Discovery and characterization of inflammation-induced electrophilic fatty acid derivatives (2009)

Electrophilic lipids are emerging as critical mediators of anti-inflammatory signaling pathways, although many biologically relevant electrophiles may still remain unknown. Nitro derivatives (NO2-FA) and á,â-unsaturated carbonyl derivatives of unsaturated fatty acids are naturally occurring electrophilic products of redox reactions, and can modulate a variety of cellular signaling processes including the transcriptional activity of the peroxisome proliferator-activated receptor-ã (PPARã). PPARã binds diverse ligands to regulate the expression of genes involved in metabolism and inflammation. Activators of PPARã include anti-hyperglycemic drugs such as thiazolidinediones (TZDs) and intermediates of lipid metabolism and oxidation that bind PPARã with very low affinity. Recently TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. In contrast, NO2-FA act as partial agonists of PPARã at nM concentrations and covalently bind PPARã via Michael addition. NO2-FA show selective PPARã modulator characteristics by inducing coregulator protein interactions distinctively different from those induced by the TZD Rosiglitazone. In further exploring the electrophilic lipidome, a new subclass of electrophilic lipid has been revealed. Using a recently developed â-mercaptoethanol (BME) alkylation reaction, followed by HPLC-MS/MS-based screening, we report six novel electrophilic fatty acid derivatives (EFADs) specifically formed during macrophage activation (RAW264.7 and THP-1 cell lines and primary macrophages treated with IFNã and LPS). The major EFADs are á,â-unsaturated oxo-derivatives of omega-3 fatty acids as confirmed by cell culture and in vitro studies and by MS/MS structural analysis. The isomers of two major EFADs were identified as 13- and 17-keto derivatives of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Purified cyclooxygenase-2 (COX-2) product profiles and treatment of activated macrophages with COX-2 inhibitors confirmed EFAD synthesis to be catalyzed by inducible COX-2, followed by hydroxy-dehydrogenase activity. EFAD production was increased 2.5 fold in activated macrophages treated with acetylsalicylic acid (ASA; aspirin). Internal standard-based quantification showed that EFADs are highly abundant electrophiles in activated macrophages, reaching intracellular concentrations as high as 350 nM. Importantly, EFADs form reversibly reactive covalent adducts with both proteins and small molecule thiols in activated macrophages, supporting a potential for post-translational protein modification-mediated cell signaling. Furthermore, synthetic isomers of EFAD-1 and -2 (17-oxo-DHA and 17-oxo-DPA, respectively) act as partial agonists of PPARã, activate Nrf2 (nuclear factor-erythroid 2-related factor 2)-dependent gene expression, and inhibit pro-inflammatory cytokine production and iNOS expression in IFNã and LPS-activated RAW264.7 cells and in primary macrophages. In conclusion, it has been demonstrated that upon activation macrophages generate omega-3 derived electrophilic signaling molecules at biologically relevant concentrations that act as autocrine mediators.