling is disrupted in response to O3, and that loss of GCN2 leads to exacerbation of inflammation, oxidative stress and lung toxicity. These studies will contribute to the growing knowledge of inflammatory mechanisms of ozone toxicity, potentially providing the development of new therapeutic interventions to reduce lung injury induced by oxidant air pollutants.
Ozone (O3) is a criteria air pollutant that causes lung injury, oxidative stress, impaired lung function, and exacerbation of pre-existing pulmonary diseases including COPD and asthma. The alveolar epithelium is a major target for O3. Damage leads to an inflammatory response characterized by an accumulation of macrophages in the lung. Our laboratory has previously demonstrated that these cells become activated and release pro-inflammatory mediators and cytotoxic oxidants that contribute to tissue injury. Biochemical pathways that regulate macrophage activation following O3 exposure are still poorly understood, and this represents the focus of my research. The integrated stress response (ISR) is a signaling pathway activated by cellular stress; it is known to downregulate protein synthesis and modulate gene transcription, resulting in restoration of cellular homeostasis or induction of apoptotic cell death. The ISR has been shown to regulate inflammatory signaling by inhibiting inflammasome activation, downregulating pro-inflammatory mediator production, and inducing macrophage phenotypic switching. The goal of my studies is to investigate the role of the ISR in macrophage proinflammatory activation and O3 toxicity. Two specific aims are proposed to test my hypothesis that ISR signaling plays a key role in regulating macrophage activation during the inflammatory response to O3. Aim 1 will determine if the ISR is activated in macrophages following O3 exposure and Aim 2 will evaluate whether ISR signaling plays a role in macrophage activation and O3 toxicity. For these studies GCN2 knockout mice (GCN2-/-) will be used. These mice have a reduced ability to handle stress due to targeted disruption of GCN2, a key kinase in the ISR pathway. C57BL/6 wild-type (WT) and GCN2-/- mice will be exposed to filtered air (FA) or O3 (0.8 ppm) in a whole-body Plexiglas chamber for 3 hr. At 24, 48 and 72 hr post-exposure, lung macrophages will be isolated and analyzed by flow cytometry to assess their phenotype; ISR proteins and gene targets will be assessed by western blotting and qPCR. Support for our hypothesis would come from findings that ISR signaling is disrupted in response to O3, and that loss of GCN2 leads to exacerbation of inflammation, oxidative stress and lung toxicity. These studies will contribute to the growing knowledge of inflammatory mechanisms of ozone toxicity, potentially providing the development of new therapeutic interventions to reduce lung injury induced by oxidant air pollutants.