The Endocannabinoid Metabolite Prostaglandin E 2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP This information is current as Receptors of September 27, 2021. Caroline Turcotte, Simona Zarini, Stéphanie Jean, Cyril Martin, Robert C. Murphy, David Marsolais, Michel Laviolette, Marie-Renée Blanchet and Nicolas Flamand J Immunol published online 3 March 2017 Downloaded from http://www.jimmunol.org/content/early/2017/03/03/jimmun ol.1601767 http://www.jimmunol.org/

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 3, 2017, doi:10.4049/jimmunol.1601767 The Journal of Immunology

The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors

Caroline Turcotte,* Simona Zarini,† Ste´phanie Jean,* Cyril Martin,* Robert C. Murphy,† David Marsolais,* Michel Laviolette,* Marie-Rene´e Blanchet,* and Nicolas Flamand*

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their by eicosanoid biosynthetic . For example, N-arachidonoyl- ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and

PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-

derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study’s aim was to define the effects of Downloaded from these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could

detect six of the documented PG-G in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by http://www.jimmunol.org/

immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G , and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo. The Journal of Immunology, 2017, 198: 000–000.

cute and chronic inflammation are characterized by leu- Historically, COX-2 was perceived as a proinflammatory , kocyte infiltration, proinflammatory mediator production, because its expression is induced by inflammatory stimuli and leads to by guest on September 27, 2021 A and tissue destruction. Non-steroidal anti-inflammatory PGE2 synthesis. This was eventually challenged by Gilroy et al. (2) drugs have long been used to limit pain and inflammatory dam- who suggested that COX-2 could have anti-inflammatory properties. It age, notably by blocking the production of PGs and thromboxane. was then shown in several murine models that COX-2 blockade Moreover, cyclooxygenase-2 (COX-2) inhibitors, which prevent the worsens inflammation or delays its resolution, at least in mice (3–5). In biosynthesis of PGE2, have been helpful at decreasing inflammation addition to PGs, COX-2 participates in the synthesis of other lipids that and inflammatory pain (1). modulate nociception and inflammation, notably PG-glycerol (PG-G) and PG-ethanolamide (PG-EA). These endocannabinoid derivatives are products of the COX-2–dependent oxygenation of 2-arachidonoyl- *Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Que´bec, De´partement de Me´decine, Faculte´ de Me´decine, Universite´ Laval, Quebec glycerol (2-AG) and , respectively. PG-EAs, or pros- City, Quebec G1V 4G5, Canada; and †Department of Pharmacology, University of tamides, preferentially activate the prostamide receptor over PG Colorado Denver, Aurora, CO 80045 receptors (6). In contrast, PGE2-G can bind to several PGE2 re- ORCIDs: 0000-0002-9430-6515 (R.C.M.); 0000-0002-8935-6657 (D.M.); 0000- 0001-5944-7200 (N.F.). ceptors to a lesser extent than PGE2 (7). Moreover, PGE2-G in- Received for publication October 17, 2016. Accepted for publication February 1, duces key signaling events that are not mimicked by PGE2 (8, 9). 2017. PGE2 is a well-established inhibitor of human neutrophil func- This work was supported by Canadian Institutes of Health Research MOP-97930 (to N.F.), tions such as leukotriene B4 (LTB4) biosynthesis, reactive oxygen the Natural Sciences and Engineering Research Council of Canada (to N.F.), and the Fonds species (ROS) production, and migration (10–14). These effects sur les Maladies Respiratoires J.-D. Be´gin–P.-H. Lavoie (to N.F.). C.T. is the recipient of a doctoral award from the Canadian Institutes of Health Research and was supported by a are the consequence of elevated cyclic cAMP and involve the EP2 research award from the Canadian Consortium for the Investigation of . receptor. Given that PGE2-G is produced by COX-2 and binds to D.M., M.L., M.-R.B., and N.F. are members of the inflammation group of the Respiratory some of the EP receptors (7), because PGE inhibits neutrophil Health Network of the Fonds de Recherche du Que´bec-Sante´. 2 functions, and because PG-Gs were shown to regulate inflammation Address correspondence and reprint requests to Dr. Nicolas Flamand, Centre de Recherche de l’ Institut Universitaire de Cardiologie et de Pneumologie de Que´bec, (15), we undertook experiments to assess whether PGE2-G and Universite´ Laval, 2725 Chemin Sainte-Foy, Room A2142, Quebec City, QC G1V PGE2-EA would also modulate human neutrophil functions. 4G5, Canada. E-mail address: [email protected] Abbreviations used in this article: AA, arachidonic acid; 2-AG, 2-arachidonoyl-glycerol; COX-2, cyclooxygenase 2; EA, ethanolamide; G, glycerol; LC-MS/MS, liquid Materials and Methods chromatography–tandem mass spectrometry; LTB4, leukotriene B4;MAFP,methyl Materials arachidonoyl fluorophosphonate; PKA, cAMP-dependent protein kinase; qPCR, quantitative PCR; ROS, reactive oxygen species; SA, serinol-amide. AH6809, 19-OH-PGB2, 2-AG, butaprost (free acid), CAY 10598, H-89, JZL184, L-902 688, methyl arachidonoyl fluorophosphonate (MAFP), Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 PGE2, PGE2-EA, PGE2-G, PGE2-serinol amide (SA), PGD2-G, PGD2-SA,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601767 2 PGE2-G INHIBITS NEUTROPHIL FUNCTIONS

PGF2a-G, sulprostone, tetrahydrolipstatin, WWL70, and the primary Ab Migration assay for MAG lipase were purchased from Cayman Chemical (Ann Arbor, m MI). Thapsigargin was obtained from Tocris Bioscience (Ellisville, MO). Migration assays were performed using 3 m pore inserts (Becton Dick- m DMSO was purchased from Sigma-Aldrich (St Louis, MO). Protease inson), as recommended by the manufacturer. In brief, 700 l of pre- inhibitor mixture tablets and adenosine deaminase were purchased from warmed (37˚C) HBSS containing 1.6 mM CaCl2 and 100 nM LTB4 were m Roche (Laval, QC, Canada). Aprotinin, leupeptin, and WWL113 were added in the lower chamber and 200 l of prewarmed neutrophil sus- 3 6 purchased from Sigma-Aldrich. DFP was purchased from BioShop pensions [37˚C, 2.5 10 cells/ml in HBSS containing 1.6 mM CaCl2 and Canada (Burlington, ON, Canada). The HRP-linked anti-mouse IgG and 5% (w/v) FBS] were added in the upper chamber of the transmigration anti-rabbit IgG secondary were obtained from Cell Signaling Technology apparatus. Neutrophils were allowed to migrate for 2 h at 37˚C. The upper (Beverly, MA). Primary Abs for ABHD6, ABHD12, and PPT1 were chambers were then removed and migrated cells in the lower chamber of purchased from Abcam (Toronto, ON, Canada). The LYPLA2 primary the migration apparatus were counted using a Scepter 2.0 handheld auto- Ab was purchased from Abnova (Taipei City, Taiwan), the ABHD16A mated cell counter. In experiments where PGE2, PGE2-G, and the type IV primary Ab was purchased from Thermo Fisher Scientific (Waltham, RO 20-1724 were used, they were added to both upper MA), and the primary Ab for CES1 was purchased from R&D Systems and lower chamber for 5 min before the addition of LTB4 in the lower (Minneapolis, MN). PMSF, RO 20-1724, palmostatin B, and the ECL chamber of the migration assay. detection kit were purchased from EMD Millipore (Billerica, MA). The magnetic bead–conjugated anti-CD16 mAb and MACS were purchased Bacterial growth assays from Miltenyi Biotec (Auburn, CA). HBSS and RPMI 1640 were ob- Escherichia coli (#25922; American Type Culture Collection) was grown tained from Wisent Laboratories (St-Bruno, QC, Canada). Dextran and overnight at 37˚C in tryptic soy broth. The obtained culture was diluted HPLC-grade methanol and acetonitrile were purchased from Fisher (1:100) in fresh media and incubated at 37˚C until an OD of 0.5 at 600 nm Scientific. ML349 was a generous contribution from Dr. L. Marnett was reached. Then 500 ml of the E. coli culture were washed and sus- (Vanderbilt University, Nashville,TN).TheHNP1-3defensinELISA pended in sodium phosphate buffer at a final concentration of 1000 CFU/ml kit was purchased from Hycult Biotech (Uden, the Netherlands). ONO- in sodium phosphate buffer. Freshly isolated human neutrophils (37˚C, Downloaded from AE2-227 was a generous gift from Ono Pharmaceutical (Osaka, Japan). 6 20 3 10 cells/ml) in HBSS containing 1.6 mM CaCl2 incubated for Cytochrome C was obtained from Bio Basic (Amherst, NY). 15 min with 10 mM of cytochalasin B then activated with 1 mM 2-AG for m m Ethics committee approval 5 min. PGE2 (1 M) or PGE2-G (1 M) were added 5 min before the addition of 2-AG. Incubations were stopped by transferring the tubes in an This work required the use of human cells from healthy volunteers and was ice-water bath. Samples were centrifuged (500 3 g; 4˚C; 5 min) and the approved by our institutional ethics committee. All the experiments were resulting supernatants were mixed with the bacterial suspensions (1:1) and conducted with the understanding and the signed consent of each partic- incubated for 4 h on a rotating plate at 37˚C. The mixtures then were http://www.jimmunol.org/ ipant. diluted (1:300) and plated on Luria–Bertani agar plates. Colonies were counted after the incubation of the agar plates overnight at 37˚C. Isolation of human neutrophils Analysis of antimicrobial peptide release Human venous blood was obtained from volunteers and collected in tubes containing K3EDTA as an anticoagulant. Eosinophil-depleted neutro- Following cell stimulation, samples were rapidly centrifuged; the super- phils were then isolated as described previously (16). In brief, platelet- natants were collected and stored at 280˚C until further analysis. Quan- rich plasma and erythrocytes were discarded by centrifugation and titation of a-defensins in the supernatants was performed using a commercially dextran sedimentation, respectively. Mononuclear cells were then sep- available ELISA kit (Hycult Biotech), according to the manufacturer’s arated from granulocytes by centrifugation on Lymphocyte Separation instructions.

Medium cushions (Corning), and residual erythrocytes were removed by guest on September 27, 2021 from the granulocyte pellets by hypotonic lysis using sterile water. Analysis of receptor and lipase expression by quantitative PCR Neutrophils were separated from eosinophils using anti-CD16–coated Total RNA extracts were prepared using TRIzol according to the manu- magnetic beads, according to the manufacturer’s instructions. The purity facturer’s instructions. cDNA was obtained by reverse using and viability of the resulting neutrophil suspensions were always the iScript Reverse Transcription Supermix from Bio-Rad, with an RNA $ 98%, as assessed by Diff-Quik staining and trypan blue exclusion, input of 1 mg per reaction. For the analysis of EP receptor expression, respectively. quantitative PCR (qPCR) assays were performed on a 7900 Fast Real-Time PCR system (Applied Biosystems) using customs RT2 Profiler qPCR Removal of endogenous adenosine Multiplex Array Kit (Qiagen). For the analysis of the different endo- To better mimic the fate of human neutrophils, adenosine deaminase (0.3 U/ml) hydrolases, qPCR was performed with the SsoAdvanced was prepared and added 10 min before the addition of the stimuli in all Universal SYBR Green Supermix on a CFX96 thermal cycler (Bio-Rad, experiments (17, 18). Mississauga, ON, Canada) according to the manufacturer’s instructions. Primers for the 18S housekeeping were obtained from Qiagen (QT00199367). Primers for all other target were designed using the Induction and analysis of LTB4 biosynthesis Primer-BLAST tool and synthetized by IDT (Coralville, IA). Primer sequences Prewarmed human neutrophil suspensions (37˚C, 5 3 106 cells/ml) in are as follows: (59→ 39): ABHD6 forward 59-CATCTGGGGGAAACAA- HBSS containing 1.6 mM CaCl2 were incubated 5 min with PGE2,PGE2- GACCA-39, ABHD6 reverse 59-TTTCCATCACTACTGAGTGCCC-39, G, PGE2-EA, PGE2-SA, PGD2-G, PGD2-SA, PGF2a-G, or the different ABHD12 forward 59-CGGATACTGAGGGAATTCCTGG-39,ABHD12re- EP receptor agonists (see figure legends for concentrations), then stim- verse 59-AGGTCTTCATGCTTCCTTCCC-39, MAG lipase forward 59- ulated with either 3 mM2-AG(5min),3mM arachidonic acid (AA) TGCCTACCATGTTCTCCACA-39, MAG lipase reverse 59-CCTCCAGT- (5 min), or 100 nM thapsigargin (10 min). In experiments where inhib- TATTGCAGTCTGG-39,CES1forward59-TGCCTTTATCCTGGCCACTC- itors were used, they were added 10 min before the addition of the 39,CES1reverse59-CTTGGGTGCACATAGGAGGG-39, PPT1 forward 59- stimulus. Incubations were stopped by adding 0.5 volume of a cold (220˚ C) TGGCATGGGATGGGTGTTTT-39, PPT1 reverse 59-GGCGTTCCTGAA- stop solution [MeOH/MeCN, 1/1 (v/v)] containing 12.5 ng of both 19-OH- CAACTTTGG-39, LYPLA2 forward 59-AAGAAGGCAGCAGAGAACATC- PGB2 and PGB2 as internal standards. Samples were denatured overnight 39,LYPLA2reverse59-CTCCCAGCACGATTCGATTG-39,ABHD16Afor- (220˚C), centrifuged (700 3 g, 10 min) to eliminate the denatured proteins, ward 59-CCCCCGGCTCTACAAAATCTAC-39, ABHD16A reverse 59- then analyzed by reversed phase HPLC using an online extraction GATAGTACGTATCCCAGGAGCTG-39. Results are expressed in a relative 2DDCT procedure (19). LTB4,20-COOH-LTB4,20-OH-LTB4,and5(S)-HETE quantification normalized to the 18S rRNA as reference gene with the 2 were quantitated using PGB2 as internal standard and are referred to method. as LTs. Liquid chromatography–tandem mass spectrometry analyses Stimulation and analysis of superoxide anion release For the analysis of PGE2-G, PGE2, and D4-PGE2, incubations were stopped Superoxide anion production by human neutrophils was assessed by by the addition of one volume of cold (220˚C) MeOH and samples were cytochrome c reduction exactly as described previously (20). PGE2, kept frozen until further processing. Samples were centrifuged (700 3 g; PGE2-EA, PGD2-G, PGE2-G, PGF2a-G, PGD2-SA, PGE2-SA, or the EP 10 min) after addition of 2 ng of the internal standards (D4-PGE2 and D4- receptor agonists were added 5 min before fMLF. EP receptor antagonists PGE2-G). The supernatants were diluted with water to a final MeOH or inhibitors were added 10 min before fMLF. concentration of 10% and loaded on solid phase extraction cartridges The Journal of Immunology 3

(Strata-X Polymeric Reversed Phase, 60 mg/1 ml; Phenomenex). Car- analysis was performed in the negative ion mode using multiple reaction tridges were washed with 2 ml water then lipids were extracted with 1 ml monitoring for the specific mass transitions of D4-PGB2 (m/z 337.20→179.05), MeOH. The eluates were dried down and reconstituted in 40 ml of HPLC LTB4 (m/z 351.2→195.1), 20-OH-LTB4 (m/z 351.2→195.1), 20-COOH-LTB4 solvent A (8.3 mM acetic acid buffered to pH 5.7 with ammonium hy- (m/z 365.3→169.1), and 5-HETE (m/z 319.2→115.1). Quantitation was per- droxide) and 20 ml of solvent B (MeCN/MeOH, 65/35, v/v). A 25 ml al- formed using standard isotope dilution curves, as previously described (21). iquot was injected on to a reversed phase HPLC column (Ascentis C18, 150 3 2.1 mm, 5 mm; Supelco) eluted at a flow rate of 200 ml/min with a Analysis of proteins by immunoblot linear gradient from 45% solvent B, increased to 75% in 12 min, from 75 Cells were lysed by sonication at 4˚C in sucrose buffer containing 10 mg/ml to 98% in 2 min, and held for 10 min at 98% B before re-equilibration to leupeptin, 10 mg/ml aprotinin, 1 mM PMSF, 3 mM DFP, and one tablet 45% B in 10 min. The HPLC system was directly interfaced into the protease inhibitor mixture (for 10 ml of buffer). Laemmli sample buffer electrospray source of a triple quadrupole mass spectrometer (API 3000; AB [62.5 mM TRIS-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromo- Sciex) and mass spectrometric analysis was performed in the negative ion phenol blue] was added to sonicated cell lysates and samples were boiled mode using multiple reaction monitoring for the specific mass transitions of for 10 min. Buffer volumes were adjusted to obtain a final concentration of m z → m z → 6 PGE2 ( / 351.3 271.2) and D4-PGE2 ( / 355.3 275.2) and in the 2 3 10 cells/50 ml of lysate for all samples. Proteins were separated by + m z positive ion mode for the ammonium adducts [M+NH4] of PGE2-G ( / SDS-PAGE on 12% polyacrylamide gels and transferred on to polyvinyl- → m z → 444.4 391.3) and D4-PGE2-G ( / 448.4 395.3). Quantitation was per- idene difluoride membranes. Transfer efficiency and equal protein loading formed using standard isotope dilution curves, as previously described (21). were verified by Ponceau Red staining. Membranes were placed in TBS- In experiments in which concentrations of JZL184 higher than 1 mM Tween buffer (25 mM Tris-HCl [pH 7.6], 0.2 M NaCl, 0.15% Tween 20) were used, LTB4 biosynthesis was analyzed by liquid chromatography– containing 5% non-fat dried milk (w/v) for 30 min at room temperature, tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring. then probed with the primary Ab (4˚C, overnight). The membranes were In brief, incubation was stopped by the addition of one volume of cold 2 revealed by chemiluminescence using a HRP-coupled secondary Ab and ( 20˚C) MeOH and samples were kept frozen until further processing. an ECL detection kit. Samples were centrifuged (700 3 g; 10 min) after addition of 2 ng of the Downloaded from internal standard (D4-PGB2). The supernatants were diluted with water to a Statistical analyses final MeOH concentration of 10% and loaded on solid phase extraction cartridges (Strata-X Polymeric Reversed Phase, 60 mg/1 ml; Phenom- The effect of the lipase inhibitors on PGE2-G hydrolysis was analyzed enex). Cartridges were washed with 2 ml water then lipids were extracted with GraphPad Prism 6. The software was used to perform one-way with 1 ml MeOH containing 2% formic acid. The eluates were dried down ANOVA with Dunnett’s test. The p values ,0.05 were considered sig- and reconstituted in 25 ml of HPLC solvent A (0.05% formic acid in water) nificant. and 25 ml of solvent B (0.05% formic acid in acetonitrile). A 25 ml aliquot was injected on to an HPLC column (Kinetec C8, 150 3 2.1 mm, 2.6 mm; http://www.jimmunol.org/ Phenomenex) eluted at a flow rate of 400 ml/min with a discontinuous Results gradient (from 10% solvent B, increased to 25% in 15 min, from 25 to 35% PGE2-G inhibits human neutrophil functions in 5 min, from 35 to 75% in 10 min, from 75 to 95% in 0.1 min and held for 5 min at 95% B before re-equilibration to 10% B in 5 min). The HPLC We first performed a series of experiments in which we assessed system was directly interfaced into the electrospray source of a triple the impact of PGE2, PGE2-G, and PGE2-EA on LT biosynthesis. quadrupole mass spectrometer (Shimadzu 8050) and mass spectrometric A 5-min pretreatment of neutrophils with PGE2-G, but not by guest on September 27, 2021

FIGURE 1. Impact of PGE2-G on human neutrophil functions. (A) Prewarmed neutrophil suspensions were treated with PGE2, PGE2-G, or PGE2-EA at the indicated concentrations for 5 min, and then stimulated with 100 nM thapsigargin for 10 min. Samples were then processed and analyzed for LTB4 biosynthesis by HPLC, as described in Materials and Methods.(B) Neutrophil suspensions were treated with PGE2, PGE2-G, or PGE2-EA at the indicated concentrations for 5 min, and then stimulated with 100 nM fMLF for 10 min. ROS production was determined as described in Materials and Methods.(C) Human neutrophil suspensions were added to the upper chambers of the transmigration apparatus, and neutrophils were allowed to migrate toward 30 nM

LTB4 for 2 h into the lower chambers, as described in Materials and Methods. RO 20-1724 (10 mM), PGE2 (10 mM), PGE2-G (10 mM), or vehicle were added to the neutrophil suspensions and the lower chamber medium 5 min before the addition of LTB4 in the lower chamber. (D and E) Prewarmed neutrophil suspensions were treated with 3 mM of PGE2 or PGE2-G for 5 min before stimulation with 3 mM 2-AG or 100 nM fMLF for another 5 min. Incubations were stopped and samples were processed (D) to assess E. coli killing or (E) to measure antimicrobial peptide release by ELISA, as described in Materials and Methods.(A–E) Results are the mean (6 SEM) of at least three individual experiments, each performed in duplicate. (C) ****p , 0.0001 versus DMSO + RO 20-1724. (E)*p , 0.05 versus 2-AG alone, **p , 0.01 versus fMLF alone. 4 PGE2-G INHIBITS NEUTROPHIL FUNCTIONS

FIGURE 2. Involvement of EP receptors on the effect of PGE2-G on neutrophils. (A) mRNA was extracted from freshly isolated neutrophils and qPCR reactions were done as described in Materials and Methods. Results are expressed in relative quantification, with 18S rRNA as a housekeeping gene using 2DDCT the 2 method. (B) Neutrophil suspensions were treated with 1 mM PGE2 or PGE2-G for 5 min then were stimulated with 100 nM fMLF for 10 min. ROS production was determined as described in Materials and Methods. AH6809 (10 mM) or ONO-AE2-227 (10 mM) were added 5 min before PGE2 or PGE2-G. (C) Freshly isolated neutrophils suspensions were treated with the PKA inhibitor H-89 (10 mM), then with DMSO, PGE2, or PGE2-G, and finally stimulated with 100 nM thapsigargin for 10 min. H-89 and PGE2/PGE2-G were respectively added 10 and 5 min before the addition of thapsigargin. LT biosynthesis was analyzed as described in Materials and Methods.(A–C) Results are the mean (6 SEM) of at least three individual experiments, each performed in duplicate. (C) ****p , 0.0001 versus PGE2 or PGE2-G without H-89. Downloaded from

PGE2-EA, prevented LTB4 biosynthesis by thapsigargin-stimulated receptor antagonist AH-6809 and the EP4 antagonist ONO-A2E- neutrophils in a concentration-dependent manner (Fig. 1A). Under 227. The inhibitory effect of PGE2 and PGE2-G on the fMLF- that experimental setting, the inhibitory effect of PGE2-G on LTB4 induced ROS production was completely prevented by the EP1/ biosynthesis was less potent than that observed with PGE2 by one EP2 receptor antagonist AH-6809 (Fig. 2B). In contrast, the EP4 order of magnitude (IC50 of 3 and 30 nM for PGE2 and PGE2-G, antagonist ONO-A2E-227 did not prevent the inhibitory effects of http://www.jimmunol.org/ respectively). A similar pattern was obtained when we assessed PGE2 or PGE2-G. Of note, the EP2 receptor agonist butaprost ROS production induced by fMLF (Fig. 1B). PGE2-G also inhibited mimicked the effects of PGE2 and PGE2-G on neutrophil activa- the migration of human neutrophils ex vivo, although this required a tion as previously described (22), whereas the EP1/3 agonist sul- higher concentration (10 mM) and the use of the type IV phos- prostone had no effect (data not shown), supporting the phodiesterase inhibitor RO 20-1724 (Fig. 1C). Finally, PGE2-G and involvement of the EP2 receptor in the effect we observed. Finally, PGE2 also inhibited the ability of 2-AG–activated neutrophil su- we confirmed that like those of PGE2, the inhibitory effects of pernatants to kill E. coli (Fig. 1D). This inhibitory effect of PGE2 PGE2-G were prevented by the cAMP-dependent protein kinase and PGE2-G correlated with an inhibition of antimicrobial peptide (PKA) inhibitor H-89, underscoring similar downstream signaling release by 2-AG– or fMLF-stimulated neutrophils (Fig. 1E). Alto- events for both lipids (Fig. 2C). by guest on September 27, 2021 gether, the results presented in Fig. 1 indicate that in contrast to Hydrolysis of PGE2-G into PGE2 by human neutrophils PGE2-EA, PGE2-G exerts potent inhibitory effects on human neu- trophil functions. Of note, neither PGE2-G or PGE2-EA stimulated In contrast to PGE2, which binds to all EP receptors, PGE2-G only any of the functional responses investigated above (data not shown). binds to the EP1,EP3, and EP4 receptors (7). Given that PGE2-G inhibits neutrophil functions in an EP2-dependent manner, we Involvement of EP receptors in the inhibitory effects of PGE2 investigated whether PGE2-G was hydrolyzed into PGE2 by hu- and PGE2-G man neutrophils. As shown in Fig. 3A, the incubation of human PGE2 mediates its effects by activating the EP receptors 1 to 4. We neutrophils with 300 nM PGE2-G resulted in a time-dependent thus analyzed EP receptor expression in human neutrophils by decrease in its levels (half-life of ∼60 min) and a concomitant qPCR array and confirmed that they mainly express the EP2 and buildup of PGE2. To confirm that this PGE2 buildup originated EP4 receptors (Fig. 2A). To establish the contribution of the EP2 from PGE2-G hydrolysis rather than de novo biosynthesis, we and EP4 receptors in the inhibitory effect of PGE2-G and PGE2 on incubated neutrophils with D4-PGE2-G. We observed a time- human neutrophils, we performed experiments with the EP1/EP2 dependent increase of D4-PGE2 levels, whereas those of PGE2

FIGURE 3. Hydrolysis of PGE2-G into PGE2 by neutrophils. Prewarmed neutrophil suspensions were treated with 300 nM of (A) PGE2-G or (B)D4- PGE2-G during the indicated time. Incubations were stopped with 0.5 ml ice-cold MeOH containing 2 ng of D4-PGE2 and/or D4-PGE2-G as an internal standard. Lipid extractions and analyses by LC-MS/MS were performed as detailed in Materials and Methods.(C) Prewarmed neutrophil suspensions were treated with PGE2 (300 nM), PGE2-G (300 nM), or butaprost (3 mM) for the indicated times before being stimulated with 100 nM thapsigargin for 10 min. Samples were analyzed for LT biosynthesis as described in Materials and Methods. Data are the mean (6 SEM) of at least three individual experiments, each performed in duplicate. The Journal of Immunology 5

did not change (Fig. 3B), demonstrating that PGE2-G is hydro- note, PGE2, PGE2-G, and PGE2-EA did not modulate the ex- lyzed into PGE2 by neutrophils. In agreement with the hydrolysis pression of COX-2 or mPGES-1, the main proteins involved in of PGE2-G into PGE2 in our neutrophil suspensions, the inhibitory PGE2 biosynthesis in neutrophils [data not shown (23)]. effect of 300 nM PGE2-G occurred over time, and reached its PG-Gs can by hydrolyzed to some extent by several that maximal effect after 5 min of preincubation (Fig. 3C), which are more or less sensitive to lipase inhibitors (Table I). In this corresponds to the buildup of midnanomolar range concentrations respect, by qPCR and immunoblot we assessed which of these of PGE2 (Fig. 3A). In contrast, PGE2 and the EP2 receptor agonist hydrolases are expressed by freshly isolated neutrophils. We were butaprost did not require any preincubation time to inhibit the AA- able to detect, by qPCR, the mRNA for all of these lipases, with induced LT biosynthesis (Fig. 3C). Altogether, this suggests that the exception of CES1. In contrast, our immunoblot data, which PGE2-G is hydrolyzed into PGE2 and that PGE2 mediates the includes a positive control for each target, indicates that only inhibitory effects of PGE2-G on human neutrophil functions. Of ABHD12 and ABHD16A are expressed at a level that allows their

Table I. Summary of PGE2-G hydrolyzing enzymes

a Enzyme (Reference) Inhibitor Sensitive? IC50 Value Reference ABHD6 (31) MAFP Yes 16.9 nM (32) . m JZL184 Yes 100 M (33) Downloaded from Palmostatin B Yes 52.5 nM (34) WWL70 Yes 70 nM (35) Tetrahydrolipstatin Yes 48 nM (32) ML349 No n/a (36) WWL113 Yes — (37) ABHD12 (31) MAFP Yes 87 nM (32) JZL184 No n/a (33) http://www.jimmunol.org/ Palmostatin B Yes 1.8 mM (34) WWL70 No n/a (35) Tetrahydrolipstatin Yes 193 nM (32) ML349 No n/a (36) WWL113 Unknown n/a n/a ABHD16A (34) MAFP Unknown n/a n/a JZL184 No n/a (33) Palmostatin B Yes 100 nM (34) WWL70 No n/a by guest on September 27, 2021 Tetrahydrolipstatin Yes 170 nM ML349 Unknown n/a n/a WWL113 Unknown n/a n/a CES1 (38) MAFP Yes — (38, 39) JZL184 Yes — (38) Palmostatin B Unknown n/a n/a WWL70 Unknown n/a n/a Tetrahydrolipstatin Unknown n/a n/a ML349 Unknown n/a n/a WWL113 Yes 46 nM (37) LYPLA2 (24) MAFP Unknown n/a n/a JZL184 Yes 29 mM (24) Palmostatin B Yes 37.7 nM (40) WWL70 No n/a (35) Tetrahydrolipstatin No n/a (41) ML349 Yes 904 nM (24, 36) WWL113 No n/a (37) MAG lipase (31, 38) MAFP Yes — (31) JZL184 Yes 8 nM (33) Palmostatin B Yes 93.3 nM (34) WWL70 No n/a (33, 35) Tetrahydrolipstatin No n/a (34, 42) ML349 Unknown n/a n/a WWL113 No n/a (37) PPT1 (39) MAFP Yes — (39) JZL184 No n/a Palmostatin B Yes — (40, 43) WWL70 Unknown n/a n/a Tetrahydrolipstatin Unknown n/a n/a ML349 Unknown n/a n/a WWL113 Unknown n/a n/a aDetermined either by activity-based protein profiling or enzymatic assays. —, not determined; n/a, not applicable. 6 PGE2-G INHIBITS NEUTROPHIL FUNCTIONS detection with this technique (Fig. 4). As for LYPLA2, we found higher concentrations (Fig. 5C). We obtained similar results when mRNA levels comparable to those of the documented positive we attempted to prevent the inhibitory effect of PGE2-G on control, the MDA-231 cell line (24). However, we were unable to thapsigargin-induced LTB4 biosynthesis (data not shown). In ad- detect the protein in our neutrophil lysates, despite getting a strong dition to showing these effects on neutrophil functions, we sought band for MDA-231 cells. Altogether, these experiments under- to confirm that the inhibitors indeed prevented PGE2-G hydrolysis score that the expression of PG-G–hydrolyzing lipases is limited into PGE2. We thus incubated neutrophils with PGE2-G for to ABHD12 and ABHD16A in neutrophils. 10 min, in the presence of 10 mM MAFP, palmostatin B, MAFP, or In an attempt to pinpoint whether ABHD12 and ABHD16A are WWL113 and measured the accumulation of PGE2 in superna- involved in the hydrolysis of PGE2-G into PGE2, we next un- tants by LC-MS/MS (Fig. 5E). We found that MAFP, JZL184, and dertook experiments using numerous hydrolase inhibitors and palmostatin B almost completely blocked the buildup of PGE2, assessed if they could prevent the inhibitory effect of PGE2-G on whereas WWL113 had a partial, statistically significant inhibitory human neutrophils. Knowing that neutrophils display a strong 2- effect (p , 0.001). Of note, none of the inhibitors prevented the AG–hydrolyzing activity that is involved in the 2-AG–induced inhibitory effects of PGE2 on the fMLF-induced ROS production LTB4 biosynthesis (25), we also assessed the impact of these in- or the AA-induced LTB4 biosynthesis (data not shown). hibitors on that biosynthetic pathway, to compare the pharmaco- logical profiles of both hydrolytic reactions. Palmostatin B, Impact of other PG-Gs and their non-hydrolysable analogs on JZL184, and MAFP were the only compounds we tested that neutrophil functions completely inhibited the 2-AG–induced LTB4 biosynthesis in a Finally, we investigated if the inhibitory effect of PGE2-G is concentration-dependent fashion (Fig. 5A, 5B). In contrast, al- mimicked by other PG-Gs. We found that like PGE2-G, PGD2-G Downloaded from though the effect PGE2-G was sensitive to MAFP and palmostatin also inhibits the AA-induced LT biosynthesis as well as the fMLF- B, it was only partially sensitive to WWL113 and JZL184 at induced ROS production, whereas PGF2a-G was ineffective http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Expression of documented PGE2-G hydrolases in human neutrophils. (A–G) Each graph and immunoblot shows data for a documented positive control (left) and human neutrophils (right). mRNA was obtained from tissues and cells with TRIzol and qPCR was performed as described in Materials and Methods. Results are expressed in relative quantification normalized to the 18S rRNA as reference gene with the 22DDCT method. For immunoblots, cells or hypothalamus samples were disrupted and analyzed as described in Materials and Methods. The input per well is the equivalent of two million cells for neutrophils and cell lines, and 30 mg of protein per well for hypothalamus lysates (HYPO). The qPCR data are the mean (6 SEM) of at least four experiments, and the Western blotting images are representative of three separate experiments. The Journal of Immunology 7 Downloaded from

FIGURE 5. Pharmacological profiles of 2-AG and PGE2-G hydrolysis in human neutrophils. (A and B) Prewarmed neutrophil suspensions were treated with the different inhibitors 5 min prior to the addition of 3 mM 2-AG for 5 min. Incubations were then stopped and LTB4 biosynthesis was analyzed as indicated in Materials and Methods.(C and D) Prewarmed neutrophil suspensions were treated with the different inhibitors 5 min prior to the addition of

1 mM PGE2-G for 5 min. Cells then were stimulated with 100 nM fMLF for 10 min. (E) Prewarmed neutrophil suspensions were treated with 10 mM of the http://www.jimmunol.org/ various inhibitors during 5 min before adding 1 mM PGE2-G for 10 min. Incubations were stopped and PGE2 levels were measured by LC-MS/MS, as described in Materials and Methods.(A–E) Data are the mean (6 SEM) of at least three independent experiments performed in duplicate. (E) ****p , 0.0001 versus vehicle (t = 10 min).

(Fig. 6). Importantly, the structurally similar but non hydro- can exert either similar or opposite effects, a phenomenon that is lysable PGE2-SA and PGD2-SA had no effect, again under- likely attributable to differential patterns of EP1–4 and PG-G re- scoring the importance of PG-G hydrolysis for their inhibitory ceptor expression and activation. by guest on September 27, 2021 effect to occur. Our data show that PGE2-G, but not PGE2-EA, inhibits every neutrophil function we tested in a similar fashion to PGE2 (Fig. 1). Discussion The EP2 antagonist AH-6809 (10 mM) and the PKA inhibitor Endocannabinoids have been classified as anti-inflammatory lipids, H-89 prevented the inhibitory effects of both PGE2 and PGE2-G, mainly because of the proinflammatory state that CB1/CB2 defi- suggesting the involvement of EP2 and PKA (Fig. 2). Given that cient mice usually display in experimental models of inflamma- PGE2-G has some binding affinity for EP4 but practically none for tory disease (26, 27). However, arachidonoyl-ethanolamide and EP2 (7), this indicates that the EP2-dependent effects we observed 2-AG do not only modulate leukocyte functions by activating the are not caused by PGE2-G but are rather the result of PGE2-G CB receptors, but also through their numerous metabolites, no- hydrolysis into PGE2. This is supported by the facts that: 1) PGE2-G tably eicosanoids, prostamides, and PG-Gs (26, 28). The cellular is hydrolyzed into PGE2 by neutrophils (Fig. 2); 2) the non- and molecular mechanisms involved in the immunomodulatory hydrolysable version of PGE2-G, PGE2-SA, does not inhibit human effects of prostamides and PG-Gs remain ill defined. In this neutrophils (Fig. 5); and 3) the effect of PGE2-G is mediated by the study, we provide evidence that: 1) the 2-AG metabolites PGE2- only EP receptor not activated by PGE2-G (Fig. 2). GandPGD2-G inhibit human neutrophil functions; 2) the effects PGE2-EA and PGE2-G are relatively stable in biological sys- of PGE2-G and PGD2-G require their hydrolysis into PGE2 and tems. Indeed, in human plasma, PGE2-EA undergoes slow PGD2, respectively; 3) the PGE2-G hydrolases ABHD12 and dehydration/isomerization into PGB2-EA, whereas PGE2-G is ABHD16A were detected by qPCR and immunoblot in human hydrolyzed with a half-life of over 10 min (30). However, the neutrophils; 4) the hydrolysis of PGE2-G into PGE2 likely in- enzymatic pathways involved in the hydrolysis of PGE2-G into volves more than one hydrolase; and 5) the effects of PGE2-G are PGE2 remain unclear. Of the seven candidate lipases that were blocked by the EP2 receptor antagonist AH-6809 and the PKA reported to hydrolyze PGE2-G (Table I), six were detected by inhibitor H-89. qPCR, but only two were detected by qPCR and immunoblot, A limited number of studies have previously evaluated the namely ABHD12 and ABHD16A (Fig. 4). Of note, we were un- bioactivity of COX-2 metabolites of endocannabinoids. Hu et al. able to detect ABHD6, MAG lipase, PPT1, and LYPLA2 in (29) observed that in rats, PGE2-G and PGE2 had similar effects neutrophils by immunoblot, despite the presence of mRNA. Given on NF-kB activity, mechanical allodynia and thermal hyper- that our experiments included documented positive controls that algesia. Interestingly, the mixture of EP receptor antagonists they did yield a band at the expected size, we can conclude that the Abs used completely blocked the effects of PGE2, but only partially we used are reliable in this setting. It is possible, however, that our blocked the effects of PGE2-G, suggesting different mechanisms failure to detect these lipases at the protein level was caused by of action. In this regard, Nirodi et al. (7) observed that PGE2-G artifacts during cell lysis and protein denaturation, even though we 2+ (but not PGE2) induces a quick, dose-dependent Ca mobilization tried three different lysis methods (sonication, immediate solubi- in RAW264.7 cells. These results indicate that PGE2 and PGE2-G lization in boiling Laemmli buffer, and hypotonic lysis), which 8 PGE2-G INHIBITS NEUTROPHIL FUNCTIONS

ent. In addition, none of the documented PGE2-G hydrolases match the pharmacological profile we found. Thus, the data we gathered did not allow us to confirm that one enzyme among our candidates (Table I) is responsible for the hydrolysis of PGE2-G by human neutrophils. Therefore, it is likely that the PGE2-G hydrolytic activity that we observed is catalyzed by more than one lipase, and/or by a lipase that has yet to be characterized. A recent study showed that the inhibition of 2-AG hydrolysis led to decreased inflammation in mice (15). This was due, at least in part, to the endocannabinoid metabolite PGD2-G, which prevented the production of IL-1b. Of note, PGE2-G and PGF2a-G did the opposite. Thus, it is possible that PG-Gs regulate inflammation through multiple mechanisms of actions and thus participate in the inflammatory process in a coordinated and timely manner. Our data indicate that human neutrophils have the ability to hydrolyze PG-Gs, notably PGE2-G and PGD2-G. Although our data support that PG-G hydrolysis leads to PG production and dampens neu- trophil functions, it remains possible that neutrophils prevent the

anti-inflammatory effects of PG-Gs by eliminating those putative Downloaded from proresolving mediators in vivo. In conclusion, we provide clear evidence that PGE2-G, but not PGE2-EA, inhibits numerous functions of human neutrophils in a concentration-dependent manner similar to PGE2. This inhibitory effect requires the hydrolysis of PGE2-G into PGE2, the subse-

quent activation of the EP2 receptor and PKA. These results also http://www.jimmunol.org/ support the view that the anti-inflammatory effects related to the endocannabinoid 2-AG might be due, at least in part, to its metabolism by the COX pathway.

Acknowledgments We thank Dr. Lawrence Marnett for generously providing the selective LYPLA2 inhibitor ML349. by guest on September 27, 2021 FIGURE 6. Impact of other PG-Gs and their non-hydrolysable analogs Disclosures on neutrophil functions. (A) Prewarmed neutrophil suspensions were The authors have no financial conflicts of interest. treated with 1 mM of PGE2-G, PGE2-SA, PGD2-G, PGD2-SA, or PGF2a-G for 5 min, then stimulated with 100 nM thapsigargin (100 nM) for 10 min.

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