Identification and Actions of the Maresin 1 Metabolome in Infectious Romain A. Colas, Jesmond Dalli, Nan Chiang, Iliyan Vlasakov, Julia M. Sanger, Ian R. Riley and Charles N. This information is current as Serhan of September 27, 2021. J Immunol published online 31 October 2016 http://www.jimmunol.org/content/early/2016/10/31/jimmun ol.1600837 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 31, 2016, doi:10.4049/jimmunol.1600837 The Journal of Immunology

Identification and Actions of the Maresin 1 Metabolome in Infectious Inflammation

Romain A. Colas,1 Jesmond Dalli,1 Nan Chiang, Iliyan Vlasakov, Julia M. Sanger, Ian R. Riley, and Charles N. Serhan

Maresin 1 (MaR1) is an immunoresolvent that governs resolution of acute inflammation, and its local metabolism in the context of infectious inflammation is of interest. In this study, we investigated the MaR1 metabolome in infectious exudates and its bioactions in regulating leukocyte responses in the context of bacterial infection. In Escherichia coli infectious exudates, MaR1 was temporally regulated with maximal levels at 4 h (2.2 6 0.4 pg/lavage). In these exudates we also identified two novel products, and their structure elucidation gave 22-hydroxy-MaR1 and 14-oxo-MaR1. Using human primary leukocytes, we found that primarily produced 22-OH-MaR1, whereas the main product was 14-oxo-MaR1. Both 22-OH-MaR1 and 14-oxo-

MaR1 incubated with human primary gave dose-dependent increases in macrophage of ∼75% at 1 pM Downloaded from 22-OH-MaR1 and ∼25% at 1 pM 14-oxo-MaR1, whereas 14-oxo-MaR1 was less active than MaR1 at higher concentrations. Together these findings establish the temporal regulation of MaR1 during infectious inflammation, and elucidate the structures and actions of two novel MaR1 further metabolites that carry bioactivities. The Journal of Immunology, 2016, 197: 000–000.

cute inflammation is a natural host protective mechanism The biosynthesis of Maresin 1 (MaR1 [7R,14S-dihydroxydocosa-

mounted by the body in response to injury or invading 4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid]), the first mediator identi- http://www.jimmunol.org/ A pathogens that when self-limited leads to homeostasis fied in this family, is initiated by human macrophage 12- (1). If uncontrolled, inflammation may become chronic and lead to to produce 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z docosahexaenoic tissue damage (2). It is now recognized that the resolution of in- acid that is then converted to 13S,14S-epoxy-docosa-4Z,7Z,9E,11E, flammation is an active process orchestrated by a new genus of 16Z,19Z-hexaenoic acid (5, 6) and subsequently to MaR1. MaR1 potent molecules known as specialized proresolving mediators displays potent leukocyte-directed actions, decreasing polymorpho- (SPMs) (3). SPMs are bioactive autacoids having both anti- nuclear (PMN) infiltration during murine peritonitis and inflammatory and proresolving properties. They are enzymatically increasing human macrophage of apoptotic PMNs. In produced from essential fatty acids, with unique stereochemistries. addition, MaR1 stimulates tissue regeneration in planaria and reduces Recently, a new family of macrophage-derived mediators from chemotherapy-induced neuropathic pain (5). It exerts protective ac- by guest on September 27, 2021 was identified and coined as maresins for tions in murine models of colitis where it reduces colonic damage, macrophage mediator resolving inflammation (4). body weight loss, and proinflammatory mediators (7). MaR1 is also organ-protective in murine acute respiratory distress syndrome (8). Given that MaR1 carries potent leukocyte-directed actions, we Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experi- mental Therapeutics and Reperfusion Injury, Harvard Institutes of Medicine, Brigham questioned whether MaR1 is produced during self-limited infec- and Women’s Hospital and Harvard Medical School, Boston, MA 02115 tions. We also investigated the further conversion of MaR1 in self- 1Current address: William Harvey Research Institute, Barts and The London School limited infectious exudates, identifying two novel MaR1 further of Medicine and Dentistry, Queen Mary University of London, London, U.K. metabolic products, and established their bioactions with human ORCIDs: 0000-0001-6328-3640 (J.D.); 0000-0003-1963-1585 (N.C.); 0000-0003- macrophages. 4627-8545 (C.N.S.). Received for publication May 12, 2016. Accepted for publication October 3, 2016. Materials and Methods This work was supported by National Institutes of Health Grants PO1 GM095467 and R01 GM38765 (to C.N.S.). Animals R.A.C., J.D., N.C., and C.N.S. conceived the study and designed the research; R.A.C., Male Friend Virus B mice (6–8 wk old) purchased from Charles River Lab- J.D., N.C., I.V., J.M.S., and I.R.R. performed experiments; R.A.C., J.D., N.C., I.V., oratories were fed ad libitum Laboratory Rodent Diet 20-5058 (Lab Diet; J.M.S., and I.R.R. analyzed data; R.A.C., J.D., N.C., I.V., J.M.S., and C.N.S. contributed Purina Mills). All animal experimental procedures were approved by the to manuscript preparation, and edited and revised the manuscript; and C.N.S. conceived Standing Committee on Animals of Harvard Medical School (protocol 02570) the overall research plan. and complied with institutional and National Institutes of Health guidelines. Address correspondence and reprint requests to Prof. Charles N. Serhan, Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Thera- Escherichia coli peritonitis peutics and Reperfusion Injury, Harvard Institutes of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, HIM 829, Boston, MA Mice were infected with live E. coli (serotype O6:K2:H1; 105 CFU). Mice 02115. E-mail address: [email protected] were euthanized and peritoneal lavages were collected at 0, 4, 12, 24, and The online version of this article contains supplemental material. 48 h after E. coli administration. Leukocyte numbers and differential Abbreviations used in this article: ECIS, electric cell-substrate impedance sensing; GPCR, counts were assessed using Turks solution and flow cytometry analysis. The rest of the lavages were placed in 2 vol of methanol and subjected to G protein–coupled receptor; LM, lipid mediator; LTB4, ; LX, ; MaR1, Maresin 1 (7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid); MRM, lipid mediator (LM) metabololipidomics. multiple reaction monitoring; MS-MS, tandem mass spectrometry; m/z, mass-to-charge ratio; PLS-DA, partial least squares-discriminant analysis; PMN, polymorphonuclear neu- Flow cytometry trophil; RT, retention time; Rv, ; SPM, specialized proresolving mediator. Murine peritoneal lavage cells were suspended in FACS buffer (5% BSA in Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Dulbecco’s phosphate buffered saline) and incubated with Fc block (15 min,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600837 2 MARESIN 1 BIOTRANSFORMATION AND ACTIONS

4˚C; BD Pharmingen) and then rat anti-mouse (from eBioscience) CD11b- matography–mass spectrometry analysis was performed with a Hewlett- PerCP/Cy5.5 (clone M1/70) and Ly6G-FITC (clone 1A8) (30 min, 4˚C) or Packard 593 mass-selective quadrupole detector and HP6890 GC system appropriate isotype controls. Staining was assessed using FACSDiva CantoII (Agilent column HP-5MS 30 m 3 0.25 mm 3 0.25 mm). Samples were (BD Biosciences) and analyzed using FlowJo (Tree Star). injected with hexane as the solvent, and the temperature program was ini- tiated at 150˚C and held for 2 min and reached 230˚C at 10 min (10˚C/min) PMN isolation and then 280˚C at 20 min (5˚C/min). Reference saturated fatty acid methyl esters carbons C –C gave the following retention times (R s; min): C , Peripheral blood human PMNs were purified using density-based Ficoll- 16 24 T 16 9.1; C18, 11.1; C20, 13.2; C22, 15.6; and C24, 18.1. These were used to Histopaque 1077-1 (Sigma) as previously described (9). PMNs were iso- calculate respective C values of 22-OH-MaR1. lated from human whole blood from healthy human volunteers giving informed consent under protocol 1999-P-001297 approved by the Partners Macrophage phagocytosis Human Research Committee. RBCs were lysed by hypotonic lysis. Experiments were conducted as previously described (9). Macrophages, Further metabolites preparation 7-d differentiated PBMCs with GM-CSF (10 ng/ml), were adhered (24 h) 4 +/+ on a 96-well plate (5 3 10 cells per well) in culture medium deprived of 14-oxo-MaR1. MaR1 (10 nM) suspended in PBS was added to isolated +/+ human macrophages (1 3 106 cells/ml) and conversion initiated by E. coli GM-CSF. Macrophages were incubated with vehicle (PBS ) alone or (50:1). Macrophages were incubated for 1 h at 37˚C. Samples were extracted compounds (MaR1, 14-oxo-MaR1, or 22-OH-MaR1) for 15 min at 37˚C, with C18 solid-phase extraction. Also, 14-oxo-MaR1 was produced by in- followed by fluorescent-labeled E. coli (BacLight; Molecular Probes, cubating MaR1 (10 mg) with Dess-Martin periodinane (10 mg/ml in Carlsbad, CA) at a 50:1 ratio (E. coli/macrophage) for 60 min. Plates were CH Cl ) for 1 h at room temperature. Reaction was stopped with NaHCO gently washed, extracellular fluorescence quenched by trypan blue, and 2 2 3 phagocytosis determined by measuring total fluorescence (excitation 495 (saturated) and Na2S2O3 (10%), and 14-oxo-MaR1 was extracted with MeOH. nm/emission 535 nm) using a fluorescent plate reader (Molecular Probes).

m Downloaded from 22-OH-MaR1. MaR1 (10 g) was dried down under N2 stream and sus- Real-time imaging pended in PBS+/+. Isolated human PMNs (50 3 106 cells/ml) were added and conversion initiated by zymosan (0.1 mg). PMNs were incubated for Macrophages were plated onto 8-well chamber slides (1 3 105 cells per 2+ 1 h at 37˚C. Samples were extracted with C18 solid-phase extraction. well in PBS ). Chamber slides were kept in a Stage Top Incubation system for microscopes equipped with a built-in digital gas mixer and Further metabolites isolation temperature regulator (TOKAI HIT model INUF-K14). MaR1 was added to macrophages (10 nM, 15 min at 37˚C) followed by BacLight Green- Biogenic 14-oxo-MaR1 and 22-OH-MaR1 were isolated by reversed phase labeled E. coli (5 3 106 CFU). Images were then acquired every 5 min for http://www.jimmunol.org/ HPLC. Liquid chromatographic analyses were performed using a Hewlett- 60 min (37˚C) with Keyence BZ-9000 (BIOREVO) inverted fluorescence Packard Series 1100 HPLC system equipped with a Poroshell column phase-contrast microscope (203 objective) equipped with a monochrome/ 3 3 m (100 mm 4.6 mm 2.7 m; Agilent) and a UV diode array detector. 14- color switching camera using BZ-II Viewer software (Keyence, Itasca, IL). oxo-MaR1 was eluted with a mobile phase consisting of methanol-water-acetic Green fluorescence intensity was quantified using BZ-II Analyzer. acid of 55:45:0.01 (v/v/v) that was ramped to 65:35:0.01 (v/v/v) over 2 min, then to 75:25:0.01 (v/v/v) over 18 min, and finally to 98:2:0.01 (v/v/v) over Electric cell-substrate impedance sensing 0.1 min. 22-OH-MaR1 was eluted with a mobile phase consisting of methanol- water-acetic acid of 50:50:0.01 (v/v/v) isocratic for 2 min, ramped to Ligand–receptor interactions were monitored by measuring impedance 57:43:0.01 (v/v/v) over 12.5 min, isocratic for 3 min, and then ramped to across cultured CHO-hBLT1 cell monolayers using an electric cell- 98:2:0.01 (v/v/v) over 5.5 min. Flow rate was maintained at 0.4 ml/min for substrate impedance sensing (ECIS; Applied Biophysics) (11) and essen- tially performed as in Krishnamoorthy et al. (12). In brief, CHO-hBLT1 both compounds. by guest on September 27, 2021 cells were plated in 8W10E+ ECIS chambers (1 3 105/well) and incubated Sample extraction and LM-metabololipidomics with MaR1 and 22-OH-MaR1 (0–100 nM) in Ham’s F-12 serum-free culture media for 10 min, followed by addition of 1 nM of LTB4 for Samples were processed as in Colas et al. (10). Internal labeled standards 10 min. d4-PGE2,d5-lipoxin (LX) A4,d8-5HETE, d4-leukotriene B4 (LTB4), and d5-resolvin (Rv) D2 (500 pg each) in 2 vol of ice-cold methanol were Statistical analysis added to each sample to facilitate quantification and sample recovery. Next, samples were held at 220˚C for 45 min to allow protein precipitation and Data are expressed as mean 6 SEM. Differences between groups were then centrifuged (1200 3 g, 4˚C, 10 min). Supernatants were collected and compared using Student t test. Criterion for statistical significance was p , brought to ,1 ml of methanol content with nitrogen gas. Samples were then 0.05. Partial least squares-discriminant analysis (PLS-DA) was performed placed into an automated extraction system (RapidTrace) and products using SIMCA 13.0.3 software (Umetrics) following mean centering and extracted. In brief solid-phase C18 cartridges were equilibrated with 3 ml of unit variance scaling of LM amount. methanol and 6 ml of H2O. Acidified samples (pH 3.5, HCl) were rapidly loaded onto the conditioned C18 columns and were washed with 4 ml of Results H2O. Next, 5 ml of hexane was added and products eluted with 9 ml of methyl formate. Products were brought to dryness and immediately sus- Endogenous production of novel metabolites derived from pended in methanol-water (50:50 v/v) for liquid chromatography tandem MaR1 during E. coli self-limited peritonitis mass spectrometry (MS-MS) automated injections. Because SPMs are temporally regulated during the time course of The liquid chromatography MS-MS system, a Shimadzu LC-20AD sterile inflammation (13) and during infection (14), we first in- HPLC and a Shimadzu SIL-20AC autoinjector (Shimadzu), paired with a QTrap 5500 (AB Sciex), was routinely used. A Poroshell column (100 mm 3 vestigated whether during self-resolving E. coli infections MaR1 4.6 mm 3 2.7 mm; Agilent) was kept in a column oven maintained at 50˚C was temporally regulated (Fig. 1). Mice were inoculated with E. coli (ThermaSphere), and LMs were eluted with a mobile phase consisting of at 105 CFU by i.p. injection that gave a self-limited inflammatory methanol-water-acetic acid of 50:50:0.01 (v/v/v) isocratic for 2 min that was response with PMN reaching maximum at 12 h, followed by decline ramped to 80:20:0.01 (v/v/v) over 9 min, kept isocratic for 3.5 min, and then until 48 h (Fig. 1A, inset). Peritoneal lavages were collected and ramped to 98:2:0.01 (v/v/v) for the next 0.1 min. This was subsequently maintained at 98:2:0.01 (v/v/v) for 5.4 min, and the flow rate was maintained LMs were investigated using LM metabololipidomics. MaR1 was at 0.5 ml/min. The QTrap 5500 was operated in negative ionization mode identified in these exudates in accordance to published criteria in- using scheduled multiple reaction monitoring (MRM) coupled with cluding RT and MS-MS fragmentation (4). MaR1 levels peaked 4 h information-dependent acquisition and an enhanced product ion scan. The after inoculation (2.2 6 0.4 pg/lavage) and gradually decreased to scheduled MRM window was 90 s. 24 h (Fig. 1A). In these exudates, we also identified two further Gas chromatography–mass spectrometry analysis metabolites of MaR1. The first gave a RT of 13.7 min and MS-MS spectrum consistent with 14-oxo-MaR1 with a characteristic parent Isolated 22-OH-MaR1 was taken to dryness using a stream of N2,suspended in MeOH (50 ml), and treated with excess ethereal diazomethane (45 min at ion of mass-to-charge ratio (m/z) 357 and daughter ion of m/z 248 room temperature), followed by N,O-bis(trimethylsilyl)trifluoroacetamide (Supplemental Table I). The second gave a RT of 10.0 min and a MS- treatment (10 min at room temperature; obtained from Supelco). Gas chro- MS fragmentation consistent with 22-OH-MaR1 with a characteristic The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Endogenous production of MaR1 and its biotransformation during E. coli self-limited peritonitis. Mice were inoculated with E. coli at 105 CFU by i.p. injection and euthanized at indicated interval. Peritoneal lavages were collected and LMs were investigated by LM metabololipidomics. (A) Time course of MaR1, 14-oxo-MaR1, and 22-OH-MaR1 levels at indicated interval. Results are expressed as pg/lavage and are mean 6 SEM. n = 3 for 4, 12, 24, and 48 h; n = 4 for 0 h. Peritoneal leukocytes were enumerated (inset). (B) MS-MS spectrum used for the identification of 22-OH-MaR1. (C) PLS- DA two-dimensional score plot. Gray ellipse denotes 95% confidence regions. (D) PLS-DA two-dimensional loading plot. LM colored in gray displayed a variable importance in projection coefficient $1. parent ion of m/z 375 and daughter ion of m/z 221 (Fig. 1B). Levels (Fig. 1D) demonstrated that the 4-h group (initiation) was charac- for 14-oxo-MaR1 peaked at 4 h (1.4 6 0.6 pg/lavage), whereas those terized by the presence of MaR1, 14-oxo-MaR1, LXB4,andRvD1, of 22-OH-MaR1 gradually increased during the initiation phase of whereas the 12-h group (peak of inflammation) had higher levels of the inflammatory response, reaching a maximum at 24 h (3.4 6 0.5 -triggered LXA4, RvD5, PGE2,andPGD2. These results es- pg/lavage). To assess the temporal regulation of MaR1 and its further tablish the MaR1 temporal profile during self-resolving E. coli in- metabolites in comparison with other SPMs and LMs, we assessed fections in relation to its further metabolites, SPM and eicosanoids. the LM profile during the course of the self-limited inflammatory response (Supplemental Fig. 1). Using PLS-DA (Fig. 1C), we in- Human leukocytes convert MaR1 to 14-oxo-MaR1 and terrogated the LM profile. Data-driven modeling such as PLS-DA is 22-OH-MaR1 useful to interrogate large biological data sets such as metabolomics. Having identified MaR1 metabolites in vivo during self-resolving Principal components were calculated from the systematic variation inflammation, we next questioned whether human leukocytes also in the data matrix consisting of the overall bioactive LM identified in converted this SPM to these novel products (Fig. 2). Given that each sample (15). Each of the intervals gave distinct clusters, with MaR1 is a macrophage product, we first assessed whether these the 4-h cluster giving a leftward shift within respect to the 0-h cluster. cells converted MaR1. Incubation of human macrophages with The 12- to 48-h clusters gave an upward and rightward shift within MaR1 (Fig. 2A, 2B) gave 14-oxo-MaR1 with RT of 13.7 min and respect to the 4-h cluster, with the 24- and 48-h clusters giving characteristic MS-MS fragmentation that matched those of en- similar coordinates on the score plot. Assessment of the loading plot dogenous 14-oxo-MaR1 produced in vivo in E. coli infectious 4 MARESIN 1 BIOTRANSFORMATION AND ACTIONS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Activated human leukocytes convert MaR1: physical properties of these new metabolites. Synthetic MaR1 was incubated with E. coli– activated human macrophages, Dess-Martin periodinane for 14-oxo-MaR1, or zymosan-activated PMN for 22-OH-MaR1 synthesis. (A) Representative chromatogram obtained by MRM of a parent ion (Q1) and a diagnostic daughter ion (Q3) (Q1/Q3 for MaR1 = 359/250, 14-oxo-MaR1 = 357/248, and 22- OH-MaR1 = 375/221). Representative MS-MS spectra used for identification of (B) MaR1, (C) 14-oxo-MaR1, and (D) 22-OH-MaR1 along with (E) characteristic in-phase UV chromophore for each compound. (A–E) Representative of three different incubations. exudates (Fig. 1, Supplemental Table I). These included the fol- selective reaction described by Dess and Martin (17) to convert lowing characteristic ions m/z 357 (M-H), 339 (M-H-H2O), 313 secondary alcohol groups into ketones (n = 3 preparations, data (M-H-CO2), 295 (M-H-CO2-H2O), 248, 245, 221, 217, and 177 not shown). Levels of 22-OH-MaR1 in these incubations were (221-CO2) (Fig. 2A, 2C). This product also gave a UV chromo- below limits in these macrophage incubations. We next investi- MeOH/Water phore with lmax at 338 nm (Fig. 2E) that is character- gated whether human neutrophils converted MaR1 to these novel istic of a triene double-bound system conjugated to a ketone (16). products. Incubation of human PMNs with MaR1 gave a peak at The presence of a ketone at carbon 14 was confirmed using a RT 10.0 min, and the MS-MS spectrum for the product under this The Journal of Immunology 5

peak gave ions with m/z 375 (M-H), 357 (M-H-H2O), 339 (M-H- 2H2O), 331 (M-H-CO2), 311 (M-H-CO2-H2O), 295 (M-H-CO2- 2H2O), 244 (262-H2O), 250, 244 (262-H2O), 226 (262-2H2O), 221, 177 (221-CO2), 159 (221-CO2-H2O), 141, 135 (153-H2O), and 113 that is consistent with 22-OH-MaR1 (Fig. 2A, 2D). This MeOH/Water product also gave a UV chromophore with lmax of 271 nm and two shoulders at 261 and 282 nm (Fig. 2E), characteristic of a conjugated triene double-bound system. To obtain further evidence for each of the proposed structures, we assessed the MS- MS spectrum of methyl ester and trimethyl silyl ether derivatives of 14-oxo-MaR1 and 22-OH-MaR1 (Supplemental Fig. 2). Real-time recording of human macrophage phagocytosis of E. coli: enhancement by MaR1 while it is biotransformed in vitro Given that MaR1 was identified in infectious exudates, we next questioned whether MaR1 regulated human macrophages phagocytosis of E. coli and whether MaR1 was further converted during this process (Fig. 3). Human macrophages were incubated with vehicle or MaR1

followed by the addition of fluorescently labeled E. coli and fluores- Downloaded from cence recorded every 5 min. MaR1 at 10 nM gave 38% increase in fluorescence, representing increased phagocytosis of E. coli,compared with macrophages incubated with vehicle at the 60-min interval (Fig. 3A, 3B). Using LM-metabololipidomics, we investigated the kinetics of conversion of MaR1 to its further metabolites in these

incubations. Macrophages were incubated with 10 nM of MaR1. At http://www.jimmunol.org/ 5 min after E. coli addition, MaR1 levels started to decrease with a 36% reduction at the 60-min interval (Fig. 3C). 14-oxo-MaR1 was present at the initial time point t = 0 with levels equivalent to 6.7 pg/ 2 3 106 cells, and its level increased to 53.0 pg/2 3 106 cells after 60 min. Levels of 22-OH-MaR1 were below the limit of detection in these incubations. Notably, E. coli incubated with MaR1 did not yield either 14-oxo-MaR1 or 22-OH-MaR1, pointing to leukocytes as the main source of these products in these ex vivo conditions. These results demonstrate that MaR1 potently regulates the clearance of bacteria by by guest on September 27, 2021 upregulating macrophage phagocytosis, and that these macrophages actively convert MaR1 to 14-oxo-MaR1 during this process. 14-oxo-MaR1 and 22-OH-MaR1 retain biological activity with human macrophages Having identified these novel products in infectious exudates and with human leukocytes, we next tested the biological activities of the MaR1 further metabolites. We investigated whether these products stimulated macrophage phagocytosis of E. coli and their relative actions com- pared with MaR1 (Fig. 4). At a concentration as low as 0.1 pM, MaR1 and its two further metabolites had similar potency and in- creased E. coli phagocytosis by ∼37% compared with macrophages incubated with vehicle. At higher concentrations, 14-oxo-MaR1 was significantly less potent, increasing phagocytosis by only ∼30% at 10 pM, whereas MaR1 increased E. coli phagocytosis ∼93% at 10 pM (Fig. 4B). These results indicate that both further metabolites retain biological actions associated with the parent molecule. FIGURE 3. MaR1 enhances human macrophage phagocytosis of E. coli and was biotransformed to new metabolites. Human macrophages were MaR1 and 22-OH-MaR1 interact with recombinant human 3 5 plated (1 10 cells per well in an eight-well chamber slide) and incu- BLT1 receptor bated with MaR1 (10 nM, black) or vehicle control (white), followed by addition of labeled E. coli to initiate phagocytosis. Fluorescent images Given that the LTB4 receptor (BLT1) is critical in the propagation of were recorded every 5 min for 60 min. (A) Increase in phagocytosis (0–60 inflammation, we next investigated whether MaR1 could regulate min, n = 3). In each experiment, four fields (320) per condition (per well) were recorded. Results are mean fluorescence intensity (MFI) of phago- cytized E. coli of four fields per well from one representative experiment. samples were subjected to LM metabololipidomics. Results are expressed as (Inset) MFI of phagocytized E. coli at 60 min, mean 6 SEM. *p , 0.05, % of time 0 for MaR1 and pg/2 3 106 cells for its further metabolites; mean 6 MaR1 versus vehicle. (B) Representative fluorescent images from one SEM. n = 5 for 0, 10, 15, 30, and 60 min; n = 4 for 5 min. Inset, E. coli experiment. Scale bars, 100 mm. (C) Human macrophages were plated incubated with MaR1 (10 nM; 60 min, 37˚C). Results are expressed as % (2 3 106 cells per well in a six-well plate) stimulated with E. coli and of time 0 for MaR1 and pg/2 3 106 cells for its further metabolites [14- incubated with MaR1 (10 nM; 60 min, 37˚C). At the indicated time points, oxo-MaR1 (A) and 22-OH-MaR1 (B)]; mean 6 SEM. n =3. 6 MARESIN 1 BIOTRANSFORMATION AND ACTIONS

FIGURE 4. MaR1 new metabolites retained biological activity with human macrophages. Human macrophages were plated (5 3 104 cells per well in a 96-well plate) and incubated with vehicle, MaR1, (A) 22-OH-MaR1, or (B) 14-oxo-MaR1 (0.1 pM to 1 nM) (15 min, 37˚C) followed by addition of fluorescent-labeled E. coli (1 h, 37˚C) (see Materials and Methods). Results are mean 6 SEM of n = 7 cell preparations, d = 3. *p , 0.05, ***p , 0.001 versus vehicle; †p , 0.05, †††p , 0.001 versus MaR1.

the response of this receptor to LTB4.WeusedanECISsystemto derived from MaR1, namely 14-oxo-MaR1 and 22-OH-MaR1. monitor impedance change upon ligand activation of the receptor. The production of these MaR1-derived molecules was also tem- Downloaded from ECIS allows for real-time monitoring of adherent cell shape change porally regulated, with 14-oxo-MaR1 levels reaching a maximum by measuring impedance across the electrodes. When cells are at 4 h and 22-OH-MaR1 levels reaching a maximum at 24 h. In- added to the ECIS arrays and attach to the electrodes, they act as cubation of human macrophages with MaR1 gave 14-oxo-MaR1, insulators increasing the impedance. The current is impeded in a whereas the main product with human neutrophils was 22-OH- manner related to the number of cells covering the electrode, the MaR1. Each of these novel molecules retained at low concentra-

morphology of the cells, and the nature of the cell attachment. When tions the bioactions of their parent SPM, that is, MaR1, with human http://www.jimmunol.org/ cells are stimulated to change their function, the accompanying macrophages. Notably, 22-OH-MaR1 gave a biphasic dose response changes in cell morphology alter the impedance. The data generated at upregulating E. coli phagocytosis by human macrophages. This are impedance versus time. Given that ligand engagement to cognate response is suggestive of the engagement of a high- and a low-affinity G protein–coupled receptor (GPCR) lead to characteristic shape receptor by 22-OH-MaR1 as observed for other proresolving medi- change, this provides an ideal system for measuring ligand–GPCR ators including RvD1 (19). In addition, both MaR1 and its metabolite interaction in vitro. In CHO cells transfected with hBLT1, LTB4 (0– 22-OH-MaR1 act at the hBLT1 receptor and impact LTB4 signaling. 100 nM) initiated impedance change with EC50 ∼0.23 nM (Fig. 5A). These results identify novel molecules and establish novel functions LTB4 activation of CHO-hBLT1 cells was abolished when cells were for the MaR1 bioactive metabolome. incubated with a BLT1 inhibitor U75302 (18) (Fig. 5B). We next Because SPMs orchestrate the resolution of inflammation, it is by guest on September 27, 2021 investigated the actions of MaR1 on CHO-hBLT1 cells. MaR1 dose- essential to gain a detailed understanding of their temporal regu- dependently increased impedance to ∼30 V (Fig.5C).Toconfirm lation during disease progression and their relationship with the that these responses were hBLT1 mediated, we incubated cells with classical proinflammatory mediators (i.e., leukotrienes and PGs). U75302 (0 nM to 1 mM) for 15 min before the addition of 100 nM MaR1 is a mediator from the 12-lipoxygenase pathway originally of MaR1 (Fig. 5D). U75302 dose-dependently inhibited the change identified in macrophages (4, 20). MaR1 biosynthesis at early time in impedance elicited by MaR1. We next questioned whether MaR1 points could result from activation of resident macrophages (21) regulated hBLT1 response to LTB4. Addition of MaR1 (1–100 nM, as well as involvement of transcellular mechanisms with leuko- 15 min) before LTB4 (1 nM) decreased LTB4-elicited response from cytes and as reported by Abdulnour et al. (8). The MaR1 ∼40 to ∼10 V,anactionthatwasfoundtobedosedependent. pathway is temporally regulated during self-limited sterile in- Because 22-OH-MaR retained the bioaction in phagocytosis, we flammation, with levels of its pathway marker 14-HDHA reaching tested this metabolite with CHO-hBLT1 cells and found that 22-OH- a maximum during the resolution phase (4). MaR1 also enhances MaR1 (1–100 nM) gave dose-dependent impedance changes, with macrophage phagocytosis of and ∼40 V increase at 100 nM (Fig. 5F). Incubation of the 22-OH-MaR1 Aggregatibacter actinomycetemcomitans (22). MaR1 may also be with CHO-hBLT1 cells also led to a dose-dependent inhibition of produced by eosinophils given that these cells express the murine LTB4-elicited impedance change (Fig. 5G). Notably, in CHO cell homolog of initiating enzyme in the MaR1 metabolome; in ad- incubation with MaR1 (10 nM, 30 min), we did not identify the dition, these leukocytes are also known to produce SPMs in- MaR1 further metabolites or a decrease in MaR1 levels, suggesting cluding , RvD5, and LXA4 during sterile peritonitis that the responses observed in the ECIS incubation were in response (23). Future studies will need to establish their trafficking during to the products added and not the local metabolism of MaR1 by the self-limited bacterial infections as well as their contributions to CHO cells (n = 3). These results demonstrated that both MaR1 and the MaR1 metabolome. 22-OH-MaR1 are partial agonists to hBLT1 and antagonize the MaR1 also potently regulates host responses to bacteria, stim- activation of hBLT1 by the potent proinflammatory mediator LTB4 ulating phagocytosis and killing of oral pathogens by human (Fig. 6). leukocytes (22). In this report, we investigated the temporal reg- ulation of MaR1 during self-limited infections. In self-resolving Discussion E. coli peritonitis, we identified MaR1 at biologically active In this report, we investigated the metabolism of MaR1, a potent concentrations (2.2 6 0.4 pg/lavage) that peaked at 4 h and sub- proresolving and antinociceptive bioactive mediator, in the context sequently decreased (Fig. 1). of infectious inflammation. MaR1 was temporally regulated in Autacoids are by definition locally produced, act locally, and exudates from self-resolving E. coli peritonitis. In these exudates are rapidly enzymatically inactivated (24). LTB4 is metabolized we also identified and elucidated structures of two new molecules by 12-hydroxydehydrogenase to 12-oxo-LTB4 (25) or by P450 to The Journal of Immunology 7

human and murine leukocytes (33). In other instances, SPM con- version may result in a partial or complete loss of bioactivity, as observed for 16-oxo-RvD2 (30), 10,11-dihydro-RvE1, 18-oxo-RvE1, and 20-carboxy-RvE1 (34), and 8-oxo-RvD1 and 17-oxo-RvD1 (29). In this work, we investigated further conversion of MaR1 during bacterial infections. In self-resolving exudates, we identified two new MaR1-derived molecules. The production of 14-oxo-MaR1 was also found to be temporally regulated with human macrophages during E. coli phagocytosis. Assessment of the biological actions of this mol- ecule with human macrophages demonstrated that 14-oxo-MaR1 was less potent than MaR1 at promoting macrophage responses to E. coli, thereby suggesting that the conversion of MaR1 to 14-oxo-MaR1 in macrophages is an inactivation route for this potent SPM. Conversely, 22-OH-MaR1, which reached a maximum during the resolution phase of the inflammatory response and was the main product with human neutrophils, was found to display similar potencies at regu- lating human macrophage responses to E. coli (Fig. 3). These suggest that conversion to 14-oxo-MaR1 is an inactivation in the MaR1

metabolome, whereas conversion to 22-OH-MaR1 does not lead to Downloaded from inactivation. The latter product may in turn undergo further conver- sion to yield a dicarboxylate, in line with the LTB4 metabolome where 20-OH-LTB4 is further converted to 22-COOH-LTB4,whichis inactive (35). Further studies will need to determine whether 22-OH- MaR1 is also converted to this product and whether this indeed is an

inactivation route. http://www.jimmunol.org/ SPMs are by definition potent in resolving inflammatory processes. Their actions are mediated by GPCRs. For example, ALX receptor mediates the actions of LXA4 and RvD1 (12) and GPR18 the actions of RvD2 (36). In addition to activating their cognate receptors, SPMs are also partial agonists or antagonists to receptors for proinflammatory LM. For example, RvE1 is a partial agonist to hBLT1, one of the LTB4 receptors, thereby regulating the actions of this potent inflammation-initiating LM (37). In this study, we found that MaR1 and its further product 22-OH-MaR1 by guest on September 27, 2021 are both partial agonists to hBLT1 and regulate the activation of this receptor by its ligand LTB4. Thus, in addition to increasing the clearance of bacteria (Fig. 4), MaR1 also regulates BLT1 activa- tion by its cognate ligand, the potent chemoattractant LTB4,a biological action retained by the MaR1 further metabolite 22-OH- MaR1. Thus, these experiments identify novel mechanisms for the regulation of inflammatory responses by the MaR1 metab- olome in governing the responses within the resolution of acute FIGURE 5. MaR1 and 22-OH-MaR1 are partial agonists of human inflammation. recombinant BLT1 receptor in vitro. Ligand–receptor interactions were In conclusion, in this study, using LM profiling, we established the monitored by measuring impedance changes in cultured CHO-hBLT1 cells temporal regulation of MaR1 in self-resolving infectious exudates. In 5 using an ECIS system (Applied Biophysics). Cells were plated (1 3 10 these exudates we also identified two novel MaR1-derived products, A per well in an eight-well chamber slide) and ( ) incubated 10 min with 14-oxo-MaR1 and 22-hydroxy-MaR1, and conducted their structure LTB (0–100 nM). (B) Cells were incubated with U75302 (0–1 mM), a 4 elucidation with the proposed structure being 7R-hydroxy,14-oxo BLT1 inhibitor, for 10 min before the addition of 1 nM of LTB4 for 10 min. (C) Cells were incubated 10 min with MaR1 (0–100 nM). Cells were in- docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid and 7R,14S,22- cubated with (D) U75302 (0–1 mM) or (E) MaR1 (0–100 nM) for 10 min trihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid, respec- before the addition of (D) 100 nM of MaR1 or (E) 1 nM of LTB4 for tively. The production of these molecules was also established with 10 min. (F) Cells were incubated 10 min with 22-OH-MaR1 (0–100 nM). human phagocytes, where 14-oxo-MaR1 was the main product Cells were incubated with (G) 22-OH-MaR1 (0–100 nM) for 10 min fol- produced by human macrophages, whereas 22-hydroxy-MaR1 was lowed by addition of 1 nM of LTB4 for 10 min. Results are mean of six primarily produced by human neutrophils. Assessment of the bio- separate tracings from n = 3 independent experiments. actions of these products demonstrated that, although 22-hydroxy- MaR1 retained the bioactions on human macrophages of its parent SPM, 14-oxo-MaR1 was less potent. MaR1 and 22-hydroxy-MaR1 20-hydroxy-LTB4 (26). PGE2 is converted by 15-PG dehydroge- were partial agonists of the recombinant hBLT1. Together these nase to 15-oxo-PGE2 (27), which is further converted by PG reduc- findings provide novel structures for developing therapeutics in tase to 13,14-dihydro-PGE2 (28). RvE1, LXA4, RvD1, AT-RvD1, bacterial infection focusing on further conversion pathways and cross and RvD2 (29–31) are also substrates for 15-PG dehydrogenase, talk between inflammatory and resolution pathways. They afford the eicosanoid oxidoreductase (32). SPM further conversion may novel leads to understanding local regulation of distinct SPM yield products that retain the proresolving actions of the parent SPM metabolomes, furnishing new insights into mechanisms leading to as, for example, 22-OH-protectin D1 that also potently regulates both disease, and establish the basis for design of MaR1 mimetics that 8 MARESIN 1 BIOTRANSFORMATION AND ACTIONS

FIGURE 6. MaR1 further metab- olome and biological actions. Downloaded from http://www.jimmunol.org/

9. Dalli, J., and C. N. Serhan. 2012. Specific lipid mediator signatures of human could resist further enzymatic conversion to give prolonged MaR1 by guest on September 27, 2021 phagocytes: microparticles stimulate macrophage efferocytosis and pro- bioactions. resolving mediators. Blood 120: e60–e72. 10. Colas, R. A., M. Shinohara, J. Dalli, N. Chiang, and C. N. Serhan. 2014. Identification and signature profiles for pro-resolving and inflammatory Acknowledgments lipid mediators in human tissue. Am. J. Physiol. Cell Physiol. 307: C39– We thank Mary Halm Small for expert assistance in manuscript preparation C54. and Chien-Yee C. Cheng for technical assistance. 11. Peters, M. F., and C. W. Scott. 2009. Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity. J. Biomol. Screen. 14: 246–255. 12. Krishnamoorthy, S., A. Recchiuti, N. Chiang, S. Yacoubian, C. H. Lee, R. Yang, Disclosures N. A. Petasis, and C. N. Serhan. 2010. Resolvin D1 binds human phagocytes The authors have no financial conflicts of interest. with evidence for proresolving receptors. Proc. Natl. Acad. Sci. USA 107: 1660– 1665. 13. Dalli, J., J. W. Winkler, R. A. Colas, H. Arnardottir, C. Y. C. Cheng, N. Chiang, References N. A. Petasis, and C. N. Serhan. 2013. Resolvin D3 and aspirin-triggered resolvin D3 are potent immunoresolvents. Chem. Biol. 20: 188–201. 1. Cotran, R. S., V. Kumar, and T. Collins, eds. 1999. Robbins Pathologic Basis of 14. Chiang, N., G. Fredman, F. Ba¨ckhed, S. F. Oh, T. Vickery, B. A. Schmidt, and Disease. W.B. Saunders Co., Philadelphia. C. N. Serhan. 2012. Infection regulates pro-resolving mediators that lower anti- 2. Tabas, I., and C. K. Glass. 2013. Anti-inflammatory therapy in chronic disease: biotic requirements. Nature 484: 524–528. challenges and opportunities. Science 339: 166–172. 15. Janes, K. A., and M. B. Yaffe. 2006. Data-driven modelling of signal- 3. Serhan, C. N., N. Chiang, and J. Dalli. 2015. The resolution code of acute in- transduction networks. Nat. Rev. Mol. Cell Biol. 7: 820–828. flammation: novel pro-resolving lipid mediators in resolution. Semin. Immunol. 16. Wainwright, S. L., and W. S. Powell. 1991. Mechanism for the formation of 27: 200–215. dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 4. Serhan, C. N., R. Yang, K. Martinod, K. Kasuga, P. S. Pillai, T. F. Porter, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid to 12-oxo intermediates. J. Biol. S. F. Oh, and M. Spite. 2009. Maresins: novel macrophage mediators with potent Chem. 266: 20899–20906. antiinflammatory and proresolving actions. J. Exp. Med. 206: 15–23. 17. Dess, D. B., and J. C. Martin. 1983. Readily accessible 12-I-5 oxidant for the 5. Serhan, C. N., J. Dalli, S. Karamnov, A. Choi, C. K. Park, Z. Z. Xu, R. R. Ji, conversion of primary and secondary alcohols to aldehydes and ketones. J. Org. M. Zhu, and N. A. Petasis. 2012. Macrophage proresolving mediator maresin 1 Chem. 48: 4155–4156. stimulates tissue regeneration and controls pain. FASEB J. 26: 1755–1765. 18. Falcone, R. C., and D. Aharony. 1990. Modulation of ligand binding to leuko- 6. Dalli, J., M. Zhu, N. A. Vlasenko, B. Deng, J. Z. Haeggstro¨m, N. A. Petasis, and triene B4 receptors on guinea pig lung membranes by sulfhydryl modifying C. N. Serhan. 2013. The novel 13S,14S-epoxy-maresin is converted by human reagents. J. Pharmacol. Exp. Ther. 255: 565–571. macrophages to maresin 1 (MaR1), inhibits hydrolase (LTA4H), 19. Orr, S. K., R. A. Colas, J. Dalli, N. Chiang, and C. N. Serhan. 2015. Proresolving actions of a new resolvin D1 analog mimetic qualifies as an immunoresolvent. and shifts macrophage phenotype. FASEB J. 27: 2573–2583. Am. J. Physiol. Lung Cell. Mol. Physiol. 308: L904–L911. 7. Marcon, R., A. F. Bento, R. C. Dutra, M. A. Bicca, D. F. Leite, and J. B. Calixto. 20. Deng, B., C. W. Wang, H. H. Arnardottir, Y. Li, C. Y. Cheng, J. Dalli, and 2013. Maresin 1, a proresolving lipid mediator derived from omega-3 polyun- C. N. Serhan. 2014. Maresin biosynthesis and identification of maresin 2, a new saturated fatty acids, exerts protective actions in murine models of colitis. anti-inflammatory and pro-resolving mediator from human macrophages. PLoS J. Immunol. 191: 4288–4298. One 9: e102362. 8. Abdulnour, R. E., J. Dalli, J. K. Colby, N. Krishnamoorthy, J. Y. Timmons, 21. Davies, L. C., M. Rosas, S. J. Jenkins, C. T. Liao, M. J. Scurr, F. Brombacher, S. H. Tan, R. A. Colas, N. A. Petasis, C. N. Serhan, and B. D. Levy. 2014. D. J. Fraser, J. E. Allen, S. A. Jones, and P. R. Taylor. 2013. Distinct bone Maresin 1 biosynthesis during -neutrophil interactions is organ-protective. marrow-derived and tissue-resident macrophage lineages proliferate at key Proc. Natl. Acad. Sci. USA 111: 16526–16531. stages during inflammation. Nat. Commun. 4: 1886. The Journal of Immunology 9

22. Wang, C. W., R. A. Colas, J. Dalli, H. H. Arnardottir, D. Nguyen, H. Hasturk, 30. Cla`ria, J., J. Dalli, S. Yacoubian, F. Gao, and C. N. Serhan. 2012. Resolvin D1 N. Chiang, T. E. Van Dyke, and C. N. Serhan. 2015. Maresin 1 biosynthesis and and resolvin D2 govern local inflammatory tone in obese fat. J. Immunol. 189: proresolving anti-infective functions with human-localized aggressive peri- 2597–2605. odontitis leukocytes. Infect. Immun. 84: 658–665. 31. Arita, M., S. F. Oh, T. Chonan, S. Hong, S. Elangovan, Y. P. Sun, J. Uddin, 23. Yamada, T., Y. Tani, H. Nakanishi, R. Taguchi, M. Arita, and H. Arai. 2011. N. A. Petasis, and C. N. Serhan. 2006. Metabolic inactivation of resolvin E1 and Eosinophils promote resolution of acute peritonitis by producing proresolving stabilization of its anti-inflammatory actions. J. Biol. Chem. 281: 22847–22854. mediators in mice. FASEB J. 25: 561–568. 32. Clish, C. B., B. D. Levy, N. Chiang, H.-H. Tai, and C. N. Serhan. 2000. Oxi- 24. Majno, G., and I. Joris. 2004. Cells, Tissues, and Disease: Principles of General doreductases in lipoxin A4 metabolic inactivation: a novel role for 15- Pathology. Oxford University Press, New York. onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in 25. Yokomizo, T., T. Izumi, T. Takahashi, T. Kasama, Y. Kobayashi, F. Sato, inflammation. J. Biol. Chem. 275: 25372–25380. Y. Taketani, and T. Shimizu. 1993. Enzymatic inactivation of leukotriene B4 by a 33. Tungen, J. E., M. Aursnes, A. Vik, S. Ramon, R. A. Colas, J. Dalli, C. N. Serhan, novel enzyme found in the porcine kidney. Purification and properties of leu- and T. V. Hansen. 2014. Synthesis and anti-inflammatory and pro-resolving kotriene B4 12-hydroxydehydrogenase. J. Biol. Chem. 268: 18128–18135. activities of 22-OH-PD1, a monohydroxylated metabolite of protectin D1. 26. Soberman, R. J., T. W. Harper, R. C. Murphy, and K. F. Austen. 1985. Identification J. Nat. Prod. 77: 2241–2247. and functional characterization of leukotriene B4 20-hydroxylase of human 34. Hong, S., T. F. Porter, Y. Lu, S. F. Oh, P. S. Pillai, and C. N. Serhan. 2008. polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 82: 2292–2295. Resolvin E1 metabolome in local inactivation during inflammation-resolution. 27. Ensor, C. M., J. Y. Yang, R. T. Okita, and H. H. Tai. 1990. Cloning and sequence J. Immunol. 180: 3512–3519. analysis of the cDNA for human placental NAD(+)-dependent 15-hydroxy- 35. Hansson, G., J. A. Lindgren, S. E. Dahle´n, P. Hedqvist, and B. Samuelsson. prostaglandin dehydrogenase. J. Biol. Chem. 265: 14888–14891. 1981. Identification and biological activity of novel omega-oxidized metabolites 28. Ensor, C. M., H. Zhang, and H. H. Tai. 1998. Purification, cDNA cloning and of leukotriene B4 from human leukocytes. FEBS Lett. 130: 107–112. expression of 15-oxoprostaglandin 13-reductase from pig lung. Biochem. J. 330: 36. Chiang, N., J. Dalli, R. A. Colas, and C. N. Serhan. 2015. Identification of 103–108. resolvin D2 receptor mediating resolution of infections and organ protection. 29. Sun, Y. P., S. F. Oh, J. Uddin, R. Yang, K. Gotlinger, E. Campbell, S. P. Colgan, J. Exp. Med. 212: 1203–1217. N. A. Petasis, and C. N. Serhan. 2007. Resolvin D1 and its aspirin-triggered 17R 37. Arita, M., T. Ohira, Y. P. Sun, S. Elangovan, N. Chiang, and C. N. Serhan. 2007. epimer. Stereochemical assignments, anti-inflammatory properties, and enzy- Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and matic inactivation. J. Biol. Chem. 282: 9323–9334. ChemR23 to regulate inflammation. J. Immunol. 178: 3912–3917. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021