Pro-resolving actions and stereoselective biosynthesis of 18S E-series resolvins in human leukocytes and murine inflammation

Abstract

E-series resolvins are antiinflammatory and pro-resolving lipid mediators derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) that actively clear inflammation to promote tissue homeostasis. Aspirin, in addition to exerting antithrombotic actions, also triggers the biosynthesis of these specialized pro-resolving mediators. Here, we used metabolomic profiling to investigate the biosynthesis of E-series resolvins with specific chiral chemistry in serum from human subjects and present evidence for new 18S series resolvins. Aspirin increased endogenous formation of 18S-hydroxyeicosapentaenoate (18S-HEPE) compared with 18R-HEPE, a known resolvin precursor. Human recombinant 5-lipoxygenase used both enantiomers as substrates, and recombinant LTA4 hydrolase (LTA4H) converted chiral 5S(6)-epoxide-containing intermediates to resolvin E1 and 18S-resolvin E1 (RvE1 and 18S-RvE1, respectively). 18S-RvE1 bound to the leukocyte GPCRs ChemR23 and BLT1 with increased affinity and potency compared with the R-epimer, but was more rapidly inactivated than RvE1 by dehydrogenase. Like RvE1, 18S-RvE1 enhanced macrophage phagocytosis of zymosan, E. coli, and apoptotic neutrophils and reduced both neutrophil infiltration and proinflammatory cytokines in murine peritonitis. These results demonstrate two parallel stereospecific pathways in the biosynthesis of E-series resolvins, 18R- and 18S-, which are antiinflammatory, pro-resolving, and non-phlogistic and may contribute to the beneficial actions of aspirin and ω-3 polyunsaturated fatty acids.

Figures

Figure 1. Chiral lipidomic separation and quantitation of isobaric monohydroxy-EPEs.

In order to detect and quantify each positional isomer without ambiguity, an MRM method was established. Signature daughter ions for each standard HEPE (parent m/z, 317) were as follows: 18-HEPE, 259; 15-HEPE, 219; 12-HEPE, 179; 5-HEPE, 115. For each enantiomer pair, the R isomer was eluted before S isomers. Each signature ion for each species was unique; only two (R and S isomers) peaks are present on each extracted ion chromatogram.

Figure 2. Aspirin enhances endogenous production of novel E-series resolvin precursors.

(A) Chiral HPLC-MS/MS analysis of E-series resolvin precursor stereoisomers from human serum. Healthy human sera, treated with or without aspirin, were collected (see inset) and 18S- and 18R-HEPE were separated by reverse-phase chiral column without derivatization and quantified by MRM, specific transition at 317→259. Results are from human subjects given aspirin; representative chromatogram of n = 3. (B) Tandem mass spectra and structural assignment of 18R-HEPE and 18S-HEPE from human serum.

Figure 3. Human 5-LOX converts both 18R- and 18S-HEPE to direct precursors of RvE2.

(A) Chiral separation of 18S- and 18R-RvE2 after 5-LOX incubation and reduction. Diastereomeric mixture of RvE2 was isolated by conventional RP-HPLC separation and further resolved by chiral HPLC. (B) Tandem mass spectra of 18R-RvE2 and 18S-RvE2 and (C) their MS/MS assignment. 18R- and 18S-RvE2 had indistinguishable MS/MS spectra.

Figure 4. Correct stereochemistry of RvE1 is essential for its bioactivity.

(A) RvE1 matching study with synthetic standard. Nonenzymatic hydrolysis products such as 6-trans 5,12,18-triHEPE or 5,6,18-triHEPE (peaks shown with solid line) have retention times different from the synthetic RvE1-spiked run (peak shown with dashed line). (B) Direct comparison of RvE1 and 5,6,18-triHEPE in zymosan-induced 2-hour murine peritonitis model. Equal doses (20 ng/mouse) of synthetic RvE1 and 5,6,18-triHEPE were administered via tail vein immediately before intraperitoneal injection of zymosan A. The cell number in the peritoneum was enumerated, and PMN numbers were compared after differential counting. Results are mean ± SEM. **P < 0.05 compared with vehicle.

Figure 5. LTA4H in RvE1 biosynthesis.

(A) Activated human PMNs were incubated with 18-HEPE, in the presence or absence of LTA4H inhibitor bestatin, and representative LC-MS/MS chromatograms, transition of 349→205 monitored, are shown. Treating PMNs with hydrolase inhibitor (250 μM bestatin) reduced RvE1 synthesis by 67.3% ± 6.2% (mean ± SEM, n = 3; P < 0.05 compared with control). (B) LC-MS/MS spectrum of RvE1 from 5-LOX (25 U) and LTA4H (2 μg) combinatorial incubation. Retention time of this product matched with synthetic RvE1. Statistically significant amounts of RvE1 were not produced with 5-LOX alone or with bestatin-treated combinatorial incubations.

Figure 6. Molecular pathways of 18S-RvE1 actions: ligand-receptor interaction and metabolic inactivation.

(A) ChemR23 activation. Increasing doses of RvE1 (diamonds) or 18S-RvE1 (squares) were incubated with the ChemR23-overexpressing reporter system, followed by β-gal substrate incubation. Luminescence was measured to obtain the RvE1 EC50, 1.37 × 10–10 M, and 18S-RvE1 EC50, 6.33 × 10–12 M, from fitted dose-response curves. (B) BLT1 antagonism. BLT1-overexpressing cells were incubated with different concentrations of 18S-RvE1 or RvE1, followed by 30 nM LTB4, and further incubated for 30 minutes. Results are mean ± SEM; n ≥ 3. P < 0.05, RvE1 versus 18S-RvE1. (C) Comparison between RvE1 and 18S-RvE1 by 15-PGDH–mediated oxidation (diamonds: RvE1, squares: 18S-RvE1) addressed in the presence of NAD+ as a cofactor. NADH was monitored as a readout of dehydrogenation. Results shown are representative of n = 3 with similar trends.

Figure 7. Direct comparison of the antiinflammatory actions of 18S and 18R E-series resolvins.

(A) Direct comparison of action at equal doses of resolvins (20 ng/mouse) in zymosan-induced murine peritonitis at 2 hours. (B) Comparison of 18S-RvE1 (black bars) and RvE1 (white bars) action at 4 hours. (C) 18S-RvE1 decreased PMN infiltration in the TNF-α–induced murine dorsal air pouch. Results are mean ± SEM; n ≥ 3. *P < 0.05, ***P < 0.001 compared with vehicle-treated mice.

Figure 8. Pro-resolving actions of 18S-RvE1.

Enhancement of (A) zymosan particle and (B) live E. coli phagocytosis: Increasing doses of RvE1 (diamonds) and 18S-RvE1 (squares) were incubated with murine macrophages, followed by addition of FITC-labeled zymosan or fluorescent E. coli. Only particles or cells completely phagocytosed by macrophages remained fluorescent and were counted. (C) Enhancement of apoptotic PMN phagocytosis by human macrophages. Two doses of RvE1 (white bars) or 18S-RvE1 (black bars) were tested. Results are mean ± SEM; n ≥ 3. *P < 0.05, **P < 0.01 compared with vehicle treatment; †P < 0.05, RvE1 versus 18S-RvE1. (D) Enhancement of microbial clearance by peritoneal resident macrophages. 18S-RvE1 or RvE1 was intraperitoneally administered immediately (100 ng) before fluorescent E. coli injection and each peritoneum lavaged 30 minutes later to measure phagocytosed bacteria by macrophages. Results are mean ± SEM; n = 6–8 for each treatment. *P < 0.05, **P < 0.01 compared with vehicle treatment.

Figure 9. Transient bioactions of 18S-RvE1.

(A) Regulation of PMN infiltration during E. coli peritonitis by 18S-RvE1 and RvE1. 18S-RvE1 or RvE1 (100 ng) was administered via tail vein 3 hours after E. coli injection to peritoneum. **P < 0.01 compared with vehicle-treated. (B) Enhancement of neutrophil removal from site of inflammation: Vehicle, 100 ng RvE1, or 18S-RvE1 was injected intraperitoneally at 8 hours after zymosan A injection, and PMNs in the peritoneum were enumerated 12 hours later (20 hours after zymosan injection). Each point is the average from 3 mice with the same treatment on the same day. Results in A and B are mean ± SEM. †P < 0.05, 18S-RvE1 versus RvE1; *P < 0.05 compared with vehicle-treated. (C) Regulation of zymosan-stimulated mouse macrophage cytokine production by RvE1 and 18S-RvE1. Cell supernatants were collected and centrifuged for multiplex cytokine quantitation. Results are averages of duplicated measurements; *P < 0.05, **P < 0.01 compared with cells exposed to zymosan alone.

Figure 10. Dual biosynthetic pathways of 18S and 18R E-series resolvins.

18R- and 18S-HEPE are endogenously generated in a stereoselective manner by enzymes such as aspirin-treated COX-2 and subsequently converted to epimeric E-series resolvins by a shared biosynthetic route. The stereochemistry of the epoxide intermediate shown is tentative, and those of RvE1, RvE2, and their 18S forms are established. See text for details.