Identification and structure determination of novel anti-inflammatory mediator resolvin E3, 17,18-dihydroxyeicosapentaenoic acid

Abstract

Bioactive mediators derived from omega-3 eicosapentaenoic acid (EPA) elicit potent anti-inflammatory actions. Here, we identified novel EPA metabolites, including 8,18-dihydroxyeicosapentaenoic acid (8,18-diHEPE), 11,18-diHEPE, 12,18-diHEPE, and 17,18-diHEPE from 18-HEPE. Unlike resolvins E1 and E2, both of which are biosynthesized by neutrophils via the 5-lipoxygenase pathway, these metabolites are biosynthesized by eosinophils via the 12/15-lipoxygenase pathway. Among them, two stereoisomers of 17,18-diHEPE, collectively termed resolvin E3 (RvE3), displayed a potent anti-inflammatory action by limiting neutrophil infiltration in zymosan-induced peritonitis. The planar structure of RvE3 was unambiguously determined to be 17,18-dihydroxy-5Z,8Z,11Z,13E,15E-EPE by high resolution NMR, and the two stereoisomers were assigned to have 17,18R- and 17,18S-dihydroxy groups, respectively, using chemically synthesized 18R- and 18S-HEPE as precursors. Both 18R- and 18S-RvE3 inhibited neutrophil chemotaxis in vitro at low nanomolar concentrations. These findings suggest that RvE3 contributes to the beneficial actions of EPA in controlling inflammation and related diseases.

FIGURE 1.

Formation of 18-HEPE metabolites from human leukocyte incubations. MRM chromatograms of the 18-HEPE incubation products with human PMN (A) and eosinophils (B) were separated by reverse-phase HPLC. 18-HEPE metabolites were monitored by MRM mode using established transitions for RvE1 (349/195 m/z) and RvE2 (333/199 m/z) as well as predicted transitions for 8,18-diHEPE (333/159 m/z), 11,18-diHEPE (333/167 m/z), 12,18-diHEPE (333/163 m/z), and 17,18-diHEPE (333/201 m/z). Peaks of each metabolite are marked by asterisks.

FIGURE 2.

12/15-LOX-dependent formation of 18-HEPE metabolites from mouse eosinophils. Lipidomic profiles of 18-HEPE incubation products of mouse eosinophils (A) and 12/15-LOX-deficient mouse eosinophils (B) were compared.

FIGURE 3.

Formation of 18-HEPE metabolites by cells expressing mouse 12/15-LOX or human 15-LOX. Lipidomic profiles of 18-HEPE (A) or arachidonic acid (B) incubation products of HEK293 cells transiently transfected with mock (white bars), mouse 12/15-LOX (black bars), or human 15-LOX (gray bars) cDNA plasmids are shown. Lipidomic profiles of 18-HEPE (C) or arachidonic acid (D) incubation products of mouse (black bars) or human (gray bars) eosinophils are shown. Relative production was determined by calculating peak area ratio of each analyte to deuterium-labeled internal standard (LTB₄-d4). Values represent mean ± S.E., n = 3–5.

FIGURE 4.

Enzymatic formation of 18-HEPE metabolites and their anti-inflammatory properties in vivo. A, reverse-phase HPLC chromatogram of the 18-HEPE incubation products with soybean 15-LOX monitored with UV absorbance at 270 nm. B, UV and tandem mass spectra of major products isolated from soybean 15-LOX incubation. Based on the MS/MS spectra, compounds I to IV were assigned as 11,18-diHEPE with corresponding fragments at m/z 333(M-H), 315(M-H-H₂O), 297(M-H-2H₂O), 271(M-H-H₂O-CO₂), 253(M-H-2H₂O-CO₂), and diagnostic fragments at m/z 275, 231(275-CO₂), and 167. Compounds V and VI were assigned as 17,18-diHEPE with corresponding fragments at m/z 333(M-H), 315(M-H-H₂O), 297(M-H-2H₂O), 271(M-H-H₂O-CO₂), 253(M-H-2H₂O-CO₂), and diagnostic ions at m/z 275, 257(275-H₂O), 245, 213(275-H₂O-CO₂), and 201(245-CO₂).

FIGURE 5.

Comparison of eosinophil-derived 18-HEPE metabolites with enzymatically generated products. Top panel, MRM chromatograms of 11,18-diHEPE (A) and 17,18-diHEPE (B) obtained from eosinophil incubation with 18-HEPE. Lower panels, MRM chromatograms of compounds I–VI obtained from soybean 15-LOX-catalyzed synthesis and co-injection of these enzymatically generated products with eosinophil derived products. Note that compounds II and III co-eluted in this liquid chromatographic condition.

FIGURE 6.

Inhibition of PMN infiltration in zymosan-induced peritonitis. A, compounds I–VI were injected intravenously (10 ng/mouse) via tail vein followed by peritoneal injection of zymosan A (1 mg/ml). After 2 h, peritoneal lavages were collected, and PMN leukocyte numbers were counted. Values represent mean ± S.E., n = 3–12, *, p < 0.05; **, p < 0.01 as compared with vehicle control. B, dose-dependent comparison of the actions of compound V (□), compound VI (■), RvE2 (●) and dexamethasone (○) on PMN infiltration. Values represent mean ± S.E., n = 4–12, *, p < 0.05; **, p < 0.01 as compared with vehicle control.

FIGURE 7.

Physical and spectroscopic properties of RvE3. A, ¹H-¹H coupling constants of conjugated triene and full structure of RvE3 (compound V and VI). B and C, reverse-phase HPLC chromatogram of synthetic 18S-HEPE or 18R-HEPE incubation products with soybean 15-LOX monitored with UV absorbance at 270 nm.

FIGURE 8.

RvE3 formation in vivo. Comparison of endogenously formed 17,18-diHEPEs in mouse inflammatory exudates with enzymatically generated RvE3 isomers. Top panel, MRM chromatogram with established transition of 333/201 m/z to monitor 17,18-diHEPEs present in murine peritoneal exudates 48 h after zymosan challenge. Middle and lower panel, MRM chromatogram obtained from co-injection of enzymatically generated 18S-RvE3 (middle panel) or 18R-RvE3 (lower panel) with 17,18-diHEPEs present in murine peritoneal exudates 48 h after zymosan challenge.

FIGURE 9.

RvE3 reduces mouse PMN chemotaxis efficiency. Effect of RvE3 on chemotaxis of mouse bone marrow PMNs toward LTB₄. A, bone marrow PMNs were incubated with 10 nm 18S-RvE3 during the assay. Images of PMNs are shown at 0 and 30 min after addition of LTB₄ (10 nm) as a chemoattractant. B, velocity of the motile cells were determined from digital time lapse movies. C, concentration dependence of 18S-RvE3 (○) and 18R-RvE3 (●) on reduced velocity of PMN chemotaxis. Values represent mean ± S.E., n ≥20 cells, *, p < 0.05; **, p < 0.01; ***, p < 0.001 as compared with vehicle control.

FIGURE 10.

Proposed scheme for biosynthesis of E series resolvins and related products. E series resolvins are generated from a common precursor 18-HEPE. EPA is converted to 18-HEPE by aspirin-acetylated COX-2 or cytochrome P450 monooxygenase. 18-HEPE is further converted via the sequential actions of lipoxygenases, which leads to formation of E series resolvins. 5-LOX expressed in PMNs converts 18-HEPE into RvE1 and RvE2. The stereochemistry of RvE1 and RvE2 was established (9, 12). In addition, 18-HEPE is converted by 12/15-LOX present in eosinophils or resident macrophages into 8,18-diHEPE, 11,18-diHEPE, 12,18-diHEPE, and 17,18-diHEPE (RvE3). The stereochemistry of the alcohols in 8,18-diHEPE and 12,18-diHEPE is depicted as tentative.