Inflammatory Generation and Pro-Resolving Signaling Actions of Conjugated Nitro-Linoleic Acid

Inflammatory responses result in the activation of pro-oxidant enzymes such as the inducible isoform of nitric oxide synthase (iNOS) and NAPDH oxidases, leading to the generation of the nitrating species nitrogen dioxide ( ● NO 2 ). Conjugated linoleic acid is a primary substrate for biological nitration in the gastric compartment giving rise to the formation of conjugated nitro-linoleic acid (NO 2 -CLA). Herein we show that macrophage activation leads to NO 2 -CLA formation both in vitro and in vivo, as determined by selective reaction monitoring analysis using isotopically labeled internal standards and confirmed by high resolution mass spectrometry. CLA nitration by activated RAW264.7 cells results not only in the dose- and time-dependent formation of the parent NO 2 -CLA compound but also in the generation of secondary electrophilic and non-electrophilic β-oxidation metabolites. The formation of two positional NO 2 -CLA isomers with different metabolization rates but similar signaling potency was also observed. Addition of either mixed or pure NO 2 -CLA positional isomers to cells in culture led to antioxidant response element (ARE) activation and to dose-dependent expression of Nrf2-regulated genes. Similarly, NO 2 -CLA also attenuated TNF-α mediated NF-κB response element activation and decreased iNOS expression and pro-inflammatory cytokine release in LPS/IFN-γ activated RAW264.7 cells. To test the physiological relevance of these observations, a mouse zymosan A peritonitis model that promotes CLA nitration was utilized. Animals were administered vehicle, CLA or synthetic NO 2 -CLA and then polymorphonuclear cell (PMN) recruitment and clearance from the peritoneal cavity was monitored. Both synthetic and in situ CLA-derived NO 2 -CLA led to decreased recruitment and enhanced PMN clearance, which coincided with higher expression of Nrf2-dependent genes in peritoneal cells. Our work demonstrates that NO 2 -CLA generation might constitute an endogenous adaptive mechanism capable of modulating the severity of the inflammatory response by favoring resolution and inducing the expression of both cytoprotective and antioxidant genes.