Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages

Abstract

Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC₄S and GSTM4. Incubation of human macrophages with LTC₄S inhibitors blocked LTC₄ and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration.

Keywords: eicosanoids; inflammation; omega-3 fatty acids; proresolving mediators; regeneration.

Fig. 1.

MCTRs are produced in lymph nodes. Peptide conjugated lipid mediators obtained from human lymph nodes following C18 solid-phase extraction were identified using LM metabololipidomics (Materials and Methods). (A) Representative multiple reaction monitoring (MRM) chromatograms of peptide conjugated lipid mediators. (B) MS-MS spectra used for identification of (Upper) MCTR1, (Middle) MCTR2, and (Lower) MCTR3. Results represent n = 7 healthy donors.

Fig. S1.

Identification of PCTR in lymph nodes. Peptide-conjugated LMs were obtained from lymph nodes using C18 solid-phase extraction and mediators were identified using LM metabololipidomics (see Materials and Methods for details). (Upper Left) MRM chromatograms for identified PCTR. Representative MS-MS spectra used in the identification of (Upper Right) PCTR1, (Lower Left) PCTR2, and (Lower Right) PCTR3. Results represent n = 7 healthy donors.

Fig. 2.

LTC₄S and GSTM4 promote MCTR biosynthesis in human macrophages. (A–E) Human macrophages were prepared from peripheral blood mononuclear cells and the expression of LTC₄S and GSTM4 determined using flow cytometry and fluorescently conjugated antibodies. (Upper) Representative histogram plot for human macrophage incubated with anti-LTC₄S or isotype antibodies. (Lower) Representative histogram plot for human macrophage incubated with anti-GSTM4 or isotype antibodies. Human macrophages (5 × 10⁶ cells/10 mL) were transfected with shRNA targeting human GSTM4, LTC₄S, or a CS-shRNA then were incubated with Escherichia coli (2.5 × 10⁷ cfu/mL) and 14S-hydroperoxy-docosahexaenoic acid (100 nM; PBS^+/+, pH 7.45, 30 min, 37 °C). Incubations were stopped and products extracted and profiled using metabololipidomics (Materials and Methods). (B) Representative MRM chromatogram for each of the mediators identified and quantified. (C) MS-MS spectrum of MCTR1. (D and E) Specific bioactive mediators quantified using Q1: M-H (parent ion) and Q3: diagnostic ion in the MS-MS (daughter ion). Results in A–C are representative of n = 4 donors, D and E are mean ± SEM, n = 4 donors. *P < 0.05, **P < 0.01 vs. CS-shRNA.

Fig. 3.

Regulation of LM SPM profiles by LTC₄ inhibitors in human macrophages. Macrophages (2 × 10⁷ cells) were incubated with vehicle (PBS containing 0.1% DMSO), MK886 (10 µM), or BAY-X-1005 (10 µM) for 20 min (room temperature, PBS containing 2% FCS, pH 7.45). Cells were then incubated with E. coli (2 × 10⁸ cfu) and incubations were quenched with 2 volumes of ice-cold MeOH containing deuterium-labeled internal standards after 45 min. Lipid mediators were extracted, identified, and quantified using LM profiling. (A) MRM chromatograms for identified mediators. (B) Representative MS-MS spectrum used in the identification of RvD1. (C) PLS-DA for identified lipid mediator profiles. (Upper) 2D loading plot. (Lower) 2D score plot. Results are representative of n = 5 healthy volunteers.

Fig. 4.

Human LTC₄S and GSTM4 convert 13S,14S-eMaR to MCTR1. (A) Human recombinant LTC₄S (40 ng/20 µL; 0.12 µM) was incubated with the indicated concentrations of (Left) 13S,14S-eMaR or (Right) LTA₄ in 25 mM Tris⋅HCl, pH 7.8, 0.05% Triton X-100, and 5 mM glutathione at room temperature. (B) Human recombinant GSTM4 (61 ng/20 µL; 0.12µM) was incubated with the indicated concentrations of (Left) 13S,14S-eMaR or (Right) LTA₄ in 25 mM Tris⋅HCl, pH 7.8, 0.05% Triton X-100, and 5 mM glutathione at room temperature. MCTR1 and LTC₄ were each identified and quantified using LC-MS-MS metabololipidomics. Results are mean of three independent experiments.

Fig. 5.

Human LTC₄S and GSTM4 are not inactivated by LTA₄ or 13S,14S-epoxy-MaR. (A and B) LTC₄S (40 ng/20 µL; 25 mM Tris⋅HCl, 5 mM reduced glutathione, and 0.05% Triton X-100, pH 7.8) was incubated with 13S,14S-epoxy-MaR (5 μM, 2 min, 37 °C) then 13S,14S-epoxy-MaR (5 μM, 2 min, 37 °C) or vehicle. Incubations were then quenched using 2 volumes of ice-cold methanol and products profiled using LM metabololipidomics. (A) MCTR1 and LTC₄ produced by LTC₄S (40 ng/20 µL; 25 mM Tris⋅HCl, 5 mM reduced glutathione, and 0.05% Triton X-100, pH 7.8) incubated with eMaR followed by eMaR or LTA₄ (5 μM, 2 min, 37 °C) then LTA₄ (5 μM, 2 min, 37 °C) or vehicle. (B) GSTM4 (61 ng/20 µL; 0.12 μM; 25 mM Tris⋅HCl, 5 mM reduced glutathione, and 0.05% Triton X-100, pH 7.8) was incubated with 13S,14S-epoxy-MaR (5 μM, 2 min, 37 °C) then 13S,14S-epoxy-MaR (5 μM, 2 min, 37 °C) or vehicle. (Left) GSTM4 was incubated with LTA₄ (5 μM, 2 min, 37 °C) then LTA₄ (5 μM, 2 min, 37 °C) or vehicle. Incubations were then quenched and products profiled as above. Results are mean ± SEM, n = three independent incubations. *P < 0.05 vs. vehicle.

Fig. 6.

Human macrophage GGT converts MCTR1 to MCTR2. KG1a cells (1 × 10⁶ cells per mL) were incubated with acivicin (2.5 mM), serine borate (45 mM), or vehicle (PBS^+/+, pH 7.45, 15 min) then MCTR1 (0.33 µM) and serum-treated zymosan (0.1 mg, 37 °C, PBS^+/+, pH 7.45, 180 min). Incubations were stopped with ice-cold methanol and products profiled using lipid mediator metabololipidomics. (A, Left) Representative MS-MS spectrum of MCTR2 and (Right) MCTRs amounts in macrophage incubations. Results are mean ± SEM, n = 4 macrophage preparations. *P < 0.05 vs. macrophages + MCTR1. (B) Time course: 4.4 nM of MCTR1 (Left) or LTC₄ (Right) were each incubated with human recombinant GGT (147 ng/20 µL, 185 mM Tris⋅HCl, pH 8.2, room temperature) for the indicated intervals. Results are mean ± SEM; n = 4 macrophage preparations. (C) Human recombinant GGT (147 ng/20 µL) was incubated with the indicated concentrations of (Left) MCTR1 or (Right) LTC₄ (185 mM Tris⋅HCl, pH 8.2, room temperature). All incubations were stopped with ice-cold methanol and extracted and products were profiled using LM metabololipidomics. Results are mean ± SEM; n = 3 independent incubations.

Fig. 7.

MCTR3 is formed by human macrophage dipeptidase from MCTR2. (A–C) KG1a cells (1 × 10⁶ cells/mL) were incubated with cilastatin sodium (2.3 mM), or vehicle (PBS^+/+, pH 7.45, 15 min) then MCTR2 (66.9 nM) or MCTR1 (83.4 nM) and serum-treated zymosan (0.1 mg, 37 °C, PBS^+/+, pH 7.45, 360 min). Incubations were stopped and extracted and products were profiled using LM metabololipidomics. (A) Representative MS-MS spectrum of MCTR3. (B and C) MCTR in macrophage incubations. Results are mean ± SEM; n = 4 independent experiments. (B) *P < 0.05 vs. KG1a cells + MCTR2. (C) *P < 0.05, **P < 0.01 vs. KG1a cells + MCTR1.

Fig. 8.

MCTR biosynthetic pathway. Structures are illustrated in most likely conformations based on biosynthetic evidence (4, 14). Stereochemistry of MCTR1, MCTR2, MCTR3, MaR1, and the maresin-epoxide intermediate are established (14, 16). The lipoxygenase responsible for 14-lipoxygenation and epoxidation reactions in human macrophages is human 12-LOX (16, 21). DPEP, dipeptidase; EH, epoxide hydrolase; LOX, lipoxygenase.