LAUSR

Growing evidence suggests that many of the worldwide health concerns today can be impacted by omega-3 fatty acids. Indeed, dietary intake of omega-3 fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is associatedwith protection against many significant pathologies, including ischemic and inflammatory diseases. EPA and DHA can be incorporated into cellular membranes to influence their biophysical properties, but can also be metabolized by enzymes such as cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochromes P450 to a diverse collection of biologically active lipid mediators.1 Discerning which of these lipid mediators are responsible for the beneficial effects of omega-3 fatty acid supplementation has been a daunting task. DHA and EPA can be metabolized into subsets of molecules including resolvins, protectins, andmaresins. Collectively termed specialized proresolving mediators (SPMs), these compounds promote resolution of inflammation in a variety of settings.2,3 To date, more than 40 distinct SPMs have been identified. Minor differences in the regioor stereochemical composition of the SPMs can significantly alter their potency and bioactivity, and influence the downstream signaling pathways activated by SPMs. SPMs are subsequently degraded to secondary metabolites which may or may not maintain proresolving properties.4 Progress on the bioactions and functional relevance of individual SPMs has been hampered by the high cost related to the complex synthesis of these compounds, which are often not commercially available. As with all endogenous mediators, it is critical to validate the precise biochemical structures, quantify the endogenous levels, and elucidate the biological actions of these proresolving mediators. In the current issue of the Journal of Leukocyte Biology, Winkler et al. directly address these concerns and elucidate the endogenous production, metabolism, and potent anti-inflammatory effects of Resolvin D4 (RvD4).5 Like other D-series resolvins, RvD4 is formed by sequential enzymatic reactions in vivo (Fig. 1); 15-LOX metabolizes DHA to 17Shydroxydocosahexaenoic acid (HDHA) and 5-LOX then generates 4S,5R,17S-trihydroxydocosahexaenoic acid (that is, RvD4). Winkler et al. reported a method for commercial scale, total chemical synthesis of RvD4 and several of the RvD4 stereoisomers and endogenous metabolites. Using liquid chromatography, tandemmass spectrometry, they validated the synthetic RvD4 as identical to endogenous RvD4 produced in vivo in human bone marrow. Commercial scale production of RvD4 provided the capability to treat mice and cells with this compound to further elucidate its biological actions, downstream signaling pathways, and utility as a possible therapeutic agent. The authors found that RvD4 was abundantly produced in vivo. Mice treated with permanent ligation of the femoral artery exhibited pronounced production of RvD4 after 24 h. Interestingly, postligation RvD4 levels were nearly 30-fold higher than those of its biosynthetic partner RvD3, which also has potent biological effects.6 In a model of leukocyte-mediated secondary organ damage, pharmacological doses of RvD4 reduced markers of lung injury after temporary hind limb ischemia. RvD4 had similar potency to RvD3 in reducing neutrophil infiltration and levels of the pro-inflammatory eicosanoids LTB4, TXB2, PGE2, and PGF2α . While RvD4 prevented neutrophil infiltration in vivo, this appears to be an indirect effect, as it failed to regulate neutrophil chemotaxis in vitro. In contrast, RvD4 promoted bacterial phagocytosis by both macrophages and neutrophils in vitro. While the identity of the putative RvD4 receptor remains unknown, stimulation of macrophage phagocytosis by RvD4 was inhibited by cholera toxin, which suggests signaling through a Gs-coupled G-protein– coupled receptor. Winkler et al. also examined the metabolic inactivation of RvD4. Incubation of RvD4 with human bone marrow–derived leukocytes produced 2 major products: 17-oxo-RvD4 and 15,16-dihydroRvD4. Eicosanoid oxidoreductase (EOR), also known as 15-hydroxyprostaglandin dehydrogenase, was shown tometabolize RvD4 entirely to 17-oxo-RvD4. Importantly, conversion to 17-oxo-RvD4 eliminated its ability to stimulate bacterial phagocytosis. Thus, confirmation of this inactivation pathway may enable development of potent SPM analogs that resist metabolic inactivation. Commercial scale synthesis facilitated validation of RvD4 as a potent anti-inflammatory SPM. This critical first step opens the door to new avenues of investigation; however, many questions remain. To date,RvD4hasbothdistinct andoverlapping featuresof otherD-series resolvins. For example, like RvD4, RvD1 stimulates bacterial phagocytosis and suppresses inflammatory eicosanoid production.7 Unlike RvD4, RvD1 directly regulates neutrophil migration. RvD1 and RvD2 have beenwidely studied; both reduce inflammation through suppression of NF-kB, histamine receptor, or ERK signaling.2 It remains to be determined whether RvD4 regulates any or all of these processes or signaling pathways in vivo. The next critically important step will be to determine the cell surface receptors through which RvD4 signals. RvD1 binds to Lipoxin