Resolvin E2: Identification and Anti-Inflammatory Actions: Pivotal Role of Human 5-Lipoxygenase in Resolvin E Series Biosynthesis

Abstract

The family of resolvins consists of omega-3 fatty acid-derived mediators, including E series resolvins generated from eicosapentaenoic acid (EPA), and carry potent anti-inflammatory properties. Here, we report the isolation, identification, and bioactions of resolvin E2 (RvE2), which is 5S,18-dihydroxy-eicosapentaenoic acid. RvE2 stopped zymosan-induced polymorphonuclear (PMN) leukocyte infiltration and displayed potent anti-inflammatory properties in murine peritonitis. We also demonstrate that human recombinant 5-lipoxygenase generates RvE2 from a common precursor of E series resolvins, namely, 18-hydroxyeicosapentaenoate (18-HEPE). Furthermore, the initial 5-hydroperoxide intermediate was also converted to a 5(6)-epoxide intermediate in RvE1 formation. These results demonstrate that RvE2, together with RvE1, may contribute to the beneficial actions of omega-3 fatty acids in human diseases. Moreover, they indicate that the 5-lipoxygenase in human leukocytes is a pivotal enzyme that can produce both pro- and anti-inflammatory chemical mediators.

Pro-resolving actions and stereoselective biosynthesis of 18S E-series resolvins in human leukocytes and murine inflammation

“…Hydrolysis of this allylic triene epoxide is the key step for RvE1 biosynthesis in order to form the 6Z,8E,10E-triene conjugation present in RvE1 and is required for its bioactivity (19). Without enzymatic activity, this 5(6)-epoxide intermediate was converted not to RvE1 but rather to either 6E,8E,10E-isomers or 5,6,18-trihydroxyeicosapentaenoate by nonenzymatic hydrolysis (20) (Figure 4A). None of these products (solid line, quartet around 11 minutes and single peak at 16.8 minutes) matched the retention time of RvE1 (dotted line, single peak at 12.1 minutes).…”

“…Thus, we next assessed the role and contribution of LTA4H in the biosynthesis of RvE1. This was addressed in several ways: First, since human PMNs biosynthesize RvE1 and RvE2 from 18-HEPE (20), RvE1 production by PMNs was tested in the presence of the LTA4H inhibitor bestatin (22). When incubated with bestatin, RvE1 biosynthesis by isolated human PMNs was decreased by 67.3% ± 6.2% ( Figure 5A).…”

“…Human 5-LOX can further convert the 5S-hydroperoxy,18-hydroxyeicosapentaenoate intermediate to a 5S(6)-epoxide-containing product (20), which becomes the direct upstream precursor of RvE1. Hydrolysis of this allylic triene epoxide is the key step for RvE1 biosynthesis in order to form the 6Z,8E,10E-triene conjugation present in RvE1 and is required for its bioactivity (19).…”

“…The biosynthesis experiments involved competitive conversion of 18-HEPE with human recombinant 5-LOX. For assessment of the potential for stereospecific preference, racemic mixtures of 18R- and 18S-HEPE were incubated with 5-LOX in the presence of required 5-LOX activators (20), followed by reduction of 5S-hydroperoxide to RvE2 and analysis by both conventional and chiral LC-MS/MS. Two 5-LOX products from racemic 18-HEPE were identified and assigned as 5,18-dihydroxyEPE by UV chromatogram at 236 nm and selective ion monitoring at m/z 333→199 ( Figure 3 stereochemically pure 18R- or 18S-HEPE.…”

“…With regard to EPA-derived E-series resolvins, the stereochemical structure of endogenous RvE1 was assigned and confirmed via total organic synthesis (19), and the E-series resolvin biosynthesis mechanism has been partially elucidated (20). The contribution of aspirin in E-series resolvin biosynthesis and identity of downstream enzymes generating EPA-derived (E-series) resolvins in a stereospecific and regiospecific manner remain of interest.…”