Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions

Abstract

The endogenous cellular and molecular mechanisms that control acute inflammation and its resolution are of wide interest. Using self-resolving inflammatory exudates and lipidomics, we have identified a new pathway involving biosynthesis of potent antiinflammatory and proresolving mediators from the essential fatty acid docosahexaenoic acid (DHA) by macrophages (MPhis). During the resolution of mouse peritonitis, exudates accumulated both 17-hydroxydocosahexaenoic acid, a known marker of 17S-D series resolvin (Rv) and protectin biosynthesis, and 14S-hydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid from endogenous DHA. Addition of either DHA or 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid to activated MPhis converted these substrates to novel dihydroxy-containing products that possessed potent antiinflammatory and proresolving activity with a potency similar to resolvin E1, 5S,12R,18R-trihydroxyeicosa-6Z,8E,10E,14Z,16E-pentaenoic acid, and protectin D1, 10R,17S-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Stable isotope incorporation, intermediate trapping, and characterization of physical and biological properties of the products demonstrated a novel 14-lipoxygenase pathway, generating bioactive 7,14-dihydroxydocosa-4Z,8,10,12,16Z,19Z-hexaenoic acid, coined MPhi mediator in resolving inflammation (maresin), which enhances resolution. These findings suggest that maresins and this new metabolome may be involved in some of the beneficial actions of DHA and MPhis in tissue homeostasis, inflammation resolution, wound healing, and host defense.

Figures

Figure 1.

Self-resolving acute inflammatory exudates. (A) Time course of PMN (dashed line; n = 4) accumulation, resolution, and hydroxydocosahexaenoic acid formation during zymosan-initiated peritonitis. Exudates were extracted for targeted lipidomics using LC/MS/MS. Hydroxydocosahexaenoic acids 17-HDHA (dotted line) and 14S-HDHA (continuous line) identified using MRM results are representative (n = 3). (B and C) Representative mass spectra for 17-HDHA (B) and 14S-HDHA (C; n = 3). amu, atomic mass unit.

Figure 2.

MΦs generate novel products. (A) Mouse-resident MΦs (5 × 106 cells/ml) incubated with DHA or 14S-HpDHA showing targeted LC/MS/MS-based mediator lipidomics. Selected ion chromatogram (m/z 359/250) of 7,14-diHDHA (II) and its transconjugated isomer (I) is shown. Selected ion chromatogram (dashed overlay; m/z 359/250) shows double dioxygenation product 7S,14S-diHDHA. (inset) FACS plot of isolated resident MΦs. (B and C) Lipid mediator lipidomics. (B) Mass spectra for 7,14-diHDHA (m/z 359; B) and corresponding isomer (C). See inset and Results for diagnostic ions (n = 3). amu, atomic mass unit.

Figure 3.

Antiinflammatory and proresolving novel MΦ products. (A) Reduction in PMN in peritonitis. Activity in methyl formate fractions from C18 extraction of isolated MΦs and 20 ng/mouse of MΦ products isolated with RP-HPLC, 20 ng/mouse PD1, or 20 ng/mouse RvE1. Results are expressed as exudate PMN means ± SEM (n = 3; *, P < 0.05 compared with zymosan plus vehicle). (B) Differential PMN versus monocyte actions. Mice were injected with 0.1 ng/mouse of the double dioxygenation product, 0.1 ng/mouse of MΦ isolate, or vehicle alone (as in A), followed by i.p injection of 1 mg zymosan to evoke peritonitis. After 2 h, leukocytes were enumerated (black bar, PMNs; hatched bar, mononuclear cells). Results are means ± SEM (n = 3; *, P < 0.05 compared with zymosan plus vehicle; †, P < 0.05 for double dioxygenation vs. MΦ isolate). (C) Reduction in peritonitis showing dose response. MΦ product isolated after HPLC isolation was injected i.v. ∼2 min before i.p. zymosan. Results are means ± SEM (n = 3; *, P < 0.05 compared with zymosan plus vehicle). (D) MaR1 enhances phagocytosis. MΦs (24-well plate, 105 cells/well) were exposed to the indicated concentrations for 15 min followed by FITC-labeled zymosan for 30 min at 37°C. Results are means ± SEM expressed as the percent increase above vehicle (n = 3; *, P < 0.05 compared with vehicle; †, P < 0.05 for double dioxygenation vs. MaR1). The closed diamond represents MaR1, and the closed square represents the double dioxygenation product 7S,14S-diHDHA. (inset) Comparison of MaR1 with other mediators (1 nM).

Figure 4.

Identification of methoxy-trapping product from MΦs. MS/MS spectrum of m/z 373 product at 10.2 min. (inset) Extracted ion chromatogram of m/z 373→263 and deduced structure. amu, atomic mass unit.

Figure 5.

Biosynthetic scheme proposed for MaR1 and related products. Stereochemistries and double-bond geometries of the new dihydroxy-containing mediators are tentative assignments and depicted in likely configurations based on biogenic synthesis, trapping, and labeling (see Results).