Significance
We recently uncovered a family of macrophage-derived molecules, coined maresin conjugates in tissue regeneration, that regulate the system’s ability to clear bacteria as well as repair and regenerate damaged tissues. In the present study, we identified enzymes involved in the formation of these potent molecules in human macrophages. These enzymes were shared with the classic cysteinyl leukotrienes, underscoring the presence of conserved biosynthetic motifs in these two functionally distinct lipid mediator families. Inhibition of these pathways upregulated the formation of several specialized proresolving mediator (SPM) families including D- and E-series resolvins. Thus, these illustrate the dynamic nature of the SPM biosynthetic pathways and provide new targets in the resolution of inflammation and regulation of tissue repair and regeneration.
Keywords: proresolving mediators, inflammation, omega-3 fatty acids, regeneration, eicosanoids
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.
Resolution of acute inflammation is an orchestrated host response to injury and/or infection that leads to the clearance of bacteria and tissue debris as well as tissue repair and regeneration (1–3). Central to the regulation of resolution responses is a novel genus of endogenous mediators termed specialized proresolving mediators (SPM) (2). They actively counterregulate production of inflammation-initiating signals including cytokines, chemokines, and lipid mediators and regulate leukocyte trafficking and phenotype as well as promote tissue repair and regeneration (1, 2, 4–6). At the site of inflammation leukocytes are key in the production of both inflammation-initiating (7, 8) and proresolving mediators (2, 4) because they carry the necessary enzymatic machinery for the stereoselective conversion of precursor essential fatty acids to the bioactive mediators.
Macrophages are central players in the acute inflammatory response governing both initiation and resolution phases (3, 4, 9–12). Distinct macrophage subtypes are involved in the regulation of these different phases of acute inflammatory responses, with macrophages from the resolution phase expressing higher levels of SPM biosynthetic enzymes (12). Recent evidence also demonstrates that lipid mediator profiles change with macrophage phenotype. Classic macrophages express higher levels of inflammation-initiating eicosanoids, whereas alternatively activated cells display higher levels of proresolving mediators (6, 13). Recently, we reported that macrophages produce a family of bioactive peptide-conjugated mediators coined maresin conjugates in tissue regeneration (MCTR) (4) and the complete stereochemistries of MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) [International Union of Pure and Applied Chemistry nomenclature: (4Z,7Z,9E,11E,13R,14S,16Z,19Z)-13-(((R)-2-amino-3-((carboxymethyl)amino)-3-oxopropyl)thio)-14-hydroxydocosa-4,7,9,11,16,19-hexaenoic acid], MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid), and MCTR3 (13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) were established (14). Each displays potent bioactions in stimulating human phagocyte functions, promotes the resolution of bacterial infections, counterregulates the production of proinflammatory mediators, and promotes tissue repair and regeneration (14).
In the proposed MCTR biosynthetic pathway (4), human macrophage 12-lipoxygenase is the initiating enzyme, converting docosahexaenoic acid to 14S-hydro(peroxy)-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid and then to 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-eMaR). The epoxide intermediate is then enzymatically converted to MCTRs. For example, in planaria, a GST catalyzes the conversion of the epoxide to MCTR1, which in turn is the proposed precursor to MCTR2 and MCTR3 (4). The identity of the enzymes that catalyze the conversion of 13S,14S-eMaR to MCTR1 and those that catalyze the formation of MCTR2 and the bioactive MCTR3 in human macrophages are of interest. This is because in addition to establishing the biosynthetic route in specific cell types, determining the identity of these enzymes provides essential information on the intrinsic competition between substrates in distinct stages for the inflammation-resolution cascade. This in turn allows for development of more targeted therapeutic strategies because it sheds light on the contribution of each of these pathways in disease pathophysiology as well as provides potential novel therapeutic leads that can focus on selective regulation of resolution pathways. Given the potent biological actions of these molecules (4) and the differential expression of MCTRs in distinct macrophage subtypes (6), here we identified MCTRs in human lymph nodes, serum, and plasma and investigated the human macrophage enzymes involved in the biosynthesis of MCTRs. Both leukotriene C4 synthase (LTC4S) and GST-mu 4 (GSTM4) catalyze the formation of MCTR1 and contribute to its biosynthesis in human macrophages. Gamma-glutamyltransferase (GGT) converts MCTR1 to MCTR2, which is then further converted to MCTR3 by a dipeptidase. Together, these results establish the MCTR biosynthetic pathway and identify the enzymes that catalyze these reactions in human macrophages.
Results
MCTRs Are Produced in Human Tissues.
To establish the production of MCTR in human systems we used liquid chromatography-tandem MS (LC-MS-MS)–based lipid mediator metabololipidomics to profile human lymph nodes, serum, and plasma. MCTR1 and MCTR3 were identified in all three human tissues, whereas MCTR2 was present in human lymph nodes and serum. Each of these molecules was identified in accordance with published criteria (4, 14), including matching retention times and MS-MS spectra (Fig. 1). We then assessed their amounts in relation to other peptide-conjugated lipid mediators, namely the cysteinyl leukotrienes (7) and protectin conjugates in tissue regeneration (PCTR) (6). MCTR levels in human serum and plasma demonstrated that serum contained significantly higher levels of MCTR1 and MCTR2 (Fig. 1, Fig. S1, and Table 1). In addition, levels for MCTR1, MCTR2, and MCTR3 in each of these tissues were comparable to those of the potent inflammation-initiating cysteinyl leukotrienes and the proresolving and tissue-regenerative PCTR (Table 1).
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.
Table 1.
MCTR in human tissue: Relation to cysLT and PCTR
| Mediator | Q1 | Q3 | Lymph node, pg/150 mg | Serum, pg/mL | Plasma, pg/mL |
| LTC4 | 626 | 189 | 6.7 ± 3.1 | 5.9 ± 3.4 | 2.3 ± 1.0 |
| LTD4 | 497 | 189 | 0.2 ± 0.1 | 0.4 ± 0.1 | 0.2 ± 0.0 |
| LTE4 | 440 | 189 | 0.5 ± 0.2 | 1.7 ± 0.4 | 0.3 ± 0.2 |
| MCTR1 | 650 | 191 | 1.1 ± 0.3 | 1.9 ± 0.5 | 0.8 ± 0.1 |
| MCTR2 | 521 | 191 | 0.3 ± 0.2 | 0.3 ± 0.1 | —* |
| MCTR3 | 464 | 191 | 1.4 ± 0.3 | 1.1 ± 0.5 | 0.3 ± 0.0 |
| PCTR1 | 650 | 231 | 2.9 ± 0.6 | 1.6 ± 0.5 | 0.6 ± 0.2 |
| PCTR2 | 521 | 231 | 1.6 ± 0.9 | 0.8 ± 0.3 | 0.4 ± 0.1 |
| PCTR3 | 464 | 231 | 1.6 ± 0.8 | 1.7 ± 0.8 | 0.7 ± 0.1 |
LMs were extracted using C18 SPE columns and products profiled using LC-MS-MS–based LM metabololipidomics. Products were identified from MS-MS spectra and quantified using MRM with calibration curves specific to each compound. Results are mean ± SEM, n = 7 for axillary human lymph nodes and serum and 10 for plasma (see Fig. S1).
Below the limit of detection (∼0.1 pg).
Role of Human Macrophage LTC4S and GSTM4 in MCTR Formation.
GST enzymes catalyze the formation of bioactive lipid mediators that are peptide-lipid conjugates (7, 8). The proposed homolog of human GSTM4 in planaria promotes formation of MCTR in planaria (4). Therefore, we investigated the expression of both GSTM4 and LTC4S in human macrophages. Using flow cytometry and fluorescently conjugated antibodies, we found that human macrophages expressed both LTC4S and GSTM4 (Fig. 2A). We next tested whether these enzymes were involved in MCTR biosynthesis. To this end, human macrophages were transfected with shRNA targeting LTC4S or GSTM4 or a control sequence. In cells transfected with the shRNA to LTC4S or GSTM4, we found >50% reduction in the expression of these enzymes compared with control scrambled (CS) shRNA (n= 4 independent experiments). We next investigated MCTR production in these cells, and using LC-MS-MS–based lipid mediator profiling found that transfection of cells with shRNA to GSTM4 led to a reduction in MCTR1 (∼60%), MCTR2 (∼60%), and MCTR3 (∼55%; Fig. 2D) compared with CS-shRNA transfection. Of note, in these incubations we also observed a significant increase in both maresin (MaR) 1 and MaR2 (Fig. 2E). Similar results were obtained when macrophages were transfected with shRNA for LTC4S (Fig. 2 D and E).
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.
To further test the role of LTC4S in MCTR biosynthesis and dynamic modulation of lipid mediator pathways in human macrophages we investigated the regulation of endogenous lipid mediator-SPM pathways by LTC4S and LT biosynthesis inhibitors. Incubation of human macrophages with MK886 significantly reduced cysteinyl leukotrienes, with LTC4 levels reduced by ∼41%, LTD4 by ∼36%, and LTE4 by ∼29%, in line with published findings (15). MCTR levels were also reduced, with MCTR1 levels reduced from 3.0 ± 0.1 pg/4 × 106 cells to 1.3 ± 0.4 pg/4 × 106 cells, MCTR2 from 1.5 ± 0.5 pg/4 × 106 cells to 0.8 ± 0.2 pg/4 × 106 cells, and MCTR3 from 2.4 ± 0.4 pg/4 × 106 cells to 0.9 ± 0.1 pg/4 × 106 cells (Fig. 3 and Table S1). In these incubations we also identified and quantified proresolving mediators from the arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid bioactive metabolomes, which were identified in accordance with published criteria including matching retention time and MS-MS fragmentation spectra (13). We also found, in these incubations, up-regulation of select proresolving mediators including resolving (Rv) D2, lipoxin (LX) A4, and LXB4. Incubation of human macrophage with another leukotriene and LTC4S inhibitor, BAY-X-1005, also significantly inhibited both the cysteinyl leukotriene (∼47%) and the MCTR (∼31%) pathways (Table 1). In addition, we found that this inhibitor regulated several biosynthetic pathways up-regulating the levels of SPM from all three bioactive metabolomes including RvD1, RvD2, MaR1, RvD5, LXA4, LXB4, and RvE1 (Fig. 3 and Table S1). Of note, in these incubations we did not observe significant regulation of the inflammation-initiating eicosanoids including LTB4 and PGE2.
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.
Table S1.
LM and SPM profiles in human macrophages with LTC4S inhibitors
| Lipid mediator | Q1 | Q3 | E. coli + vehicle | E. coli + MK886 | E. coli + BAY-X-1005 |
| DHA bioactive metabolome | |||||
| RvD1 | 375 | 233 | 1.4 ± 0.4 | 1.6 ± 0.2 | 2.4 ± 0.2* |
| RvD2 | 375 | 141 | 6.7 ± 0.6 | 10.8 ± 1.9* | 9.2 ± 0.8* |
| RvD3 | 375 | 147 | 1.0 ± 0.3 | 0.8 ± 0.2 | 0.6 ± 0.2 |
| RvD4 | 375 | 101 | 0.4 ± 0.2 | 0.4 ± 0.1 | 0.4 ± 0.2 |
| RvD5 | 359 | 199 | 2.1 ± 0.6 | 1.8 ± 0.5 | 3.9 ± 1.4 |
| RvD6 | 359 | 101 | 2.7 ± 0.3 | 3.0 ± 0.3 | 2.8 ± 0.4 |
| AT-RvD1 | 375 | 141 | 1.0 ± 0.4 | 0.3 ± 0.1* | 1.0 ± 0.4 |
| AT-RvD3 | 375 | 147 | 1.6 ± 0.7 | 0.9 ± 0.2 | 1.7 ± 0.5 |
| PD1 | 359 | 153 | 0.8 ± 0.2 | 1.2 ± 0.2 | 0.9 ± 0.2 |
| MCTR1 | 650 | 231 | 3.0 ± 0.5 | 1.7 ± 0.4* | 1.2 ± 0.4* |
| MCTR2 | 521 | 231 | 1.5 ± 0.1 | 0.8 ± 0.2* | 0.8 ± 0.2* |
| MCTR3 | 464 | 231 | 2.4 ± 0.3 | 0.9 ± 0.1* | 1.5 ± 0.3* |
| MaR1 | 359 | 221 | 2.6 ± 0.8 | 1.2 ± 0.4 | 3.4 ± 0.8 |
| MaR2 | 359 | 191 | 3.2 ± 0.8 | 3.9 ± 1.0 | 1.9 ± 0.3 |
| EPA bioactive metabolome | |||||
| RvE1 | 349 | 161 | 1.3 ± 0.2 | 1.3 ± 0.4 | 1.8 ± 0.3 |
| RvE2 | 333 | 159 | 3.0 ± 0.8 | 2.0 ± 0.4 | 2.3 ± 0.8 |
| RvE3 | 333 | 201 | 1.8 ± 0.7 | 1.9 ± 0.8 | 2.6 ± 0.7 |
| AA bioactive metabolome | |||||
| LXA4 | 351 | 217 | 0.6 ± 0.2 | 1.0 ± 0.1* | 1.3 ± 0.1* |
| LXB4 | 351 | 221 | 9.1 ± 0.7 | 13.2 ± 2.6 | 21.6 ± 2.2* |
| 5S,15S-diHETE | 335 | 235 | 76.8 ± 7.8 | 67.8 ± 14.3 | 80.2 ± 13.1 |
| AT-LXA4 | 351 | 217 | 76.8 ± 7.8 | 67.8 ± 14.3 | 80.2 ± 13.1 |
| AT-LXB4 | 351 | 221 | 230.1 ± 6.9 | 244.6 ± 30.4 | 263 ± 10.5* |
| LTB4 | 335 | 195 | 16.6 ± 3.0 | 13.6 ± 4.7 | 15.6 ± 3.8 |
| LTC4 | 626 | 189 | 2.6 ± 0.4 | 1.5 ± 0.4* | 1.6 ± 0.4* |
| LTD4 | 497 | 189 | 4.5 ± 0.4 | 2.9 ± 0.4* | 2.5 ± 0.3* |
| LTE4 | 440 | 189 | 15.3 ± 1.4 | 10.9 ± 1.3* | 11.3 ± 1.3* |
| PGD2 | 351 | 189 | 634.5 ± 44.5 | 548.8 ± 47.5 | 612.8 ± 39.5 |
| PGE2 | 351 | 189 | 675.2 ± 220.7 | 669.2 ± 242.9 | 743.1 ± 257.5 |
| PGF2α | 353 | 193 | 2,980.5 ± 281.0 | 2,915.4 ± 236.1 | 2,954.2 ± 277.9 |
| TxB2 | 369 | 169 | 26,306.7 ± 3084.0 | 24,841.9 ± 1126.8 | 24,848.4 ± 1545.4 |
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% (vol/vol) 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. LMs were extracted, identified, and quantified using LM profiling. Results are means ± SEM and expressed as picograms per 2 × 107 cells. n = 5 cell preparations. *P < 0.05 vs. E. coli + vehicle group.
Given that LTC4S catalyzes the reaction of glutathione with LTA4 in the cysteinyl leukotriene biosynthetic pathway (7) we questioned whether this enzyme was responsible for catalyzing the reaction of 13S, 14S-eMaR with glutathione. Incubation of human recombinant (hr) LTC4S with increasing concentrations of synthetic 13S, 14S-eMaR yielded MCTR1, a reaction that gave a maximum reaction rate (Vmax) of 6.7 ± 1.6 mmol/min and a Michaelis Menten constant (Km) of 70.5 ± 33.5 µM (Fig. 4A, Left) and a kcat/Km of 1.2 ± 0.4 M−1⋅s−1. For direct comparison, conversion of LTA4 to LTC4 by hrLTC4S gave a Vmax of 8.7 ± 1.5 mmol/min and a Km of 31.2 ± 13.8 µM (Fig. 4A, Right) with a calculated kcat/Km of 4.4 ± 1.4 M−1⋅s−1. Incubation of hrGSTM4 with 13S, 14S-eMaR also yielded MCTR1 with a Vmax of 5.1 ± 0.2 mmol/min and a Km of 42.5 ± 3.8 µM (Fig. 4B, Left) and a kcat/Km of 1.4 ± 0.1 M−1⋅s−1, whereas incubation of LTA4 with GSTM4 gave Vmax of 1.6 ± 0.6 mmol/min and Km of 98.0 ± 6.6 µM (Fig. 4B, Right) and a kcat/Km of 2.1 ± 0.2 M−1⋅s−1. Together these results demonstrate that LTC4S and GSTM4 each convert 13S, 14S-eMaR to MCTR1 in human macrophages. In addition, GSTM4 gave higher affinity to 13S, 14S-eMaR, whereas LTC4S has a higher affinity to LTA4.
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.
Allylic epoxides such as 13S, 14S-eMaR and LTA4 can directly interact with biosynthetic enzymes, regulating their activity, as in the case of LTA4 hydrolase that is inactivated by its substrate LTA4 inhibiting the production of LTB4 (7, 16). Thus, we next questioned whether these epoxides regulated the activity of either LTC4S or GSTM4. Incubation of LTC4S with 13S, 14S-eMaR did inhibit the conversion of 13S, 14S-eMaR to MCTR1, as evidenced by a doubling in MCTR1 levels in incubations with a second addition of the epoxide compared with incubations where only vehicle was added (Fig. 5 A and B). Addition of LTA4 to hrLTC4S also did not interfere with the conversion of LTA4 to LTC4. Similar results were also obtained with hrGSTM4 (Fig. 5C).
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.
Macrophage GGT Converts MCTR1 to MCTR2.
MCTR1 is the proposed precursor to MCTR2 via the conversion of glutathione to cysteinyl-glycinyl (4). GGT enzymes are involved in the conversion of LTC4 to LTD4 by cleaving γ-glutamyl from the glutathione moiety in LTC4 (7). Thus, we next tested whether MCTR1 was a precursor to MCTR2 and the role of GGT in catalyzing this step in human macrophages. Incubation of human macrophages with MCTR1 and either acivicin or serine borate, two GGT enzyme inhibitors, significantly reduced the MCTR2 and MCTR3 and significantly increased MCTR1. These results implicate GGT in macrophage production of MCTR2 (Fig. 6A).
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.
To further test this, we incubated hrGGT with MCTR1 and assessed the kinetics of conversion to MCTR2. MCTR1 was rapidly converted to MCTR2 with 50% maximal kinetics similar to those observed for the conversion of LTC4 to LTD4 (Fig. 6B). Having found that hrGGT converts MCTR1 to MCTR2, we next assessed the catalytic efficiencies of hrGGT. MCTR1 gave a Vmax of 8.1 ± 0.4 mmol/min a Km of 4.6 ± 1.0 µM, and a kcat/Km of 6.0 ± 0.6 M−1⋅s−1 for hrGGT. For direct comparison, LTC4 gave a Vmax of 8.9 ± 0.8 mmol/min, a Km of 18.7 ± 5.0 µM, and a kcat/Km of 1.6 ± 0.4 M−1⋅s−1 (Fig. 6C), suggesting that GGT has a higher affinity for MCTR1 than LTC4. Together these results indicate that MCTR1 is a precursor to MCTR2 via enzymatic conversion by GGT as demonstrated in human macrophages and using recombinant human enzyme.
MCTR3 Is Produced by Dipeptidase Enzymes in Human Macrophages.
We next assessed whether MCTR2 is a precursor to MCTR3, a step that would involve the cleavage of the cysteinyl-glycinyl bond. Given that dipeptidase enzymes are responsible for catalyzing this reaction in the cysteinyl leukotriene biosynthetic pathway (7) we next questioned whether this enzyme(s) is responsible for MCTR3 formation in human macrophages. For this purpose, human macrophages were incubated with MCTR2 in the presence or absence of cilastatin sodium, a dipeptidase enzyme inhibitor, and the formation of MCTR3 using lipid mediator (LM) metabololipidomics was assessed (Fig. 7). In cells incubated with cilastatin sodium we found significantly higher MCTR2 levels and significantly lower MCTR3 (Fig. 7B). These results suggest that macrophage dipeptidase catalyzes the conversion of MCTR2 to MCTR3.
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.
To further evaluate the role of dipeptidases in MCTR biosynthetic pathway, human macrophages were incubated with MCTR1 in the presence or absence of cilastatin sodium, and the levels of MCTR1, MCTR2, and MCTR3 were assessed (Fig. 7C). In these incubations all three molecules were identified, with statistically significant increases in MCTR2 and decreases in MCTR3 in the presence of the dipeptidase inhibitors, indicating that dipeptidase contributes to the conversion of MCTR2 to MCTR3 in human macrophages. Together, these results support the proposed biosynthetic pathway, in which the epoxide 13S, 14S-eMAR is converted to MCTR1, which is then converted to MCTR2 and subsequently to MCTR3 (Fig. 8).
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.
Discussion
In the present paper we establish the MCTR production in human tissues and biosynthetic pathway with human macrophages together with recombinant enzymes. Using material prepared by total organic synthesis, we found that 13S, 14S-eMaR is converted to MCTR1, a step that in human macrophages is catalyzed by both LTC4S and GSTM4. Cleavage of the γ-glutamyl moiety of MCTR1 by GGT yields MCTR2. This mediator is then a precursor in the biosynthesis of MCTR3, where in human macrophages the cysteinyl-glycinyl bond is cleaved by a dipeptidase enzyme. Using LM metabololipidomics, we profiled human tissues identifying MCTR in human plasma, serum, and lymph nodes at concentrations (0.5–4.5 pM) commensurate with their known bioactive ranges (4, 14).
Tissue repair and regeneration are essential in the reestablishment of barrier function and return to homeostasis (1–3, 10, 17). Macrophages are central in orchestrating these responses, with cells of the alternative activated lineage being primarily linked with tissue repair and regeneration (2, 17, 18). In this context identification of MCTRs as macrophage-derived mediators with potent tissue protective and regenerative actions (4, 14) provides leads into pathways and mechanisms that control reestablishment of functions to damaged tissues. We also recently found that alternatively activated human macrophages produce higher levels of MCTRs than classically activated macrophages (6), underscoring the potential role of this pathway in tissue and organ repair in human tissue.
Bioactive mediators are produced via the stereoselective conversion of essential fatty acids that give rise to molecules with defined stereochemistries (2, 7). Hence, identifying the enzymes responsible for the formation of lipid mediators is of fundamental importance. This is because establishing the identity of these enzymes allows for a better appreciation of their biological roles during both health and disease. In the present study, we demonstrated that two enzymes from the GST family, GSTM4 and LTC4S, catalyze the formation of MCTR1 from 13S,14S-eMAR (Figs. 2–5). Both of these enzymes also catalyze the conversion of LTA4 to LTC4, a lipid mediator that displays potent vasoactive and smooth muscle constricting actions (7). Of note, the two enzymes displayed different affinities to these substrates and whereas LTC4S displayed a higher affinity to LTA4, GSTM4 displayed a higher affinity toward 13S,14S-eMAR (Fig. 4). These findings suggest that in addition to substrate availability, the relative expression of the two enzymes in one cell type may determine the balance between the inflammation-, contraction-, and stress-initiating LTC4 (7) vs. the tissue-regenerative pathway of MCTRs. The second enzyme in the MCTR biosynthetic pathway that was identified in this report is GGT, which catalyzes the conversion of MCTR1 to MCTR2 (Fig. 6). This enzyme, and the third enzyme identified in the present study, the dipeptidase enzyme(s) that catalyzes the conversion of MCTR2 to MCTR3, are also shared with the cysteinyl leukotriene pathway (Figs. 6 and 7). Of note, substrate affinity for the GGT enzyme to MCTR1 was higher than to LTC4 (Fig. 6), a finding that further underscores the role of these enzymes in determining the macrophage lipid mediator phenotype.
In summation, in the present experiments using primary human macrophages, stereochemically defined materials prepared using total organic synthesis and human recombinant enzymes, we establish the MCTR biosynthetic pathway and precursor–product relationship(s) for MCTR1, MCTR2, and MCTR3. The identification of these potent proresolving and tissue-regenerative immunoresolvents in other human organs and tissues, including lymph nodes and serum, suggests that these pathways and mediators may be of interest in other human tissues. Given the differential affinity of enzymes identified herein to the cysteinyl leukotriene and MCTR pathways, their relative expression at sites of injury and/or inflammation may also assist in understanding disease processes. In addition, they also provide leads for targeted therapeutic strategies that may preferentially inhibit formation of inflammation-initiating cysteinyl leukotriene and up-regulate SPM formation.
Materials and Methods
Human Tissues and Cells.
This study was conducted in accordance with Partners Human Research Committee Protocols 1999P001297 and 1999P001279 and a protocol approved by Barts and the London Research Ethics Committee [London (QMREC 2014:61)]. Informed consent was obtained from all participants.
LM Metabololipidomics.
Human lymph nodes (∼150 mg) were defrosted on ice and carefully weighed, then 1 mL ice-cold methanol was added to each (see Table S2 for patient demographics and tissue source). Fresh serum and plasma (1 mL) were obtained from healthy donors and 4 mL of ice-cold methanol was added to each sample. Five hundred picograms of internal standards d5-LTC4, d5-LTD4, d5-LTE4, d4-LTB4, d4-PGE2, d5-RvD2, and d5-LXA4 were added to each sample to facilitate identification and quantification. Samples were then kept at −20 °C for 1 h to allow for protein precipitation and products isolated as detailed in SI Materials and Methods.
Table S2.
Patient demographics and tissue source for human axillary lymph nodes
| Age (years) | Sex | Diagnosis | Tissue status | Race | Source |
| 80 | Male | Crohn’s disease | Normal | White | OSU Tissue Procurement Services |
| 53 | Male | Bacteremia, right lower extremity cellulitis, rhabdomyolysis, acute kidney injury, acute blood loss anemia, morbid obesity, hypertension, hyperlipidemia, venous stasis | Normal | Black | OSU Tissue Procurement Services |
| 27 | Female | Irritable bowel syndrome, chronic constipation | Normal | White | OSU Tissue Procurement Services |
| 28 | Female | Crohn’s disease | Normal | White | OSU Tissue Procurement Services |
| 89 | Male | Cardiorespiratory failure, end-stage Alzheimer’s disease | Normal | White | Science Care |
| 64 | Female | Stage 4 breast cancer | Normal | White | Science Care |
| 89 | Male | Cardiorespiratory failure, end stage Alzheimer’s disease | Normal | White | Science Care |
Samples were obtained from a commercial source from postmortem human subjects.
Incubation Conditions: Enzymes.
HrLTC4S (40 ng/20 µL; Origene) and hrGSTM4 (61 ng/20 µL; Creative Biomart) were suspended in 25 mM Tris⋅HCl containing 5 mM reduced glutathione and 0.05% Triton X-100 (pH 7.8) and incubated with 13S, 14S-eMaR or LTA4 (0.3, 1, 3, 10, 30, and 100 μM) at room temperature for 2 min. The incubations were quenched using 2 volumes of MeOH and were profiled using lipid mediator metabololipidomics. Synthetic epoxide eMaR was prepared as in ref. 16, and MCTR1, 2, and 3 were prepared as in ref. 19.
hrLTC4S (40 ng/20 µL; Origene) was suspended in 25 mM Tris⋅HCl containing 5 mM reduced glutathione and 0.05% Triton X-100 (pH 7.8). This was incubated with 13S,14S-eMaR (5 μM, 2 min, 37 °C) then with 13S,14S-eMaR (5 μM, 2 min, 37 °C) or vehicle. Incubations were then quenched using 2 volumes of ice-cold methanol and products profiled using lipid mediator metabololipidomics. In separate experiments hrLTC4S (0.12 μM; 25 mM Tris⋅HCl, 5 mM reduced glutathione, and 0.05% Triton X-100, pH 7.8), was incubated with LTA4 from Cayman Chemical (5 μM, 2 min, 37 °C) then with LTA4 (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.
HrGSTM4 (61 ng/20 µL; Creative Biomart) was suspended in 25mM Tris⋅HCl containing 5 mM reduced glutathione and 0.05% Triton X-100 (pH 7.8). This was incubated with synthetic 13S,14S-epoxy-MaR (5 μM, 2 min, 37 °C) then with 13S,14S-epoxy-MaR (5 μM, 2 min, 37 °C) or vehicle. Incubations were then quenched and products profiled as above. Also, 61 ng/20 µL GSTM4 (25 mM Tris⋅HCl, 5 mM reduced glutathione, and 0.05% Triton X-100, pH 7.8) was incubated with LTA4 (5 μM, 2 min, 37 °C) then with LTA4 (5 μM, 2 min, 37 °C) or vehicle. Incubations were then quenched and products profiled as above.
MCTR1 (4.4 nM) and LTC4 (4.4 nM) were suspended separately in Tris⋅HCl (185 mM, pH 8.2) and were incubated with GGT (147 ng/20 µL; Lee Biosolutions) for a total of 10 min. Aliquots were taken at predetermined intervals, placed in two volumes of ice-cold methanol, and mediator levels determined. GGT (147 ng/20 µL) was suspended in 185 mM Tris⋅HCl (pH 8.2) and incubated with 13S, MCTR1 or LTC4 (0.3, 1, 3, 10, 30, and 100 μM) at room temperature for 2 min. All incubations were stopped using 2 volumes of MeOH, extracted, and profiled using LM metabololipidomics. Human lymph nodes (deidentified) were purchased from Science Care and Ohio State University (OSU) Tissue Procurement Services.
Statistics.
All results are expressed as means ± SEM. Differences between groups were compared using Student t test (two groups). The criterion for statistical significance was P < 0.05. Sample sizes for each experiment were determined on the variability observed in preliminary experiments and prior experience with the experimental systems. The criterion for statistical significance was P < 0.05. Partial least squares discriminant analysis (PLS-DA) was conducted as described in ref. 20 with mediators and macrophage lineage markers giving variable importance in projection scores greater than 1 taken as displaying significant correlation.
SI Materials and Methods
LM Profiling.
Cell incubations and enzyme incubations were placed in 2 volumes of methanol containing 500 pg d5-LTC4, d5-LTD4, d5-LTE4, d4-LTB4, d4-PGE2, d5-RvD2, and d5-LXA4, which were added to facilitate identification and quantification. For quantification molecules deuterium-labeled internal standards with physical and chromatographic properties similar to those of the mediators of interest were used. Samples were then held at −20 °C for 45 min to allow for protein precipitation and then centrifuged (1,200 × g at 4 °C for 10 min). Products were extracted using solid-phase extraction and eluted using methanol. Eluted isolates were then brought to dryness in a stream of nitrogen and suspended in methanol:water (50:50; vol/vol). The LC-MS-MS system was operated (4) with the following modifications: a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector paired with a QTrap 5500 (AB Sciex) were used. A Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7 μm; Agilent Technologies) was kept in a column oven maintained at 50 °C (ThermaSphere model TS-130; Phenomenex), and LMs were eluted with a mobile phase consisting of methanol:water:acetic acid at 55:45:0.1 (vol:vol:vol) over 5 min, then to 80:20:0.1 (vol:vol:vol) for 2 min, then isocratic 80:20:0.1 (vol:vol:vol) for the next 3 min and ramped to 98:2:0.1 (vol:vol:vol) over 3 min. This was maintained at 98:2:0.1 (vol:vol:vol) for 3 min, and the flow rate maintained at 0.65 mL/min. QTrap 5500 was operated in positive ionization mode using scheduled MRM coupled with information-dependent acquisition and enhanced product ion scan.
Incubation Conditions: Cells.
Human macrophages (deidentified) were prepared from peripheral blood mononuclear cells (4) purchased from Children’s Hospital Boston Blood Bank and the National Health Services Blood and Transplant services. Briefly, monocytes were isolated from peripheral blood of healthy volunteers using dextran sedimentation and density centrifugation with Histopaque 1077–1. Cells were then incubated in 10-cm dishes for 45 min at 37oC in PBS (containing calcium and magnesium) and nonadherent cells were washed using PBS (without calcium and magnesium). Cells were then incubated in RPMI containing human serum in the presence of GM-CSF (20 ng/mL) for 7 d. For shRNA knockdown, human MΦ (5 × 106 cells per 10 mL) were transfected with shRNA plasmids for LTC4S (TR319192; Origene), GSTM4 (TR304180; Origene), or with CS-shRNA (5 µg) using Jet-Pei transfection reagent following the manufacturer’s instructions (Polyplus-transfection SA). Seventy-two hours later, macrophages were harvested, suspended in PBS containing calcium and magnesium (pH 7.45), and incubated with 100 nM of 14S-hydroperoxy-docosahexaenoic acid (prepared as in ref. 4) and E. coli (2.5 × 107 cfu/mL) for 30 min at 37 °C.
In select experiments human macrophages (2 × 107 cells) were suspended in 500 µL PBS containing calcium and magnesium and 2% (vol/vol) FCS (pH 7.45). These were incubated with MK886 (10 µM), BAY-1X-1005 (10 µM), or vehicle (PBS containing 0.1% DMSO) for 20 min at room temperature. E. coli (2 × 108 cfu) were then added and cells were kept at 37 oC for 45 min. Incubations were then with 2 volumes of ice-cold methanol containing deuterium-labeled internal standards and LMs were profiled.
Cells from the human macrophage cell line KG1a (1 × 106 cells per mL; American Type Culture Collection) were suspended in PBS containing calcium and magnesium and incubated with vehicle (PBS containing 0.01% EtOH), acivicin (2.5 mM), or serine borate (45 mM) (37 °C, pH 7.45, 15 min, 37 °C), then with MCTR1 (0.33 μM) and serum-treated zymosan (0.1 mg, 37 °C) for 180 min. KG1a cells (1 × 106 cells per mL) were suspended in PBS containing calcium and magnesium and incubated with or without cilastatin sodium (Tocris) (2.3 mM, 37 °C in pH 7.45, 15 min) and then incubated with MCTR1 (66.9 nM) or MCTR2 (83.4 nM) for 6 h at 37 °C.
Acknowledgments
We thank Mary Halm Small for expert assistance in manuscript preparation. This work was supported in part by NIH Grants R01GM38765 and P01GM095467 (to C.N.S.) and the European Research Council under the European Union’s Horizon 2020 research and innovation programme Grant 677542 (to J.D.).
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. E.A.D. is a Guest Editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1607003113/-/DCSupplemental.
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