Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust biosynthesis of 2-arachidonoyl- glycerol and its congeners in human myeloid leukocytes

Caroline Turcotte1 | Anne-Sophie Archambault1 | Élizabeth Dumais1 | Cyril Martin1 | Marie-Renée Blanchet1 | Elyse Bissonnette1 | Nami Ohashi2 | Keiko Yamamoto2 | Toshimasa Itoh2 | Michel Laviolette1 | Alain Veilleux3 | Louis-Philippe Boulet1 | Vincenzo Di Marzo1,3,4 | Nicolas Flamand1
1Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Département de médecine, Faculté de médecine, Université Laval, Québec City, QC, Canada
2Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, Machida, Japan
3École de nutrition, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Québec City, QC, Canada
4Joint International Unit between the National Research Council (CNR) of Italy and Université Laval on Chemical and Biomolecular Research on the Microbiome and its Impact on Metabolic Health and Nutrition (UMI-MicroMeNu), Institute of Biomolecular Chemistry, CNR, Pozzuoli, Italy

Nicolas Flamand, Centre de recherche de l’IUCPQ, Département de médecine, Faculté de médecine, Université Laval, 2725 Chemin Sainte-Foy, Room A2142, Québec City, QC G1V 4G5, Canada.
Email: [email protected]

Funding information
Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC), Grant/Award Number: RGPIN-2015-04728; Canada Excellence Research Chairs, Government of Canada (CERC)

Abbreviations: 2-AG, 2-arachidonoyl-glycerol; 2-DHG, 2-docosahexaenoyl-glycerol; 2-DPG, 2-docosapentaenoyl-glycerol; 2-EPG, 2-eicosapentaenoyl- glycerol; 2-LG, 2-linoleoyl-glycerol; 2-OG, 2-oleoyl-glycerol; 2-PG, 2-palmitoyl-glycerol; AA, arachidonic acid; AEA, anandamide or N-arachidonoyl- ethanolamine; AMs, alveolar macrophages; BAL, bronchoalveolar lavage; DAG, diacylglycerol; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; eCB, endocannabinoid; EPA, eicosapentaenoic acid; FAAH, fatty acid amide hydrolase; GPCR, G protein-coupled receptor; LA, linoleic acid; LPA, lysophosphatidic acid; LT, leukotriene; MAFP, methylarachidonoyl-fluorophosphonate; MAG, monoacylglycerol; MBOAT, membrane bound O-acyl transferase; NAE, N-acyl-ethanolamine; OA, oleic acid; PA, palmitic acid; PAF, platelet activating factor; PL, phospholipase; UFA, unsaturated fatty acid.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2020 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology

The FASEB Journal. 2020;00:1–13.

wileyonlinelibrary.com/journal/fsb2 | 1


2-Arachidonoyl-glycerol (2-AG) is a bioactive lipid and endo- cannabinoid (eCB) activating the cannabinoid receptors CB1 and CB2. As such, it modulates several physiological processes including appetite, pain, and adipogenesis.1-3 2-AG also mod- ulates immune cell functions, usually leading to decreased in- flammatory responses, at least in mice,4,5 very much likely by activating the CB2 receptor.6,7 Two main strategies are currently investigated to, respectively, mimic or promote the anti-inflam- matory effects of 2-AG: the use of CB2 receptor agonists or the use of 2-AG hydrolysis inhibitors, which would enhance 2-AG half-life in vivo. While both strategies are attractive, using 2-AG hydrolysis inhibitors in a context of inflammation might have the additional benefit of decreasing eicosanoid levels because leukocytes efficiently hydrolyze 2-AG and its metabolites from the cyclooxygenase-2 and 15-lipoxygenase pathways.8-12
The synthesis of 2-AG by human leukocytes is not well documented. Furthermore, efforts to induce 2-AG biosyn- thesis in leukocytes led to 2-AG levels lower than those re- quired to activate the CB2 receptor, for which 2-AG has a Ki of
~145 nM.13-16 The classic 2-AG biosynthetic pathway involves
two enzymatic steps. First, a phospholipase C (PLC) will cleave a phosphatidylinositol-4,5-bisphospate containing arachidonic acid (AA) in the sn-2 position into inositol-1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). Next, the obtained DAG will be hydrolyzed by DAG lipase α or β into 2-AG.17-19 While the expression profile of DAG lipase α or β in leukocytes is ill defined, DAG lipase β blockade in murine peritoneal macro- phages, led to a significant decrease in 2-AG.20 Two alternative routes for the biosynthesis of 2-AG have been documented. The first one likely involves phospholipase D (PLD), as it uses phosphatidic acid as a precursor for DAG synthesis, which is then converted into 2-AG by DAG lipases.21 The second one is the dephosphorylation of lysophosphatidic acid (LPA) into 2-AG.22 The importance of these alternative pathways in 2-AG biosynthesis in vivo remains to be elucidated in the periphery. Herein, we assessed whether human leukocytes were a significant source of 2-AG as well as the underlying mech- anism involved. We report that 2-AG hydrolysis inhibition strikingly prolongs 2-AG half-life, allowing to correctly

assess the 2-AG biosynthetic capabilities of leukocytes. We also report that the classic 2-AG biosynthetic pathway (PLC/ DAG lipase) does not lead to an important 2-AG biosynthesis in human leukocytes; that instead AA stimulates a robust bio- synthesis of 2-AG; and that unsaturated fatty acids (UFAs) stimulate the synthesis of their monoacylglycerols (MAGs) and 2-AG congeners. The UFA-induced MAG biosynthetic pathway was found in neutrophils, eosinophils, and mono- cytes but not in alveolar macrophages (AMs), lymphocytes, platelets, or erythrocytes. This biosynthetic pathway is in- sensitive to DAG lipase inhibitors, sensitive to acyl-CoA synthase and transferase inhibitors, and is preceded by the biosynthesis of a corresponding LPA intermediate.

2.1 | Materials
Dextran and mass spectrometry-grade methanol and ace- tonitrile were purchased from Fisher Scientific. 1-AG-d5, 2-AG, 2-AG-d8, AA, AA-d8, leukotriene (LT) LTB4, Platelet- activating factor (PAF), fMet-Leu-Phe (fMLP), A23187, R848, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (n-3) (DPA), linoleic acid (LA), oleic acid (OA), palmitic acid (PA), 2-palmitoyl-glycerol (2-PG), 2-oleoyl-glycerol (2-OG), 2-linoleoyl-glycerol (2-LG), meth- ylarachidonoyl-fluorophosphonate (MAFP), triascin C, and JZL184 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Lymphocyte separation medium was purchased from Corning (Corning, NY, USA). Palmostatin B was pur- chased from EMD Millipore (Billerica, MA, USA). Strata-X columns for solid phase lipid extraction were purchased from Phenomenex (Torrance, CA, USA). The magnetic bead-con- jugated anti-CD16 and anti-CD14 mAb and MACS columns were purchased from Miltenyi Biotec (Auburn, CA, USA). Adenosine deaminase (ADA) was purchased from Roche (Laval, QC, Canada). 1/2-Eicosapentaenoyl-glycerol (2-EPG), 1/2-docosapentaenoyl-glycerol (n-3) (2-DPG), and 1/2-doc- osahexaenoyl-glycerol (2-DHG) were either provided by Dr Samuel Fortin from SCF pharma or purchased from Nu-Chek

Prep (Waterville, MN, USA). The primary antibody for DAG lipase α was purchased from Abcam (Toronto, ON, Canada) and the anti-DAG lipase β and anti-mouse HRP-linked second- ary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Aprotinin, leupeptin, and thimerosal were purchased from Sigma-Aldrich (St-Louis, MO, USA). LPA species containing either DHA or EPA were obtained by chemical synthesis as documented previously.23

2.2 | Ethics committee approval
This work required the use of human cells from volunteers and was approved by our institutional ethics committee. All the experiments were conducted with the understanding and the signed consent of each participant.

2.3 | Isolation of human leukocytes
For the isolation of neutrophils, eosinophils, lymphocytes, and monocytes, human venous blood was obtained from healthy or rhinitic volunteers and collected in tubes contain- ing K3EDTA as anticoagulant. Leukocytes were isolated as described previously with some modifications.24 In brief, the blood was centrifuged and the plasma was discarded. Erythrocytes were sedimented with 3% dextran, and granu- locytes were separated from PBMCs using a discontinuous gradient. The PBMC layer was harvested and monocytes and lymphocytes were separated using a magnetic bead-conju- gated anti-CD14, according to the manufacturer’s instructions. Residual erythrocytes were eliminated from the granulocyte pellet by hypotonic lysis with sterile water. Eosinophils were separated from neutrophils using anti-CD16-conjugated magnetic beads according to the manufacturer’s instructions. The purity and viability of the resulting leukocyte suspen- sions were always ≥98%, as assessed by counting 500 cells by Diff Quik staining and trypan blue exclusion, respectively. Human AMs were obtained by bronchoalveolar lavage (BAL)
of healthy volunteers as previously described.25 The BAL fluid containing leukocytes was recovered and centrifuged (4°C, 350g, 10 minutes). Supernatants were discarded and cells were washed twice with cold HBSS then their viability was assessed by trypan blue exclusion. Viability and purity of AMs were always greater than 95%, as assessed by enumerating 500 cells with stained with trypan blue and Diff Quick staining, respectively.

2.4 | Cell stimulations
Cells were suspended in HBSS containing 1.6 mM CaCl2 and preheated at 37°C for 10 minutes. To better mimic their fate, adenosine deaminase (0.3 U/mL) was added 10 minutes

before the addition of the stimuli in all experiments involving neutrophils.10,26 Inhibitors were added 5 minutes before the stimuli and/or the fatty acids, at the concentrations detailed in the legends. In experiments assessing their impact on eCB biosynthesis, PAF, fMLP, and LTB4 were added to the cell suspensions simultaneously with the fatty acids. For the analysis of MAGs and N-acyl-ethanolamines (NAEs) by LC-MS/MS, incubations were stopped by the addition of one volume of cold (−20°C) MeOH containing 0.01% acetic acid and 2 ng of 1-AG-d5 and as an internal standard. Samples were then kept at −20°C until further processing.

2.5 | Analysis of DAG lipase expression by immunoblot
For the analysis of DAG lipase protein expression, cells were lysed with NP-40 in an hypotonic lysis buffer containing 10 µg/ mL leupeptin, 10 µg/mL aprotinin, 1 mM PMSF, 3 mM DFP, and 1 tablet protease inhibitor cocktail (for 10 mL of buffer). Laemmli sample buffer (5×; 62.5 mM TRIS-HCl [pH 6.8],
2% SDS, 10% glycerol, 0.01% bromophenol blue) was added to cell lysates and samples were boiled for 10 minutes. Buffer volumes were adjusted to obtain a final concentration of 2 × 106 cells/50 µL of lysate for all cell types except for AMs, which were adjusted to 5 × 105 cells/50 µL. Proteins were separated by SDS-PAGE on 12% polyacrylamide gels and transferred onto PVDF membranes. Transfer efficiency and equal protein load-
ing were confirmed by Ponceau Red staining. Membranes were placed in TBS-Tween buffer (25 mM Tris-HCl [pH 7.6], 0.2 M NaCl, 0.15% Tween 20) containing 5% nonfat dried milk (w/v) for 30 minutes at room temperature, then incubated with the pri- mary antibody (4°C, overnight). The membranes were revealed by chemiluminescence using a HRP-coupled secondary antibody and an ECL detection kit (EMD Millipore; Billerica, MA, USA).

2.6 | Analysis of MAGs, NAEs and LPA by liquid chromatography—Tandem mass spectrometry (LC-MS/MS)
For the analysis of MAGs and NAEs, the denatured samples containing the ISTD were thawed and centrifuged (1000g; 10 minutes) to remove cellular debris, then supernatants were diluted with water to a final MeOH concentration of 10%, and maintained at pH 3 by the addition of acetic acid. Samples were loaded on solid phase extraction cartridges (Strata-X Polymeric Reversed Phase, 60 mg/1 mL, Phenomenex). Cartridges were washed with 2 mL of acidified water and li- pids were eluted with 1 mL of MeOH. The eluates were evapo- rated to dryness under a stream of nitrogen. For the analysis of LPA, the denatured samples containing the ISTD were acidi- fied with acetic acid (0.1 M final concentration). Lipids then

were extracted from the denatured samples by adding 1 mL of chloroform, vortexing for 1 minute, and centrifuging at 4000g for 5 minutes without brakes. This was repeated three times. The organic phases were collected, pooled, and evaporated to dryness under a stream of nitrogen. For the quantification of MAGs in human plasma, samples were extracted as docu- mented before27 with slight modifications. 200 µL of plasma samples were mixed with 300 µL of TRIS (pH 7.4, 50 mM). Toluene (2 mL) containing the ISTD was then added to the samples, vortexed for 1 minute, centrifuged at 4000g for 5 min- utes without brakes. Samples then were placed in an ethanol- dry-ice bath (−80°C) to freeze the aqueous phase (bottom). The organic phase (top) was then collected and evaporated to dryness under a stream of nitrogen. Samples were reconsti- tuted in 25 µL of HPLC solvent A (H2O with 0.05% acetic acid and 1 mM NH +) and 25 µL of solvent B (MeCN/H O,

TABLE 1 Specific mass transitions and retention times of the metabolites analyzed by LC-MS/MS

95/5, v/v, with 0.05% acetic acid and 1 mM NH4 ). A 25 µL of

aliquot was injected onto an RP-HPLC column (Kinetex C8, 150 × 2.1 mm, 2.6 µm, Phenomenex). Quantification was per- formed on a Shimadzu 8050 triple quadrupole mass spectrom- eter using the same LC program as described previously.28
Quantification was achieved by generating calibration curves using pure standards and analyzed on the LC-MS/ MS system three times. The slope was then calculated using the ratio between the peak areas of the compound and its standard (1-AG-d5 for MAGs, AEA-d4 for anandamide or N-arachidonoyl-ethanolamine (AEA) and C17:1-LPA for the various LPA species). The mass transition and retention times of each compound are provided in Table 1.

2.7 | Statistical analyses
Statistical analyses (one-way ANOVA with Dunnett’s multi- ple comparisons test) were done using the GraphPad Prism 7 software. P values < .05 were considered significant.

3.1 | Expression of DAG lipase α and β
Previous attempts to stimulate 2-AG production by human immune cells were somewhat disappointing, leading to eCB levels below those required to activate the CB2 recep- tor.13,15,17 This raised the possibility that key biosynthetic en- zymes, notably the DAG lipases, might be absent or poorly expressed. In the first series of experiments, the expression of DAG lipases was thus assessed by immunoblot in human leukocytes. A sharp difference between the expression pat- terns of DAG lipase α and β were found . DAG lipase α was detected in the hypothalamus samples, which were included as a positive control. In contrast, DAG lipase α

was usually absent from our leukocyte preparations, with the exception of some eosinophil and AM samples in which we detected a weak signal. As for DAG lipase β, it was found in eosinophils, monocytes, and AMs while being almost absent in the hypothalamus samples, neutrophils, and lymphocytes. These data support the concept that the DAG lipase pathway might not be involved in the biosynthesis of 2-AG by all leu- kocyte types, particularly in neutrophils and lymphocytes.

3.2 | 2-AG biosynthesis by leukocytes stimulated with PLC-activating agonists
Considering the obtained DAG lipases expression patterns , we postulated that human leukocytes, especially eosinophils, monocytes, and AMs, might be capable of gen- erating 2-AG following PLC activation. Leukocytes hydro- lyze 2-AG and its metabolites within seconds to minutes8,9,12

1 Expression of DAG lipases in human leukocytes. Freshly isolated cell preparations were denatured and protein samples were analyzed by immunoblot for DAG lipases α and β.
Experiments were performed on three volunteers per cell type and all three gels are shown. AMs, alveolar macrophages; EOS, eosinophils; HYPO, hypothalamus; LYM, lymphocytes; MONO, monocytes; NEU, neutrophils and the efficacy of 2-AG hydrolysis inhibitors at enhancing 2-AG half-life overtime has not been documented in human leukocytes. Thus, we first compared the effects of three 2-AG hydrolysis inhibitors with varying selectivity, namely MAFP, Palmostatin B, and JZL184, to determine which one would most effectively increase 2-AG half-life in neutrophil suspensions. We selected these three compounds because they consistently inhibited 2-AG hydrolysis in each leuko- cyte types compared to other compounds.12 In absence of inhibitor, ~90% of the added 2-AG in our neutrophil suspen- sions had disappeared after one minute ( 2A). JZL184, Palmostatin B, and MAFP all prolonged 2-AG half-life in neutrophils ( 2A). MAFP, at 1 µM, was the most effi- cient with ~75% of 2-AG remaining after 15 minutes. MAFP (100 nM) and Palmostatin B (10 µM) had comparable inhibi- tory effect on 2-AG hydrolysis with ~50% 2-AG remaining after 15 minutes. JZL184 (10 µM) was the least efficient with ~35% 2-AG remaining after 5 minutes and basically no 2-AG left at 10 minutes. Consequently, we selected MAFP to prevent 2-AG hydrolysis in our experimental model, in the hope of better characterizing the possible biosynthesis of 2-AG by human leukocytes. We next investigated whether leukocytes could generate 2-AG in response to two agonists previously documented to activate the PLC (and DAG lipase) pathway in leukocytes: the calcium ionophore A23187 or PAF.13,29 The stimulations were performed in the presence of MAFP, in order to document the impact of increasing 2-AG
half-life and to correctly assess the biosynthetic capabilities of leukocytes. PAF did not significantly stimulate a robust biosynthesis of 2-AG in any cell type. A23187 stimulated the biosynthesis of 2-AG in monocytes and AMs, although the obtained levels were modest ( 2B-E). These data indicate that although A23187 has a stimulatory effect on 2-AG biosynthesis, human leukocytes do not generate large amounts of 2-AG via the classic biosynthetic pathway in- volving the sequential actions of PLC and DAG lipases.

3.3 | Arachidonic acid stimulates a robust biosynthesis of 2-AG in human neutrophils
We next treated human neutrophils with a larger panel of inflammatory effectors, notably AA (the product of 2-AG hydrolysis), TLR agonists and the recognized neutrophil acti- vator fMLP. In absence of MAFP, human neutrophils did not synthesize detectable 2-AG levels (detection limit of 25 fmol) in response to fMLP, LPS, or the TLR 7/8 agonist R848 while they synthesized modest amounts of 2-AG in response to AA ( 3A). When these experiments were repeated in pres- ence of MAFP, 2-AG was synthesized in very limited amounts upon stimulation with G protein-coupled receptor (GPCR) or TLR agonists. In contrast, MAFP-treated neutrophils synthe- sized large amounts of 2-AG in response to AA . The effect of AA was concentration-dependent

2 Biosynthesis of 2-AG induced by PAF or A23187. A, Pre-warmed neutrophil suspensions (37°C, 5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 were treated with DMSO, MAFP, Palmostatin B (Palm B), or JZL184 at the indicated concentration for 5 minutes then incubated with 1 µM 2-AG for up to 15 minutes. B-E, Pre-warmed leukocyte suspensions (37°C, 5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 were treated with 1 µM MAFP for 5 minutes then treated with either DMSO, PAF (1 µM), or A23187 (100 nM) for 15 minutes. A-E, Incubations were stopped by adding 0.5 volume of cold (−20°C) MeOH containing 2 ng 1-AG-d5 as internal standard. Samples were processed and analyzed for 2-AG levels as described in Materials and methods. Data are the mean (±SEM) of 3-4 independent experiments

3 Impact of AA on the biosynthesis of 2-AG by human neutrophils. Pre-warmed human neutrophil suspensions (37°C,
5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 were utilized for all experiments. Cells were incubated with (A) DMSO, (B-E) 1 µM MAFP or (F) JZL184, Palmostatin B or MAFP for 5 minutes before the addition of the stimuli. A,B, Cells were stimulated with fMLP (1 µM), R848 (10 µM), PAF (1 µM), A23187 (100 nM), or AA (10 µM) for 15 minutes. C, Cells were stimulated with AA for 15 minutes at the indicated concentrations. D, Cells were stimulated with 10 µM AA for the indicated times. E, Cells were stimulated with 10 µM of AA for 15 minutes.
F, Cells were stimulated with 10 µM AA during 15 minutes. A-F, Incubations were stopped by the addition of 0.5 mL of cold (−20°C) MeOH containing 2 ng 1-AG-d5 as internal standard. Samples then were processed and analyzed for 2-AG and other MAGs by LC-MS/MS as described in Material and methods. Data are the mean (±SEM) of three or four independent experiments

and kinetic experiments unraveled that 2-AG biosynthesis/ac- cumulation was maximal at 15 minutes, after AA had disap- peared from the cell preparations ( 3D). AA selectively induced the biosynthesis of 2-AG, as no other MAG was de- tected ( 3E). We next performed an additional set of experiments in which we compared the efficacy of JZL184, Palmostatin B, and MAFP to unmask the AA-induced 2-AG biosynthesis. In these experiments, AA stimulated the bio- synthesis of 2-AG in presence of the three inhibitors ( 3F). However, MAFP was the most efficient inhibitor at un- masking the AA-induced 2-AG synthesis. Importantly, these data perfectly fitted with the ability of the three inhibitors to prevent 2-AG hydrolysis in neutrophils (2A), further consolidating that MAFP was the inhibitor of choice to better assess the AA-induced biosynthesis of 2-AG by neutrophils. As for the eCB AEA, it was not detected in large amounts, from being below detection limit in untreated neutrophils to 0,28 ± 0.05 pmol/million cells in AA-stimulated neutrophils (n = 3), even though MAFP would have blocked its hydroly- sis by inhibiting the fatty acid amide hydrolase (FAAH).30,31 Thus, the treatment of human neutrophils with AA led to a robust stimulation of 2-AG, while having modest effects on AEA levels. Consequently, we did not explore the biosynthe- sis of AEA and other NAEs any further.

3.4 | Inhibition of the AA-induced 2-AG biosynthesis in human neutrophils by acylation inhibitors
Since 2-AG biosynthesis was maximal after most of the AA was cleared from the incubation media (3D), we pos- tulated that AA was acylated into cellular membranes then released from the cell membranes to yield a MAG. To verify
this, experiments were performed in which human neutrophils were treated with the fatty acyl-CoA synthase inhibitor trias- cin C32 or the acyl-CoA transferase inhibitor thimerosal.33-35 In agreement with the working hypothesis, triascin C and thi- merosal both inhibited the AA-induced 2-AG biosynthesis by
~90% (A). To further explore this recycling pathway, another set of experiments was done in which neutrophils were stimulated with AA-d8 (4C) instead of AA ( 4B). AA-d8 led to an almost inexistent biosynthesis of 2-AG, which was replaced by an equivalent biosynthesis of 2-AG-d8 ( 4B,C). Altogether, these data indicate that in neutrophils, some of the exogenously-added AA is acylated into phospholipids and then further processed into 2-AG.

3.5 | Biosynthesis of MAGs by UFAs
While 2-AG is the MAG binding to the CB2 receptor with the highest affinity, other MAGs can activate DMSO-differentiated HL60 cells (a cell model of neutrophil), which express the CB2 receptor, with equal or better potency and efficacy than AEA.7 This, combined with the results presented in 4, prompted us to determine whether other fatty acids would stimulate the biosynthesis of their MAG counterparts. Human neutrophils were thus treated with various fatty acids for 15 minutes then processed for MAG quantification.  5A shows that neu- trophils metabolized UFAs into MAGs with varying efficacy, DHA being the most efficiently metabolized. Long chain fatty acids were better transformed than shorter chains fatty acids (C22>C20>C18). In addition, for a given fatty acid chain length, an increased in the number of double bonds led to a bet- ter efficacy (DHA vs DPA; EPA vs AA; LA vs OA). Of note, the 16-carbon saturated fatty acid palmitic acid did not induce the biosynthesis of palmitoyl-glycerol.

 4 Impact of reacylation inhibitors and AA-d8 on the AA-induced 2-AG biosynthesis by neutrophils. A-C, Pre-warmed human neutrophil suspensions (37°C, 5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 were pre-incubated with 1 µM MAFP for 5 minutes

then stimulated with 10 µM AA or AA-d8 for 15 minutes. A, Triascin C or thimerosal were added 2 minutes before the addition of AA. A-C, Incubations were stopped by the addition of 0.5 mL of cold (−20°C) MeOH containing 2 ng 1-AG-d5 as internal standard. Samples were processed and analyzed for 2-AG and 2-AG-d8 levels as described in Material and methods. The data are the mean (±SEM) of at least three independent experiments

 5 Impact of fatty acids on the biosynthesis of monoacylglycerols by human leukocytes. Pre-warmed human leukocyte suspensions (37°C, 5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 were pre-incubated with 1 µM MAFP for 5 minutes then stimulated with 10 µM of the different fatty acids for 15 minutes. Incubations were stopped by the addition of 0.5 volume of cold (−20°C) MeOH containing 2 ng 1-AG-d5 as internal standard. Samples then were processed and analyzed for monoacylglycerol levels as described in Methods. Data are the mean (±SEM) of at least three independent experiments
3.6 | Eosinophils and monocytes, but not AMs nor lymphocytes, also metabolize UFAs into MAGs
In the next series of experiments, the hypothesis that other leukocytes could also biosynthesize 2-AG and other MAGs in response to fatty acids was tested. UFAs also induced the biosynthesis of their MAG derivatives in human eosinophils and human monocytes (5B,C) but to a lesser extent than neutrophils. In contrast, human lymphocytes and human AMs only produced trace amounts of MAGs in response to UFAs ( 5D,E). Finally, AA, LA, or OA did not induce the synthesis of their respective MAGs by platelets or eryth- rocytes (data not shown). Again, palmitoyl-glycerol was not detected in any cell type treated with palmitic acid, suggest- ing that saturated fatty acids do not undergo this MAG bio- synthetic pathway. Altogether, these data raise the possibility that the UFA-mediated synthesis of 2-AG and other MAGs is restricted to leukocytes from the myeloid lineage.

3.7 | Possible mechanism of UFAs-induced biosynthesis of MAGs in leukocytes
The poor stimulatory effect of GPCR agonists such as PAF and fMLP on the biosynthesis of 2-AG by leukocytes, and the striking effect of triascin C and thimerosal on the AA-induced 2-AG biosynthesis in neutrophils which we found here not to express DAG lipases compared to other leukocytes, sug- gested that AA stimulated 2-AG biosynthesis independently of the PLC/DAG lipase pathway (1-3). In further support to this hypothesis, the PLC inhibitor U73122 and the DAG lipase inhibitors KT109 and KT172, at 10 µM, did not inhibit the AA-induced 2-AG biosynthesis in neutrophils (data not shown), supporting the concept that AA induces the biosynthesis of 2-AG in a PLC/DAG lipase-independent manner and very likely via its remodeling into phospholipids.
Apart from the PLC/DAG lipase pathway, the biosynthesis of 2-AG (and possibly other MAGs) was documented to occur via to other pathways: a PLD-mediated cleavage of phos- phatidic leading to DAG, which is then converted into 2-AG
by DAG lipases21; or by the conversion of LPA into 2-AG by a
phosphatase.22 Given the barely detectable expression of DAG lipases in neutrophils (1), the lack of effect of DAG li- pase inhibitors, and the inability of fMLP to stimulate 2-AG biosynthesis in neutrophils (fMLP being the most recognized GPCR activating PLD in neutrophils36,37), we concluded that the PLD/DAG lipase pathway was not involved in the AA- induced 2-AG biosynthesis. The next experiments were thus focused on testing whether AA stimulated 2-AG biosynthesis via a LPA intermediate. Human neutrophils were thus treated with AA for different times and the levels of AA-LPA were assessed. AA stimulated the biosynthesis of AA-LPA ( 6). While 2-AG levels are maximal at 15 minutes ( 3C), AA-LPA levels peaked at 5 minutes then declined overtime ( 6A). Other fatty acids also induced the biosynthe- sis of the corresponding LPA species in human neutrophils ( 6B). The biosyntheses of the different LPA species followed the same trend as their MAGs counterparts , with longer fatty acid chains leading to more important

6 Impact of AA and other unsaturated fatty acids on LPA biosynthesis. A-C, Pre-warmed human leukocyte suspensions (37°C,

5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 (A) were treated with 10 µM AA for the indicated times; or (B,C) were treated with 10 µM of UFAs for 5 minutes. A-C, Incubations were stopped by the addition of 0.5 volume of cold (−20°) MeOH containing 10 ng C17:1-LPA. Samples were processed and analyzed for LPA content as documented in Material and Methods. Data are the mean (±SEM) of 3-4 independent experiments

biosynthesis of the corresponding LPAs. Moreover, the treat- ment of neutrophils with AA and AA-d8 led to the biosynthe- sis of AA-LPA and AA-d8-LPA, respectively, indicating that the newly biosynthesized LPA originated from the added fatty acid (6C). Importantly, thimerosal and triascin C both inhibited the AA-, the EPA-, and the DHA-stimulated LPA biosyntheses to the same extent than their MAGs (data not shown). Finally, several phosphatase inhibitors were tested to evaluate whether they could inhibit the putative dephos- phorylation of LPA into 2-AG. Sodium orthovanadate, AlF , propranolol, bromoenol lactone and XY-14, which all inhibit LPA dephosphorylation38-41 failed to prevent AA-induced 2-AG synthesis (data not shown).

3.8 | AA-induced 2-AG biosynthesis is decreased by pro-inflammatory effectors
As mentioned above, our attempts at stimulating the biosyn- thesis of 2-AG by agonists stimulating the PLC/DAG lipase pathway did not lead to a robust biosynthesis of 2-AG and other MAGs, as opposed to the UFA-stimulated 2-AG (and other MAGs) biosynthesis . We next ex- amined whether these two pathways interacted with each other by treating human neutrophils with AA in combination with PAF, fMLP, or LTB4. All three GPCR agonists signifi- cantly decreased the AA-induced 2-AG biosynthesis (7). PAF was the most potent with ~45% inhibition, followed by fMLP (~35%) and LTB4 (~20%). Interestingly, PAF had a comparable effect on the EPA-stimulated biosynthesis of 2-EPG, while having little no effect on the OA-induced 2-OG biosynthesis ( 7B). PAF is a recognized neutro- phil activator. As such, it induces an important mobilization of Ca2+ ions from internal stores.26,42 As a result, this acti- vates enzymes capable of metabolizing fatty acids, notably

the 5-lipoxygenase.43 We thus postulated that the decreased 2-AG biosynthesis in presence of PAF/AA (vs AA alone) was be the consequence of a greater metabolism of AA and EPA by the 5-lipoxygenase in neutrophils. In agreement with the latter, the combination of PAF with either AA or EPA led to a 6-fold increase in LTB4 and a 26-fold increase in LTB5 biosynthesis ( 7C), indicating that the inhibitory effect of PAF displays on the AA-induced 2-AG biosynthesis and the EPA-induced 2-EPG biosynthesis is likely the con- sequence of increased metabolism of exogenously-added AA and EPA into 5-lipoxygenase metabolites, thereby diminish- ing the amount of UFA being acylated and undergoing the MAG biosynthetic pathway we have unmasked. This also in- dicates that during the course of acute inflammation in which polyunsaturated fatty acids are present simultaneously with pro-inflammatory effectors such as PAF, MAG biosynthesis might be diminished by a pro-inflammatory entourage to the advantage of other pro-inflammatory effectors such as leu- kotrienes and prostaglandins.

3.9 | Levels of MAGs in human plasma
The levels of 2-AG in human plasma have been documented in humans, varying from 1 to 20 nM.44 However, the lev- els of the other MAGs documented herein have never been documented completely, although some studies reported the presence of 2-OG, 2-EPG, and 2-DHG.45,46 Herein, we quan- titated the levels of MAGs derived from PA, OA, LA, AA, EPA, DPA, and DHA in the plasma from fasting healthy vol- unteers (five women, five men), matched for age (29 ± 12 and 30 ± 12, respectively; mean ± SD) and body mass index (21,89 ± 1,22 vs 22,43 ± 1.41; mean ± SD). The MAGs we
investigated herein were all detected in the plasma ( 7D). 2-OG, 2-PG,and 2-LG were the most abundant. There

 7 Impact of PAF, fMLP, and LTB4 on the UFA-induced 2-AG and other MAG biosynthesis by human neutrophils and MAG levels in plasma. A-C, Pre-warmed human neutrophil suspensions (37°C, 5 × 106 cells/mL) in HBSS containing 1.6 mM CaCl2 were treated with MAFP (1 µM) for 5 minutes. UFAs (10 µM) were then added simultaneously with DMSO or 1 µM PAF, fMLP, or LTB4 for 15 minutes. Incubations were stopped by the addition of 0.5 mL of cold (−20°C) MeOH containing 2 ng 1-AG-D5 and LTB4-d4 as internal standard. Samples were processed for LC-MS/MS analysis of eCB-Glycerols as described in Material and methods. One-way ANOVA with Dunnett’s multiple comparisons test were performed using the GraphPad Prism 7 software. **P < .01; ****P < .0001 (vs AA alone). D, Platelet-rich plasma samples were processed and analyzed for their content in the different MAGs as documented in Methods. Results are the mean (±SEM) of 5 different donors for each group. No statistically significant difference was found between men and women (two-way ANOVA with Sidak’s multiple comparison test)

was no statistically significant difference between men and women for any of the MAGs we measured.

Endocannabinoids are natural anti-inflammatory media- tors, at least in mice, and the pharmacological blockade of their degradation has great potential to treat inflammatory diseases, notably by preventing leukocytes of hydrolyzing them. However, whether human leukocytes are an important source of eCBs has been elusive. In this paper, we show that:
(a) DAG lipase expression differs from a leukocyte subset to the other with DAG lipase β being more expressed than DAG lipase α, (b) leukocytes do not synthesize large amount of 2-AG via the PLC/DAG lipase pathway, (c) The non-se- lective 2-AG hydrolysis inhibitors MAFP and palmostatin B increase the half-life of 2-AG for a longer duration than JZL184 in neutrophils, (d) circulating myeloid leukocytes bi- osynthesized 2-AG and other MAGs in response to UFAs, (e) the UFA-mediated MAG synthesis is not inhibited by DAG lipase inhibitors but is inhibited by acyl-CoA synthase and an

acyl-transferase inhibitors, (f) the UFA-mediated biosynthe- sis of MAGs is preceded by the buildup of their LPA conge- ners, (g) pro-inflammatory effectors activating PLC inhibit the AA-induced 2-AG biosynthesis, and (h) human plasma contains each of the investigated MAGs, some of which were never documented before.
The expression of the different DAG lipases by human leu- kocytes was ill defined. DAG lipase α was only found in trace amounts in leukocytes, notably eosinophils and AMs, in sharp contrast with human hypothalamus samples. This finding was expected, as DAG lipase α was described as the main 2-AG
biosynthetic enzyme in mouse brain.18,19 As for DAG lipase
β, it was found in larger levels in eosinophils, monocytes and AMs ( 1) vs low/undetectable levels in neutrophils and lymphocytes, supporting that these leukocytes do not synthe- size 2-AG in a DAG lipase-dependent fashion, as underscored in  2. The latter finding is in line with data showing the importance of this enzyme in 2-AG synthesis by murine macrophages.20 The A23187-induced 2-AG biosynthesis by monocytes is likely explained by the fact that they express DAG lipase β, although this remains to be confirmed using selective DAG lipase inhibitors. However, using this rationale,

we would have expected similar findings in eosinophils, which instead produced less than 0.1 pmol/106 cells in response to PLC activation ( 2C). Given that GPCR agonists inhibit 2-AG synthesis by neutrophils ( 7), GPCR activation was bound to fail to induce 2-AG (and other MAG) biosyn- thesis in leukocytes. We did not detect large amounts of DAG lipase β in neutrophils nor lymphocytes compared to eosino- phils, supporting that these leukocytes do not synthesize 2-AG in a DAG lipase-dependent fashion, as underscored
6. Combined with the inhibitory effects of PAF, fMLP, and LTB4 on the AA-induced 2-AG biosynthesis, we conclude that the DAG lipase pathway does not mediate a robust biosynthe- sis of 2-AG by human inflammatory cells.
Our data also underscore the importance of hydrolase in- hibition when studying eCB biosynthesis in leukocytes. The data presented herein, combined with our previous work,
show that human leukocytes are experts at hydrolyzing 2-AG.8,9,12 Indeed, in absence of hydrolase inhibitors, we did not observe detectable amounts of 2-AG in stimulated neutro- phils with the exception of AA, which led to a minimal 2-AG
biosynthesis ( 2). The use of MAFP unraveled that AA induces a robust biosynthesis of 2-AG by neutrophils. These findings might provide an explanation for the low amounts of 2-AG produced by human immune cells in previous studies,
in which 2-AG hydrolysis was not prevented.13,15,17 However,
we show that most leukocytes do not biosynthesize substan- tial levels of 2-AG in response to agonists recognized to acti- vate the PLC pathway, either in the absence or in the presence of MAFP. One study showed that human lung macrophages stimulated with LPS synthesize more 2-AG in the presence of a MAG lipase inhibitor but this increase of approximately 40% is modest compared to the impact MAFP had in our bio- synthetic route involving UFAs.14
Following the structure elucidation of prostaglandins and leukotrienes, AA has been typecast as a villain. The stimula- tory effect it has on 2-AG biosynthesis by leukocytes might thus be surprising and counter-intuitive. However, the data provided herein indicate that the biosynthetic route involved in the AA-induced 2-AG biosynthesis is clearly different from the classical GPCR-PLC-PLA2 pathway involved in eico- sanoid biosynthesis. Indeed, the use of the pharmacological inhibitors triascin C and thimerosal indicate that AA is trans- formed into AA-CoA, then incorporated either into a glycer- ol-moiety or into lysophospholipids, then further metabolized into 2-AG ( 4). This biosynthetic route, while incom- plete, is also supported by the data obtained with AA-d8, the latter almost exclusively stimulating the synthesis of 2-AG-d8 ( 4C). Thimerosal is known to effectively block AA acy- lation in human neutrophils by inhibiting two acyltransferases, namely membrane bound O-acyl transferase 5 (MBOAT5) and MBOAT7.33 Given the sharp inhibitory effect of thimerosal on the AA-induced 2-AG biosynthesis, it is very likely that one, perhaps both of these enzymes are involved in this process.

The metabolism of AA into 2-AG does not involve the DAG lipase pathway, but might involve LPA as an intermedi- ate (6). The dephosphorylation of AA-LPA to produce 2-AG was indeed described in rat brains,22 but its importance in 2-AG (and other MAG) biosynthesis in humans had not been investigated. In our experimental model, AA-LPA bio- synthesis preceded that of 2-AG, with maximal levels being detected at 5 and 15 minutes, respectively ( 3 and 6). This suggests than a LPA buildup occurs first, which could be followed by its dephosphorylation into 2-AG overtime. However, we were unable to prevent the AA-induced 2-AG biosynthesis using a wide array of LPA phosphatase inhibi- tors. Furthermore, the addition of exogenous AA-LPA did not lead to the synthesis of 2-AG (data not shown). Altogether, the LPA data indicate the following possible outcomes: (a) the buildup of LPA preceding MAG biosynthesis is coincidental,
(b) LPA dephosphorylation to MAG occurs via a phospha- tase that is insensitive to the inhibitor used, and/or (c) LPA dephosphorylation occurs within the cells and exogenous- ly-added LPA do not enter the cells and is not susceptible to dephosphorylation. Therefore, the involvement of LPA in this process requires further investigation. Moreover, the fact that the UFA-induced biosynthesis of MAGs and the correspond- ing LPA species occurs to a significantly higher extent when starting with polyunsaturated vs saturated fatty acids, that is, with fatty acids that are normally esterified to the 2-position of (lyso) phospholipids, clearly points to a biosynthetic mech- anism that, via phospholipid remodeling, increases the levels of potential 2-acyl-(lyso)phospholipid precursors for MAGs.
The plasmatic levels of 2-AG we report here are in the low nM range, in line with previous studies.27,44 The MAGs from the ω-3 fatty acids EPA, DPA, and DHA were the least abun- dant among the compounds we detected. Diet is known to
have a direct effect on the fatty acid content found in the cir- culation and therefore, it could influence the levels of MAGs as well.47 Given that we show here that circulating leukocytes isolated from the peripheral blood convert UFAs into MAGs,
the dietary intake of fatty acids is probably a determinant of plasma eCB levels. Another possible determinant of 2-AG and other MAG levels in the blood might be their degrada- tion by 2-AG hydrolases, which is also a reaction that occurs
rapidly in the presence of leukocytes.8,9,12 However, we also
show that the use of a lipase inhibitor (MAFP), drastically reduces this 2-AG (and other MAG) hydrolysis and allows for an impressive build-up in fatty acid-stimulated leuko- cytes. Therefore, the therapeutic use of hydrolase inhibitors would allow the eCB tone to be increased and for eCBs to have higher bioactive potential.
Altogether, our data show that human myeloid leuko- cytes biosynthesize significant amounts of 2-AG and other MAGs. This biosynthesis is preceded by the biosynthesis of a LPA intermediate, although the involvement of this inter- mediate in the biosynthesis of MAGs remains to be proven.

Our data also suggests that the development of MAG hy- drolysis inhibitors might turn human myeloid leukocytes into anti-inflammatory effectors by increasing their ability to synthesize anti-inflammatory 2-AG (and other MAGs) and eventually activate the endocannabinoidome receptors, notably the anti-inflammatory CB2 receptor as well as other anti-inflammatory receptors for other MAGs.
This work was supported by grants to NF from the Natural Sciences and Engineering Research Council of Canada and the Canada Excellence Research Chair on the Microbiome- Endocannabinoidome Axis in Metabolic Health held by VD. CT and ASA are the recipients of doctoral awards from the Canadian Institutes of Health Research and ÉD is the re- cipient of a Master award from the Centre de recherche de l’IUCPQ foundation. We would like to thank Johane Lepage, Joanne Milot, and Hélène Villeneuve for performing the blood sampling procedures and Sophie Castonguay-Paradis for preparing and analyzing the plasma samples of healthy volunteers. CT, ASA, ÉD, ML, LPB, and NF are members of the Quebec Respiratory Health Network. CT, ASA, ÉD, CM, AV, VD, and NF are associated to the Canada Excellence Research Chair on the Microbiome-Endocannabinoidome Axis in Metabolic Health.
Authors declare they have no conflict of interest.

CT, ASA, ML, MRB, AV, VD, and NF designed research. CT, ASA, ÉD, ML, KY, TI, NO, LPB, and CM performed re- search. KY, NO, and TI contributed new reagents. CT, ASA, ÉD, and NF analyzed data. CT and NF wrote the original draft of the paper. All authors were involved in reviewing, editing, and revising the paper.
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