Ozone, oxysterols, and lung inflammation Grant uri icon

abstract

  • Ozone (O3) continues to be of great public health concern, with about 36% of the U.S. population living in areas classified as ?nonattainment? of the National Ambient Air Quality Standard for O3 (0.075ppm averaged over 8hrs). Epithelial cells (ECs) are major targets for inhaled O3, resulting in pro-inflammatory mediator production and cytotoxicity. O3 is particularly reactive with unsaturated lipids, including cholesterol, which is a primary constituent of cell membranes and epithelial lining fluid. The reactions of lipids with O3 and oxygen free radicals are complex and we show here that electrophilic oxysterols are formed in humans in vivo and in ECs in vitro. We also demonstrate that O3-derived oxysterols react with nucleophilic centers, particularly lysine residues, thus forming protein adducts affecting pro-inflammatory signalling. Formation of oxysterol-protein adducts presents a novel biochemical basis for O3-induced biological effects, which will be the focus of this application. EC-derived signals determine the immunophenotype of resident pulmonary immune cells, such as pulmonary macrophages. We have demonstrated that O3 exposure of macrophages co-cultured with ECs, but not macrophages exposed alone, results in activation of the inflammasome, decreased phagocytosis and modified surface marker expression. Similarly, we show data here indicating that O3-derived oxysterols increase innate immune phenotypes in macrophages, which are similar immunophenotypes observed in O3-exposed humans in vivo. Oxidized lipids are known to modify macrophage function, yet how O3-derived oxysterols formed at or near the EC surface modify macrophage immunophenotypes is unknown. The last step during cholesterol synthesis converts 7-dehydroxy cholesterol (7-DHC) to cholesterol via 7-DHC reductase (7-DHCR). 7-DHC is significantly more susceptible to oxidation and oxysterol generation than cholesterol. Enhancing 7-DHC levels increases O3-induced oxysterol generation and inflammation. Specifically, the small molecule antidepressant drug aripiprazole (APZ)/Abilify increases 7-DHC levels and sensitizes to O3-induced inflammation in vitro. APZ/Abilify consistently ranks among the most prescribed drugs in the US, with almost 9 million prescriptions filled per month. Understanding how availability of 7-DHC and drugs like APZ/Abilify potentially sensitizes to O3-induced adverse health effects will be examined in this application. The overall hypothesis is that O3-derived oxysterols adduct to cellular proteins, thus activating pro inflammatory signaling cascades, modifying macrophage function and that these responses are enhanced by small molecule antidepressant drugs. The following specific aims are intended to test this central hypothesis. SA1: Characterize the O3-derived oxysterol-protein ?adductome? and identify mechanisms through which O3-derived oxysterols affect pro-inflammatory responses. Using a proteomic screen of the lipid-protein adductome formed in EC after stimulation of O3-derived oxysterols, we identified several NF-kB regulatory molecules, including NLRP2, which will be initially examined here. SA2: Identify how O3-derived oxysterols affect macrophage function. Using alkynyl-tagged cholesterol and our EC-macrophage co-culture system we will track oxysterol transfer between ECs and macrophages and determine their effects on macrophage function. SA3: Determine how modulating pulmonary 7-DHC levels affect O3-induced inflammation. Using in vitro and translational in vivo experimental models, we will investigate how increased levels of 7-DHC sensitized to O3-induced pro-inflammatory signaling and examine the resulting lipid-protein adductome in ECs. Impact The guiding hypothesis of this research is that exposure of the airways to O3 generates oxysterols at or near the epithelial surface that adduct to cellular signaling proteins

date/time interval

  • April 2018 - March 2023