Contributions of the Three CYP1 Monooxygenases to Pro-Inflammatory and Inflammation-Resolution Lipid Mediator Pathways This information is current as of September 26, 2021. Senad Divanovic, Jesmond Dalli, Lucia F. Jorge-Nebert, Leah M. Flick, Marina Gálvez-Peralta, Nicholas D. Boespflug, Traci E. Stankiewicz, Jonathan M. Fitzgerald, Maheshika Somarathna, Christopher L. Karp, Charles N. Serhan and Daniel W. Nebert Downloaded from J Immunol 2013; 191:3347-3357; Prepublished online 16 August 2013; doi: 10.4049/jimmunol.1300699 http://www.jimmunol.org/content/191/6/3347 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/08/20/jimmunol.130069 Material 9.DC1 References This article cites 60 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/191/6/3347.full#ref-list-1 by guest on September 26, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Contributions of the Three CYP1 Monooxygenases to Pro-Inflammatory and Inflammation-Resolution Lipid Mediator Pathways Senad Divanovic,*,1 Jesmond Dalli,†,1 Lucia F. Jorge-Nebert,‡,1 Leah M. Flick,* Marina Ga´lvez-Peralta,‡ Nicholas D. Boespflug,* Traci E. Stankiewicz,* Jonathan M. Fitzgerald,† Maheshika Somarathna,‡ Christopher L. Karp,*,2,3 Charles N. Serhan,†,2 and Daniel W. Nebert‡,2 All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography- Downloaded from UV coupled with tandem mass spectrometry–based lipid-mediator metabololipidomics to identify and quantify three lipid- mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(2/2) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple- knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosa- noids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout http://www.jimmunol.org/ mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid–derived leukotriene B4 (LTB4) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid–derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahex- aenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid–derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB4 levels were shown to parallel increased neutrophil numbers, and 20-hydroxy–LTB4 formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation. The Journal of Immunology, 2013, 191: 3347–3357. by guest on September 26, 2021 ipid mediators (LMs) derived from polyunsaturated fatty mation. Some are more involved in the initiation phase, whereas oth- acids (Fig. 7A) include .150 chemicals, many of which ers participate more in the resolution phase. L have potent bioactivity (1). The v-6 fatty acid, arachidonic Oxidative biosynthesis of LMs is carried out in multiple steps by acid (AA), is converted to 12 known classes of LMs: PGs, prostacy- specific arachidonate lipoxygenases (encoded by six human genes clins, thromboxanes, leukotrienes (LTs), epoxyeicosatrienoic acids [ALOX5, ALOX12, ALOX12B, ALOX15, ALOX15B,andALOXE3] (EETs), hydroxyeicosatetraenoic acids (HETEs), dihydroxyeicosate- and seven mouse genes [Alox5, Alox12, Alox12b, Alox12e, Alox15, traenoic acids (DHETEs), hydroperoxyeicosatetraenoic acids, v-and Alox8,andAloxe3]). In addition to the cyclooxygenases (COX1, v-1 alcohols, lipoxins, hepoxilins, and eoxins (2–4). AA is well COX2; official names PG G synthase-1 and -2) and arachidonate known to be converted by cytochrome P450 to EETs (5), some of lipoxygenases, there is considerable evidence (2–4) suggesting that which are active in inflammation (6). In contrast, the v-3 fatty acids cytochrome P450 (CYP) monooxygenases participate in the oxi- docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are dative biosynthesis and inactivation of LMs. Members of the CYP1, converted to resolvins (E-series and D-series), neuroprotectins, and CYP2, CYP3, and CYP4 gene families, as well as CYP5A1 and maresins (2, 3, 7–9). Many v-6 and v-3 LMs participate in inflam- CYP8A1, are involved in biosynthesis and further inactivation of *Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Address correspondence and reprint requests to Prof. Daniel W. Nebert, Department Research Foundation, Cincinnati, OH 45229; †Department of Anesthesiology, Peri- of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, operative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Cincinnati, OH 45267-0056. E-mail address: [email protected] Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA The online version of this article contains supplemental material. 02115; and ‡Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, OH 45267-0056 Abbreviations used in this article: AA, arachidonic acid; CYP, cytochrome P450; ddH O, doubly distilled water; DHA, docosahexaenoic acid; DHETE, dihydroxyei- 1S.D., J.D., and L.F.J.-N. contributed equally to this work and should be considered 2 cosatetraenoic acid; EET, epoxyeicosatrienoic acid; EPA, eicosapentaenoic acid; first authors. HDHA, hydroxydocosahexenoic acid; HEPE, hydroxyeicosapentaenoic acid; HETE, 2The Karp, Serhan, and Nebert laboratories contributed equally to this project. hydroxyeicosatetraenoic acid; LC-MS-MS, liquid chromatography coupled with tan- dem mass spectrometry; LM, lipid mediator; LTA , leukotriene A ;LTB, leukotri- 3Current address: The Bill and Melinda Gates Foundation, Seattle, WA. 4 4 4 ene B4; LXA4, lipoxin A4; MRM, multiple-reaction monitoring; PD1, protectin D1; Received for publication March 18, 2013. Accepted for publication July 19, 2013. RvE2, resolvin E2; TKO, Cyp1a1/1a2/1b1(2/2) triple-knockout on .99.8% C57BL/ 6J background; WT, C57BL/6J wild-type. This work was supported by Cystic Fibrosis Foundation Research Development Program Center Component II Grant (to C.L.K.), National Institutes of General Medical Sciences Ó (Grant P01 GM095467 to C.N.S.), and National Institute of Environmental Health Sci- Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 ences (Grant T32 ES016646 to M.G.-P. and Grants R01 ES008147, R01 ES014403, and P30 ES06096 to D.W.N.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300699 3348 CYP1-DEPENDENT LIPID MEDIATOR SIGNATURES DURING INFLAMMATION LMs (10). Although some P450-mediated specific reactions of Animals bioactive LMs have been described (reviewed in Ref. 4), the vast Generation of the TKO mouse line was described (17). TKO animals were majority remains to be determined, largely because of the tech- backcrossed for .10 generations onto C57BL/6J to ensure a genetic back- nical challenges involved in identifying LMs, particularly with ground that was .99.8% similar (19) to WT C57BL/6J mice. All TKO mice regard to stereochemistry. used in the experiments were age matched with WT controls. We found that The CYP1 gene family encodes three enzymes (CYP1A1, mice provided with regular laboratory chow gave highly variable results. Therefore, both WT and TKO mice were maintained, from prior to con- CYP1A2, and CYP1B1) in both human and mouse that are evo- ception, on flavone-free Laboratory Chow Diet 5001. This diet is designed to lutionarily highly conserved, suggesting that mouse CYP1 data assure minimal inherent biological variation in long-term studies because it may likely be extrapolated to human CYP1 functions. In contrast, is enriched with v-3 essential fatty acids (http://www.labdiet.com); indeed, the CYP2, CYP3, and CYP4 families are far more complex due to the 5001 diet provided us with highly reproducible data. Results appeared to be more consistent in males than in females; subsequently, for all experi- multiple gene-duplication events followed by “genetic drift” during ments described in this article we studied males only, between 50