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)

Two circulating neutrophil populations in acute inflammation in mice.

Recent studies indicate that neutrophils are heterogeneous and may have an immunosuppressive role in addition to their well-known phagocytic and bactericidal function. This study examined neutrophil subpopulations in the circulation, peritoneum, spleen and bone marrow from mice at various time points after induction of acute inflammation. MATERIAL, TREATMENT AND METHODS: Female C57BL/6 mice were injected intraperitoneally with lipopolysaccharide (LPS). Blood, peritoneal, spleen and bone marrow cells were collected and counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in serum and peritoneal fluid were determined by ELISA. Neutrophil numbers in the circulation decreased following administration of LPS but reached similar numbers to those prior to inflammation at 8 h. At that time point, two distinct neutrophil populations were present in the circulation. These two neutrophil populations differed in size, granularity and expression of CD11b and Ly6G. Few neutrophils were recruited into the peritoneum until 24 h after administration of LPS at a time when the neutrophils in the circulation had increased their expression of the chemokine receptor CXCR2. Induction of acute inflammation leads to the appearance of two circulating neutrophil subpopulations, which may differ in their activation state and function.Icelandic Research Fund University of Iceland Research Fun

Dietary fish oil increases the proportion of a specific neutrophil subpopulation in blood and total neutrophils in peritoneum of mice following endotoxin-induced inflammation.

To access publisher's full text version of this article. Please click on the hyperlink in Additional Links field.Omega-3 polyunsaturated fatty acids may have beneficial effects in inflammation, where neutrophil migration and activation are of importance. The effects of dietary fish oil on neutrophil numbers and subpopulations in healthy mice and mice with endotoxin-induced inflammation were determined. Mice were fed a control diet with or without 2.8% fish oil, and half of them were injected intraperitoneally with endotoxin. Blood, peritoneal lavage, bone marrow and spleen were collected. Expression of cell surface molecules was analyzed by flow cytometry, and chemokine concentrations were determined by enzyme-linked immunosorbent assay. Dietary fish oil did not alter the proportion of total neutrophils in blood but increased the proportion of a specific subpopulation of neutrophils 48 h following endotoxin administration. This subpopulation of neutrophils expressed higher levels of CD11b, Ly6G and major histocompatibility complex-II, suggesting a different role for these neutrophils in the inflammatory response. Dietary fish oil did not affect neutrophil numbers in the peritoneum of healthy mice, but 12 h after endotoxin administration, there were fewer neutrophils in the peritoneum of mice fed the fish oil diet than in mice fed the control diet. However, 48 h after endotoxin administration, mice fed the fish oil diet had more neutrophils in peritoneum than mice fed the control diet. These results indicate that, although dietary fish oil may delay recruitment of neutrophils from blood to the peritoneum early in inflammation, it has the potential to increase the number of peritoneal neutrophils later, which may be of benefit as impaired neutrophil migration and activation have been associated with immunosuppression late in inflammation.Icelandic Research Fund, University of Iceland Research Fun

Dietary fish oil decreases the proportion of classical monocytes in blood in healthy mice but increases their proportion upon induction of inflammation.

Fish oil can have beneficial effects in health and disease. In healthy individuals, reduction of the inflammatory status may be of benefit, whereas in patients with systemic inflammation, such as sepsis, it is important to diminish the immunosuppression that is thought to contribute to poor outcome. The objective of this study was to determine the effects of dietary fish oil on monocytes/macrophages in blood, bone marrow, spleen, and peritoneum and chemokine concentrations in blood and peritoneum in healthy mice and mice with endotoxin-induced inflammation. Mice were fed a Western-type diet without fish oil (C) or with 2.8% fish oil (FO) for 6 wk and then either killed (healthy mice) or injected i.p. with endotoxin (LPS) and killed after 3, 8, 12, 24, or 48 h. Blood, bone marrow, spleen, and peritoneal lavage were collected. Expression of cell surface molecules and chemokine receptors was analyzed by flow cytometry and chemokine concentrations measured by ELISA. Healthy mice in the FO group had lower proportions of classical monocytes in blood than healthy mice in the C group. LPS administration increased the proportion of classical monocytes in blood in mice in the FO group but not in those in the C group. Healthy mice in the FO group had lower serum concentrations of CCL2 than mice in the C group, but in inflamed mice, CCL2 concentrations were higher in the FO group than in the C group. These results indicate that dietary fish oil can attenuate the inflammatory status in homeostasis but intensify the immune response upon inflammation.Icelandic Research Fund University of Icelan

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

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

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