Abstract
Maresins are a new family of anti-inflammatory and pro-resolving lipid mediators biosynthesized from docosahexaenoic acid (DHA) by macrophages. Here we identified a novel pro-resolving product, 13R,14S-dihydroxy-docosahexaenoic acid (13R,14S-diHDHA), produced by human macrophages. PCR mapping of 12-lipoxygenase (12-LOX) mRNA sequence in human macrophages and platelet showed that they are identical. This human 12-LOX mRNA and enzyme are expressed in monocyte-derived cell lineage, and enzyme expression levels increase with maturation to macrophages or dendritic cells. Recombinant human 12-LOX gave essentially equivalent catalytic efficiency (kcat/KM) with arachidonic acid (AA) and DHA as substrates. Lipid mediator metabololipidomics demonstrated that human macrophages produce a novel bioactive product 13,14-dihydroxy-docosahexaenoic acid in addition to maresin-1, 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1). Co-incubations with human recombinant 12-LOX and soluble epoxide hydrolase (sEH) demonstrated that biosynthesis of 13,14-dihydroxy-docosahexaenoic acid (13,14-diHDHA) involves the 13S,14S-epoxy-maresin intermediate produced from DHA by 12-LOX, followed by conversion via soluble epoxide hydrolase (sEH). This new 13,14-diHDHA displayed potent anti-inflammatory and pro-resolving actions, and at 1 ng reduced neutrophil infiltration in mouse peritonitis by ∼40% and at 10 pM enhanced human macrophage phagocytosis of zymosan by ∼90%. However, MaR1 proved more potent than the 13R,14S-diHDHA at enhancing efferocytosis with human macrophages. Taken together, the present findings demonstrate that macrophages produced a novel bioactive product identified in the maresin metabolome as 13R,14S-dihydroxy-docosahexaenoic acid, from DHA via conversion by human 12-LOX followed by sEH. Given its potent bioactions, we coined 13R,14S-diHDHA maresin 2 (MaR2).
Highlights
The acute inflammatory response is the host protection against harmful stimuli, including pathogen infections, irritants and cell damage [1]
Flow-cytometry assessment using fluorescence-labeled antibody against human 12LOX demonstrated about 10 fold increase in protein expressions in M0, M1 M2 and Immature dendritic cells (iDC), and 19 fold increase in mature dendritic cells (mDC), compared to MC (Fig. 1b), indicating that expression of human 12-LOX protein is enhanced with differentiation of MC to macrophages and dendritic cells
We identified and cloned the human macrophage 12-LOX involved in the biosynthesis and bioactive maresin metabolome, and found a new member of the maresin family produced from docosahexaenoic acid (DHA)
Summary
The acute inflammatory response is the host protection against harmful stimuli, including pathogen infections, irritants and cell damage [1]. New families of lipid mediators, derived from n-3 polyunsaturated fatty acid (PUFA), that are biosynthesized during the resolution phase of acute inflammation and coined resolvins and protectins, were uncovered that potently stop inflammation promoting catabasis and return to homeostasis [2]. Macrophage mediators in resolving inflammation, are a family of lipid mediators biosynthesized by macrophages from DHA via human 12-lipoxygenase (12-LOX) [4]. Maresin 1 (7R,14Sdihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid or Ma R1), the first member of this family identified, displays potent anti-inflammatory and pro-resolving actions inhibiting neutrophil infiltration in vivo, and stereoselectively stimulating macrophage phagocytosis and efferocytosis [4,5]. The complete stereochemistry of MaR1 has been established, and MaR1 displays potent tissue regenerative as well as anti-nociceptive actions [5]
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