Implication of the anti-inflammatory bioactive lipid prostaglandin D2-glycerol ester in the control of macrophage activation and inflammation by ABHD6 Mireille Alhouayeka,b,1, Julien Masqueliera,1, Patrice D. Canic, Didier M. Lambertb, and Giulio G. Mucciolia,2 aBioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Université catholique de Louvain, B-1200 Brussels, Belgium; bMedicinal Chemistry Research Group, Louvain Drug Research Institute, Université catholique de Louvain, B-1200 Brussels, Belgium; and cMetabolism and Nutrition Research Group, Walloon Excellence in Life sciences and BIOtechnology (WELBIO), Louvain Drug Research Institute, Université catholique de Louvain, B-1200 Brussels, Belgium Edited by Charles N. Serhan, Brigham and Women’s Hospital /Harvard Medical School, Boston, MA, and accepted by the Editorial Board September 5, 2013 (received for review July 24, 2013) Proinflammatory macrophages are key mediators in several pathol- MAGL controls around 80% of 2-AG hydrolysis, it remains ogies; thus, controlling their activation is necessary. The endocanna- unclear whether this occurs in other tissues (13, 14). Because of binoid system is implicated in various inflammatory processes. Here this control of brain 2-AG levels, inhibition of MAGL leads to we show that in macrophages, the newly characterized enzyme increased central levels of 2-AG and therefore to undesirable α β / -hydrolase domain 6 (ABHD6) controls 2-arachidonoylglycerol psychotropic side effects due to activation of the CB1 cannabinoid (2-AG) levels and thus its pharmacological effects. Furthermore, receptor (15). More recently, other enzymes have been impli- we characterize a unique pathway mediating the effects of 2-AG cated in 2-AG hydrolysis, such as the newly annotated enzyme through its oxygenation by cyclooxygenase-2 to give rise to the α/β-hydrolase domain 6 (ABHD6) (16–19). Here, we demonstrate fl anti-in ammatory prostaglandin D2-glycerol ester (PGD2-G). Pharma- the anti-inflammatory effects of inhibiting ABHD6 and thus in- cological blockade of cyclooxygenase-2 or of prostaglandin D syn- creasing 2-AG levels, without the side effects of MAGL inhibition. thase prevented the effects of increasing 2-AG levels by ABHD6 Classically, 2-AG binds to and activates two G-protein–coupled – inhibition in vitro, as well as the 2-AG induced increase in PGD2-G receptors, termed cannabinoid receptors 1 and 2 (CB1 and CB2); levels. Together, our data demonstrate the physiological relevance however, it may also activate the peroxisome proliferator-activated of the interaction between the endocannabinoid and prostanoid receptors (PPARs) (20). Alternatively, this endogenous lipid, like systems. Moreover, we show that ABHD6 inhibition in vivo allows arachidonic acid, may also be oxidized by cyclooxygenase (COX) for fine-tuning of 2-AG levels in mice, therefore reducing lipopoly- enzymes to produce prostaglandin glycerol esters (PG-Gs) (21). saccharide-induced inflammation, without the characteristic central So far, the biological effects of these PG-Gs are not fully eluci- side effects of strong increases in 2-AG levels obtained following dated. In this study, we also demonstrate that following increased monoacylglycerol lipase inhibition. In addition, administration of 2-AG levels in macrophages, COX-2–mediated production of fl PGD2-G reduces lipopolysaccharide-induced in ammation in mice, PGD -G increases, resulting in the anti-inflammatory effects ob- fi 2 thus con rming the biological relevance of this 2-AG metabolite. This served with 2-AG (Fig. S1). We also put forth, in vivo, the anti- points to ABHD6 as an interesting therapeutic target that should be inflammatory properties of PGD2-G. relevant in treating inflammation-related conditions, and proposes PGD -G as a bioactive lipid with potential anti-inflammatory proper- 2 Significance ties in vivo. COX-2 | prostaglandin synthase | glyceryl prostaglandin | FAAH | 2-Arachidonoylglycerol (2-AG) is an endogenous bioactive lipid anandamide implicated in numerous (patho)physiological processes. 2-AG classically activates the cannabinoid receptors, and its activity is terminated by enzymatic hydrolysis. The main enzyme studied acrophages are key players in innate and adaptive immune in this context is monoacylglycerol lipase (MAGL). Although its Mresponses to bacterial infections or noxious agents. Their inhibition, to increase 2-AG levels, has proven beneficial, it is role during inflammatory processes is to eliminate the threat and fl hindered by psychotropic side effects due to drastically elevated protect the body (1, 2). Macrophages exhibiting an in ammatory brain 2-AG. Here we show the anti-inflammatory effects of in- fl phenotype secrete proin ammatory mediators and reactive ox- hibition of another 2-AG hydrolase, α/β-hydrolase domain 6, fl ygen and nitrogen species that in uence the polarization of T- without the side effects associated with MAGL inhibition. We helper cells, further drive the inflammatory response, and acti- also show that 2-AG decreases macrophage activation and this vate various antimicrobial mechanisms (3). Under persistence of effect is not mediated by its classical receptors. Furthermore, the proinflammatory phase, inflammation becomes chronic, thus we demonstrate that a cyclooxygenase-2–derived metabolite of deleterious (3). Macrophages are key mediators in the immu- 2-AG, prostaglandin D2-glycerol ester, is responsible for the nopathology of metabolic inflammation and autoimmune dis- documented effects. eases such as inflammatory bowel disease (IBD) and rheumatoid arthritis (2, 4). Thus, in these pathologies, proinflammatory Author contributions: M.A. and G.G.M. designed research; M.A., J.M., and G.G.M. performed macrophage responses must be controlled and reduced. research; G.G.M. contributed new reagents/analytic tools; D.M.L. and P.D.C. provided re- agents; M.A., J.M., and G.G.M. analyzed data; and M.A., J.M., P.D.C., D.M.L., and G.G.M. The endocannabinoid 2-arachidonoylglycerol (2-AG) is in- wrote the paper. volved in various (patho)physiological processes and exerts nu- The authors declare no conflict of interest. fi merous bene cial actions (ranging from pain modulation to This article is a PNAS Direct Submission. C.N.S. is a guest editor invited by the Editorial reduction of anxiety) (5–11). The activity of this bioactive lipid Board. depends on its endogenous levels, tightly controlled by the 2-AG 1M.A. and J.M. contributed equally to this work. hydrolyzing enzymes (12). Monoacylglycerol lipase (MAGL) is 2To whom correspondence should be addressed. E-mail: [email protected]. thought to be the primary enzyme responsible for 2-AG me- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tabolism. Although this has been proven in the brain, where 1073/pnas.1314017110/-/DCSupplemental. 17558–17563 | PNAS | October 22, 2013 | vol. 110 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1314017110 Downloaded by guest on September 24, 2021 Results The results obtained in J774 cells were confirmed further in ABHD6, but Not MAGL, Inhibition Reduces Macrophage Activation. primary peritoneal macrophages, a more physiological model, as Lipopolysaccharide (LPS), a component of the cell wall of Gram- well as in other macrophage-like cell lines, such as RAW264.7 negative bacteria, is a potent macrophage activator (22). Macro- cells and the BV2 microglial cell line. Indeed, treatment of these phages activated with LPS release several proinflammatory cyto- cells with WWL70 significantly increased 2-AG levels (Fig. 1H kines, such as IL-1β, IL-6, and TNF-α, and produce nitric oxide and Fig. S5A), thus leading to a decrease in LPS-induced mac- I J B D (Fig. S2A) and prostaglandins (PGs). rophage activation (Fig. 1 and and Fig. S5 and ), as To assess the implication of ABHD6 in 2-AG metabolism by measured by mRNA expression of IL-1β as well as PG pro- macrophages, J774 macrophages incubated with increasing doses duction. Quantitative PCR (qPCR) for mRNA expression of fi of the selective ABHD6 inhibitor WWL70 (17, 23, 24) were MAGL and ABHD6 con rmed the absence of MAGL in the D–F stimulated with LPS. WWL70 induced a dose-dependent in- RAW264.7 cells, as well as the presence of ABHD6 (Fig. S4 ). crease in 2-AG levels (Fig. 1A), with an EC50 of 0.3 μM. In- creasing exogenous or endogenous 2-AG levels by exposing the The Effect of 2-AG and ABHD6 Inhibition Is Independent of Cannabinoid Receptors or PPARs. 2-AG is an agonist at both the CB and CB cells to increasing concentrations of 2-AG and WWL70 resulted 1 2 cannabinoid receptors (20). To determine whether the effect of 2- in a dose-dependent decrease of IL-1β mRNA expression (Fig. AG on LPS-induced macrophage activation is CB /CB de- 1B), with an IC of the same order of magnitude for the two 1 2 50 pendent, we used selective antagonists of these receptors, namely compounds: 6 μM for 2-AG and 1 μM for WWL70. Additionally, SLV319 (SLV, 1μM) and SR141716A (SR1, 1μM) for CB and 2-AG did not decrease macrophage activation when the cells 1 SR144528 (SR2, 1μM) for CB . Coincubation of these antagonists were assessed at 24 h, whereas WWL70 retained its efficacy (Fig. 2 with either 2-AG or WWL70 in the J774 and RAW264.7 cell lines S3A). This probably is a result of the lower stability of 2-AG in did not block their effect on macrophage activation (Figs. S3D and the medium or its hydrolysis by ABHD6 in the absence of the S5E). To further confirm that the decrease observed following inhibitor, thus strengthening the rationale to use enzyme inhib- ABHD6 inhibition is not cannabinoid receptor dependent, itors to increase this bioactive lipid’s levels and make use of its we used two potent CB1/CB2 agonists, HU210 (100 nM) and therapeutic potential. The effect of WWL70 and 2-AG was CP55,940 (100 nM) (20).