Lipoxin A4 yields an electrophilic 15-oxo metabolite that mediates FPR2 receptor-independent anti-inflammatory signaling.

The enzymatic oxidation of arachidonic acid is proposed to yield trihydroxytetraene species (termed lipoxins) that resolve inflammation via ligand activation of the formyl peptide receptor, FPR2. While cell and murine models activate signaling responses to synthetic lipoxins, primarily 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (lipoxin A4, LXA4), there are expanding concerns about the biological formation, detection and signaling mechanisms ascribed to LXA4 and related di- and tri-hydroxy ω-6 and ω-3 fatty acids. Herein, the generation and actions of LXA4 and its primary 15-oxo metabolite were assessed in control, LPS-activated and arachidonic acid supplemented RAW 264.7 macrophages. Despite protein expression of all enzymes required for LXA4 synthesis, both LXA4 and its 15-oxo-LXA4 metabolite were undetectable. Moreover, synthetic LXA4 and the membrane permeable 15-oxo-LXA4 methyl ester that is rapidly de-esterified to 15-oxo-LXA4, displayed no ligand activity for the putative LXA4 receptor FPR2, as opposed to the FPR2 ligand WKYMVm. Alternatively, 15-oxo-LXA4, an electrophilic α,β-unsaturated ketone, alkylates nucleophilic amino acids such as cysteine to modulate redox-sensitive transcriptional regulatory protein and enzyme function. 15-oxo-LXA4 activated nuclear factor (erythroid related factor 2)-like 2 (Nrf2)-regulated gene expression of anti-inflammatory and repair genes and inhibited nuclear factor (NF)-κB-regulated pro-inflammatory mediator expression. LXA4 did not impact these macrophage anti-inflammatory and repair responses. In summary, these data show an absence of macrophage LXA4 formation and receptor-mediated signaling actions. Rather, if LXA4 were present in sufficient concentrations, this, and other more abundant mono- and poly-hydroxylated unsaturated fatty acids can be readily oxidized to electrophilic α,β-unsaturated ketone products that modulate the redox-sensitive cysteine proteome via G-protein coupled receptor-independent mechanisms.