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A2825 - Ozone-Derived Oxysterols Form Adducts with NLRP2, Potentially Contributing to Inflammation in Airway Epithelial Cells
Author Block: A. Speen1, J. Hoffman2, P. W. Clapp2, H. Kim3, N. Porter3, I. Jaspers4; 1Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 2University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 3Vanderbilt University, Nashville, TN, United States, 4Univ of North Carolina At Chapel Hill, Chapel Hill, NC, United States.
RATIONALE: Despite the abundance of studies examining the adverse health effects of ozone (O3) exposure and its association with increased airway inflammation, the mechanisms mediating these responses have yet to be fully described. Previous studies, including our own, have shown that exposure to O3 results in the formation of electrophilic lipid peroxidation products, notably through the oxidation of cholesterol in epithelial cells. These electrophilic oxysterols are capable of forming covalent linkages with nucleophilic centers of proteins, particularly lysine residues, thus altering cellular signaling pathways. Much remains to be learned about the identity of the reactive species and the range of potential proteins modified by the oxysterols in the lung, as well as the biological consequences. METHODS: Airway epithelial cells were exposed to the O3-derived oxysterol Secosterol-A and 2D LC-MS/MS shotgun proteomics was used to generate a database of oxysterol-protein adducts. NLRP2 was among oxysterol-adducted proteins and bio-orthogonal cycloaddition was used to confirm oxysterol-NLRP2 adduct formation. Utilizing qPCR and western blotting, abundance of NLRP2 in airway epithelial cells was compared with other prominently studied NLR proteins. In addition, O3-induced expression of NLRP2 and association with other inflammasome proteins was determined. To determine the role of NLRP2 in O3-induced inflammation, we knocked down NLRP2 expression in human bronchial airway cells followed by analysis of inflammasome complex formation and inflammatory mediator production. RESULTS: NLRP2 is the most abundantly expressed member of the NLRP family in human airway epithelial cells. Oxysterol-NLRP2 adduction occurred in the leucine-rich-repeat region, which regulates the activity in other NLRP-inflammasome complexes. We also show that exposure of epithelial cells to O3 increases NLRP2 expression, activates caspase-1, and enhances markers of inflammasome assembly, suggesting that O3 leads to the formation and activation of NLRP2 inflammasome complexes. Knockdown of NLRP2 reduces O3-induced inflammation and caspase cleavage, further supporting the notion that oxysterol-NLRP2 adduct formation could be mediating O3-induced inflammation. CONCLUSIONS: We show for the first time that NLRP2 is highly abundant in airway epithelial cells and a key component of O3-induced inflammation. Our data demonstrate that formation of oxysterol-protein adducts is a novel mechanism mediating O3-induced pro-inflammatory responses. Funded By: R21-ES024666, T32-ES007126, T32 CA201159-01