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Contributions of the Three CYP1 to Pro-Inflammatory and -Resolution 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

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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 © 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 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., 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 –derived B4 (LTB4) and decreased 5S-hydroxyeicosatetraenoic acid; decreased –derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahex- aenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased –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 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 , 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 (encoded by six human clins, thromboxanes, (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 (COX1, v-1 alcohols, , , and eoxins (2–4). AA is well COX2; official names PG G synthase-1 and -2) and arachidonate known to be converted by 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 families, as well as CYP5A1 and (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, A4; MRM, multiple-reaction monitoring; PD1, protectin D1; Received for publication March 18, 2013. Accepted for publication July 19, 2013. RvE2, 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 and the past 65 million years, because humans and mice had a common 100 d of age. All animal experiments were approved by and conducted in ancestor. This resulted in the having 16 functional accordance with the National Institutes of Health standards for the care and CYP2 –coding genes, 4 CYP3 protein–coding genes, and 12 use of experimental animals and the University of Cincinnati Medical CYP4 protein–coding genes compared with the mouse genome, Center Institutional Animal Care and Use Committee. which has 50 functional Cyp2 protein–coding genes, 9 Cyp3 pro- Zymosan challenge tein–coding genes, and 20 Cyp4 protein–coding genes (11). An acute inflammatory response was induced with 1 mg zymosan/mouse Lipoxygenases insert both atoms (12), whereas P450 mono- (20). Zymosan (insoluble carbohydrate from yeast cell wall) was freshly insert one atom (13–15), of diatomic oxygen into prepared (2 mg/ml) in sterile 0.9% NaCl, and 0.5 ml was injected i.p. into Downloaded from substrates to form the products (Fig. 7B). Another important dis- each mouse; zero-time controls were not treated. At the appropriate time tinction between and P450 reactions points (0, 6, and 9 h), each peritoneal cavity was washed twice with 5 ml PBS (Cellgro); all baseline peritoneal cells at time zero, as well as all is that, although lipoxygenases occasionally produce epoxides (e.g., peritoneal exudates at 6 and 9 h following zymosan challenge, were har- [LTA4] formation by ALOX5), the major product is vested and centrifuged (560 3 g for 6 min). Supernatant fractions were a fixed-chirality hydroperoxide; in contrast, P450 monooxygenases collected and mixed (2:1) with cold methanol (Sigma-Aldrich); samples 2 can generate racemic mixtures of internal-monohydroxy products, were stored at 80˚C until metabololipidomics analysis. Cell pellets were http://www.jimmunol.org/ terminal-monohydroxy products, and epoxides that (following hy- resuspended in 1 ml cold PBS, and total cells were quantified. In addition, 80 ml cells was used to prepare histology slides at baseline, whereas an drolysis) often proceed to form racemic mixtures of dihydroxy additional dilution of 1:20 was used to prepare histology slides following products (Fig. 7B). zymosan challenge; the amount of lavage exudate recovered per mouse Ultimately, among many other functions (4, 10), AA-derived was quantified and used for normalization of total cell counts/mouse. For LMs are more likely to be involved in the proinflammatory phase, each group, three to eight mice (WT and TKO) were studied individually, and each experiment was repeated at least two more times. whereas the DHA-derived bioactive metabolome and EPA-derived bioactive metabolome are LMs that orchestrate the resolution Cell quantification phase of self-limited inflammatory responses. In addition, the lo- Following centrifugation (560 3 g for 6 min), total cell counts were cation of many of these metabolic reactions usually is highly tissue quantified from peritoneal lavage at baseline or lavage of peritoneal exu- by guest on September 26, 2021 and/or cell type specific (2–4). dates following zymosan challenge, as well as from cells isolated from One approach to resolving the challenging problem of identifying peripheral blood or bone marrow. Differential cell counts were quantified m which CYP participates in which step(s) of the LM cascade following cytospin analysis (on 80 l total cell exudate at baseline, an additional 1:20 dilution for conditions involving zymosan challenge) and involved in acute inflammation is to use Cyp-knockout mouse lines subsequent Diff-Quik staining (Siemens). We quantified 400 cells/slide to in combination with the most advanced metabololipidomics (16) determine the percentages of neutrophils, monocytes/macrophages, mast for separating and identifying as many unique LM metabolites cells, eosinophils, and lymphocytes. as possible. The present study capitalizes on this approach. The Sample extraction and LM metabololipidomics Cyp1a1/1a2/1b1(2/2) triple-knockout (TKO) mouse (4, 17, 18) has all three highly conserved members of the mammalian Cyp1 gene All samples for LC-MS-MS–based analyses were extracted using SPE columns (16). Briefly, columns were equilibrated with one column volume family genetically deleted. We compared TKO mice with C57BL/6J of methanol and two volumes of doubly distilled water (ddH2O). Prior to wild-type (WT) mice during zymosan-induced peritonitis. In this extraction, 500 pg deuterium-labeled internal standards d8–5S-HETE, study, we did not investigate the classical EET products produced d4– (LTB4), and d4-PGE2 were added to facilitate quantifi- by P450 from AA in the liquid chromatography coupled with tan- cation of sample recovery. Sample supernatant fractions were diluted with ∼ dem mass spectrometry (LC-MS-MS) profiles; rather, we focused 10 volumes of ddH2O, acidified (pH 3.5), and immediately loaded onto a SPE column. After loading, columns were washed with one volume of on their roles in lipoxygenase and pathways. Re- neutral ddH2O and one volume of hexane. sults from the current study indicate that CYP1 monooxygenases Samples were eluted with 6 ml methylformate and taken to dryness using play a highly significant role in the regulation of at least eight key Speedvac or nitrogen stream. Samples were subsequently suspended in steps during LM biosynthesis and their further metabolism; thus, methanol/water for LC-MS-MS. The liquid chromatography-UV coupled with tandem mass spectrometry system includes QTrap 3200 equipped with these findings provide potentially useful new therapeutic targets for a Shimadzu SIL-20AC auto-injector and LC-20AD binary pump or QTrap treating inflammation and its natural resolution. 5500 (ABSciex), equipped with an Agilent HP1100 binary pump. An Agilent Eclipse Plus C18 column (50 mm 3 4.6 mm 3 1.8 mmor100mm3 4.6 mm 3 1.8 mm) was used with a gradient of methanol/water/acetic acid Materials and Methods of 60:40:0.01 (v/v/v) to 100:0:0.01 at 0.5-ml/min flow rate. Chemicals To monitor and quantify the levels of the various LMs, we developed a multiple-reaction-monitoring (MRM) method with signature ion fragments Zymosan A was obtained from Sigma-Aldrich (St. Louis, MO). Liquid for each molecule. Identification was conducted using published criteria (16), chromatography–grade solvents were purchased from Fisher Scientific. with at least six diagnostic ions. Calibration curves were obtained using Agilent Eclipse Plus C18 columns (50 3 4.6 3 1.8 mm) and C18 SPE synthetic and authentic LM mixtures; these included d8–5S-HETE, d4-LTB4, columns were bought from Waters. Synthetic standards for LC-MS-MS d4-PGE2, 15-HETE, 12-HETE, 5-HETE, lipoxin A4 (LXA4), lipoxin B4, quantification and deuterated internal standards were procured from Cay- LTB4,PGE2,PGD2,PGF2a, thromboxane B2, resolvin E1, resolvin E2 man Chemicals. All chemicals and reagents represented the highest avail- (RvE2), 18–hydroxyeicosapentaenoic acid (HEPE), 15-HEPE, 12-HEPE, 5- able grades. HEPE, resolvin D1, resolvin D2, resolvin D5, protectin D1 (PD1), MaR1, The Journal of Immunology 3349

17–hydroxydocosahexenoic acid (HDHA), 14-HDHA, 7-HDHA, and 4- Results HDHA at 12.5, 25, 50, and 100 pg (Supplemental Table I). Linear calibration Peritoneal cell numbers and cell types curves for each were obtained with r2 values in the range of 0.98–0.99. Quantification was carried out based on peak area of the MRM transition and In an earlier study, peritoneal immune cell numbers and types were the linear calibration curve for each compound. Reverse-phase chiral LC- found to be similar at baseline between WT and TKO mice (17). In MS-MS was conducted, as described (21). contrast, following i.p. zymosan, an exaggerated increase in peritoneal Human and mouse neutrophils exudate total cell number, neutrophils, and monocyte/macrophages was observed in TKO mice (17). In the current study, zymosan Neutrophils were isolated as described (22), with minor modifications. challenge also led to significant increases in total cell numbers in Specifically, human neutrophils were isolated from peripheral blood; mouse neutrophils were purified from femoral and tibial bone marrow, as peritoneal exudates of TKO mice compared with WT mice at 6 h described (23). Cells were collected in ice-cold Ca2+-andMg2+-free (.14-fold)and9h(∼10-fold),withsignificantlyincreasedneutro- HBSS (Life Technologies) supplemented with 0.1% BSA (Sigma-Aldrich); phil accumulation 6 and 9 h after challenge and significantly in- neutrophils were purified using a discontinuous Percoll (GE Healthcare) creased monocyte/macrophage accumulation at 6 h after challenge gradient. Subsequently, neutrophils were layered onto Histopaque 1119 (Fig. 1). Significant differences between WTand TKO mice were not (Sigma-Aldrich), centrifuged at 650 3 g for 20 min, and stopped without brake, to separate RBCs. Purified neutrophils were collected and found for the numbers of eosinophils, mast cells, or lymphocytes. washed with HBSS, 0.1% BSA, centrifuged for 5 min at 400 3 g, and re- 2+ 2+ Comparison of WT versus TKO levels of LMs at baseline suspended in PBS with Ca and Mg prior to being stimulated with LTB4. The purity of isolated neutrophils was determined by cytospin analysis, To determine the contributions of CYP1 monooxygenases in LM followed by Diff-Quik stain (Siemens). Isolation of human neutrophils was biosynthetic pathways and LM profiles in vivo, we initially quantified performed at Cincinnati Children’s Hospital Medical Center; all participants Downloaded from provided written informed consent and the study was approved by the LM levels in untreated mice (Fig. 2). Illustrations of representative Cincinnati Children’s Hospital Medical Center Institutional Review Board. chromatography (Fig. 2A) unequivocally demonstrate that each of the bioactive LMs was identified using strict reported criteria Incubation of neutrophils with LTB4 (Materials and Methods). Of the 24 LMs screened, 11 were detected Cells (1 3 106) were plated in a flat-bottom 96-well plate and stimulated in baseline peritoneal cells of WT mice but not TKO mice: PGD2, with 500 nM LTB4 at 37˚C and 5% CO2. Following a 30-min incubation, PGE2,LTB4, 12-HETE, 15-HETE derived from AA (Fig. 2B–D); the reaction was stopped by the addition of 200 ml cold methanol (Sigma- http://www.jimmunol.org/ PD1, 14-HDHA, 17-HDHA derived from DHA (Fig. 2E, 2F); and Aldrich), and cells and cell lysates were snap-frozen. Human neutrophils 12-HEPE, 15-HEPE, and 18-HEPE derived from EPA (Fig. 2G). were used as a LTB -responsive positive control. 4 Statistically significant differences were not found between TKO Statistical analysis and WT peritoneal cell LMs without zymosan challenge. All assays were performed in duplicate or triplicate, and average values Comparison of WT versus TKO levels of LMs in exudate 6 h from each mouse were considered one independent determination. Statis- into inflammation tical differences were assessed by the Student pair-wise t test or x2 anal- ysis. Data were normally distributed and are presented as mean 6 SEM. Of the LMs screened via targeted LC-MS-MS–based lipidomics, ,

All p values 0.05 were regarded as statistically significant. 17 were identified in peritoneal exudates from WT mice, but not by guest on September 26, 2021

FIGURE 1. Peritoneal cell parameters from baseline cells versus acute inflammatory exudates, comparing WT with TKO mice. (A) Peritoneal cell numbers and cell types were compared at time 0 and at 6 and 9 h following i.p. zymosan administration. Data are mean 6 SEM. (B) Representative Diff- Quik staining of cells from WT and TKO mice at the three time points. Baseline (neat cells); for 6 and 9 h following zymosan challenge, a 1:20 dilution of cells was used for quantification (original magnification 340; original magnification 310 for insets). **p . 0.01, ***p , 0.001, Student two-tailed t test (n = 3 mice/genotype). 3350 CYP1-DEPENDENT LIPID MEDIATOR SIGNATURES DURING INFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 2. Baseline peritoneal LM levels are not significantly different between untreated WT and TKO mice. Peritoneal lavage samples were obtained from untreated WT or TKO mice, and LM levels were assessed by LM metabololipidomics. (A) Representative MRM chromatograms of LMs identified in peritoneal exudates. LM levels for AA-derived PGs (B), leukotrienes and lipoxins (C), and monohydroxy-ETE pathway markers (D). LM levels for DHA- derived resolvins, protectins, and maresins (E) and monohydroxy-DHA pathway markers (F). (G) LM levels for EPA-derived resolvins and monohydroxy- by guest on September 26, 2021 EPA pathway markers. Data are mean 6 SEM (n = 3 mice/group). ETE, Eicosatetraenoic acid.

TKO mice, treated with zymosan for 6 h (data not shown). In LTB4 metabolism ex vivo addition to those 11 identified in baseline peritoneal cells (Fig. 2), We chose to explore LTB4 metabolism further in peritoneal and bone 6 additional LMs and pathway markers were found following marrow neutrophils ex vivo. A proof-of-principle experiment was 6 h of inflammation. These included PGF2a, LXA4, 5-HETE, 4- first carried out with human neutrophils. When the substrate LTB4 HDHA, RvE2, and 5-HEPE. Moreover, compared with WT mice, was added to human peripheral blood neutrophils, ∼4-fold more 20- zymosan-treated TKO mice revealed a trend toward increased COOH-LTB4 was produced compared with 20-OH-LTB4 (Fig. 4A, levels of AA-derived PGs and LTB4, as well as decreased levels of 4E). The confirmed mass-to-charge ratio of the parent compound 12-HETE, DHA-derived PD1 and 14-HDHA, and EPA-derived LTB was m/z 335 (Fig. 4B); it was m/z 351 for 20-OH-LTB (Fig. 15-HEPE (data not shown). 4 4 4C) and m/z 365 for 20-COOH-LTB4 (Fig.4D).Thus,thisexvivo Comparison of WT and TKO levels of LMs in exudate 9 h into experiment with human neutrophils validates the identification of inflammation LTB4 and two additional metabolites that were reported earlier to be Of the LMs and pathways profiled, 16 were identified in the peri- produced by a member of the human CYP4 family (24). toneal exudate of WT mice, but not TKO mice, treated with zymosan Peritoneal and bone marrow neutrophils were then isolated from for 9 h (Fig. 3). LXA4, which was found at low levels at 6 h into TKO and WT mice that had received 4 h of zymosan treatment. inflammation, was no longer detectable at 9 h. Although LTB4 lev- Before incubation with the substrate LTB4, peritoneal neutrophils elsincreaseddramaticallybetween6and9hofzymosantreatment from TKO mice displayed significant increases in LTB4 levels in TKO mice, seven other LMs were decreased to a statistically compared with those from WT neutrophils (Fig. 5A, 5B). After significant extent in TKO mice compared with WT mice (Fig. 3, incubation with the substrate LTB4, TKO mice showed about two Supplemental Table I). thirds as much 20-OH-LTB4 in both peritoneal neutrophils (Fig. Therefore, of the three functionally distinct LM metabolomes 5C) and bone marrow neutrophils (Fig. 5D) relative to WT mice. screened (i.e., AA-, DHA- and EPA-bioactive metabolomes), sta- Curiously, although a significant difference in 20-OH-LTB4 tistically significant alterations were obtained in eight LMs and levels was found between TKO- and WT-elicited peritoneal neu- pathway markers during inflammation in TKO mice compared with trophils, the 20-COOH-LTB4 (downstream oxidized metabolite) WT mice: TKO mice exhibited an ∼3.9-fold increase in LTB4 levels; was not detected in either TKO or WT mice (Fig. 5A). In contrast, an ∼7.5-fold decrease in PD1 levels; 1.7- to 2.3-fold decreases in 5- high levels of 20-COOH-LTB4 were clearly demonstrable with HETE, PD1, 14-HDHA, 17-HDHA, 12-HEPE, 15-HEPE, and 18- human neutrophils (Fig. 4A, 4D, 4E); these data indicate that mice HEPE levels; and a 1.3-fold decrease in 5-HETE levels (Fig. 3). apparently do not have the CYP4 enzyme that is equivalent in The Journal of Immunology 3351

FIGURE 3. LM levels from peritoneal exudates are elevated in TKO mice 9 h fol- lowing zymosan treatment. LM levels for AA-derived PGs (A), leukotrienes and lipo- xins (B), and monohydroxy–ETE pathway markers (C). LM levels for DHA-derived resolvins, protectins, maresins (D), and monohydroxy–DHA pathway markers (E). (F) Downloaded from LM levels for EPA-derived resolvins and monohydroxy–EPA pathway markers. Data are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001 versus WT, Student two-tailed t test (n = 3 mice/genotype). ETE, Eicosate- traenoic acid. http://www.jimmunol.org/ by guest on September 26, 2021

function to the human CYP4 enzyme responsible for 20-COOH- inflammation (13). We started with the hypothesis that the absence LTB4 formation (24). In fact, the amount of 20-COOH-LTB4 in of all three CYP1 enzymes would alter the LM metabololipidomics human neutrophils was ∼4-fold greater than the amount of 20- profile at precise steps during inflammation and its resolution. OH-LTB4 (Fig. 4E). Specifically, if an LM was increased in TKO relative to WT, this To determine the contribution of CYP1 enzymes to LM bio- suggests that one or more of the CYP1 enzymes might be critical in synthesis, we next assessed the production of LM precursors by a downstream metabolic step in this pathway (i.e., absence of CYP1 peritoneal exudate leukocytes. We found that 14-HDHA, 17- would lead to a build-up in levels of the upstream LM). In contrast, HDHA, 12-HETE, and 15-HETE levels were significantly lower in if a specific LM was decreased in TKO relative to WT, this suggests TKO leukocyte incubations compared with those of WT leukocyte that one or more of the CYP1 enzymes is critical in an upstream incubations (Fig. 6). Finally, using reverse-phase chiral LC-MS- event involved in formation of the metabolite in this pathway (i.e., MS (21), we investigated the levels of R/S-enantiomers for each of absence of CYP1 would lead to a diminution of the downstream the monohydroxy acids, which were significantly decreased in LM). Elucidation of P450 enzyme specificity for any particular TKO peritoneal leukocyte incubations. This analysis demonstrated step in the initiation phase or resolution phase of inflammation an ∼59 6 9% decrease in the levels of 14-HDHA, 17-HDHA, 12- should aid in developing novel drugs for treatment of various forms HETE, and 15-HETE; these four metabolites in TKO carrying the of acute inflammation, as well as chronic inflammatory disorders, hydroxyl group in the R-enantiomeric position in TKO leukocyte such as cardiovascular disease, rheumatoid arthritis, periodontitis, and incubations, when compared with that in WT leukocyte incuba- Alzheimer’s disease (1, 25). tions. For these four metabolites carrying the hydroxy group in the Leukocyte trafficking within the site of inflammation is a dy- S-enantiomeric position, we found an ∼62 6 14% diminution in namic process regulated by chemical cues produced at the site the levels of 14-HDHA, 17-HDHA, 12-HETE, and 15-HETE. These of injury and/or infection. In this study, we focused on the initia- results provide further evidence for the contribution of CYP1 tion phase of the inflammatory response, using metrics that map enzymes to LM precursor biosynthesis. leukocyte-recruitment dynamics in response to a self-limited challenge (26). Our results demonstrate that TKO mice display Discussion altered leukocyte recruitment dynamics in response to a self- In the current study, we examined the combined contributions of limited challenge, with elevated neutrophil recruitment follow- the three CYP1 enzymes in LM pathways involved during acute ing zymosan challenge and significantly higher exudate monocyte/ 3352 CYP1-DEPENDENT LIPID MEDIATOR SIGNATURES DURING INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Human peripheral blood neutrophils preferentially convert LTB4 to 20-COOH-LTB4 more than 20-OH-LTB4.(A) Representative MRM traces for LTB4, 20-OH-LTB4, and 20-COOH-LTB4. Accompanying tandem mass spectrometric spectra used for the rigorous identification of LTB4 (B), 20- OH-LTB4 (C), and 20-COOH-LTB4 (D). (E) 20-OH-LTB4 and 20-COOH-LTB4 levels in human peripheral blood neutrophils following ex vivo incubation without (2) and with (+) the substrate LTB4. macrophage levels at the peak of inflammation compared with with a foreign chemical (e.g., PAH or dioxin), CYP1 levels in im- their WT littermates. These results, together with the finding that mune cells become dramatically increased. exudate proinflammatory LM levels (vide infra) were elevated in After 6 h of zymosan challenge, TKO peritoneal exudates TKO mice, indicate that TKO mice display a proinflammatory showed a trend of increased PGD2, PGE2, and PGF2a, along with phenotype. Our present findings are in agreement with those made a 2.3-fold increase in LTB4 levels compared with WT mice; in in models of nonresolving (27) and delayed-resolving (28) inflam- contrast, relative to WT mice, TKO exudates showed an ∼6-fold mation; these data underscore the contribution of CYP1 enzymes decrease in PD1 levels and noticeable decreases in 12-HETE, 14- to inflammation and its timely resolution. HDHA, and 15-HEPE. Following 9 h of zymosan-initiated in- Interestingly, statistically significant differences in LM levels flammation, TKO exudates demonstrated statistically significant between TKO and WT mice were not found in peritoneal cells at differences from WT exudates with regard to eight chemicals: baseline; they were only found after zymosan stimulation. This strikingly increased LTB4 levels and strikingly decreased PD1 observation is consistent with the well-known fact that CYP1 levels levels, as well as decreased levels of 5-HETE, 14-HDHA, 17- in immune tissues are very low or negligible under basal conditions HDHA, 12-HEPE, 15-HEPE, and 18-HEPE. The reason for the (29). After zymosan stimulation, an endogenous inducer of CYP1, diminished levels of AA-derived 5-HETE in TKO inflammatory believed to be one or more LMs, or after treatment of the animal exudates after 9 h of zymosan (Fig. 3) is not known; the small The Journal of Immunology 3353

FIGURE 5. Impairment of further metabo- lism of LTB4 in TKO neutrophils. Neutrophils from peritoneal exudates and bone marrow were obtained from WT and TKO mice following 4 h of zymosan treatment. Cells were counted and incubated with LTB4.(A) Representative MRM traces for LTB4 and 20-OH-LTB4 obtained from WT and TKO leukocyte incubations. (B)LTB4 in peritoneal neutrophils before incubation with substrate. 20-OH-LTB4 levels in peritoneal neutrophils (C) and 20-OH-LTB4 levels in bone marrow neutrophils (D), following incubation B D with the substrate LTB4. Data in ( – ) are mean Downloaded from 6 SEM (n = 3 mice/group). Note the differences in values on the y-axis between (B) versus (C) and (D). *p , 0.05. http://www.jimmunol.org/

decrease in 5-HETE could be caused by various factors. One with the subsequent observation of increased LTB4 levels in TKO possibility is that decreased overall ALOX5 activity is coupled mice following zymosan challenge. with more efficient conversion of 5-hydroperoxyeicosatetraenoic TKO peritoneal and bone marrow neutrophils ex vivo show acid to LTB4; another possibility is that at least a portion of 5- a diminished capacity to metabolize LTB4, but this effect seems to by guest on September 26, 2021 HETE formation represents the direct metabolism of AA by CYP1 be small relative to the ∼4-fold increase in LTB4 observed in (30, 31). peritoneal exudates from TKO mice. It is not known with certainty These results suggest that, prior to challenge, the contribution of whether this increase reflects a requirement of CYP1 for LTB4 the three CYP1 enzymes to LM biosynthesis and/or further me- oxidation in vivo in these mice or whether it reflects some com- tabolism is not significant, indicating that resident peritoneal cells pensatory decreased level of a mouse enzyme equivalent in func- do not express functionally appreciable amounts of these enzymes tion to that of the human CYP4 monooxygenase that is known to at levels that would regulate LM levels in the absence of challenge. metabolize LTB4 in neutrophils (24). Curiously, CYP4A14 and Upon challenge, leukocytes are recruited to the peritoneal cavity, in CYP4A10 mRNA expression was found to be 2–3-fold lower in particular polymorphonuclear leukocytes, which possess the CYP1 liver of Cyp1a2(2/2) single-knockout mice compared with WT enzymes and lead to alterations in the exudate LM profiles in WT mice (34); however, when TKO liver was compared with WT liver and TKO mice. Future studies will need to address the contribution under more rigorous microarray conditions in a later study (17), no of the CYP1 enzymes and the role(s) of dynamic cellular traffic at mouse CYP4 mRNA was found to be significantly upregulated or the site of inflammation and their contribution to proresolving downregulated. mediator biosynthesis during inflammation resolution. Alternatively, increased LTB4 levels in TKO mice might be We conclude that one or more CYP1 enzymes participate sig- explained by the increase in neutrophils present in TKO exudates nificantly in at least eight specific metabolic steps of LM pathways rather than by LTB4 catabolism. Nevertheless, there appears to be in the acute inflammatory response. Furthermore, our data indicate an intriguing CYP1-mediated effect on TKO cellular dynamics in that CYP1 monooxygenases contribute to the overall balance of the peritoneal cavity as a result of inflammation; the current study local autacoids (i.e., deletion of an inactivation pathway can give did not determine the mechanism of this effect with certainty. In rise to accumulation of a specific LM during the time course of both any event, CYP1 participation in the step from LTB4 to 20-OH- the initiation and resolution phases of inflammation in the intact LTB4 appears to be very likely. animal). It is not clear that differences in LTB4 metabolism in neutrophils AA-derived LTB4 is known to be a potent chemoattractant, gen- ex vivo (Fig. 5) can account for the increased LTB4 observed in erated from activated innate immune cells, such as neutrophils, the intact mouse; this is only one of several possible explanations. macrophages, and mast cells (32) with mice lacking either 5-LOX For example, LTB4 could be higher because the higher number of or LTA4 ; these two enzymes are responsible for bio- neutrophils present in the TKO exudate might produce greater synthesis of this potent mediator, displaying decreased leukocyte amounts of LTB4. Another possibility is that LTB4 levels are recruitment following zymosan challenge (33). Thus, our finding greater as the result of compensatory upregulation of lipo- of zymosan-elicited increases in the number of these three cell xygenases or effects secondary to the clearly altered state of in- types in TKO exudates compared with WT exudates is consistent flammation that exists in the TKO peritoneal cavity. Even in 3354 CYP1-DEPENDENT LIPID MEDIATOR SIGNATURES DURING INFLAMMATION

FIGURE 6. Contribution of CYP1 enzymes to LM biosynthesis following zymosan challenge. The four most significant changes in the generation of LM precursors by peritoneal exudate leukocytes included 14-HDHA, 17-HDHA (A), and 12-HETE and 15-HETE (B) levels; these metabolites were significantly lower in TKO mice compared with WT mice. n = 3 mice. *p , 0.05, **p , 0.01 versus WT.

isolated neutrophils ex vivo, it remains to be determined with by 12-hydroxydehydrogenase (PGR/LTB4DH) to produce 12-oxo- absolute certainty that any of the CYP1 enzymes is directly re- LTB4 (36, 37). sponsible for altered LM metabolism. The role of CYP1 enzymes (Fig. 7B) in the inactivation of LTB4 Conversion of 20-OH-LTB4 to 20-COOH-LTB4 in human in mouse neutrophils is of interest and the main point of the neutrophils is the main route of LTB4 inactivation (24); this experiments described in this article. We first investigated the finding was also corroborated in Fig. 4, whereas this enzymatic pathway known for LTB4 inactivation in human neutrophils that reaction does not appear to occur in mouse neutrophils (Fig. 5). It express the human orthologs of these enzymes (24). In this study, Downloaded from has been known for .2 decades that even one altered amino acid we showed that human neutrophils converted LTB4 to the P450- residue can dramatically modify P450 substrate specificity (35); mediated metabolites 20-OH-LTB4 and 20-COOH-LTB4, as re- this is the major reason why substrate specificities of various P450 ported elsewhere (24). Formation of these two metabolites are enzymes differ between mouse and human, especially among described first, to follow sequential metabolic steps regulated by members of the CYP2, CYP3, and CYP4 families (11), in which distinct enzymes. The formation of 20-OH-LTB4 occurs first and orthologs between the two species cannot be determined conclu- is primarily regulated by CYP1 enzymes, which, in humans, is http://www.jimmunol.org/ sively. In addition to v-oxidation pathways, an alternative dehy- then converted by CYP4 enzymes to 20-COOH-LTB4 (24). For drogenation pathway was described for the inactivation of LTB4 a direct comparison, we isolated mouse neutrophil populations that involves the dehydrogenation of the hydroxyl group on C-12 obtained from two distinct sites: peritoneum (collected in response by guest on September 26, 2021

FIGURE 7. Introduction to poly- unsaturated fatty acids, lipoxygenase versus monooxygenase reactions, and LM biosynthesis pathways. (A) Origin of v-6 and v-3 LMs. (B) Diagram showing the mechanisms for lipoxygenase versus P450-mon- ooxygenase reactions. The Journal of Immunology 3355 to zymosan challenge) and bone marrow polymorphonuclear Compared with WT mice, TKO mice exhibited ∼2-fold lower leukocytes; each converted LTB4 to 20-OH-LTB4. Of note, 20- levels of LXA4 at 6 h, but not at 9 h (Fig. 3), of zymosan chal- COOH-LTB4 was not produced in these incubations from two lenge. This finding suggests that LXA4 formation from 15S- mouse neutrophil populations, unlike human neutrophils. Quan- hydroxy-5(6)-epoxy-ETE, at least initially, might involve con- tification of both LTB4 and 20-OH-LTB4 levels in these incuba- tributions from CYP1 enzyme(s); subsequently, other oxidative tions demonstrated that, in the absence of the CYP1 enzymes enzymes might compensate to continue producing sufficient LXA4 (in TKO mice), there was an accumulation of LTB4 with a higher levels after 9 h of zymosan-initiated inflammation. Of possible recovery rate. These results indicate that CYP1 enzymes are im- relevance, LXA4 itself (44) as well as two downstream metabo- portant in the inactivation of this initiating signal, which is also lites of LTA4,5S,6R-DHETE and 5S,6S-DHETE (45), were sug- proinflammatory via v-hydroxylation in mice. This is in line with gested to act as endogenous ligands for aryl hydrocarbon receptor, the elevated LTB4 levels found within inflammatory exudates from a involved in regulating all three CYP1 genes TKO mice, in which we also found elevated polymorphonuclear (4, 46). Aryl hydrocarbon receptor–dependent activity is likely to leukocyte recruitment to the peritoneum upon zymosan challenge. be required during the acute inflammatory response in these peri- Although LTB4 biosynthesis in self-limited inflammatory exu- toneal exudate cells. dates is not restricted to neutrophils, in this model LTB4 produc- In addition to the TKO mouse described in this article, numerous tion coincides with increased exudate neutrophil levels (38); in P450-knockout mouse lines have been generated, including 2/2 2/2 2/2 the absence of CYP1 enzymes, LTB4 levels accumulated at the Cyp1b1( ) (47), Cyp2e1( ) (48), Cyp2j5( ) (49), and site of inflammation, which led to the exaggerated inflammatory Cyp4a10(2/2) (50) single knockouts; Cyp1a1/1a2(2/2), Cyp1a1/ 2 2 2 2 response (Fig. 1). Together, these findings suggest that CYP1 1b1( / ), and Cyp1a2/1b1( / ) double knockouts (51); and, more Downloaded from enzymes in phagocytes exert a pivotal role in inflammation res- recently, ablation of the entire Cyp2c (52), Cyp2d (53), and Cyp3a olution. (54, 55) gene subfamily clusters. Experiments similar to the cur- The absence of CYP1 in TKO mice results in decreased for- rent study should now be possible to identify participation of the mation of the DHA-derived 17S-HDHA. It is noteworthy that, in specific CYP1A1, CYP1A2, CYP1B1, CYP2, CYP3, and CYP4 human polymorphonuclear leukocytes (39), double dioxygenation enzymes in distinct steps of the LM metabolic pathways during

via a second lipoxygenase produces 10,17-diHpDHA, which is inflammation. Most likely, as we found with the CYP1 enzymes, http://www.jimmunol.org/ then reduced by activity to form 10S,17S-dihydrox- numerous LM steps will be altered in the absence of the CYP2, ydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid, an isomer of CYP3, and CYP4 monooxygenases. Moreover, we predict that neuroprotectin/PD1 (40). It also is possible to produce this PD1 redundancy of CYP enzymes for many of these LM biosynthesis isomer via a second sequential P450 step (e.g., P450-mediated steps will likely be found during the acute inflammatory response. 17S-HDHA conversion to the 10,17-diHDHA isomer of PD1). Although mouse and human CYP1 enzymes display quite The double geometry of this isomer is different from an enzymatic similar substrate specificities and inducers that upregulate CYP1 epoxide-dependent pathway and epoxide-hydrolase reaction (25, expression (46), substrate specificities vary to a much greater 40), which can then produce PD1 and other specialized pro- degree in the CYP2, CYP3, and CYP4 families because many resolving mediators as their main route in human leukocytes. gene-duplication events have occurred such that one cannot assign by guest on September 26, 2021 Also, during the resolution phase of inflammation, the absence of orthologs between the mouse and human genomes (11). Hence, CYP1 in TKO mice leads to decreased formation of DHA-derived we predict that “humanized” P450 mouse lines, in which the PD1. “knocked-in” human-specific CYP2, CYP3,orCYP4 gene is ex- Finally, the current study shows that CYP1 contributes to the pressed in place of the mouse ortholog, will provide the ultimate levels of EPA-derived 15-HEPE and 12-HEPE, as well as in- tool for further understanding the role of each human CYP2, volvement in the oxidative step from EPA-derived 18–hydro- CYP3, and CYP4 monooxygenase during LM biosynthesis/ peroxyeicosapentaenoic acid to 18R-HEPE. This latter step is inactivation. Furthermore, the use of such tools would directly a bottleneck to the generation of resolvins of the E-series. Thus, confirm the clinical importance of each P450 enzyme in a specific CYP1 ablation might also affect production of a number of pro- LM biosynthetic step. Identification of such steps should be useful resolving LMs, in addition to the ones mentioned above that in- in the future for drug targeting and, as such, promise a strong thera- clude the AA-derived lipoxins, as well as the D-series resolvins. peutic potential. CYP1 ablation appears to supply the required precursors (i.e., 17- Humanized P450 mouse lines include hCYP1A1/1A2 (56–58), HDHA and 15-HETE) that are then converted by lipoxygenases to hCYP1B1 (F. Gonzalez, personal communication), hCYP2C9 (52), D-series resolvins and lipoxins, respectively (41–43). hCYP2D6 (59), hCYP2E1 (60), and hCYP3A4 (61). Of note, the Comparing WT and TKO mice, metabololipidomic analysis of first humanized hCYP2D6 (59) and hCYP3A4 (61) mouse lines the products made ex vivo by exudate leukocytes demonstrated had the human gene inserted into the mouse genome that included a significant reduction in TKO LM precursors (i.e., 17-HDHA, 14- all of the mouse Cyp2d and Cyp3a genes. HDHA, 15-HETE, 12-HETE, and 5-HETE) (Fig. 6). These results It should be mentioned that, in the current study, we screened for are in line with the hypothesis that deletion of CYP1 enzymes is changes in the profiles and levels of several novel LMs and pathway responsible, at least in part, for the differences found in exudate markers from the AA metabolome (PGs, leukotrienes, lipoxins) LM levels between WT and TKO mice. and the DHA and EPA metabolomes (resolvins, protectins, and Although we found a significant decrease in 18-HEPE pro- maresins), as well as their pathway markers relevant during the duction in TKO mice, this did not correlate with a parallel decrease acute inflammatory response and its resolution. Whether CYP1 in RvE2 levels, an observation that may reflect a difference in the monooxygenases participate in metabolic steps (Fig. 7) involving further distal conversion of RvE2 in TKO mice, which leads to an any of the .125 other LMs during inflammation have not been apparent accumulation of this mediator in the inflammatory exu- assessed in these TKO mice. Moreover, the current study moni- dates. Although we did not see any detectable differences between tored the levels of basal peritoneal cells versus zymosan-initiated TKO and WT resolvin levels in the current study, this may reflect inflammatory exudates. Dozens of other cell types, plus numerous issues related to detection limits for these mediators. LM-mediated physiological and pathological stimuli (38, 39, 44), 3356 CYP1-DEPENDENT LIPID MEDIATOR SIGNATURES DURING INFLAMMATION in addition to zymosan-initiated inflammation, also remain to be 4. Nebert, D. W., and C. L. Karp. 2008. Endogenous functions of the aryl hydro- carbon receptor (AHR): intersection of cytochrome P450 1 (CYP1)-metabolized scrutinized via this approach in future studies. and AHR biology. J. Biol. Chem. 283: 36061–36065. In summary, our hypothesis was that global ablation of all three 5. Proctor, K. G., J. R. Falck, and J. Capdevila. 1987. Intestinal vasodilation by CYP1 enzymes combined, and comparison of genetically modified epoxyeicosatrienoic acids: arachidonic acid metabolites produced by a cyto- chrome P450 monooxygenase. Circ. Res. 60: 50–59. TKO mice with WT mice, would uncover a number of disruptions 6. Panigrahy, D., M. L. Edin, C. R. Lee, S. Huang, D. R. Bielenberg, in the signature profiles of LM pathways during an acute in- C. E. Butterfield, C. M. Barne´s, A. Mammoto, T. Mammoto, A. Luria, et al. flammatory response. Hence, to this end, we compared basal CYP1 2012. Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice. J. Clin. 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Compared with WT mice, TKO mice also show substantial 16. Dalli, J., and C. N. Serhan. 2012. Specific lipid mediator signatures of human http://www.jimmunol.org/ phagocytes: microparticles stimulate macrophage efferocytosis and pro- differences in cell population in response to zymosan. It is con- resolving mediators. Blood 120: e60–e72. ceivable that, at one or more of these metabolic steps, CYP1 en- 17. Dragin, N., Z. Shi, R. Madan, C. L. Karp, M. A. Sartor, C. Chen, F. J. Gonzalez, zymes might clear metabolites related to the zymosan challenge and D. W. Nebert. 2008. Phenotype of the Cyp1a1/1a2/1b12/2 triple-knockout mouse. Mol. Pharmacol. 73: 1844–1856. rather than metabolizing LMs directly; failure to clear those 18. Nebert, D. W., M. Ga´lvez-Peralta, Z. Shi, and N. Dragin. 2010. Inbreeding and metabolites in the absence of CYP1 might lead to an alteration in epigenetics: beneficial as well as deleterious effects. Nat. Rev. Genet. 11: 662. the local cellular response that could, in turn, produce a change in 19. Nebert, D. W., T. P. Dalton, G. W. Stuart, and M. J. Carvan, III. 2000. “Gene- swap knock-in” cassette in mice to study allelic differences in human genes. LM mediator production by cyclooxygenases and ALOXs. Ann. N. Y. Acad. Sci. 919: 148–170. Therefore, future experiments in zymosan-challenged mice 20. Kolaczkowska, E., M. Chadzinska, A. Scislowska-Czarnecka, B. Plytycz,

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