WWL70 attenuates PGE 2 production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism

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1 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 DOI /s RESEARCH Open Access WWL70 ttenutes PGE 2 production derived from 2-rchidonoylglycerol in microgli y ABHD6-independent mechnism Mikiei Tnk 1, Sen Morn 2, Jie Wen 1, Kwme Affrm 3,4, Tinghu Chen 1, Aviv J. Symes 3,4 nd Yumin Zhng 1,4* Astrct Bckground: α/β-hydrolse domin 6 (ABHD6) is one of the mjor enzymes for endocnninoid 2-rchidonoylglycerol (2-AG) hydrolysis in microgli cells. Our recent studies hve shown tht selective ABHD6 inhiitor WWL70 hs ntiinflmmtory nd neuroprotective effects in niml models of trumtic rin injury nd multiple sclerosis. However, the role of ABHD6 in the neuroinflmmtory response nd the mechnisms y which WWL70 suppresses inflmmtion hs not yet een elucidted in rective microgli. Methods: The hydrolytic ctivity nd the levels of 2-AG in BV2 cells were mesured y rdioctivity ssy nd liquid chromtogrphy coupled with tndem mss spectrometry (LC-MS/MS). The expression of cyclooxygense-2 (COX-2) nd prostglndin E 2 (PGE 2 ) synthses in microgli treted with lipopolyscchride (LPS) with/without WWL70 ws determined y western lot nd quntittive RT-PCR. The conversion of 2-AG to PGE 2 or PGE 2 -glyceryl ester (PGE 2 -G) ws ssessed y enzyme-linked immunossy (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE 2 production ws ssessed using phrmcologicl inhiitors nd smll interfering RNA (sirna). The effect of WWL70 on PGE 2 iosynthesis ctivity in the microsome frction from BV2 cells nd experimentl utoimmune encephlopthy (EAE) mouse rin ws lso exmined. Results: We found tht WWL70 suppressed PGE 2 production in LPS-ctivted microgli vi cnninoid receptorindependent mechnisms, lthough intrcellulr levels of 2-AG were elevted y WWL70 tretment. This reduction ws not ttriutle to WWL70 inhiition of ABHD6, given the fct tht downregultion of ABHD6 y sirna or use of KT182, n lterntive ABHD6 inhiitor filed to suppress PGE 2 production. WWL70 ttenuted the expression of COX-2 nd PGES-1/2 leding to the downregultion of the iosynthetic pthwys of PGE 2 nd PGE 2 -G. Moreover, PGE 2 production from rchidonic cid ws reduced in the microsome frction, indicting tht WWL70 lso trgets PGE 2 iosynthetic enzymes, which re likely to contriute to the therpeutic mechnisms of WWL70 in the EAE mouse model. Conclusions: WWL70 is n nti-inflmmtory therpeutic gent cple of inhiiting PGE 2 nd PGE 2 -G production, primrily due to its reduction of COX-2 nd microsoml PGES-1/2 expression nd their PGE 2 iosynthesis ctivity in microgli cells, s well s in the EAE mouse rin. Keywords: Endocnninoid,2-AG,Archidoniccid,ABHD6,COX-2,PGE 2,PGE 2 -glyceryl ester, PGE synthse, BV2 cells, Microgli * Correspondence: yumin.zhng@usuhs.edu 1 Deprtment of Antomy, Physiology nd Genetics, Uniformed Services University of the Helth Sciences, 4301 Jones Bridge Rod, Bethesd, MD 20814, USA 4 Neuroscience Progrm, Uniformed Services University of the Helth Sciences, 4301 Jones Bridge Rod, Bethesd, MD 20814, USA Full list of uthor informtion is ville t the end of the rticle The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 2 of 15 Bckground 2-Archidonoylglycerol (2-AG) nd N-rchidonoylethnolmine, lso known s nndmide (AEA), re the two principl endogenous lignds for cnninoid type 1 (CB1) receptor expressed primrily in neurons [1] nd the CB2 receptor mostly expressed in microgli nd strocytes, lthough they re lso found in neuronl cells in the rin stem nd other regions in the centrl nervous system (CNS) [2, 3]. A growing ody of evidence hs indicted tht modultion of the endocnninoid system plys n essentil role in meliorting the development of severl neurologicl diseses such s multiple sclerosis [4, 5], trumtic rin injury [6], nd neuropthic pin [7]. Activtion of CB1 nd/ or CB2 receptors contriutes to the suppression of inflmmtion nd reduction of glutmte excitotoxicity [8, 9]. In ddition to functioning s full gonist for CB1 nd CB2 receptors, 2-AG is lso precursor for rchidonic cid (AA) nd its derivtive prostglndins (PGs; PGD 2, PGE 2, PGF 2α, PGI 2, nd TXA 2 ) in the rin [10, 11]. Although PGs re lipid meditors involved in cell homeostsis, they lso medite the inflmmtory response in vriety of pthologicl conditions [12]. There is consensus tht AA is minly provided from diet or derived from glycerophospholipids ctlyzed y phospholipse A 2 [13]. AA is further metolized to PGs y cyclooxygenses (COXs) nd PG synthses. However, recent studies hve shown tht inhiition or deletion of the primry 2-AG hydrolytic enzyme monocylglycerol lipse (MAGL) significntly reduces the rin levels of AA nd PGs, suggesting tht 2-AG plys n essentil role in the production of PGs especilly in the CNS [14]. The mjority of the endocnninoid-hydrolyzing enzymes elong to the serine hydrolse superfmily [15]. Using novel technology tht detects ctive serine hydrolses [16], WWL70 ws reveled to e specific inhiitor for α/β-hydrolse domin 6 (ABHD6), 2-AG hydrolytic enzyme in the rin [17]. The inhiitory property of WWL70 on ABHD6 ws further demonstrted y severl lter studies [18 20]. Although the results from our lortory nd others hve shown tht only ~4% of 2-AG hydrolysis in the mouse rin nd spinl cord is ttriutle to ABHD6 under physiologicl conditions [21], ABHD6 contriutes significntly to the 2-AG hydrolysis in microgli/mcrophges, in which there is no or little expression of MAGL [18, 19]. Recently, we hve found tht WWL70 exerts therpeutic effects in mouse models of trumtic rin injury [22] nd experimentl utoimmune encephlomyelitis (EAE) [23] through ttenuting microglil ctivtion, COX-2 expression, nd the impired function of the lood-rin rrier. In ddition, WWL123, derivtive of WWL70 with high permeility to the lood-rin rrier, ws lso protective in mouse model of epilepsy [24]. However, the exct mechnism y which inhiition of ABHD6 results in the reduction of inflmmtory response remins elusive. In this study, we found tht tretment with WWL70 sustntilly reduced PG production in BV2 nd primry microgli cells. Surprisingly, this reduction ws not due to inhiition of ABHD6 y WWL70 ut ws ttriutle to its interference with the metolic pthwy from AA to PGE 2. Moreover, we found tht WWL70 inhiited microsoml PGE 2 synthesis ctivity, which is the primry cuse for PGE 2 reduction. Thus, we report here novel inhiitory ction of WWL70 on microgli y modulting PGE 2 iosynthesis ctivity. Methods Cell culture BV2 microglil cells [25] were cultured in complete DMEM contining 5% het-inctivted fetl ovine serum (Life Technologies, Grnd Islnd, NY) under humidified 5% CO 2 environment. The cells were mintined y medium chnge every other dy. One dy prior to experiment, cells were plted on 24-well pltes to rech 90% confluence t the eginning of experiment. For primry microgli culture, Sprgue Dwley rts were purchsed from Chrles River Lortories (Frederick, MD) nd housed in the niml fcility of the Uniformed Services University of the Helth Sciences (USUHS). All niml protocols were pproved y the USUHS Institutionl Animl Cre nd Use Committee. P2 Sprgue Dwley rt pups (mle nd femle) were scrificed nd their rins removed. Mixed glil cultures were then prepred from the cortices fter creful removl of the meninges nd rin stem. The cortices were then triturted y seril triturtion with 10-ml pipette, 18-G needle (3 ), 22-G needle (3 ), nd finlly, 25-G needle (2 ) in culture medium (DMEM contining 10% fetl ovine serum, 1% glutmx, nd 1% ntiiotic-ntimycotic). After triturtion, the suspension ws filtered using 70-micron mesh nd then pelleted t 168 g for 10 min t room temperture. Cells were resuspended in culture medium, seeded into T75 flsks, nd incuted in CO 2 incutor. The medium ws replced every 2 3 dys. After 14 or 15 dys in culture, primry microgli were hrvested y differentil shking on n oritl shker for 1 h t 200 rpm in CO 2 incutor. The medium, contining the detched microgli, ws collected nd centrifuged t 671 g for 5 min t room temperture. Cells were then resuspended with DMEM contining 10% norml horse serum, 1% glutmx, nd 1% streptomycin/penicillin nd trnsferred to uncoted pltes t density of cells/ml. Regents KT182, n ABHD6 inhiitor, nd HT-01, the ctivitysed protein profiling (ABPP) proe specific for

3 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 3 of 15 ABHD6, were kindly provided y Drs. Hsu nd Crvtt [26]. 2-Archidonoylglycerol [glycerol-1,2,3-3 H] ws from Americn Rdioleled Chemicls Inc. (Sint Louis, MO). Cyclooxygense inhiitor ssy kits including COX inhiitor nd recominnt COX-1 or COX-2 ctivity ssy kit were from Cymn Chemicl (Ann Aror, MI). sirna (FlexiTue Mm_hd6_3 nd Allstrs Negtive Control) nd HiPerfect trnsfection regent were from QIAGEN (Vlenci, CA). WWL70, methyl rchidonyl fluorophosphonte (MAFP), SR (SR1), SR (SR2), 2-AG, 2-AG-d 8,ndAAwerepurchsed from Cymn Chemicl. Other regents including lipopolyscchride (LPS) were purchsed from Sigm- Aldrich (St. Louis, MO). PGE 2 enzyme immunossy A multi-well cell culture plte ws prepred 1 or 2 dys prior to the test. The cell culture medium ws replced with pre-wrmed medium contining the ABHD6 inhiitor WWL70 (10 μm) nd incuted for 15 min. The cells were treted with 10 μm of 2-AG for 15 min, followed y ddition of 100 ng/ml LPS for BV2, or 2 ng/ml LPS for primry microgli. After incution for 18 h, the culture medium ws collected. Before ddition to the enzymelinked immunossy (EIA), the medium ws centrifuged t 5000 rpm for 2 min with tle top centrifuge to exclude residul cells. To determine the role of WWL70 on PGE 2 production in vivo, EAE ws induced y sucutneous injection of myelin oligodendrocyte glycoprotein peptide (MOG ) in 8-week-old femle C57BL/6 mice, nd the clinicl score ws ssessed s we reported recently [23]. WWL70 (10 mg/kg, i.p.) ws given strting t the disese onset nd then once dy until the end of the test. The mouse forerin t 3 weeks postimmuniztion ws dissected nd kept frozen t 80 C until use. The forerin tissue ws homogenized with one-fifth volume of 0.02% trifluorocetic cid (TFA) nd one volume of cetonitrile using Potter homogenizer t 4 C. Homogente ws dispersed completely in 2 ml of cetonitrile y vortex nd stored t 4 C overnight. Deris ws excluded y centrifugtion t 2000g for 5 min, nd then the superntnt ws evported under the nitrogen gs streming in wter th (pproximtely 35 C). After reconstitution with the EIA uffer, the levels of PGE 2 were mesured following the mnufcturer s protocol (Cymn Chemicl, Ann Aror, MI). 2-AG hydrolysis ctivity ssy Hydrolysis ctivity of 2-AG ws ssessed s previously descried [27]. For hydrolysis ctivity in intct cells, BV2 cells in 24-well plte were pre-incuted with or without inhiitor for 30 min t 37 C, followed y wshing with pre-wrmed DMEM plus 0.15% ftty cid-free BSA. The cells were incuted with 0.2 ml of 10 μm rdioleled 3 [H]-2-AG (33 nci) in DMEM contining 0.15% ftty cid-free BSA. After 2 min of incution, the rection ws terminted y mixing with 0.4 ml chilled methnol. The cell extrct ws trnsferred to silnized glss tue, followed y mixing with 0.4 ml of chloroform y rief vortexing. The mixture ws centrifuged t 3000g for 5 min to seprte the queous nd orgnic phses. Aliquot of the queous phse ws mixed with scintilltion cocktil nd mesured in scintilltion counter LS6500 (Beckmn Coulter, Bre, CA). For hydrolysis ctivity in memrne frction, BV2 cell homogente ws centrifuged t 1400g for 5 min to remove the deris, then ultrcentrifuged t 100,000g t 4 C for 30 min. The pellet ws resuspended with PBS, nd protein concentrtion ws determined y DC protein ssy kits using BSA s stndrd (Bio-Rd). Four hundred microliters of the memrne frction (20 μg) ws preincuted in silnized glss tue with or without 10 μm of WWL70 for 5 min t 37 C, then mixed with 100 μl of10μm 3 [H]-2-AG (33 nci) in PBS contining 0.15% ftty cid-free BSA. After 2 min of incution, the rection mixture ws mixed with chilled 2 ml methnol/ chloroform (1:1). Rdioctivity in the queous phse ws mesured s descried ove. Activity-sed protein profiling nd western lotting One milligrm per milliliter of the memrne frction prepred s ove ws pre-incuted with WWL70 t indicted concentrtions for 10 min t room temperture, then mixed with 1 μm HT-01, serine hydrolse proe tht specificlly recognizes ctive ABHD6 enzymes [26], t 37 C for 40 min. The rection mixture ws dded to the SDS-PAGE smple uffer nd heted t 95 C for 5 min. Approximtely 10 μg of the protein ws loded on SDS- PAGE. The gel ws scnned with fluorescence imger, Fuji FLA-5100 (Fujifilm, Edison, NJ) with n excittion wvelength t 473 nm using FITC mode. The intensity of the fluorescent nd is proportionl to the mount of ctive ABHD6. Susequently, the gel ws trnsferred onto PVDF memrne for western lotting with nti-clnexin s loding control for the memrne frction. For western lotting, cell lyste or microsome frction ws prepred with RIPA uffer contining 150 mm NCl, 50 mm Tris-HCl (ph 8.0), 1% Triton X-100, 0.5% N deoxycholte, 0.1% SDS, 1 mm EDTA, 1 mm EGTA, 1mMN 3 VO 4,1mMβ-glycerophosphte, nd protese inhiitor cocktil (Roche Applied Sciences) for 5 min on ice, followed y centrifugtion t 12,000g for 5 min t 4 C to remove the deris. Trnsferred PVDF memrne ws pre-incuted with 5% BSA in PBS % Tween- 20 (PBST) for 30 min, then incuted with ntiodies ginst β-ctin (AC-74, Sigm-Aldrich) t 1:1000, clnexin (T-40, Snt Cruz) t 1:1000, microsoml prostglndin E2 synthse (mpges)-1 (#160140, Cymn

4 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 4 of 15 Chemicl) t 1:500, mpges-2 (#160145, Cymn Chemicl) t 1:500, nd COX-2 (#160106, Cymn Chemicl) t 1:250 in PBST t 4 C overnight. The trnsferred memrne ws rected with secondry ntiody conjugted with horserdish peroxidse (Bio-Rd) t 1:2500 for 2 h, followed y visuliztion with ECL regent (Thermo Scientific) using the LAS3000 imger (Fujifilm). The reltive intensity of COX-2 to β-ctin ws quntified using ImgeJ softwre (Ntionl Institutes of Helth), nd the fold chnge compred to the LPS tretment lone ws presented. LC-MS/MS nlysis for 2-AG Smple preprtion for 2-AG quntifiction ws crried out sed on method pulished previously [28]. BV2 cells (90% confluence) in 10-cm dishes were treted with WWL70 (10 μm) or MAFP (10 μm) for 1 h. After rinsing with PBS once, the cells were collected y centrifugtion t 5000g for 2 min. The pellet ws suspended with 0.1 ml of 0.02% TFA nd 1 nmole of 2-AG-d 8 y pipetting nd dispersed in 4 ml of cetonitrile in silnized glss tue to precipitte the deris overnight t 20 C. The superntnt fter centrifuged t 5000g for 5 min ws trnsferred to new glss tue nd evported under nitrogen gs strem in mild hot wter th (pproximtely 35 C). 2-AG ws resuspended with 0.1 ml of cetonitrile nd stored t 80 C until mss nlysis. An HPLC system (1200 Series, Agilent Technologies, Snt Clr, CA) ws used with reverse phse gurd column (Wide Pore C18 (ODS), 4 2 mm ID; Phenomenex, Torrnce, CA), nd the column (Sephsil Peptide C18, 5 u, ST, mm ID; Phrmci Biotech (Amershm), Pisctwy, NJ) ws mintined t 40 C. The moile phse ws composed of solvent A: 0.2% formic cid in wter, nd solvent B: 0.2% formic cid in methnol; the following grdient ws used: 62% A/38% B isocrtic for 30 s, rmp to 90% B in 60 s, isocrtic t 90% B for 18.5 min, rmp ck to 62% A/38% B in 60 s, nd re-equilirte t 62% A/38% B for 8 min. The flow rte ws 0.4 ml/min. The HPLC output ws directed into the TuroV electrospry ioniztion (ESI) source of Q-Trp 4000 mss spectrometer (AB Sciex, Frminghm, MA). The injection volume ws 20 μl. LC-MS/MS nlysis ws performed in positive mode with the ion source temperture of 600 C, spry voltge of 5.5 kv, nd declustering potentil of 45 V. Multiple rection monitoring (MRM) ws performed on the trnsitions m/z for 2-AG nd for 2-AGd 8. Clirtion curves of the rtio of nlyte (2-AG) to internl stndrd (2-AG-d 8 ) pek res vs. concentrtion rtio (t lest seven different 2-AG concentrtions t single fixed 2-AG-d 8 concentrtion) were generted y liner lest squres fitting nd used to clculte the 2-AG concentrtions in the smples (converting the oserved y-vlue (pek re rtio) of the smples into x-vlues (concentrtion rtios with fixed internl stndrd concentrtion). LC-MS/MS for PGE 2 nd PGE 2 -G The method for LCMS nlysis of PGE 2 nd PGE 2 -glycerol in positive ion mode ws derived from different previously pulished procedure [29]. BV2 cells pretreted with or without 0.1 μg/ml of LPS for 8 h were rinsed once with pre-wrmed serum-free medium (Opti- MEM, Thermo Fisher Scientific), then incuted with Opti-MEM contining 10 μm 2-AG, 10 μm WWL70, or oth for 30 min in CO 2 incutor. The medium ws collected nd centrifuged t 1000g for 5 min to remove floting cells nd stored t 80 C until use. One milliliter of medium ws mixed with 1.4 ng PGE 2 -d4 nd 0.5 ng PGE 2 -G-d5 (Cymn Chemicl) nd cidified y glcil cetic cid to 1% t finl concentrtion. It ws loded to solid-phse extrction column (Osis HLB 1 cc, Wters Corportion, Milford, MA) equilirted with 0.5% cetic cid nd methnol. After wshing with 0.5% cetic cid followed y 0.5% cetic cid with 15% methnol, PGs were eluted with 1.5 ml of methnol. After evportion in nitrogen streming under pproximtely 30 C, the PGs were reconstituted with cetonitrile/wter (1:2). The sme LCMS system descried ove ws used with Higgins Anlyticl TARGA C18 3 μm mm column (Nest Group Inc., Southorough, MA). The HPLC grdient ws s follows: the column ws equilirted t 80% 5 mm mmonium formte in wter (uffer A)/20% 4:1 cetonitrile/wter with 5 mm mmonium formte (uffer B); fter smple injection, the grdient for elution of nlytes went from 20 to 80% uffer B in 15 min. The column ws re-equilirted y rmping to 100% uffer B in 1 min, holding t 100% uffer B for 2 min to flush, rmping ck down to the initil HPLC uffer composition (80%/20% A:B) in 2 min, nd holding t the initil uffer composition for 8 min efore the next injection. The flow rte ws 0.2 ml/min, the column temperture ws 40 C, nd the injection volume ws 25 μl. The ion source temperture ws 550 C, nd the declustering potentil ws 40 V. Since the HPLC uffer contined mmonium ions, the precursor ions for PGE 2 nd PGE 2 -glycerol were mmonium dducts (M + 18). The following MRM trnsitions were used: for PGE 2, precursor ion mss D to frgment ion mss D (collision energy (CE) 12.5 V) nd D (CE 15 V); for d4-pge 2,374.3 to D (CE 12.5 V) nd D (CE 15 V); for PGE 2 -glycerol, to D (CE 12.5 V) nd D (CE 26 V); nd for d5-pge 2 -glycerol, to D (CE 12.5 V) nd D (CE 26 V).

5 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 5 of 15 qrt-pcr Totl RNA from BV2 cells or primry microgli treted with regents for 8 h ws isolted using TRIzol (Life Technologies) ccording to the mnufcturer s protocol. Five hundred nnogrms of RNA were used for cdna synthesis using MAXIMA First Strnd cdna synthesis kit with dsdnase (Thermo Fisher Scientific) following the mnufcturer s protocol. The Cq vlue of negtive control without reverse trnscription ws >40 due to the tretment with DNse. Complementry DNA from 10 ng RNA ws employed for qpcr in the presence of 250 nm of gene-specific primers (mpges-1 forwrd, 5 -TGTCCAA ATCCTGTCTTCCA-3, reverse,5 -GGTTCTGGAGCAC ACCCTAT-3 ; mpges-2 forwrd, 5 -GAAATGGCTG- CAGAATTGAA-3, reverse, 5 -AAGGAGAATGGTGCT CCAAG-3 ; COX-2forwrd,5 -GAAATGGCTGCAGAA TTGAA-3, reverse,5 -AAGGAGAATGGTGCTCCAAG- 3 ; ABHD6 forwrd, 5 -CATTCCAATCCTGGCATTTG TTG-3, reverse, 5 -ATGGTGTGCGTAGCGAACTT-3 ; nd GAPDH forwrd, 5 -AGGTCGGTGTGAACGGATT TG-3, reverse,5 -TGTAGACCATGTAGTTGAGGTCA- 3 ) usingpowersybrgreenpcrmstermix(lifetechnologies) in 12 μl rection mixture. Therml cycling condition ws 95 C for 10 min, followed y 40 cycles of 95 C 15 s nd 60 C 60 s performed y LightCycler 480 II (Roche Life Science, Indinpolis, IN). Gene-specific PCR mplifiction ws confirmed y the melting curve profile using LightCycler 480 system progrm showing 80 to 85 C of single melting temperture for ll the genes tested. GAPDH ws used s reference gene ecuse its Cq vlue ws constnt mong the cells tested (Cq vlue is 15.7 ± 0.1, men ± SD). The PCR mplifiction linerity ws confirmed using seril diluted cdna showing the consistent mplifiction in the concentrtion rnge tested (r 2 >0.998). sirna trnsfection BV2 cells in 24-well plte (50 to 70% confluent) were trnsfected with sirna for the mouse ABHD6 gene (Mm_hd6_3, QIAGEN) or the negtive control using HiPerfect trnsfection regent ccording to the mnufcturer s protocol. Briefly, 37.5 ng sirna ws mixed with 100 μl DMEM without FBS, then dded 6 μl of the trnsfection regent nd incuted for 10 min t room temperture. The sirna complex suspension ws dded to the cells drop-wise. Totl RNA ws isolted from the cells fter 1 dy of incution nd qrt-pcr ws performed to exmine the expression of ABHD6 nd GAPDH. Otherwise, the trnsfected cells fter 2 dys of incution were used to prepre memrne frction for ABPP to ssess ABHD6 hydrolytic ctivity, followed y western lot with the clnexin ntiody s loding control. Enzyme ctivity ssy The COX ssy ws crried out using the COX inhiitor screening ssy kit from Cymn Chemicl. Briefly, ovine recominnt COX-1 or humn recominnt COX-2 ws pre-incuted in 0.1 M Tris-HCl (ph 8.0) nd EDTA (1 mm) uffer with WWL70 (10 μm), SC- 560 (0.33 μm), or Dup-697 (0.3 μm) for 5 min t 27 C. Then, 10 μm of AA ws dded nd incuted for 1 min t 27 C. The rection ws stopped y dding 1 N HCl. Stnnous cid ws then dded to convert PGH 2 to the stle product PGF 2α. The resultnt mixture ws pplied to prostglndin EIA to mesure the totl mount of prostglndins including PGE 2 nd PGF 2α. The PGE 2 iosynthesis ssy ws performed using microsomes derived from BV2 cells treted with LPS for 8 h or rin tissue from EAE mouse t dy 21 post-immuniztion [23]. BV2 cells were suspended with 0.1 M potssium phosphte (ph 7.4), 0.25 M sucrose, 1 mm β-glycerophosphte, 1 mm sodium vndte, 1 μm MAFP, nd protese inhiitor cocktil (Roche) nd disrupted y soniction for 15 s, while the rin tissue ws homogenized with Potter homogenizer with the potssium phosphte uffer t 4 C. These homogentes were centrifuged t 1400g for 10 min t 4 C to remove the deris, followed y further centrifugtion t 10,000g for 10 min t 4 C. The superntnt ws ultrcentrifuged t 170,000g for 1 h t 4 C to precipitte the microsome frction. The pellet ws suspended with potssium phosphte uffer contining glutthione (2.5 mm) y rief soniction. One hundred microgrms per milliliter of BV2 microsomes ws pre-incuted with WWL70, MK886 (3 μm), SC-560 (1 μm), or NS398 (10 μm) for 5 min t 23 C, then mixed with 10 μm ofaafor1mint23 C. 500μg/ml rin microsomes were incuted with 10 μm of AA for 2 min t 23 C. The rection ws stopped y mixing with stnnous cid (5 mg/ml in 0.1 N HCl) to dectivte the enzyme nd convert intermedite PGH 2 to PGF 2α, followed y the mesurement of PGE 2 concentrtion y EIA s descried ove. The ctivity ws determined fter sutrction with the mount of PGE 2 in the microsome frction incuted without sustrte. Sttistics All dt re expressed s men ± SD. The GrphPd Prism 7 (GrphPd Softwre Inc., Sn Diego, CA) ws used for sttisticl nlysis. The sttisticl comprison mong the drug-treted groups (different drugs or the sme drug t vrious concentrtions) ws performed using one-wy ANOVA followed y Turkey s test; while in experiments with oth LPS, LPS + 2-AG, or LPS + AA groups, two-wy ANOVA ws performed. An unpired t test ws used for dt comprison etween two groups. Sttisticl significnce ws set t P < 0.05, with single, doule, nd triple sterisks to denote p < 0.05, 0.01, nd 0.001, respectively.

6 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 6 of 15 Results WWL70 inhiited 2-AG hydrolysis nd elevted 2-AG level in BV2 cells vi inhiition of ABHD6 2-AG hydrolysis to AA in BV2 cell memrne ws confirmed y deuterted AA genertion using LC-MS/MS (Additionl file 1: Figure S1). Becuse AA cn e rpidly metolized, we mesured the simultneously produced glycerol derived from 2-AG using rdioctive ssy. In the presence of WWL70, 2-AG hydrolysis in BV2 cells ws decresed y 40%, while the generl serine hydrolse c Fig. 1 Inhiition of ABHD6 y WWL70 incresed the 2-AG levels in BV2 cells. BV2 cells were treted with WWL70 or MAFP for 30 min nd then incuted with 3 [H]-2-AG t 37 C for 2 min. Cell lystes were mixed with MeOH/CHCl 3 to seprte the orgnic from queous phse. The mount of 3 [H]-glycerol, the product of 2-AG hydrolysis, ws mesured in the queous phse. Inhiitors were dded t either 1 or 10 μm. Dt re represented s men ± SD (n = 3).Triple sterisks denote p < compred to the drug-treted groups. The memrne frction from BV2 cells ws incuted with WWL70 for 10 min, rected with HT-01 proe (1 μm) t 37 C for 40 min, nd then pplied to SDS-PAGE. The gel ws scnned to detect ctive ABHD6, followed y western lotting with n nti-clnexin ntiody. c BV2 cells were incuted with 10 μm of WWL70 or MAFP for 1 h efore hrvest. Cell lipids were extrcted with cetonitrile plus 0.02% TFA together with 2-AG-d 8 s n internl stndrd. 2-AG ws identified nd quntified with LC-MS/MS sed on the rtio to the internl stndrd. Dt re represented s men ± SD (n =6).Doule nd triple sterisks denote p < 0.01 nd 0.001, respectively, compred to control Fig. 2 WWL70 locked PGE 2 production independently on CB receptor signling. BV2 cells were incuted with WWL70 for 15 min, efore ddition of 2-AG (10 μm) for 15 min, nd susequent ddition of LPS (100 ng/ml). After 18 h, the culture medium ws tested for PGE 2 y EIA. BV2 cells were treted with LPS, WWL70 with/without CB1R ntgonist SR1 (SR141716A), or CB2R ntgonist SR2 (SR144528) for 18 h efore the culture medium ws ssyed for PGE 2 content. There were no significnt differences in the mount of PGE 2 with or without ntgonist. Representtive experiment, men ± SD (n = 3), out of three similr independent experiments. Single nd triple sterisks denote p < 0.05 nd 0.001, respectively, compred to the drug-treted groups

7 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 7 of 15 inhiitor, methyl rchidonyl fluorophosphonte (MAFP) [30], lmost completely inhiited the hydrolytic ctivity (Fig. 1). This result is consistent with previous report [19] indicting tht ABHD6 is one of the min 2-AG hydrolytic enzymes in BV2 cells. ABPP is recently developed technology to detect serine hydrolses using proe tht specificlly inds to the ctive enzymes ut not the inctive or the inhiitor ound forms [15]. Using the HT-01 proe, we found tht WWL70 t 1 μm nd10μm drmticlly reduced the nd intensity of ABHD6 (Fig. 1). Next, we exmined whether inhiition of ABHD6 could lter the intrcellulr levels of 2-AG with LC-MS/MS. At 1 h fter WWL70 (10 μm) tretment, 2-AG ws incresed y 20% compred to untreted cells (Fig. 1c). Tretment with MAFP (10 μm) lso significntly incresed 2-AG levels (Fig. 1c). c c Fig. 3 WWL70 suppressed COX-2 expression. Western lots of BV2 cell lystes with different tretment protocols, showing COX-2 expression with β-ctin controls. The lots shown here re representtive of three independent experiments. BV2 cells were incuted with synthetic CB receptor gonist, WIN55,212-2, or 2-AG for 15 min, efore ddition of LPS (0.1 μg/ml) for 18 h. Single nd doule sterisks denote p < 0.05 nd 0.01, respectively, compred to the LPS tretment lone (men ± SD, n = 3). BV2 cells were treted with AA or endocnninoids t the indicted concentrtions for 15 min followed y incution with LPS for 18 h. Single, doule, nd triple sterisks denote p < 0.05, 0.01, nd 0.001, respectively, when compred to the LPS tretment lone (men ± SD, n =3).c BV2 cells were treted with WWL70 for 15 min, followed y the ddition of 2-AG for further 15 min, efore ddition of LPS for 18 h. Reltive COX-2 expression levels to β-ctin were shown under ech lot fter normlizing with the LPS-treted conditions. Single sterisk denotes p <0.05(men±SD, n =3). WIN refers to WIN55,212-2, nd Noldn refers to 2-rchidonyl glyceryl ether Fig. 4 WWL70 suppressed microsoml PGESs nd COX-2 mrna expression. qrt-pcr of mpges-1 (), mpges-2 (), or COX-2 (c) mrna isolted from BV2 cells treted with WWL70 (10 μm) for 15 min, efore ddition of 2-AG (10 μm) for 15 min, nd then LPS (0.1 μg/ml) for 18 h. Dt re represented s reltive expression to control condition fter normliztion with GAPDH (men ± SD, n = 4). Single nd doule sterisks denote p < 0.05 nd 0.01, respectively

8 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 8 of 15 WWL70 reduced PGE 2 production in LPS-ctivted BV2 cells It hs een reported tht the hydrolysis of 2-AG y MAGL is one of the mjor sources of AA nd its derivtive PGs in the mouse rin [10, 14]. Becuse ABHD6, ut not MAGL is the primry 2-AG hydrolytic enzyme in BV2 cells [18, 19], we tested the possiility tht WWL70 could reduce PGE 2 production in BV2 cells. Tretment with LPS (100 ng/ml) for 18 h incresed PGE 2 production, nd this increse ws completely locked y WWL70 t either 1 or 10 μm (Fig. 2). To further determine if PGE 2 production is ffected y 2-AG metolism, cells were incuted for 18 h in the presence of 2-AG nd LPS. Exogenous ddition of 2-AG together with LPS cused sevenfold increse of PGE 2 production compred to cells incuted with LPS lone, suggesting tht the incresed PGE 2 ws derived from 2-AG. In the presence of 2-AG, the production of PGE 2 ws reduced sustntilly y 1 nd 10 μm WWL70 (Fig. 2). To determine whether PGE 2 reduction y WWL70 is medited y cnninoid receptors, cells were coincuted with the CB1 receptor ntgonist SR141716A (SR1) or the CB2 receptor ntgonist SR (SR2). Neither CB1 nor CB2 ntgonist reversed the PGE 2 reduction y WWL70, suggesting tht cnninoid receptor signling ws not involved in the inhiitory effect of WWL70 (Fig. 2). The expression levels of COX-2 nd mpges in LPS-ctivted BV2 cells were reduced y WWL70 Since COX-2 is involved in the metolism of AA to PGE 2, the potentition of 2-AG on PGE 2 production in LPS-ctivted BV2 cells might e due to the incresed expression of COX-2. To test this possiility, BV2 cells were treted with LPS for 18 h in the presence of different concentrtions of 2-AG. Expression of COX-2 ws induced y LPS nd further potentited y 2-AG (Fig. 3). However, the CB1 nd CB2 cnninoid receptor gonist WIN55,212-2 drmticlly suppressed the expression of COX-2. This result suggests tht the expression of COX-2 is differentilly regulted y 2-AG nd the other synthetic cnninoid receptor gonists. To determine the ction of other endocnninoid (ecb) lignds, AEA, which ctivtes either CB receptors with comprle efficiency to 2- AG nd is hydrolyzed to AA y ftty cid mide hydrolse, c Fig. 5 The highly selective ABHD6 inhiitor KT182 did not reduce PGE 2 production. Activity-sed protein profiling with the ABHD6-specific proe HT-01 shows reduced ABHD6 ctivity in the memrne frction of BV2 cells in the presence of KT182. Using memrne frction from BV2 cells, tretment with KT182, WWL70, or MAFP for 5 min showed decresed hydrolysis of 2-AG y quntifying relesed rdioctive glycerol s descried in Fig. 1. Reltive 2-AG hydrolysis s compred to the non-treted group re shown (men ± SD; n =3).c BV2 cells were incuted with DMEM plus WWL70 (10 μm) or KT182 (100 nm) for 15 min, efore ddition of 10 μm 2-AG or AA for 15 min, followed y ddition of LPS (100 ng/ml) for 18 h. Culture medium ws ssyed for PGE 2 y EIA (men ± SD, n =3).Single, doule, ndtriple sterisks denote p < 0.05, 0.01, nd 0.001, respectively

9 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 9 of 15 ws lso tested in LPS-treted BV2 cells nd shown to upregulte the expression of COX-2. Addition of AA lso potentited the expression of COX-2, wheres the Noldin ether, which is n unhydrolyzle ecb lignd, did not show ny increse (Fig. 3). These results suggest tht COX-2 upregultion y ecbs ws dependent on the production of AA from their hydrolysis. In line with this notion, tretment with WWL70 reduced the increse of COX-2 y 2-AG (Fig. 3c), suggesting tht the reduced COX-2 expression ws likely due to the limited production of AA. Consistently, the enhnced mrna expression of mpges-1 nd mpges-2 (Fig. 4, ) y LPS ws lso potentited y 2-AG nd reduced y WWL70 similr to tht of COX-2 (Fig. 4c). c PGE 2 reduction y WWL70 ws independent of ABHD6 inhiition To further exmine whether phrmcologicl inhiition of ABHD6 leds to the reduced PGE 2 levels, recently developed ABHD6 inhiitor KT182 ws tested. KT182 is derivtive of piperidyl-1,2,3-trizole ure inhiitors with high potency nd selectivity to trget ABHD6 [26]. Activity-sed protein profiling using KT182-treted BV2 memrne frction showed strong reduction in ctive ABHD6 t 1 nd 10 nm of KT182 (Fig. 5), which is consistent with previously pulished report tht the IC 50 of this inhiitor is less thn 5 nm [26]. The inhiition of 2-AG hydrolysis ctivity y KT182 in the BV2 memrne frction ws similr to tht of WWL70 (Fig. 5). However, unlike WWL70 which reduced the production of PGE 2 in LPS-ctivted BV2 cells, PGE 2 production ws not ffected y KT182 in the presence or sence of either 2-AG or AA (Fig. 5c). These dt suggest tht the inhiition of PGE 2 production y WWL70 might e independent of ABHD6 inhiition. Next, we tested whether ABHD6 knockdown could ffect PGE 2 production. 24 h fter sirna trnsfection, the mrna levels of ABHD6 were reduced y pproximtely 80% compred to the control sirna-trnsfected cells (Fig. 6). The ctivity of ABHD6 in BV2 cells fter 2 dys sirna trnsfection ws lso drmticlly reduced (Fig. 6). Different from the results with WWL70, ut consistent with those with KT182, knockdown of ABHD6 did not decrese PGE 2 production in BV2 cells treted with LPS in the sence or presence of 2-AG. On the other hnd, the production of PGE 2 ws slightly incresed in siabhd6 cells possily due to the differences in cell popultion fter trnsfection. The reduction of PGE 2 y WWL70 ws similr in oth control nd ABHD6 sirna-trnsfected cells (Fig. 6c). These results suggest tht the inhiition of PGE 2 production y WWL70 is independent of its inhiition of ABHD6. Rther, the inhiitory ction of WWL70 is likely due to its Fig. 6 ABHD6 sirna knockdown did not reduce PGE 2 production. qrt-pcr of ABHD6 trnscript normlized to GAPDH, 24 h fter trnsfection with ABHD6-specific sirna. Memrne frction prepred from the trnsfected cells fter 48 h trnsfection ws rected with the ABHD6-specific HT-01 proe for 30 min to detect ctive ABHD6 enzyme, followed y western lotting of clnexin s loding control. c Cell cultures fter 48 h trnsfection were replced with fresh medium with LPS (0.1 μg/ml), WWL70 (10 μm), or 2-AG (10 μm). The culture medium ws collected fter 18 h incution nd employed PGE 2 EIA. Dt re nlyzed with two-wy ANOVA nd represented s men ± SD (n =3). Doule or triple sterisks denote p < 0.01 or 0.001, respectively interference with the iosynthetic pthwy from AA to PGE 2, in which COXs nd PGE 2 synthses re involved. WWL70 inhiited PGE 2 synthesis We exmined whether WWL70 cn modulte PGE 2 production y inhiition of the ctivity of PGE 2 iosynthetic enzymes, rther thn through suppression of COX-2 nd PGES expression. Eighteen hours fter tretment with LPS to ctivte the PGE 2 iosynthetic pthwy, the medium ws replced with fresh medium contining WWL70 with or without either 2-AG or AA, nd then incuted for short time period (30 min). The production of PGE 2 ws reduced y WWL70 in the sence or the presence of 2-AG or AA with similr dose-response (Fig. 7). KT182 tretment did not lter the mount of PGE 2 generted, except for the cotretment with 2-AG (Fig. 7). Following the 30-min drug tretment, the expression of mpges-1 nd mpges-2 ws not chnged (see western lot imges in Fig. 7). These results suggest tht the enzymtic ctivity of COX-2 or PGES is inhiited y WWL70, ut not y KT182. WWL70 inhiited PGE 2 -G production We further exmined the possiility tht WWL70 inhiits PGE 2 -G production, since the iosynthetic pthwys for synthesis of PGE 2 -G nd PGE 2 overlp. BV2 cells

10 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 10 of 15 Fig. 7 WWL70 inhiited PGE 2 production in LPS-ctivted BV2 cells. Cells were treted with LPS (0.1 μg/ml) for 18 h to ctivte PGE 2 iosynthesis efore replcement with fresh DMEM contining () 10μM 2-AG or AA nd WWL70 (1 or 10 μm) or () 10μM 2-AG or AA nd WWL70 (10 μm) or KT182 (100 nm). The culture medium fter 30 min incution ws employed PGE 2 EIA. Using the microsome frction, mpges-1, mpges-2, nd β-ctin protein expression fter 30 min incution with different tretment conditions ws exmined y western lot s shown elow the EIA results. Dt re represented s men ± SD (n = 3). Single nd triple sterisks denote p < 0.05 nd 0.001, respectively ctivted y LPS were susequentilly incuted with non-serum contining medium with 2-AG or WWL70 for 30 min. The PGs in the medium were nlyzed with LC- MS/MS. Consistent with previous results, PGE 2 production ws incresed y LPS tretment nd oosted in the presence of 2-AG, wheres WWL70 decresed PGE 2 production (Fig. 8). PGE 2 -G ws detected only under the conditions in the presence of 2-AG, ut not in the sence of 2-AG, indicting tht the PGE 2 -G ws derived from 2-AG (Fig. 8). Tretment with WWL70 reduced the PGE 2 -G significntly in the cells treted with LPS nd 2-AG (Fig. 8). WWL70 trgets PGE 2 synthesis pthwy in microsomes To exmine whether WWL70 directly inhiits the ctivity of COX enzymes, COX ctivity ws ssyed in vitro in the presence of WWL70 using ovine COX-1 nd humn COX-2 recominnt proteins. WWL70 did not hve ny effect on the ctivities of COX-1 or COX-2, while their ctivities were completely locked y the selective COX-1 nd COX-2 inhiitors SC560 nd Dup697, respectively (Additionl file 2: Figure S2). Since PGE 2 iosynthetic enzymes re minly loclized in microsomes, we exmined PGE 2 production ctivity in microsome frction of BV2 cells ctivted y LPS for

11 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 11 of 15 Fig. 8 Determintion of WWL70 inhiitory effect on PGE 2 nd PGE 2 -G productions using LC/MS/MS. BV2 cells treted with LPS (0.1 μg/ml) for 8 h were replced with fresh non-serum medium contining 2-AG with/ withoutwwl70ndincutedfor30min.pgsinthemediumws extrcted with SPE, then employed LC-MS/MS together with internl stndrds PGE 2 -d 4 nd PGE 2 -G-d 5 to determine the quntity of PGE 2 ()ndpge 2 -G (). nd denotes not detected. Dt re represented s men ± SD (n =3or4) 8 h. Microsome frction ws incuted with 10 μm AA for 1 min, followed y mesurement of PGE 2 (Fig. 9). The enzyme ctivity ws reduced to 60% y WWL70, wheres the mpges-1 inhiitor MK886, the COX-1 inhiitor SC-560, nd the COX-2 inhiitor NS398 COX-2 inhiitor reduced the enzymtic ctivity to 55, 39, nd 36%, respectively. The dose-response study indicted tht the IC 50 of WWL70 to inhiit the PGE 2 iosynthesis is out 100 nm (Fig. 9), which is comprle to its C 50 for ABHD6 inhiition (70 nm) [17]. WWL70 suppressed the PGE 2 production in primry microgli cells The effect of WWL70 on PGE 2 production ws lso exmined in rt primry microgli cells. Consistent with the results otined in BV2 cells, overnight WWL70 Fig. 9 WWL70 inhiited microsoml PGE 2 iosynthetic pthwy. Microsome from LPS-ctivted BV2 cells ws pre-incuted with WWL70 (3 μm), the mpges-1 inhiitor MK886 (3 μm), the COX-1 inhiitor sc-560 (1 μm), or the COX-2 inhiitor NS398 (10 μm) for 5 min, then incuted with 10 μm AA for 1 min t 23 C. The rection mixture fter incution ws employed PGE 2 EIA (). Dose-dependent inhiition of WWL70 ws exmined to determine its IC 50 on PGE 2 synthesis (). Dt re presented s men ± SD (n =3 6). Triple sterisks denote p < 0.001, respectively tretment olished PGE 2 production in LPS-ctivted microgli, wheres KT182 did not hve ny inhiitory effects (Fig. 10). The ddition of CB1 nd CB2 receptor ntgonists did not reverse the inhiitory effect of WWL70 on PGE 2, suggesting tht the effect of WWL70 ws not medited y cnninoid receptor ctivtion. Primry microgli lso replicted the effect in BV2 cells of short-term drug tretment (30 min) on the reduction of PGE 2 production in LPS-stimulted cells supplemented with either 2-AG or AA (Fig. 10). Cells were treted with LPS for 18 h nd then switched to the medium contining WWL70 or KT182 for 30 min prior to PGE 2 EIA. PGE 2 production ws enhnced pproximtely tenfold in the presence of either 2-AG or AA,

12 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 12 of 15 Fig. 10 WWL70 inhiited PGE 2 in primry microgli. PGE 2 EIA of medi from primry microgli. Cells were treted with LPS for 18 h in the presence or sence of WWL70, KT182, or CB1 or CB2 receptor ntgonist. Microgli were treted with LPS for 18 h efore medi were replced with DMEM contining WWL70 (10 μm), KT182 (0.1 μm), AA (10 μm), or 2-AG (10 μm) nd incuted for 30 min. The dt re represented s men ± SD (n =3). Single nd triple sterisks denote p < 0.05 nd 0.001, respectively indicting tht the exogenous 2-AG nd AA were metolized to PGE 2. Agin, WWL70 ut not KT182 inhiited PGE 2 production. WWL70 suppressed PGE 2 production in EAE mouse rin We hve recently reported tht WWL70 reduces microgli ctivtion, demyelintion, nd xonl injury nd meliortes clinicl symptoms in the EAE mouse model [23]. To determine whether WWL70 could lso inhiit PGE 2 production in vivo, we exmined EAE mouse rin tissue t dy 21 post-immuniztion, t tht time point, the clinicl scores re significntly reduced (Fig. 11). The levels of PGE 2 in the EAE mice rin tissues incresed y 1.5-fold compred to the control mice (Fig. 11), which re similr to the previous study [31]. Tretment with WWL70 reversed the incresed production of PGE 2 (Fig. 11). Consistently, the PGE 2 iosynthetic ctivity in microsome frction from the rin tissue ws lso incresed in the EAE-vehicle group nd reduced y WWL70 tretment (Fig. 11c). These results suggest tht the reduced PGE 2 production my contriute to the nti-inflmmtory effect of WWL70 in the EAE mouse model. Discussion WWL70, n ABHD6 selective inhiitor, possesses ntiinflmmtory nd neuroprotective effects in LPSinduced rin inflmmtion, trumtic rin injury, nd multiple sclerosis [18, 22, 23]. Although ABHD6 is expressed in microgli nd mcrophges, nd WWL70 cn suppress microgli/mcrophge ctivtion, whether ABHD6 is the primry trget for the ction of WWL70 remins elusive. In this study, we found tht WWL70 tretment inhiited 2-AG hydrolysis nd incresed the intrcellulr levels of 2-AG. WWL70 lso significntly reduced the production of PGE 2 in LPS-ctivted BV2 nd primry microgli cells. However, the incresed PGE 2 production ws not ffected y nother newly developed ABHD6 inhiitor KT182, nd the inhiitory effect of WWL70 ws lso oserved in BV2 cells with ABHD6 knockdown. These results suggest tht the inhiitory effect of WWL70 on PGE 2 production in LPSctivted microgli is not dependent on its inhiition of ABHD6 ctivity. Tretment with WWL70 reduced the expression of oth COX2 nd mpges, the metolic enzymes crucil for PGE 2 production from AA. Furthermore, WWL70 directly inhiited the enzymtic ctivity of microsoml PGE 2 iosynthesis. It hs recently een demonstrted tht the hydrolysis of 2-AG y MAGL contriutes significntly to the rin levels of AA [14]. Inhiition or deletion of MAGL hs therpeutic effects in niml models of Prkinson s disese [14] nd Alzheimer s disese [32] y locking AA nd PG production, ut not y cnninoid-receptormedited signling. These recent findings highlight lterntive therpeutic mechnisms of ecb-hydrolyzing enzyme inhiitors. Although MAGL is the primry enzyme for 2-AG hydrolysis in the rin, MAGL seems to ply minor role in microglil cells nd mcrophges ecuse of its low expression [18, 19]. Insted, ABHD6 is one of the mjor hydrolyzing enzymes in mcrophges s ABHD6 inhiition cused twofold increse in 2-AG concentrtion [18]. We found tht the increse in 2-AG y WWL70 ws reltively mild in BV2 cells (Fig. 1c), possily ecuse the mximl inhiition y 10 μm of WWL70 only ccounts for 40% of the totl 2-AG hydrolysis (Fig. 1). This result suggests tht ecb signl potentition y incresed 2-AG my not e mjor fctor to the nti-inflmmtory effect of WWL70 in microgli. It is of note tht the exogenously dded 2-AG incresed COX-2 protein expression in LPS-ctivted BV2 cells, wheres the commonly used synthetic CB receptor gonist WIN55,212-2 hd n opposite effect on COX-2 expression (Fig. 3). The increse of COX-2 expression

13 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 13 of 15 c Fig. 11 WWL70 tretment ttenuted PGE 2 production in EAE mouse rin. EAE mouse model ws induced y immuniztion with the MOG peptide. Intrperitonel injection of WWL70 ws strted t the disese onset nd then once dy until the end of experiment. The clinicl scores t dy 21 in EAE mice in vehicle nd drug-treted groups were shown (). Mouse forerin ws dissected nd used for mesuring PGE 2 concentrtion (n =10 11) (). Microsome frction prepred from the rin tissue ws pplied to PGE 2 synthetic ctivity ssy (n =4 5) (c). Dt re represented s men ± SD. Single, doule, nd triple sterisks denote p < 0.05, 0.01, nd 0.001, respectively y 2-AG is unlikely due to the ctivtion of CB receptors. Similrly, AEA tht is lso metolized to AA, or AA itself, incresed the expression of COX-2 in LPSctivted BV2 cells, wheres 2-rchidonyl glyceryl ether (Noldin ether), n unhydrolyzle ecb lignd [33], hd no effect. These results indicte tht ddition of AEA nd 2-AG cn increse AA production nd fcilitte eicosnoid signling in rective microgli through the incresed expression of COX-2. Microsoml PGES1 nd PGES2 were lso incresed in LPS-ctivted microgli nd further potentited y 2-AG. WWL70 suppressed COX-2 nd mpges1/2 expression in the sence or presence of 2-AG (Fig. 4). Tken together, these results suggest tht the suppression of the metolic enzymes to generte PGE 2 contriutes to the nti-inflmmtory effects of WWL70. Recently, it hs een reported tht WWL70 in mouse mcrophges modultes the production of vrious PGs including PG-glycerol esters (PG-Gs) [18], which re the oxygention product of 2-AG y COX-2 nd PG synthses [34]. These results suggest tht WWL70 lso modultes the PGE 2 -G metolism y incresing the vilility of 2-AG nd ltertion of COX-2 nd mpges. Consistently, we lso found tht the genertion of PGE 2, s well s PGE 2 -G, in LPS-ctivted microgli ws enhnced y the ddition of 2-AG, nd this increse ws significntly inhiited y WWL70 (Fig. 8). Severl studies hve shown tht PGE 2 -G triggers proinflmmtory response [35 37], while PGD 2 -G hs n nti-inflmmtory effect [18]. It is possile tht WWL70 interferes with the PGE 2 -G synthetic pthwy, which in turn promotes the production of PGD 2 -G y the incresed vilility of 2-AG (Fig. 12). It is still uncler how vrious PG-Gs cn modulte the pro- nd ntiinflmmtory responses nd how they differ from the ctions of PGs, such s PGD 2, PGE 2, nd PGF 1α. WWL123, derivtive of WWL70, hs een shown to e protective in the niml model of epileptic seizures [24]. We hve recently shown tht the eneficil effect of WWL70 in the mouse model of trumtic rin injury [22] is t lest in prt due to its ctivtion of CB1 nd CB2 receptors, wheres in the experimentl utoimmune encephlomyelitis mouse model, the therpeutic effect of WWL70 is chiefly medited y the

14 Tnk et l. Journl of Neuroinflmmtion (2017) 14:7 Pge 14 of 15 Additionl files Additionl file 1: Figure S1. Conversion of 2-AG to AA in memrne frction. Memrne frction from BV2 cells ws incuted with 100 μm of2-ag-d8for0,1,or5mint37 C.AA-d8extrctedwith cetonitrile ws pplied to LC-MS/MS. Mss spectrogrms for the MRM trnsition from m/z 312 to m/z 93 is shown t three time points. Trnsition peks of ech time point re shown in inset represented s men ± SD (n = 3). (PPTX 53 k) Additionl file 2: Figure S2. Effect of WWL70 in recominnt COX ssy. Ovine recominnt COX-1 (A) or humn recominnt COX-2 (B) ws pre-incuted in 0.1 M Tris-HCl (ph 8.0) nd EDTA (1 mm) uffer with WWL70 (10 μm), SC-560 (0.33 μm), or Dup-697 (0.3 μm) for 5 min t 27 C. Then, 10 μm of AA ws dded nd incuted for 1 min t 27 C. The rection mixture ws pplied for prostglndins EIA to mesure totl mount of prostglndins. (PPTX 42 k) Fig. 12 Potentil ctions of WWL70 on PGE 2 nd PGE 2 -G production in microgli. 2-AG is minly metolized to rchidonic cid, which in turn converts to PGE 2 y ctlysis of COX-1/2 nd mpgess. WWL70 inhiits ABHD6 therey incresing the levels of 2-AG nd reduces the production of PGE 2 y inhiiting PGE 2 synthetic enzymes. 2-AG cn lso e oxygented y COX-2 nd then converted to PGE 2 glycerol ester y mpgess, which is lso inhiited y WWL70 ctivtion of CB2 receptors, s ssessed y the use of the selective CB2 receptor ntgonist nd the CB2 receptor knockout mice [23]. Our present study suggests tht the eneficil effect of WWL70 in these niml models might e lso due to the reduced expression of COX-2 nd PGES nd the inhiition of the enzymtic ctivity for PGE 2 production. Indeed, we found tht the IC 50 of WWL70 for PGE 2 synthesis is out 100 nm, which is comprle to its inhiition on ABHD6 (70 nm) reported previously [17]. It hs een demonstrted tht mpges1 is loclized in microgli in the EAE mouse spinl cord nd tht PGE 2 is criticl lipid meditor in microgli cusing neuroinflmmtion through ctivtion of EP 2 receptor [38]. In LPS-ctivted BV2 microgli, we oserved tht the expression of EP 2 receptor ws upregulted nd reduced y WWL70 tretment (dt not shown). It is likely tht the eneficil effect of WWL70 is lso linked to the signling medited y EP 2 receptor or other PGE 2 receptors. Conclusions Our dt suggest tht WWL70 cn enhnce the cnninoid signling nd reduction of the eicosnoid signling in microgli nd my serve s n inhiitory modultor for the metolism from 2-AG to PGE 2 (Fig. 12). Thus, we speculte tht WWL70 might e effective for the tretment of EAE nd other neuroinflmmtory nd neurodegenertive diseses in which ctivtion of the COX-2-PGES-PGE 2 xis is mjor contriutor to the pthogenic mechnisms. Arevitions 2-AG: 2-Archidonoylglycerol; AA: Archidonic cid; ABHD6: α/β- Hydrolse domin 6; ABPP: Activity-sed protein profiling; AEA: N-rchidonoylethnolmine or nndmide; CB: Cnninoid; CNS: Centrl nervous system; COX-2: Cyclooxygense-2; EAE: Experimentl utoimmune encephlomyelitis; ecb: Endocnninoid; EIA: Enzyme-linked immunossy; LC-MS/MS: Liquid chromtogrphy coupled with tndem mss spectrometry; LPS: Lipopolyscchride; MAGL: Monocylglycerol lipse; MOG: Myelin oligodendrocyte glycoprotein; mpges: Microsoml prostglndin E2 synthse; Noldin ether: 2-Archidonyl glyceryl ether; PG-Gs: Prostglndin glycerol esters; PGs: Prostglndins; SR1: SR141716A; SR2: SR144528; TFA: Trifluorocetic cid; WIN: WIN55,212-2 Acknowledgements The uthors thnk Dr. Brin Cox for his creful reding nd comments on the mnuscript. Funding This work ws supported y grnts from the Ntionl Multiple Sclerosis Society (RG3741), the Defense Medicl Reserch nd Development Progrm ( ), the Militry Center of Excellence Reserch Awrd ( ), nd the Center for Neuroscience nd Regenertive Medicine ( ). Avilility of dt nd mterils All rw dt re summrized in the ody of the mnuscript. The dt used in the nlyses presented here re freely ville for review upon request from the corresponding uthor. Authors contriutions MT designed nd performed the experiments nd nlyzed the dt. SM performed the LC-MS/MS nlysis. KA nd TC prepred the primry cultures of microgli. MT nd JW mde the figures. MT, AS, nd YZ wrote the mnuscript. YZ designed the experiments nd secured funding for the studies. All uthors red nd pproved the finl mnuscript. Competing interests The uthors declre tht they hve no competing interests. Consent for puliction Not pplicle. Ethics pprovl All niml protocols were pproved y the USUHS Institutionl Animl Cre nd Use Committee. Author detils 1 Deprtment of Antomy, Physiology nd Genetics, Uniformed Services University of the Helth Sciences, 4301 Jones Bridge Rod, Bethesd, MD 20814, USA. 2 Biomedicl Instrumenttion Center, Uniformed Services University of the Helth Sciences, 4301 Jones Bridge Rod, Bethesd, MD 20814, USA. 3 Deprtment of Phrmcology nd Moleculr Therpeutics,