Maresin 1 biosynthesis during platelet–neutrophil interactions is organ-protective Raja-Elie E. Abdulnoura,1, Jesmond Dallib,1, Jennifer K. Colbya, Nandini Krishnamoorthya, Jack Y. Timmonsa, Sook Hwa Tana, Romain A. Colasb, Nicos A. Petasisc, Charles N. Serhanb, and Bruce D. Levya,b,2 aPulmonary and Critical Care Medicine and bCenter for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and cDepartment of Chemistry, University of Southern California, Los Angeles, CA 90089 Edited by Derek William Gilroy, University College London, London, United Kingdom, and accepted by the Editorial Board October 10, 2014 (received for review April 17, 2014) Unregulated acute inflammation can lead to collateral tissue injury 12-lipoxygenase that may be capable of generating the 13S,14S- in vital organs, such as the lung during the acute respiratory distress epoxy-maresin intermediate and participating in MaR1 production syndrome. In response to tissue injury, circulating platelet–neutro- at sites of vascular inflammation. Here, we provide evidence for phil aggregates form to augment neutrophil tissue entry. These a MaR1 biosynthetic route during platelet–neutrophil interactions early cellular events in acute inflammation are pivotal to timely that is operative in vivo in a murine model of ARDS to restrain resolution by mechanisms that remain to be elucidated. Here, we inflammation and restore homeostasis of the injured lung. identified a previously undescribed biosynthetic route during hu- man platelet–neutrophil interactions for the proresolving mediator Results maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z- To determine if platelets can participate in vascular MaR1 bio- hexaenoic acid). Docosahexaenoic acid was converted by platelet synthesis, platelets were isolated from fresh human whole blood, 12-lipoxygenase to 13S,14S-epoxy-maresin, which was further trans- then incubated with d5-DHA in the presence or absence of au- formed by neutrophils to MaR1. In a murine model of acute respira- tologous peripheral blood neutrophils, and activated with either tory distress syndrome, lipid mediator metabololipidomics uncovered platelet-activating factor (PAF) or the platelet-specific agonist MaR1 generation in vivo in a temporally regulated manner. Early thrombin. MaR1 biosynthesis was assessed by lipid mediator MEDICAL SCIENCES MaR1 production was dependent on platelet–neutrophil interactions, (LM) metabololipidomics (SI Methods). Small amounts of MaR1 and intravascular MaR1 was organ-protective, leading to decreased – lung neutrophils, edema, tissue hypoxia, and prophlogistic mediators. were identified in platelet d5-DHA incubations by matching MS/ A B Together, these findings identify a transcellular route for intravascular MS fragmentation patterns and retention times (Fig. 1 and ). maresin 1 biosynthesis via platelet–neutrophil interactions that regu- Coincubation of platelets with neutrophils in the presence of lates the extent of lung inflammation. PAF markedly increased MaR1 generation (Fig. 1A) in a neu- trophil-dependent manner (Fig. 1C); platelet activation with maresin | platelet | inflammation | lung | resolution PAF (Fig. 1A) or thrombin (Fig. 1C) or increased platelet ne critical function of acute inflammatory responses is de- Significance Olivery of leukocytes to sites of tissue injury to protect the host and restore homeostasis (1, 2). Activated vascular endo- Neutrophil accumulation is fundamental to acute inflammation. In thelial cells recruit leukocytes by direct interaction (primary response to tissue injury, circulating neutrophil–platelet aggre- capture) or via platelets (secondary capture) that engage circu- gates (N-PAs) form for secondary capture. Counterregulation of lating leukocytes rapidly after tissue injury (3, 4). If excessive, acute inflammatory processes by specialized proresolving medi- leukocyte recruitment and activation can injure healthy host ators is essential to mitigate collateral injury to healthy bystander bystander tissues and cause organ damage (2, 5)—a pathobiology tissue. Here, we identified a biosynthetic route in human platelets common to several diseases, including the acute respiratory for the proresolving mediator maresin 1 (MaR1) that is amplified distress syndrome (ARDS), a devastating condition of acute lung during platelet–neutrophil interactions. In a self-resolving murine inflammation with excess morbidity and mortality and without model of acute lung injury, N-PAs rapidly formed and a MaR1 available medical therapy (6). Platelet–neutrophil interactions counterregulatory circuit was engaged to restrain N-PAs and are integral to the early inflammatory response in ARDS (7, 8). acute inflammation and restore homeostasis of the injured lung. In health, acute inflammatory responses are terminated by Author contributions: R.E.A., J.D., C.N.S., and B.D.L. designed research; R.E.A., J.D., J.K.C., specialized proresolving mediators (SPMs) that restrain the in- N.K., J.Y.T., S.H.T., R.A.C., C.N.S., and B.D.L. performed research; N.A.P. contributed new flammatory response and signal for resolution (2). Early cellular reagents/analytic tools; R.E.A., J.D., N.K., R.A.C., C.N.S., and B.D.L. analyzed data; and and biochemical events in acute inflammation are pivotal to R.E.A., J.D., J.K.C., N.K., J.Y.T., S.H.T., R.A.C., N.A.P., C.N.S., and B.D.L. wrote the paper. timely resolution (9, 10). A new family of SPMs of macrophage Conflict of interest statement: C.N.S. is an inventor on patents (resolvins) assigned to origin was recently uncovered in murine peritonitis and human Brigham and Women’s Hospital (BWH) and licensed to Resolvyx Pharmaceuticals. C.N.S. was the scientific founder of Resolvyx Pharmaceuticals and owns founder stock in the macrophages and coined maresins (macrophage mediators in re- company. C.N.S.’s interests were reviewed and are managed by the Brigham and solving inflammation) (11). Maresins are biosynthesized via initial Women’s Hospital and Partners HealthCare in accordance with their conflict of in- lipoxygenation and molecular oxygen insertion at the carbon 14 terest policies. B.D.L. is an inventor on patents (resolvins) assigned to BWH and licensed to position of docosahexaenoic acid (DHA) followed by epoxi- Resolvyx Pharmaceuticals. B.D.L.’s interests were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict dation and oxygen incorporation at the carbon 7 position from of interest policies. amoleculeofH2O (11). The complete stereochemistry of maresin This article is a PNAS Direct Submission. D.W.G. is a guest editor invited by the Editorial 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hex- Board. aenoic acid) (12) and its biosynthesis via a 13S,14S-epoxy-maresin 1R.E.A. and J.D. contributed equally to this work. intermediate (13S,14S-epoxy-docosa-4Z,7Z,9E,11E,16Z,19Z-hex- 2To whom correspondence should be addressed. Email: [email protected]. aenoic acid) (13) were recently established. In addition to mac- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rophages, human and murine platelets contain arachidonate 1073/pnas.1407123111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1407123111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 ACB d -MaR1 364 5 100 175 * * PA F 221 Thrombin 100 D 250 H+ 141 Neutrophils and Platelets D D H+ 150 D OH OH D H+ 113 neutrophils) 125 * 6 COO- 100 75 d5-MaR1 50 Platelets 302 m/z 346 Relative Abundance (%) Relative Abundance (%) 364>250 25 221 250 284 141 177 320 364>250 113 206 232 328 0 (pg/5x10 production-MaR1 0 5 0 2 4 6 8 101214161820 22 100 140 180 220 260 300 340 380 d 1:1 1:5 1:10 Time (min) m/z, Da Neutrophil to Platelet ratio DEF G 359 100 100 100 x 5.0 4 Neutrophils MaR1 Neutrophils MaR1 plus plus 221 141 * O 250 H+ + 3 OOH 109 H COOH OH OH COO 315 14S-HpDHA 13S,14S-epoxy-maresin H+113 2 297 COO- 323 331 1 Relative Abundance (%) Abundance Relative MaR1 (pg/ml) MaR1 Relative Abundance (%) Relative Abundance (%) 161.2 277 341 109 141 221232 113 177 279 206 250 0 0 0 2.0 4.0 6.0 8.0 10.012.014.0 16.018.0 20.0 22.0 0 2.0 4.0 6.0 8.0 10.012.014.0 16.018.0 20.0 22.0 100 140 180 220 260 300 340 380 IgG Psel-Ab Time (min) Time (min) m/z, Da Fig. 1. MaR1 is produced by N-PAs via transcellular biosynthesis. (A–C) MaR1 production by isolated human peripheral blood platelets incubated with d5- DHA before incubation with or without neutrophils in the presence of PAF or thrombin. Levels of d5-MaR1 were assessed by liquid chromatography (LC)–MS/MS metabololipidomics. (A) Representative multiple reaction monitoring (MRM) chromatograms (m/z = 364–250) and (B) MS/MS spectrum from PAF-treated samples used for the identification of d5-MaR1. (C)d5-MaR1 levels in incubations with different neutrophil-to-platelet ratios. Results for A and B are rep- resentative of n = 3 healthy donor blood isolations. Results for C are mean ± SEM for n = 3 healthy donor blood isolations. *P < 0.05. (D–F) Human peripheral blood neutrophils were isolated by density centrifugation. Cells were suspended at 1 × 108 in PBS with 10 mg/mL BSA, then incubated with (D)10μM 14HpDHA or (E)10μM 13S,14S-eMaR and 0.1 mg of serum-treated zymosan (15 min, 37 °C, pH 7.45). (F) Representative MS/MS spectrum used for the identification of MaR1 (7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid). (G) Levels of MaR1 in fresh human whole blood pretreated with anti-human P-selectin or isotype control antibody after treatment with PAF (500 nM).