Maresin Biosynthesis and Identification of Maresin 2, a New Anti-Inflammatory and Pro-Resolving Mediator from Human Macrophages

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)

Human 12-LOX converts AA and DHA with essentially equivalent efficiency.

<p>Increasing concentrations of AA or DHA (1 to 50 µM) were mixed with 12-LOX (0.1 µM, pH 8.0, R.T.) in the presence or absence of CaCl₂ (2 mM). Initial rates were monitored and plotted versus substrate at indicated concentrations. Each point represents mean±SEM from n = 3 separate experiments.</p

MaR1 and MaR2 (13R,14S-diHDHA) display potent anti-inflammatory and proresolving actions: direct comparison.

<p>(A) Mouse peritonitis: exudate PMN numbers <i>in vivo</i>. Male mice (6–8 weeks) were administered <i>i.v</i>. MaR1, 13R,14S-diHDHA (1 ng/mouse each) or vehicle prior to <i>i.p</i>. administration of zymosan (0.1 mg/mouse). Peritoneal exudates were collected, and PMNs enumerated using both light microscopy and flow cytometry. Results are mean ± SEM. n = 3 mice per treatment from three separate experiments (*<i>P</i><0.05 <i>vs</i>. vehicle). Enhanced phagocytosis of (B) opsonized zymosan or (C) apoptotic PMN. Human macrophages were seeded in 96-well plates (5×10⁴ cells/well) and incubated with vehicle (PBS containing 0.1% ethanol), MaR1 or 13R,14S-diHDHA (PBS^+/+, pH 7.45, 37°C, 15 min). (B) FITC-labeled zymosan (5×10⁵ particles/well) or (C) fluorescently labeled apoptotic PMN (1.5×10⁵ cells/well) were added and cells incubated for an additional 60 min (pH 7.45, 37°C). Non-phagocytosed zymosan or apoptotic PMN were washed, extracellular florescence quenched and phagocytosis quantified. Results are mean ± SEM. n = 3 separate human macrophage preparations (*<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001 <i>vs</i>. vehicle; #<i>P</i><0.05, ###<i>P</i><0.001 <i>vs</i>. MaR1).</p

Biosynthesis of 13R,14S-diHDHA in coincubations with human recombinant 12-LOX and sEH.

<p>(A) MRM chromatography for ion pair 359>221. DHA (10 µM) was incubated with 12-LOX (0.2 µM) in the absence or presence of 2 U sEH (20 mM Tris, pH 8.0, 100 mM KCl, 37°C, 10 min). (B) MS/MS spectra employed in the identification of 13,14S-diHDHA (peak II) and 7S,14S-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-docosahexaenoic acid (7S,14S-diHDHA) (peak I). Results are representative of n = 3.</p

Human macrophage endogenous production of 13R,14S-diHDHA.

<p>(A) MRM chromatography for ion pair 359>221 (m/z). Human macrophages were incubated with opsonized zymosan (DPBS+/+, pH 7.45, 15 min, 37°C). Incubations were stopped with 2 volumes of ice-cold methanol, and products were assessed by lipid mediator metabololipidomics. (B) MS-MS spectra. <i>Inset</i>, diagnostic ions used for identification and corresponding possessed structure fragments. Results are representative of n = 3.</p

Human macrophage 12-LOX produces 14S-HpDHA.

<p>(A) Chiral chromatography of synthetic 14R-HDHA and 14S-HDHA (upper panel). DHA (5 µM) was incubated with human macrophage 12-LOX (0.2 µM, 10 min, 37°C, pH 8). Products were extracted (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102362#s2" target="_blank">methods</a> for details) and subject to chiral high performance liquid chromatography-tandem mass spectrometry (m/z, 343>205) (lower panel). (B) MS-MS spectrum of 14S-HDHA from human macrophage 12-LOX incubations (lower panel in A).</p

Parameters of Michaelis-Menten kinetics.^*

<p>*Initial rate at each substrate concentration was fitted into Michaelis-Menten equation to determine K_M and k_cat. Results are mean ± SEM.</p

Human macrophages and dendritic cells express human 12-LOX.

<p>(A) Expression levels of 12-LOX mRNA in human monocytes (MC), M0, M1, M2 macrophages, immature dendritic cells (iDC) and mature dendritic cells (mDC) were assessed by quantitative PCR. (B) 12-LOX protein expression levels were assessed by flow cytometry. In the left panel, a representative histogram shows 12-LOX present in each cell type. In the right panel, results are mean fluorescent intensity (MFI) of 12-LOX protein levels for each cell type, expressed as mean±SEM of 4 separate cell preparations (*<i>P</i><0.05, **<i>P</i><0.01).</p

Proposed biosynthetic scheme for MaR1 and MaR2.

<p>Human macrophage 12-LOX converts DHA to the 13S,14S-epoxy-maresin intermediate, and via soluble epoxide hydrolase this intermediate is converted to MaR2. See text for details and stereochemical assignments.</p