Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels Or Distribution in Macrophages

cells

Article Cellular Plasmalogen Content Does Not Inﬂuence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling

Patricia Lebrero 1,2, Alma M. Astudillo 1,2 , Julio M. Rubio 1,2, Lidia Fernández-Caballero 1, George Kokotos 3 , María A. Balboa 1,2 and Jesús Balsinde 1,2,*

1 Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Cientíﬁcas (CSIC), Universidad de Valladolid, 47003 Valladolid, Spain 2 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 28029 Madrid, Spain 3 Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, 15771 Athens, Greece * Correspondence: [email protected]; Tel.: +34-983-423-062

Received: 11 July 2019; Accepted: 30 July 2019; Published: 31 July 2019

Abstract: Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A2γ (cPLA2γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A2 forms such as cytosolic phospholipase A2α, calcium-independent phospholipase A2β, or secreted phospholipase A2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA2γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

Keywords: arachidonic acid; eicosanoids; inﬂammation; phospholipid remodeling; phospholipase A2; monocytes/macrophages

1. Introduction Arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid; AA) is the precursor of the eicosanoids, a large family of compounds with key roles in the initiation and resolution of inflammation [1]. Since AA is not found in free fatty acid form in cells but esterified into the sn-2 position of membrane glycerophospholipids, the participation of phospholipase A2 enzymes that liberate the fatty acid constitutes a limiting step for the synthesis of eicosanoids, a process which also depends on the expression levels and activity of the AA-metabolizing enzymes cyclooxygenases and lipoxygenases [2–5]. AA, which is usually the major polyunsaturated fatty acid in the membranes of innate immune cells, is not uniformly distributed among membrane glycerophospholipids. Rather, marked

Cells 2019, 8, 799; doi:10.3390/cells8080799 www.mdpi.com/journal/cells Cells 2019, 8, 799 2 of 20 differences exist in the distribution of AA across several different phospholipid molecular species. Monocytes and macrophages exhibit a characteristic distribution of AA between phospholipid classes, with ethanolamine phospholipids (PE) constituting the richest AA-containing class, followed by choline glycerophospholipids (PC), and phosphatidylinositol (PI) [6–8]. Among particular molecular species within phospholipid classes, the ethanolamine plasmalogens are markedly enriched with AA [6–11]. This asymmetric distribution of AA in cells is also key for eicosanoid regulation because, depending on the original phospholipid source of the AA, certain eicosanoids may be produced in preference over others. For example, production of lipoxygenase metabolites in ionophore-activated human neutrophils [12] and zymosan-stimulated mouse peritoneal macrophages [8] appears to be strikingly associated with the mobilization of AA from PC, not PE or PI. By inference, these findings imply that not all AA-containing phospholipid pools may be accessible to the relevant phospholipase A2 affecting the AA release. Thus, depending on conditions, AA compartmentalization among different membrane pools may constitute a third limiting step for eicosanoid synthesis. Incorporation of AA into cellular phospholipids does not primarily occur via de novo phospholipid synthesis but at a later step via fatty acid recycling of the sn-2 position of glycerophospholipids in the so-called Lands pathway [5,11–15]. In this pathway, the 2-lysophospholipids generated by constitutively active phospholipase A2 enzymes such as the calcium-independent group VIA enzyme (iPLA2β)[16–20] are utilized by AA-selective coenzyme A (CoA)-dependent acyltransferases to incorporate AA into phospholipids. Afterward, a further remodeling step, primarily involving direct transacylation reactions between phospholipids, serves to place the AA in the appropriate phospholipid pools [5,11–14]. These reactions proceed without energy expenses as no fatty acid activation by CoA is involved and are believed to be responsible for the asymmetric distribution of AA between phospholipid classes and molecular species and, especially, for the very high content of this fatty acid in ethanolamine plasmalogens [5,11–14]. CoA-independent transacylase (CoA-IT; EC 2.3.1.147) is a major enzyme involved in phospholipid AA remodeling in most cells. This enzyme catalyzes the CoA-independent transfer of AA and other polyunsaturates from, primarily, diacyl-PC to lyso-PE or lyso-PC [11–14]. CoA-IT manifests marked affinity for lysophospholipid acceptors containing an ether bond in the sn-1 position of the glycerol backbone, particularly ethanolamine lysoplasmalogens (1-alkenyl-2-lyso-glycerophosphoethanolamine) and alkyl-lyso-PC (1-alkyl-2-lysoglycerophospho-choline). This circumstance may explain why the AA content in ether-containing PC and PE species is generally higher than in their diacyl counterparts. Although the gene sequence of CoA-IT has not been identified so far, its activity has been relatively well-characterized in broken cell preparations, and pharmacological inhibitors have been identified [21–23]. The enzyme can also be followed in whole cells by determining the pattern of radiolabeling of PC versus PE. Like all phospholipase A2 enzymes, CoA-IT cleaves the sn-2 position of phospholipids, but it cannot be regarded as a bona fide phospholipase A2 enzyme because it does not produce a free fatty acid [13,14]. Given that some of the better known phospholipase A2s such as cytosolic group IVA phospholipase A2 (cPLA2α) or iPLA2β exhibit CoA-IT activity in in vitro assays [16], the proposal has been made that, in vivo, the CoA-IT reaction may represent an unidentified function of an otherwise described phospholipase A2 enzyme [14]. Based primarily on biochemical commonalities, i.e., membrane-bound, calcium-independent, group IVC cytosolic phospholipase A2 (cPLA2γ) has been suggested as a likely candidate [14]. cPLA2γ overexpression studies have provided in vivo evidence that the enzyme modulates phospholipid fatty acid composition, although it was not clarified whether the remodeling reactions involved were CoA-dependent or -independent [24]. In previous work from our laboratory, we used advanced mass spectrometry-based lipidomic approaches to study phospholipase A2-regulated phospholipid fatty acid metabolism (release and reincorporation mechanisms) in monocytes and macrophages responding to stimuli of the innate immune response [6–8,25–29]. In this work, we have employed similar approaches to unveil novel regulatory features of CoA-IT-mediated phospholipid remodeling responses. To characterize further the role of plasmalogen forms in these processes, we have taken advantage of Cells 2019, 8, 799 3 of 20 the plasmalogen-deficient RAW cell variants generated by Zoeller and coworkers [30–32]. Our previous studies with these cells uncovered essential roles for ethanolamine plasmalogens in regulating phagocytosis [33] and in the execution of lipopolysaccharide (LPS)-primed responses [29]. In the current work, we show that overall phospholipid AA remodeling is essentially independent of the amount of plasmalogen present in the cells, suggesting that compartmentalization of AA in innate immune cells may depend primarily on the relative distribution of the fatty acid between classes (PE versus PC versus PI) rather than on specific molecular species within classes (i.e., alkenyacyl versus alkylacyl versus diacyl species). Moreover, our results also implicate cPLA2γ in regulating phospholipid AA remodeling.

2. Materials and Methods

2.1. Reagents Cell culture medium was from Molecular Probes-Invitrogen (Carlsbad, CA, USA). Organic solvents (Optima® LC/MS grade) were from Fisher Scientiﬁc (Madrid, Spain). Lipid standards were from Avanti (Alabaster, AL, USA) or Cayman (Ann Arbor, MI, USA). Silicagel G thin-layer chromatography plates were from Macherey-Nagel (Düren, Germany). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (180 Ci/mmol) was from PerkinElmer (Boston, MA, USA). The group IVA phospholipase A2 (cytosolic phospholipase A2α; cPLA2α) inhibitor pyrrophenone [34] was synthesized and provided by Dr. Alfonso Pérez (Department of Organic Chemistry, University of Valladolid). The group VIA phospholipase A2 (calcium-independent phospholipase A2β; iPLA2β) inhibitors FKGK18 and GK436 and the secreted phospholipase A2 inhibitor GK241 were synthesized in the Kokotos laboratory [35–37]. All other reagents were from Sigma-Aldrich (Madrid, Spain).

2.2. Cell Culture RAW264.7 macrophage-like cells and their ether phospholipid-deficient variants RAW.12 and RAW.108 (generously provided by Dr. R. A. Zoeller, Boston University) [30–32] and P388D1 macrophage-like cells (MAB clone, generously provided by Dr. E. A. Dennis, University of California at San Diego) [38–40] were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine at 37 ◦C in a humidified atmosphere of 5% CO2, as previously described [41,42]. Mouse peritoneal macrophages from Swiss mice (University of Valladolid Animal House, 10–12 weeks old) were obtained by peritoneal lavage using 5 mL cold phosphate-buffered saline and cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, as described elsewhere [43,44]. All procedures involving animals were undertaken under the supervision of the Institutional Committee of Animal Care and Usage of the University of Valladolid (No. 907046) and are in accordance with the guidelines established by the Spanish Ministry of Agriculture, Food, and Environment and by the European Union. All experiments were conducted in serum-free media. When activated cells were used, the stimulus (150 µg/mL yeast-derived zymosan) was added 1 h after incubating the cells in serum-free media for the times indicated. Zymosan was prepared exactly as described [43,44]. Only zymosan batches that demonstrated no measurable endogenous phospholipase A2 activity, as measured by in vitro assay under different conditions [45,46], were used in this study. Cell protein content was quantified according to Bradford [47], using a commercial kit (BioRad Protein Assay). To generate classically (M1) or alternatively (M2) polarized macrophages, the cells were treated with LPS (250 ng/mL) plus interferon-γ (500 U/mL) or with interleukin-4 (20 ng/mL) plus interleukin-13 (20 ng/mL) for 8 h, respectively [48]. When required, radiolabeling of the cells with [3H]AA was achieved by including 0.25 µCi/mL [3H]AA during the overnight adherence period (20 h). Labeled AA that had not been incorporated into cellular lipids was removed by washing the cells four times with serum-free Cells 2019, 8, 799 4 of 20 medium containing 0.5 mg/mL albumin. The determination of [3H]AA release from activated cells was carried out exactly as described [41,42,49].

2.3. Quantitative PCR Total RNA was extracted from the cells with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and 2 µg RNA was reverse transcribed using random primers and oligo d(T) and Moloney murine leukemia virus reverse transcriptase (Ambion, Austin, TX, USA). Quantitative PCR was carried out with an ABI 7500 machine (Applied Biosystems, Carlsbad, CA, USA) using Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA). Cycling conditions were as follows: 1 cycle at 95 ◦C for 3 min and 40 cycles at 95 ◦C for 12 s, 60 ◦C for 15 s, and 72 ◦C for 28 s. The replicates were averaged, and fold induction was determined in ∆∆Ct-based fold-change calculations, with cyclophilin A as a control [50]. Primer sequences are available upon request.

2.4. Small Interfering RNA (siRNA) Transfection The procedure for transient transfection of RAW264.7 cells with siRNAs targeting group IVC phospholipase A2 (cytosolic phospholipase A2γ; cPLA2γ) was adapted from Valdearcos et al. [51]. Briefly, 3 105 cells were transfected with siRNAs (20 nM) (sequence: 5 -(GGAGGAGAGA × 0 GAGGAAGAGAA)TT-30; Eurofins Genomics, Ebersberg, Germany) in the presence of 5 µl/mL LipofectamineTM RNAiMAX (Invitrogen) under serum-free conditions for 5 h. Afterward, 5% serum was added and the cells were maintained at normal culture conditions for 24 h. A scrambled siRNA was used as a negative control (sequence: 50-UGGUUUACAUGUCGACUAA-30).

2.5. Gas Chromatography/Mass Spectrometry (GC/MS) Analyses Total lipids from approximately 107 cells were extracted according to Bligh and Dyer [52]. After addition of internal standards, phospholipids were separated from neutral lipids by thin-layer chromatography, using n-hexane/diethyl ether/acetic acid (70:30:1, v/v/v) [53]. The phospholipid classes (PC, choline-containing phospholipids; PE, ethanolamine-containing phospholipids; PI, phosphatidylinositol; and PS, phosphatidylserine) were separated twice with chloroform/methanol/28% (w/w) ammonium hydroxide (60:37.5:4, v/v/v) as the mobile phase [54]. Fatty acid methyl esters were obtained from the various lipid fractions by transmethylation with 0.5 M KOH in methanol for 60 min at 37 ◦C[55–59]. Analysis was carried out using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C mass-selective detector operated in electron impact mode (EI, 70 eV), equipped with an Agilent 7693 autosampler and an Agilent DB23 column (60 m length 0.25 mm internal diameter × 0.15 µm ﬁlm thickness). Data analysis was carried out with the Agilent G1701EA MSD Productivity × Chemstation software, revision E.02.00 [55–59] (Agilent Technologies, Santa Clara, CA, USA).

2.6. Liquid Chromatography/Mass Spectrometry (LC/MS) Analyses of Phospholipids Lipids were extracted according to Bligh and Dyer [52], and internal standards were added. The samples were redissolved in 50 µL of hexanes/2-propanol/water (42:56:2, v/v/v), and 40 µL was injected into an Agilent 1260 Infinity high-performance liquid chromatograph equipped with an Agilent G1311C quaternary pump and an Agilent G1329B autosampler (Agilent Technologies, Santa Clara, CA, USA). The column was a FORTIS HILIC (150 3 mm, 3 µm particle size) (Fortis Technologies, Geston, UK), × protected with a Supelguard LC-Si (20 mm 2.1mm) cartridge (Sigma-Aldrich, Madrid, Spain). × The mobile phase consisted of a gradient of solvent A (hexanes/2-propanol, 30:40, v/v) and solvent B (hexanes/2-propanol/20 mM ammonium acetate in water, 30:40:7, v/v/v). The gradient started at 75% A from 0 to 5 min, then decreased from 75% A to 40% A at 15 min and from 40% A to 5% A at 20 min, was held at 5% until 40 min, and increased to 75% at 41 min. The column was then re-equilibrated by holding at 75% A for an additional 14 min before the next sample injection. The flow rate through the column was fixed at 400 µL/min, and this flow entered into the electrospray ionization interface of an AB/Sciex QTRAP Cells 2019, 8, x FOR PEER REVIEW 5 of 20

Cellsinjection.2019, 8 ,The 799 flow rate through the column was fixed at 400 μL/min, and this flow entered into5 ofthe 20 electrospray ionization interface of an AB/Sciex QTRAP 4500 hybrid triple quadropole mass spectrometer operated in negative ion mode (Applied Biosystems, Carlsbad, CA, USA). Source 4500parameters hybrid were triple as quadropole follows: ion mass spray spectrometer voltage, operated−4500 V; incurtain negative gas, ion 30 modepsi; nebulizer (Applied gas, Biosystems, 50 psi; Carlsbad, CA, USA). Source parameters were as follows: ion spray voltage, 4500 V; curtain gas, 30 psi; desolvation gas, 60 psi; and temperature, 425 °C. Phospholipid species were− detected as [M–H]− ions nebulizer gas, 50 psi; desolvation gas, 60 psi; and temperature, 425 C. Phospholipid species were except for choline phospholipids, which were detected as [M+CH◦ 3COO−]− adducts and were detectedidentified as by [M–H] comparison− ions except with prev for cholineiously published phospholipids, data [6–8,25–29]. which were detected as [M+CH3COO−]− adducts and were identified by comparison with previously published data [6–8,25–29]. 2.7. Liquid Chromatography/Mass Spectrometry (LC/MS) Analyses of Eicosanoids 2.7. Liquid Chromatography/Mass Spectrometry (LC/MS) Analyses of Eicosanoids Analysis of eicosanoids by LC/MS was carried out exactly as described elsewhere [8,27], using Analysis of eicosanoids by LC/MS was carried out exactly as described elsewhere [8,27], an Agilent 1260 Infinity high-performance liquid chromatograph equipped with an Agilent G1311C using an Agilent 1260 Inﬁnity high-performance liquid chromatograph equipped with an Agilent quaternary pump and an Agilent G1329B Autosampler, coupled to an API2000 triple quadrupole G1311C quaternary pump and an Agilent G1329B Autosampler, coupled to an API2000 triple mass spectrometer (Applied Biosystems, Carlsbad, CA, USA). Quantification was carried out by quadrupole mass spectrometer (Applied Biosystems, Carlsbad, CA, USA). Quantiﬁcation was carried integrating the chromatographic peaks of each species and by comparing with an external calibration out by integrating the chromatographic peaks of each species and by comparing with an external curve made with analytical standards [8,27]. calibration curve made with analytical standards [8,27].

2.8. Measurement of Phospholipid Arachidonate Remodeling This was carried out exactly as described by Pérez Pérez et al. [60]. [60]. Briefly, Brieﬂy, the cells were pulse labeled with 1 nM [33H]AA (0.25 μCi/mL) for 15 min at 37 °C. The cells were then washed with medium with 1 nM [ H]AA (0.25 µCi/mL) for 15 min at 37 ◦C. The cells were then washed with medium containing 0.50.5 mgmg/mL/mL bovine bovine serum serum albumin albumin to removeto remo theve the non-incorporated non-incorporated label. label. Afterward, Afterward, the cells the cells were placed in serum-free medium and incubated at 37 °C for the indicated periods of time. were placed in serum-free medium and incubated at 37 ◦C for the indicated periods of time. After lipid extraction,After lipid phospholipidextraction, phospholipid classes were separatedclasses were by thin-layerseparated chromatography by thin-layer chromatography as indicated above. as Theindicated spots above. corresponding The spots to corresponding each phospholipid to each class phospholipid were cut out class and were assayed cut out for radioactivityand assayed for by liquidradioactivity scintillation by liquid counting. scintillation Scheme counting.1 provides Scheme a graphical 1 provides description a graphical of this description kind of experiment. of this kind of experiment.

Scheme 1. Measurement of arachidonic acid (AA) movement from choline glycerophospholipids (PC) toScheme ethanolamine 1. Measurement phospholipids of arachidonic (PE). acid (AA) movement from choline glycerophospholipids (PC) to ethanolamine phospholipids (PE). 2.9. Statistical Analysis 2.9. Statistical Analysis All experiments were carried out at least three times with incubations in duplicate or triplicate, and theAll dataexperiments are expressed were carried as means out atSEM. least Statisticalthree times analysis with incubations was carried in outduplicate by Student’s or triplicate,t-test, ± withand thep < data0.05 takenare expressed as statistically as means significant. ± SEM. Statistical analysis was carried out by Student’s t-test, with p < 0.05 taken as statistically significant. 3. Results 3. Results 3.1. AA Distribution in RAW264.7 Cells and Plasmalogen-Deficient Variants 3.1. AAFigure Distribution1A compares in RAW264.7 the Ce phospholipidlls and Plasmalogen-Deficient fatty acid composition Variants of RAW264.7 cells and theFigure plasmalogen-deficient 1A compares the variantsphospholipid RAW.12 fatty and RAW.108,acid composition as assessed of byRAW264.7 GC/MS. Fattycells acidsand the are designatedplasmalogen-deficient by their number variants of carbon RAW.12 atoms, and and RAW.108, their number as assessed of double by bonds GC/MS. are designatedFatty acids after are a colon. To differentiate isomers, the n x (n minus x) nomenclature is used, where n is the number − Cells 2019, 8, x FOR PEER REVIEW 6 of 20

Cells 2019, 8, 799 6 of 20 designated by their number of carbon atoms, and their number of double bonds are designated after a colon. To differentiate isomers, the n−x (n minus x) nomenclature is used, where n is the number of carbonsof carbons of a given of a fatty given acid fatty and acid x is and an xinteger is an integerwhich, which,subtracted subtracted from n, fromgives n,the gives position the position of the of last doublethe last bond double of bondthe molecule. of the molecule. The AA content The AA was content very wassimilar very in similarall three in cell all types three celltested. types Also, tested. no significantAlso, no significant variations variations were detected were detectedin any ot inher any fatty other acid, fatty including acid, including the polyunsaturates the polyunsaturates of the of n–3 theseries.n–3 When series. the When various the variousphospholipid phospholipid classes we classesre separated were separated and analyzed and analyzed for AA content, for AA content, PE was PEfound was to found constitute to constitute the major the AA-containing major AA-containing class in all class three in types, all three representing types, representing almost halfalmost of totalhalf AA ofpresent total AAin these present cells. in The these fatty cells. acid The was fatty found acid at wascomparable found at levels comparable in PI and levels PC, and in PI lower and PC, amountsand lowerwere found amounts in PS. were No foundother phospholipid in PS. No other class phospholipid contained significant class contained amounts significant of AA (Figure amounts 1B). ofThe AA relative (Figure 1distributionB). The relative of AA distribution among ofph AAospholipid among phospholipidclasses of RAW264.7 classes of cells RAW264.7 and its cells plasmalogen-deficientand its plasmalogen-deficient variants, with variants, PE predominatin with PE predominating,g, is consistent iswith consistent previous with data previous for other data phagocyticfor other cells phagocytic such as human cells such monocytes as human [6] and monocytes murine peritoneal [6] and murine macropha peritonealges [7,8]. macrophages Collectively, [ 7,8]. theseCollectively, results show these that results cellular show plasmalogen that cellular conten plasmalogent influences content neither influences cellular neither AA levels cellular nor AA the levels relativenor thedistribution relative distributionof the fatty acid of the among fatty acidphosphol amongipid phospholipid classes. classes.

60 10

RAW 264.7 RAW 264.7 A RAW.12 B RAW.12 50 RAW.108 RAW.108 8

40 6

4 20

2 10 Fatty Acid (nmol/mg protein) AA Content (nmol/mg protein)

0 0 PC PE PI PS 14:0 16:0 18:0 20:0 16:1n-x 16:1n-7 18:1n-9 18:1n-7 18:2n-6 20:1n-9 20:3n-9 20:3n-6 20:5n-3 22:4n-6 22:6n-3 20:4 n-6

FigureFigure 1. Phospholipid 1. Phospholipid fatty fattyacid composition acid composition of RAW of RAW 264.7 264.7cells and cells plasmalogen-deficient and plasmalogen-deficient variants: variants: (A) The(A) Theprofiles profiles of ofmajor major fatty fatty acids acids in in RAW RAW 264.7264.7 cellscells (open (open bars), bars), RAW.12 RAW.12 (light (light brown), brown), and RAW.108and RAW.108(dark (dark brown) brown) were determinedwere determined in the in phospholipid the phospholipid fraction fraction by GC-MS by GC-MS after convertingafter converting the fatty the acid fattyglyceryl acid glyceryl esters intoesters fatty into acid fatty methyl acid esters. methyl 16:1 esters.n-x denotes 16:1n a-x mix denotes of the na-9 mix and ofn-10 the isomers, n-9 and which n-10 elute isomers,together. which ( Belute) The together. phospholipid (B) The classes phospholipid of RAW classes 264.7 of cells RA (openW 264.7 bars), cells RAW.12(open bars), (light RAW.12 turquoise), (lightand turquoise), RAW.108 and (dark RAW.108 turquoise) (dark were turquoise) separated, were and theirseparated, AA content and wastheir determinedAA content by was GC /MS. determinedResults by are GC/MS. shown asResults means are S.E.M.shown (nas= means3). ± S.E.M. (n = 3). ± The Thedistribution distribution of AA of AAbetween between phospholipid phospholipid molecular molecular species species was wasmeasured measured by LC/MS by LC /MS (Figure(Figure 2). Fatty2). Fatty chains chains within within phospholipids phospholipids are designated are designated by their by number their number of carbon of atoms, carbon and atoms, theirand number their numberof double of doublebonds are bonds designated are designated after a aftercolon. a colon.A designation A designation of O- ofbefore O- before the first the firstfatty fatty chainchain indicates indicates that that the the sn-1 positionposition is is ether-linked, ether-linked, whereas whereas a P- designationa P- designation indicates indicates a plasmalogen a plasmalogenform (sn-1 form vinyl ( ethersn-1 linkage)vinyl ether [61 ].linkage) The results [61]. showed The results that PE showed plasmalogens that PE constituted plasmalogens the major constitutedreservoir the of major this fatty reservoir acid in of RAW this fatty 264.7 acid cells in (Figure RAW2 264.7). High cells amounts (Figure of 2). AA High were amounts also found of AA in one wereparticular also found PI species,in one particular namely PI(18:0 PI species,/20:4), name and inly diacyl-PEPI(18:0/20:4), species. and in Lower diacyl-PE AA amounts species. were Lower found AA inamounts several PCwere and found PS species in several (Figure PC2). Interestingly,and PS species despite (Figure the plasmalogen2). Interestingly, deficiency despite of RAW.12the plasmalogenand RAW.108 deficiency cells, theof RAW.12 AA distribution and RAW.108 by the cells, phospholipid the AA distribution class in these by the cells phospholipid was preserved class due to in thesea compensatory cells was preserved elevation due of AA to a in compensatory diacyl species ofelevation PE and PCof AA compared in diacyl to wildspecies type of RAWPE and 264.7 PCcells compared(Figure to2). wild type RAW 264.7 cells (Figure 2).

Cells 2019, 8, 799 7 of 20 Cells 2019, 8, x FOR PEER REVIEW 7 of 20

0.0 0.2 0.4 0.6 0.8 1.0 1.2 012345

RAW 264.7 RAW 264.7 PC(O-16:0/20:4) RAW.12 PE(P-16:0/20:4) RAW.12 RAW.108 RAW.108

PC(O-18:0/20:4) PE(P-18:0/20:4)

PC(O-18:1/20:4) PE(P-18:1/20:4)

PC(16:0/20:4) * PE(16:0/20:4) * * *

PC(16:1/20:4) * PE(18:0/20:4) * * *

PC(18:0/20:4) * PE(18:1/20:4) * * *

PC(18:1/20:4) * PI(16:0/20:4) *

PC(18:2/20:4) * PI(16:1/20:4) *

PS(18:0/20:4) PI(18:0/20:4)

PS(18:1/20:4) PI(18:1/20:4)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 012345 Species (nmol/mg protein) Species (nmol/mg protein) FigureFigure 2. AA-containing phospholipid species in RAW 264. 264.77 cells and plasmalogen-deficient plasmalogen-deficient variants: TheThe profileprofile ofof AA-containingAA-containing PC,PC, PSPS (left(left panel),panel), PE,PE, PIPI (right(right panel)panel) waswas determined for RAW 264.7264.7 cellscells (open(open bars), RAW.12 cellscells (light(light purplepurple bars),bars), and RAW.108 cellscells (dark(dark purple bars) by LC/MS. LC/MS. ResultsResults areare shownshown asas meansmeans ± S.E.M.S.E.M. (n(n= = 3).3). *pp << 0.05,0.05, significantly significantly differe differentnt from the corresponding ± speciesspecies ofof RAW264.7RAW264.7 cells.cells.

3.2.3.2. Importance of Plasmalogen ContentContent for Phosphospholipid AAAA RemodelingRemodeling InIn mammalian cells, cells, plasmaloge plasmalogenn enrichment enrichment with with AA AA is thought is thought to occur to occur primarily primarily via CoA- via CoA-IT-mediatedIT-mediated reactions, reactions, which which transfer transfer a fatty a fattyacyl moiety acyl moiety from froma phospholipid a phospholipid donor, donor, primarily primarily AA- AA-containingcontaining PC PCspecies, species, to a to lysophospholipid a lysophospholipid acce acceptor,ptor, very very often often an an ethanolamine ethanolamine lysoplasmalogen, lysoplasmalogen, withoutwithout using CoA or forming forming a a free free fatty fatty acid acid intermediate intermediate [11–14]. [11–14 ].This This reaction reaction also also appears appears to be to beinstrumental instrumental forfor AA AA mobilization mobilization responses, responses, as asinhi inhibitionbition of ofCoA-IT CoA-IT leads leads to tomarked marked inhibition inhibition of 3 ofAA AA release release [8,23,62]. [8,23, 62To]. characterize To characterize this ro thisute, RAW route, 264.7 RAW cells 264.7 were cells labeled were with labeled [3H]AA with for [ 15H]AA min forand, 15 after min extensive and, after washing extensive to remove washing non-incorpor to remove non-incorporatedated fatty acid, the fattymovement acid, theof labels movement between of labelsphospholipid between classes phospholipid was analyzed. classes Immediately was analyzed. after Immediately the 15-min labeling after the period, 15-min PC labeling was the period, major 3 3 PC[3H]AA-containing was the major [ H]AA-containingphospholipid, followed phospholipid, by PI and followed PE. by[3H]AA PI and incorporation PE. [ H]AA incorporationinto PS was intoconsiderably PS was considerably lower (Figure lower 3A). The (Figure amount3A). Theof labeled amount AA of in labeled PC underwent AA in PC a underwentrapid decrease a rapid with decreasetime, which with was time, paralleled which was by paralleledan increase by ofan similar increase magnitude of similar of magnitude AA in PE, ofreflecting AA in PE, the reflecting action of theCoA-IT. action Levels of CoA-IT. of labeled Levels AA of labeled in PI and AA inPS PI remained and PS remained unchanged unchanged along the along time the course time courseof the ofexperiment. the experiment. To make To direct make comparisons direct comparisons between between various conditions various conditions and in accord and in with accord previous with 3 previouswork [55] work we have [55] defined we have the defined time at the which time atthe which amount the of amount [3H]AA of in [ PCH]AA equals in PC that equals in PE that as the in PE as the “remodeling time” and found it to be 21 4 min (mean S.E.M., n = 6). Importantly, “remodeling time” and found it to be 21 ± 4 min (mean± ± S.E.M., n = 6).± Importantly, examination of examinationthe rate of AA of the remodeling rate of AA remodelingfrom PC to from PE PCin tothe PE plasmalogen-deficient in the plasmalogen-deficient variants variants RAW.12 RAW.12 and andRAW.108 RAW.108 revealed revealed essentially essentially the the same same kinetics kinetics as asin inRAW RAW 264.7 264.7 cells cells and, and, hence, hence, nearly identical remodelingremodeling timestimes (Figure(Figure3 3B).B). Thus, Thus, these these results results indicate indicate that that phospholipid phospholipid AA AA remodeling remodeling from from PC PC to PE is not influenced by the cellular plasmalogen content. For comparative purposes, remodeling experiments under identical conditions were also carried out using another murine macrophage-like

Cells 2019, 8, 799 8 of 20

Cells 2019, 8, x FOR PEER REVIEW 8 of 20 to PE is not inﬂuenced by the cellular plasmalogen content. For comparative purposes, remodeling experiments under identical conditions were also carried out using another murine macrophage-like cell line, P388D1, and using resident murine peritoneal macrophages. In keeping with previous cell line, P388D , and using resident murine peritoneal macrophages. In keeping with previous estimates [63–65],1 the remodeling time of P388D1 cells was found to be similar to that of RAW 264.7 estimatescells and their [63–65 variants], the remodeling and considerably time of lower P388D than1 cells that was of found murine to peritoneal be similar macrophages to that of RAW (Figure 264.7 cells3B). and their variants and considerably lower than that of murine peritoneal macrophages (Figure3B).

60 350

300 50 AB PE 250 40 200 PI 30 30 20 PC 20 Remodeling Time (min) AA in phospholipids (%) 10 10 PS 0 0 0 20 40 60 80 100 120

Time (min) MRPM P388D1 RAW.12 RAW.108 RAW264.7

FigureFigure 3.3. PhospholipidPhospholipid AA AA remodeling remodeling in inRAW RAW 264.7 264.7 cells cells and and plasmalogen-deficient plasmalogen-deﬁcient variants: variants: (A) (RAWA) RAW 264.7 264.7 cells cells were were pulse-labeled pulse-labeled with with [3H]AA, [3H]AA, washed, washed, and andincubate incubatedd without without label label for forthe theindicated indicated periods periods of oftime. time. Phospholipids Phospholipids were were sepa separatedrated into into classes classes by by thin-layer thin-layer chromatography. chromatography. TheThe radioactivityradioactivity incorporatedincorporated intointo eacheach phospholipidphospholipid classclass waswas determineddetermined byby scintillationscintillation countingcounting andand isis givengiven asas aa percentagepercentage ofof thethe radioactivityradioactivity presentpresent inin phospholipids.phospholipids. ((BB)) AAAA remodelingremodeling waswas analyzedanalyzed forfor didifferentﬀerent cellcell types,types, andand thethe remodelingremodeling timetime (time(time atat whichwhich thethe radioactivityradioactivity contentcontent ofof PCPC equalsequals thatthat ofof PE)PE) waswas determined.determined. ResultsResults areare shown shown as as means means ±S.E.M. S.E.M.(n (n= = 66 forfor panelpanel A;A; nn= = 33 ± forfor panelpanel B).B). MRPM,MRPM, mousemouse residentresident peritonealperitoneal macrophages.macrophages. 3.3. Role of Plasmalogens in Functional Responses of Macrophages to Receptor Stimulation 3.3. Role of Plasmalogens in Functional Responses of Macrophages to Receptor Stimulation In previous work from our laboratory, we took advantage of the RAW 264.7 cells and theIn plasmalogen-deficientprevious work from variantsour laboratory, RAW.12 andwe took RAW.108 advantage to establish of the the RAW key roles 264.7 of ethanolaminecells and the plasmalogensplasmalogen-deficient in regulating variants the phagocytic RAW.12 activity and RAW.10 [33] and8 into theestablish execution the of key LPS-primed roles of responsesethanolamine [29] ofplasmalogens macrophages. in regulating Here, we extendedthe phagocytic our studies activity on [33] the and role in of the plasmalogens execution of inLPS-primed cellular physiology responses by[29] studying of macrophages. other functional Here, responseswe extended of macrophages. our studies Figureon the4 showsrole of the plasmalogens quantitative analysisin cellular of eicosanoidsphysiology producedby studying by other zymosan-stimulated functional responses cells. of The macrophages. profile of eicosanoids Figure 4 shows produced the quantitative was similar, bothanalysis qualitatively of eicosanoids and quantitatively, produced by to zymosan-stim that previouslyulated reported cells. by The Buczynski profile of et al.eicosanoids [66]. Only produced products ofwas the similar, cyclooxygenase both qualitatively pathway and were quantitatively, found, with to PGD that2 previouslyconstituting reported the major by eicosanoidBuczynski et detected. al. [66]. EicosanoidOnly products production of the wascyclooxygenase the same in thepathway RAW 264.7 were cells found, as in with the plasmalogen-deficient PGD2 constituting the variants major (Figureeicosanoid4), thus detected. suggesting Eicosanoid that plasmalogen production status was doesthe same not influence in the theRAW eicosanoid 264.7 cells biosynthetic as in the responseplasmalogen-deficient of the macrophages. variants (Figure 4), thus suggesting that plasmalogen status does not influence the eicosanoid biosynthetic response of the macrophages.

Cells 2019, 8, x FOR PEER REVIEW 9 of 20 Cells 2019, 8, 799 9 of 20

90 ctrl (unstim) RAW 264.7

cells) RAW.12 6 RAW.108 60

30 Eicosanoid (pmol / 10 0 PGE2 PGD2 PGF2a 12-HHT 11-HETE 15-HETE dhk-PGE2 dhk-PGD2 15d-PGD2

Figure 4. Eicosanoid production by RAW 264.7 cells and plasmalogen-deﬁcient variants: RAW 264.7 cellsFigure (open 4. Eicosanoid bars), RAW.12 production cells (light by green RAW bars), 264.7and cells RAW.108 and plasmalogen-deficient cells (dark green bars) variants: were stimulatedRAW 264.7 withcells 150 (openµg/ mLbars), zymosan RAW.12 for cells 8 h. (light Afterward, green extracellularbars), and RAW.108 media was cells removed (dark andgreen analyzed bars) were for eicosanoidstimulated levels with by150 LC μ/MS.g/mL Eicosanoids zymosan for produced 8 h. Afterward, by untreated extracellular RAW 264.7 media cells (crosshatched was removed bars) and

areanalyzed shown forfor comparison.eicosanoid levels Results by areLC/MS. shown Ei ascosanoids means producedS.E.M. (n =by3). untreated PGE2, prostaglandin RAW 264.7 cells E2; ± 12,14 PGD2,(crosshatched prostaglandin bars) are D2 ;shown PGF2a, for prostaglandin comparison. FResu2α; 15d-PGD2,lts are shown 15-deoxy- as means∆ ± S.E.M.-prostaglandin (n = 3). PGE2, D2; dhk-PGE2,prostaglandin 13,14-dihydro-15-keto-prostaglandin E2; PGD2, prostaglandin D2; PGF2a, E2; dhk-PGD2, prostaglandin 13,14-dihydro-15-keto- F2α; 15d-PGD2, 15-deoxy- prostaglandinΔ12,14- Dprostaglandin2; 11-HETE, 11-hydroxyeicosatetraenoic D2; dhk-PGE2, 13,14-dihydro-15-keto-prostaglandin acid; 12-HHT, 12-hydroxyheptadecatrienoic E2; dhk-PGD2, 13,14-dihydro-15- acid; 15-HETE, 15-hydroxyeicosatetraenoicketo- prostaglandin D acid.2; 11-HETE, 11-hydroxyeicosatetraenoic acid; 12-HHT, 12- hydroxyheptadecatrienoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid. In the next series of experiments, we treated the cells with LPS/interferon-γ or interleukin-4/interleukin-13 to induce polarization/activation of the macrophages to pro-inflammatory In the next series of experiments, we treated the cells with LPS/interferon-γ or interleukin- (M1) or anti-inflammatory (M2) phenotypes [48], respectively, and changes in the gene expression levels 4/interleukin-13 to induce polarization/activation of the macrophages to pro-inflammatory (M1) or of various markers associated to each phenotype were assessed by qPCR. Figure5 shows that there were anti-inflammatory (M2) phenotypes [48], respectively, and changes in the gene expression levels of no differences between RAW 264.7 cells and the plasmalogen-deficient variants in the expression levels of various markers associated to each phenotype were assessed by qPCR. Figure 5 shows that there any of the genes assayed. were no differences between RAW 264.7 cells and the plasmalogen-deficient variants in the These results, along with our previous data [29,33], underscore the differential involvement of expression levels of any of the genes assayed. plasmalogens in some, but not all, responses of macrophages, thus reflecting some sort of biological These results, along with our previous data [29,33], underscore the differential involvement of specificity of this kind of phospholipids. plasmalogens in some, but not all, responses of macrophages, thus reflecting some sort of biological specificity of this kind of phospholipids.

Cells 2019, 8, 799 10 of 20 Cells 2019, 8, x FOR PEER REVIEW 10 of 20

50 100 150 200 2000 0 5 10 15 100 150 AB RAW 264.7 RAW 264.7 RAW.12 RAW.12 RAW.108 RAW.108 Cxcl1

Cox2 Fizz1

Il1b Arg1

Il6 Mrc1

Il12b Mrc2

Nos2 Tgfb

Tnfa Ym1

50 100 150 200 2000 0 5 10 15 100 150 Relative mRNA Expression Relative mRNA Expression FigureFigure 5.5. MacrophageMacrophage polarizationpolarization through thethe classicclassic pro-inﬂammatorypro-inflammatory (M1) andand alternativealternative (M2)(M2) pathways:pathways: RAWRAW 264.7 264.7 cells cells (open (open bars) bars and) and their their plasmalogen-defective plasmalogen-defectiv variantse variants RAW.12 RAW.12 (light (light red bars) red andbars) RAW.108 and RAW.108 (dark red(dark bars) red were bars) treated were treated for 8 h fo withr 8 h LPS with (250 LPS ng (250/mL) ng/mL) plus interferon- plus interferon-γ (500 Uγ/ mL)(500 (U/mL)A) or with (A) or interleukin-4 with interleukin-4 (20 ng/ mL)(20 ng/mL) plus interleukin-13 plus interleukin-13 (20 ng /(20mL) ng/mL) (B) to ( generateB) to generate classically classically (M1) or(M1) alternatively or alternatively (M2) polarized (M2) polarized macrophages, macrophages, respectively. respectively. Afterward, Afterward, the expression the expression of the indicated of the markersindicated was markers studied was by qPCR.studied Data by qPCR. are relative Data toare basal rela expressiontive to basal levels expression and normalized levels and to normalized cyclophilin A.to cyclophilin A representative A. A representative experiment is experiment shown, and is sh theown, data and are the expressed data are asexpressed means asS.E.M. means of± S.E.M. three ± individualof three individual replicates. replicates.

3.4.3.4. Studies Utilizing AA-enriched CellsCells AsAs aa consequence consequence of of continuous continuous growth growth in culture,in culture, cell cell lines lines are known are known to exhibit to exhibit diminished diminished levels oflevels polyunsaturated of polyunsaturated fatty acids, fatty including acids, including AA, compared AA, compared to normal to cells normal [67,68 ].cells For [67,68]. example, For RAW example, 264.7 cellsRAW contain 264.7 cells 2–3-fold contain less AA2–3-fold than murineless AA resident than mu peritonealrine resident macrophages peritoneal [ 58macrophages]. Since CoA-IT-driven [58]. Since fattyCoA-IT-driven acid remodeling fatty acid reactions remodeling determine reactions the distribution determine the of AAdistribution among membrane of AA among phospholipids, membrane wephospholipids, reasoned that we the reasoned much that faster the remodeling much faster observed remodeling in RAWobserved 264.7 in cellsRAW compared 264.7 cells tocompared normal macrophagesto normal macrophages could be related could tobe theirrelated relative to their deficiency relative indeficiency AA. To test in AA. this possibility,To test this RAWpossibility, 264.7 µ cellsRAW and 264.7 the cells plasmalogen-deficient and the plasmalogen-deficient variants were vari culturedants were in mediacultured supplemented in media supplemented with 25 M with AA (complexed25 μM AA (complexed with bovine with serum bovine albumin serum at albumin a 2:1 ratio) at a for 2:1 48ratio) h. This for 48 procedure h. This procedure resulted inresulted the cells in increasingthe cells increasing their AA their content AA by conten aboutt by 2–3-fold, about thus2–3-fold, reaching thus levelsreaching comparable levels comparable to those found to those in normalfound in peritoneal normal peritoneal macrophages macrophages [58]. Figure [58].6A compares Figure 6A the compares distribution the of distribution AA between of phospholipid AA between classesphospholipid in the AA-enrichedclasses in the cells. AA-enriched Importantly, cells. all Im ofportantly, the AA-containing all of the phospholipidAA-containing classes phospholipid in wild typeclasses RAW in 264.7wild type cells andRAW their 264.7 variants cells and incorporated their variants exogenous incorporated AA to a similarexogenous extent. AA This to a resulted similar inextent. the relativeThis resulted distribution in the relative profile distribution of the fatty profile acid by of class the fatty being acid preserved, by class being i.e., PE preserved, comprising i.e., approximatelyPE comprising 50% approximately of cellular AA 50% and of PI cellular constituting AA theand second PI constituting richest AA the phospholipid, second richest followed AA 3 closelyphospholipid, by PC. Afterfollowed the AA-enrichment closely by PC. period,After the the AA- cellsenrichment were spiked period, with [theH]AA cells and were the spiked movement with of[3H]AA AA from and PC the to movement PE was followed of AA at from different PC to times. PE was Notably, followed under at thesedifferent conditions, times. Notably, the remodeling under processthese conditions, was markedly the remodeling slowed in both process wild was type markedly and plasmalogen-deficient slowed in both wild variants, type withand plasmalogen- a remodeling timedeficient of approx. variants, 2 h, with i.e., a comparable remodeling to time that of of approx. resident 2 peritonealh, i.e., comparable macrophages to that (cf of. Figuresresident3 Bperitoneal and6B). Thesemacrophages data suggest (cf. Figure that endogenous 3B and Figure cellular 6B). These levels data of AA, sugg notest plasmalogen that endogenous status, cellular determines levels the of rateAA, ofnot remodeling plasmalogen of AAstatus, among determines phospholipids. the rate of remodeling of AA among phospholipids.

Cells 2019, 8, x FOR PEER REVIEW 11 of 20 Cells 2019, 8, 799 11 of 20

50 AA-untreated * A RAW 264.7 * * RAW.12 40 RAW.108

30 * * * 20 * * *

10 * * * AA Content (nmol/mg protein) 0 PC PE PI Total 180

150 B

120

30 Remodeling Time (min)

0 RAW 264.7 RAW.12 RAW.108

Figure 6. Phospholipid AA remodeling in AA-enriched cells: (A) RAW 264.7 cells (open bars), Figure 6. Phospholipid AA remodeling in AA-enriched cells: (A) RAW 264.7 cells (open bars), RAW.12 cells (light grey bars), and RAW.108 cells (dark grey bars) were incubated in media containing RAW.12 cells (light grey bars), and RAW.108 cells (dark grey bars) were incubated in media 25 µM exogenous AA for 48 h. Afterward, cellular AA content in phospholipids was analyzed by containing 25 μM exogenous AA for 48 h. Afterward, cellular AA content in phospholipids was GC/MS. The AA content of otherwise untreated RAW 264.7 cells (cross-hatched bars) is shown for analyzed by GC/MS. The AA content of otherwise untreated RAW 264.7 cells (cross-hatched bars) is comparison. (B) AA remodeling was analyzed in the AA-enriched cells, and the remodeling time shown for comparison. (B) AA remodeling was analyzed in the AA-enriched cells, and the (time at which the radioactivity content of PC equals that of PE) was determined. Results are shown remodeling time (time at which the radioactivity content of PC equals that of PE) was determined. as means S.E.M. (n = 3). * p < 0.05, significantly different from AA-untreated cells. Results are± shown as means ± S.E.M. (n = 3). *p < 0.05, significantly different from AA-untreated cells. A key step in phospholipid AA remodeling is the continuous generation of lysophospholipid A key step in phospholipid AA remodeling is the continuous generation of lysophospholipid moieties by phospholipase A2 enzymes, which act as fatty acid acceptors for the subsequent moieties by phospholipase A2 enzymes, which act as fatty acid acceptors for the subsequent CoA-IT- CoA-IT-mediated transacylation reaction. Since macrophage activation by receptor-directed agonists, mediated transacylation reaction. Since macrophage activation by receptor-directed agonists, including zymosan, results in noticeable elevations of lysophospholipid levels compared to including zymosan, results in noticeable elevations of lysophospholipid levels compared to unstimulated cells [8,27,44,69,70], we investigated whether the activation state of the cells, with its unstimulated cells [8,27,44,69,70], we investigated whether the activation state of the cells, with its attendant elevation of cellular lysophospholipid levels, exerted any influence on their phospholipid attendant elevation of cellular lysophospholipid levels, exerted any influence on their phospholipid AA remodeling rate. To this end, the cells were labeled with [3H]AA for 15 min and, after extensive AA remodeling rate. To this end, the cells were labeled with [3H]AA for 15 min and, after extensive washing, they were untreated (control incubations) or treated with zymosan (150 µg/mL) for different washing, they were untreated (control incubations) or treated with zymosan (150 μg/mL) for different time periods and the transfer of label from PC to PE was measured. The experiments were carried out time periods and the transfer of label from PC to PE was measured. The experiments were carried in otherwise untreated cells and in exogenous AA-treated cells, and both wild type and plasmalogen out in otherwise untreated cells and in exogenous AA-treated cells, and both wild type and variants were used. No effect of cell activation could be ascertained on phospholipid AA remodeling plasmalogen variants were used. No effect of cell activation could be ascertained on phospholipid under any of these conditions. The remodeling time of zymosan-activated cells was the same as that AA remodeling under any of these conditions. The remodeling time of zymosan-activated cells was of resting cells, either using wild-type cells, the plasmalogen-deficient variants, or cells preloaded the same as that of resting cells, either using wild-type cells, the plasmalogen-deficient variants, or with exogenous AA. Thus, phospholipid AA remodeling rate is independent of the activation state of cells preloaded with exogenous AA. Thus, phospholipid AA remodeling rate is independent of the the cells. activation state of the cells.

3.5. Phospholipase A2 Inhibition Studies 3.5. Phospholipase A2 Inhibition Studies Earlier studies attempting to identify the origin of the lysophospholipid acceptors used in Earlier studies attempting to identify the origin of the lysophospholipid acceptors used in the the CoA-IT-mediated transacylation reaction took advantage of PLA2 inhibitors available at that time; however,CoA-IT-mediated given the transacylation uncertain speciﬁcity reaction of sometook advantage of the inhibitors of PLA used,2 inhibitors these studies available did notat that provide time; however, given the uncertain specificity of some of the inhibitors used, these studies did not provide unambiguous responses [71,72]. Recently, much more selective phospholipase A2 inhibitors with improvedunambiguous properties responses have been[71,72]. developed Recently, [73 much] and wemore have selective used these phospholipase to re-evaluate A2 the inhibitors involvement with improved properties have been developed [73] and we have used these to re-evaluate the of phospholipase A2 isoforms. The inhibitors used were pyrrophenone for cPLA2α (at least 3 orders of involvement of phospholipase A2 isoforms. The inhibitors used were pyrrophenone for cPLA2α (at

Cells 2019, 8, 799 12 of 20 Cells 2019, 8, x FOR PEER REVIEW 12 of 20 magnitudeleast 3 orders more of magnitude potent for more cPLA potent2α than for for cPLA iPLA2α2 βthanor sPLAfor iPLA2 enzymes)2β or sPLA [342 ];enzymes) FKGK18 [34]; for iPLAFKGK182β (atfor least iPLA 2002β and(at 400least times 200 moreand 400 potent times for more iPLA 2potentβ than forfor cPLAiPLA22αβ andthan sPLA for 2cPLAs, respectively)2α and sPLA [352];s, GK436,respectively) also for [35]; iPLA GK436,2β (at leastalso 1000-foldfor iPLA2β more (at least potent 1000-fold for iPLA more2β than potent for cPLAfor iPLA2α, and2β than no appreciablefor cPLA2α, eandffect no on appreciable sPLA2 enzymes) effect [on36]; sPLA and2 GK241 enzymes) for sPLA[36]; 2and(inhibits GK241 IIA for and sPLA V2 forms, (inhibits lacking IIA and appreciable V forms, inhibitionlacking appreciable against cPLA inhibition2α, iPLA against2β, or cPLA any2 otherα, iPLA sPLA2β, 2orform) any other [37]. sPLA However,2 form) neither [37]. However, of these inhibitorsneither of had these any inhibitors measurable had e anyffect measurable on the remodeling effect on time the of remodeling RAW 264.7 time cells, of thus RAW providing 264.7 cells, further thus additionalproviding supportfurther additional for the lack support of involvement for the lack of cPLAof involvement2α, iPLA2β of, or cPLA sPLA2α2, -IIAiPLA/V2β in, or phospholipid sPLA2-IIA/V AAin phospholipid remodeling. AA remodeling. Recently,Recently, it it has has been been speculated speculated that that a a lesser lesser known known member member of of the the group group IV IV phospholipase phospholipase A A2 2 familyfamily ofof enzymes, enzymes, i.e., i.e., the the group group IVC IVC form, form, also also known known as as cytosolic cytosolic phospholipase phospholipase A A2γ2γ(cPLA (cPLA22γγ),), couldcould bebe involvedinvolved in phospholipid AA AA remodeling remodeling [14]. [14]. This This suggestion suggestion is ismade made primarily primarily on onthe 2+ thebasis basis of the of thebiochemical biochemical properties properties of cPLA of cPLA2γ (i.e.,2γ (i.e., Ca2+ Ca-independent,-independent, manifests manifests measurable measurable CoA- CoA-independentindependent transacylation transacylation activity activity in vitro,in vitro and, and permanently permanently associated associated with with membranes) membranes) [14]. [14 To]. Tobegin begin to address to address this this question, question, conditions conditions were were established established to achieve to achieve silencing silencing of cPLA of2 cPLAγ by siRNA2γ by siRNAtechnology technology in RAW in 264.7 RAW cells. 264.7 Since cells. we Since have we been have unable been unableto find toreliable find reliable antibodies antibodies against againstmurine murinecPLA2γ cPLA, the efficiency2γ, the effi ofciency siRNA of knockdown siRNA knockdown was judged was by judged qPCR. by Using qPCR. this Using technique, this technique, we were weable were to achieve able to as achieve much as a much 70–75% as adecrease 70–75% in decrease cPLA2γ inmRNA cPLA under2γ mRNA our conditions under our (Figure conditions 7A). (FigureStrikingly,7A). Strikingly,even with even this withincomplete this incomplete cPLA2γcPLA silencing,2γ silencing, cells still cells exhibited still exhibited clear cleardefects defects in AA in AAremodeling remodeling from from PC PC to toPE, PE, reflected reflected by by a astatistica statisticallylly significant significant increase increase of their remodelingremodeling timetime (Figure(Figure7 B).7B). Thus, Thus, these these data data provide provide strong strong evidence evidence for for the the involvement involvement of of cPLA cPLA2γ2γin in phospholipid phospholipid AAAA remodeling remodeling in in macrophages. macrophages. Conversely, Conversely, upon upon stimulation stimulation with zymosan,with zymosan, cells deficient cells deficient in cPLA2 γin releasedcPLA2γ released similar amounts similar amounts of AA as of control AA as cellscontrol (Figure cells8 (FigureA), thus 8A), indicating thus indicating that the that enzyme the enzyme is not ais significant not a significant regulator regulator of the of AA the release AA release response. response. In keeping In keeping with with previous previous data data [8,41 [8,41,42,74–,42,74–77], AA-release77], AA-release experiments experiments conducted conducted in parallel in parallel in thein the presence presence of of the the phospholipase phospholipase A A2 2inhibitors inhibitors describeddescribed aboveabove confirmedconfirmed thethe rolerole ofof cPLA cPLA2α2αbut but notnotof of sPLA sPLA22or oriPLA iPLA22ββ inin AAAA release,release, asas onlyonly inhibitioninhibition of of cPLA cPLA2α2αresulted resulted in in blockade blockade of theof the response response (Figure (Figure8B). In8B). these In these experiments, experiments, we also we used also bromoenolused bromoenol lactone lactone (BEL) for(BEL) comparative for comparative purposes purposes with data with in bibliographydata in bibliography [32,74]. BEL[32,74]. is a BEL widely is a usedwidely irreversible used irreversible inhibitor inhibitor of calcium-independent of calcium-independent phospholipase phospholipase A2s, withA2s, with little little or no or e noffect effect on calcium-dependenton calcium-dependent enzymes enzymes [78 ,[78,79],79], although although with with potential potential o ffoff-target-target e ffeffectsects depending depending on on cell cell typetype [ 80[80,81].,81]. In In our our hands, hands, BEL BEL did did not not have have any any eff ecteffect on zymosan-stimulatedon zymosan-stimulated AA AA release release (Figure (Figure8B). Collectively,8B). Collectively, these resultsthese results provide provide evidence evidence for separate for separate roles for roles the group for the IV group cytosolic IV familycytosolic members family cPLAmembers2α and cPLA cPLA2α and2γ in cPLA cellular2γ in AAcellular homeostasis; AA homeostasis; while the whil formere the former regulates regulates AA release AA release but not but phospholipidnot phospholipid AA remodeling,AA remodeling, the latter the latter does does the opposite. the opposite.

A B

) 6 -3 6 1.00

5 5

0.75 4 4 *

3 0.50 3

2 2

Relative mRNA Level mRNA Relative 0.25 PC 1 PC 1 cPLA γ Silenced Control PE 2 PE

H]AA in H]AA phospholipids(dpm x 10 0 0 0.00 3 [ 0 20406080100120 γ 0 20406080100120 Ctrl cPLA2 Time (min) Time (min) Silenced

FigureFigure 7. 7.cPLA cPLA22γγ rolerole inin phospholipidphospholipid AAAAremodeling: remodeling: ((AA)) PCRPCR analysisanalysis of of cPLA cPLA2γ2γexpression expression inin RAWRAW 264.7 264.7 cells. cells. ((BB)) PhospholipidPhospholipid AAAA remodelingremodeling waswas analyzedanalyzed inin cellscells treatedtreated with with a a scrambled scrambled siRNAsiRNA (Control) (Control) or or siRNA siRNA targeting targeting cPLA cPLA2γ2γ(cPLA (cPLA2γ2γSilenced). Silenced). ResultsResults areareshown shownas asmeans means ± S.E.M.S.E.M. ± (n(n= =5). 5). TheThe asteriskasterisk inin thethe FigureFigure7 B7B right right panel panel denotes denotes that that the the remodeling remodeling time time of of cPLA cPLA2γ2γ-silenced-silenced cellscells is is signiﬁcantly significantly di differentﬀerent from from that that of of control control cells cells (* (*pp< < 0.05).0.05).

Cells 2019, 8, 799 13 of 20 Cells 2019, 8, x FOR PEER REVIEW 13 of 20

No inhibitor Pyrrophenone GK241 A 12 B 8 8 8

10 6 6 6 8

6 4 4 4 * 4 AA Release (%) AA Release (%) 2 2 2 2

0 0 0 0 Ctrl Zym Ctrl Zym Ctrl Zym

BEL FKGK18 GK436 Control

Silenced 8 8 8 γ 2

cPLA 6 6 6

4 4 4 AA Release (%) 2 2 2

0 0 0 Ctrl Zym Ctrl Zym Ctrl Zym FigureFigure 8. 8. StimulatedStimulated AAAA mobilizationmobilization inin RAWRAW 264.7264.7 cells:cells: ( A))[ [3H]AA-labeledH]AA-labeled cells, cells, treated treated with with a

ascrambled scrambled siRNA siRNA (Control) (Control) or siRNA forfor cPLAcPLA22γ (cPLA(cPLA22γ Silenced), werewere leftleft untreateduntreated(dark (dark red red bars)bars) or or treated treated with with 150 150µ μgg/mL/mL zymosan zymosan (dark (dark yellow yellow bars) bars) for for 1 1 h. h. ( B(B)) The The cells, cells, labeled labeled with with [ 3[3H]AA,H]AA, werewere preincubated preincubated withwith eithereither 2 µμM pyrrophenone, 10 10 μµMM GK241, GK241, 5 5μµMM bromoenol bromoenol lactone lactone (BEL), (BEL), 10 10μMµM FKGK18, FKGK18, 5 5μµMM GK436, GK436, or or neither neither (no (no inhibitor) inhibitor) for 30 mi min.n. Afterward they werewere untreateduntreated (Ctrl)(Ctrl) oror treated treated with with 150 150µ gμ/g/mLmL zymosan zymosan for for 1 h.1 h. Results Results are are shown shown as as means means S.E.M.± S.E.M. (n (n= =3). 3). * p*p< <0.05, 0.05, ± signiﬁcantlysignificantly di differentﬀerent from from zymosan-stimulated zymosan-stimulated cells cells in in the the absence absence of of inhibitors. inhibitors.

4. Discussion 4. Discussion PhospholipidPhospholipid AAAA remodelingremodeling isis necessary necessary forfor cells cells of of the the innate innate immune immune system system to to distribute distribute fatty acid within the appropriate cellular pools for its subsequent mobilization by phospholipase A2 fatty acid within the appropriate cellular pools for its subsequent mobilization by phospholipase A2 enzymes.enzymes. This This is is a a key key aspect aspect in in eicosanoideicosanoid regulation regulation because because the the nature nature and and amount amount of of eicosanoids eicosanoids producedproduced under under activation activation conditions conditions maymay ultimately ultimatelydepend depend on on the the composition composition and and subcellular subcellular localization of the phospholipid pool where the AA-hydrolyzing phospholipase A2 acts [1,2]. In innate localization of the phospholipid pool where the AA-hydrolyzing phospholipase A2 acts [1,2]. In immuneinnate immune cells, ether cells, phospholipids, ether phospholipids, particularly particularly the ethanolamine the ethanolamine plasmalogens, plasmalogens, are strikingly are strikingly enriched inenriched AA, which in AA, is thought which is to thought be due to thebe due fact to that the these fact that phospholipids these phospholipids are major are acceptors major acceptors for fatty acidfor fatty transfer acid reactions transfer betweenreactions phospholipids between phospholip that helpids shapethat help the finalshape distribution the final distribution of the fatty of acid the infatty cells acid [11 –in14 cells]. While [11–14]. the While enrichment the enrichment of ether phospholipids of ether phospholipids with AA with suggests AA suggests a key role a forkey this role kindfor this of phospholipids kind of phospholipids in AA homeostasis, in AA homeostasis, their role their still remainsrole still relatively remains relatively obscure. Inobscure. fact, receptor In fact, stimulationreceptor stimulation of AA mobilization of AA mobilization in plasmalogen-deficient in plasmalogen-deficient cells is similar cells to is that similar of normal to that cells of [normal29,32], and none of the major AA-releasing phospholipase A2s described to date have been found to exhibit cells [29,32], and none of the major AA-releasing phospholipase A2s described to date have been anyfound particular to exhibit preference any particular for substrates preference containing for substrates an sncontaining-1 ether bond an sn-1 [16 ether]. Although bond [16]. these Although data suggested that plasmalogens may not be essential for the phospholipase A2-mediated AA mobilization these data suggested that plasmalogens may not be essential for the phospholipase A2-mediated AA processmobilization itself, process the possibility itself, the remained possibility that remained they are stillthat instrumentalthey are still forinstrumental placing the for fatty placing acid the in thefatty appropriate acid in the subcellular appropriate localizations subcellular via localizations phospholipid via transacylation phospholipid reactions.transacylation Unexpectedly, reactions. theUnexpectedly, results presented the results in this presented study show in thatthis thisstudy is alsoshow not that the this case. is We also find not no the di ffcase.erence We between find no plasmalogen-deficientdifference between plasmalogen-deficient and otherwise normal and cells, ot thusherwise suggesting normal thatcells, plasmalogen thus suggesting status hasthat noplasmalogen influence on status phospholipid has no influence AA remodeling. on phospholipid It should AA be remodeling. noted in this It regardshould thatbe noted the relative in this regard that the relative distribution of AA among phospholipid classes (i.e., PE versus PC versus PI) is maintained in the plasmalogen-deficient cells because, in these cells, there is a compensatory

Cells 2019, 8, 799 14 of 20 distribution of AA among phospholipid classes (i.e., PE versus PC versus PI) is maintained in the plasmalogen-deficient cells because, in these cells, there is a compensatory elevation of AA levels in diacyl species. This is an important concept because it suggests that, in terms of overall AA distribution, it is the substituent at the sn-3 position of the phospholipid (i.e., ethanolamine versus choline versus inositol versus serine) and not the chemical nature of the sn-1 bond (acyl versus alkyl versus alkenyl) that determines the incorporation of AA. Consistent with this notion, AA transacylation reactions involve only ethanolamine- and choline-containing phospholipids, thus indicating some sort of specificity at the level of the sn-3 substituent. While the lack of sn-1 influence on the AA transacylation reaction in cells is unanticipated, it should be noted that CoA-IT, the enzyme catalyzing these transacylation reactions, may use 1-acyl-PE just as well as 1-alkenyl-PE [11–14] and this, in fact, may allow to explain satisfactorily why the plasmalogen-deficient variants are able to compensate their deficiency by directing the AA to diacyl phospholipids. On the other hand, the finding that plasmalogen-deficient cells and normal cells mobilize AA similarly in spite of their composition being so different at the molecular species level provides strong support to the idea that cells can sustain critical reactions with many different lipid compositions rather than a single composition [82]. Furthermore, our results also raise the intriguing possibility that the enrichment of plasmalogens with AA might not be necessarily related to regulatory aspects of AA homeostasis and eicosanoid metabolism but to biophysical effects and interaction of the phospholipid with other membrane components to sustain different biological responses. Our collective findings showing that plasmalogens participate in the execution of some responses [29,33] but not in others (this study) are fully consistent with this view. AA-containing ethanolamine plasmalogens are frequently found as components of specific membrane microdomains called lipid rafts [83–85]. The relative plasmalogen content within these domains may affect key properties such as fluidity, tendency to fusion, packing, thickness, and density, thereby influencing the biological behavior of membrane rafts in membrane transport and transmembrane signaling [83–85]. CoA-IT catalyzes the enzymatic step that is unique to the phospholipid AA remodeling pathway, i.e., the direct transfer of AA from a phospholipid donor to a lysophospholipid acceptor in the absence of CoA or ATP [11–14]. CoA-IT has been defined as a membrane-bound, calcium-independent enzyme. Based on biochemical and mechanistic commonalities, Yamashita and coworkers suggested that CoA-independent transacylation reactions in cells are catalyzed by (an) enzyme(s) of the phospholipase A2 family and speculated that group IVC cytosolic phospholipase A2 (also called cytosolic phospholipase A2γ, cPLA2γ) is a possible candidate [14,86,87]. The same authors noted that the cPLA2γ-catalyzed transacylase reaction works better when the AA donor is lysoPC instead of AA-containing diacyl-PC. On the other hand, Stewart et al. [88] noted that the enzyme has higher lysophospholipase activity than phospholipase A2 activity. However, conditions of in vitro specificity assays are not necessarily translatable to the in vivo situation, where compartmentalization of substrates and products and the presence of competing enzymes may dramatically modify the specificities reported. By using siRNA technology to specifically knock down cellular expression levels of cPLA2γ, we have tested experimentally the proposed involvement of cPLA2γ in regulating phospholipid AA remodeling. Our results clearly indicate that this is the case, as cells which were made deficient in cPLA2γ transfer AA from PC to PE significantly more slowly than control cells. These results constitute, to the best of our knowledge, the first report attributing cellular AA remodeling activity to a well-characterized molecular entity, i.e., the cPLA2γ enzyme. Thus, the finding is significant because it makes now possible to apply molecular biology approaches, such as overexpression or deletion, which should significantly expand our knowledge about the cellular and molecular regulation of phospholipid AA remodeling reactions. At this point, we cannot indicate whether cPLA2γ participates in the CoA-independent transacylation reaction by providing the lyso acceptors that initiate the reaction, by directly catalyzing the fatty acid transacylation, or by acting at both levels. Clearly, further work should be conducted to clarify these mechanistic issues and whether the enzyme is constitutively active or its activity increases after agonist stimulation [24]. Also, since our data clearly show that Cells 2019, 8, 799 15 of 20

selective inhibition of cPLA2γ by siRNA slows but does not eliminate phospholipid AA remodeling, it seems likely that other enzymes in addition to cPLA2γ operate as CoA-IT in cells, and further work should be undertaken to identify them. Another striking feature of this work is the finding that the rate of AA remodeling from PC to PE appears to be independent of the state of activation of the cells. We could not observe any change in the rate of AA remodeling from PC to PE in zymosan-stimulated cells compared to unstimulated cells. This finding was somehow unexpected because lysophospholipid availability initiates phospholipid AA remodeling in cells and zymosan-activated macrophages increase their intracellular lysophospholipid content as a consequence of cPLA2α activation [8,27,29]. Lack of an effect of cell stimulation on CoA-IT-mediated phospholipid remodeling is at variance with previous results in phorbol ester-stimulated platelets [89], tumor necrosis factor α-stimulated neutrophils [90], and antigen-stimulated mast cells [91] but is consistent with work in neutrophils on the regulation of platelet-activating factor synthesis via transacylation reactions, where it was found that CoA-IT activity did not increase as a consequence of cell activation but that regulation occurred at the level of increased substrate availability [92]. In this regard, we also report in this paper that increasing the cellular levels of AA results in decreased rates of CoA-mediated phospholipid AA remodeling. It is thus tempting to speculate with a scenario wherein an AA-containing phospholipid that is present in the AA-enriched cells but not in the otherwise normal cells may act to regulate CoA-IT-mediated transacylation reactions by directly impinging on the enzyme and/or by regulating substrate availability. We have previously shown that a short-lived AA-containing phospholipid produced by activated cells, namely 1,2-diarachidonoyl-glycerophosphoinositol, is able to regulate macrophage responses to innate immune stimuli [7].

5. Conclusions We demonstrate in this work that, although plasmalogens constitute the major reservoirs of AA in normal cells, CoA-IT-mediated phospholipid AA remodeling reactions responsible for the asymmetric distribution of the fatty acid in the various phospholipid pools are not influenced by the plasmalogen content of cells. Compartmentalization of AA in cells appears to depend primarily on headgroup composition of membrane phospholipids rather than on specific molecular species. In addition, our work implicates cPLA2γ as a major enzyme involved in phospholipid AA remodeling. Taken together, our results provide new information to understand better the regulatory processes underlying cellular AA availability, which may be used to develop valid strategies to manipulate eicosanoid metabolism and signaling pathways in innate immunity and inflammation.

Author Contributions: Conceptualization, J.B.; methodology, P.L., A.M.A., J.M.R., L.F.-C., and G.K.; investigation, P.L., A.M.A., J.M.R., and L.F.-C.; writing—original draft preparation, J.B.; writing—review and editing, P.L., A.M.A., G.K., M.A.B., and J.B.; supervision, M.A.B. and J.B.; project administration, M.A.B. and J.B.; funding acquisition, M.A.B. and J.B. Funding: This research was funded by the Spanish Ministry of Economy, Industry, and Competitiveness, grant number SAF2016-80883-R. P.L. was supported by a predoctoral fellowship from the University of Valladolid (Plan de Formación de Personal Investigador). CIBERDEM is an initiative of Instituto de Salud Carlos III. Acknowledgments: We are indebted to Raphael A. Zoeller (Boston University) for providing us with plasmalogen-deficient RAW.12 and RAW.108 cells. We thank Montse Duque for the excellent technical assistance. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Abbreviations: AA: arachidonic acid; CoA-IT, coenzyme A-independent transacylase; GC/MS, gas chromatography coupled to mass spectrometry; LC/MS, liquid chromatography coupled to mass spectrometry; LPS, bacterial lipopolysaccharide; cPLA2α, group IVA cytosolic phospholipase A2α; cPLA2γ, group IVC cytosolic phospholipase A2γ; iPLA2β, group VIA calcium-independent phospholipase A2β; sPLA2, secreted phospholipase A2; PC, choline-containing phospholipids; PE, ethanolamine-containing phospholipids; PI, phosphatidylinositol; PS, phosphatidylserine. Cells 2019, 8, 799 16 of 20

References

1. Dennis, E.A.; Norris, P.C. Eicosanoid storm in infection and inﬂammation. Nat. Rev. Immunol. 2015, 15, 511–523. [CrossRef][PubMed]

2. Astudillo, A.M.; Balboa, M.A.; Balsinde, J. Selectivity of phospholipid hydrolysis by phospholipase A2 enzymes in activated cells leading to polyunsaturated fatty acid mobilization. Biochim. Biophys. Acta 2019, 1864, 772–783. [CrossRef][PubMed]

3. Guijas, C.; Rodríguez, J.P.; Rubio, J.M.; Balboa, M.A.; Balsinde, J. Phospholipase A2 regulation of lipid droplet formation. Biochim. Biophys. Acta 2014, 1841, 1661–1671. [CrossRef][PubMed] 4. Astudillo, A.M.; Balgoma, D.; Balboa, M.A.; Balsinde, J. Dynamics of arachidonic acid mobilization by inﬂammatory cells. Biochim. Biophys. Acta 2012, 1821, 249–256. [CrossRef][PubMed] 5. Pérez-Chacón, G.; Astudillo, A.M.; Balgoma, D.; Balboa, M.A.; Balsinde, J. 2009. Control of free arachidonic

acid levels by phospholipases A2 and lysophospholipid acyltransferases. Biochim. Biophys. Acta 2009, 1791, 1103–1113. [CrossRef][PubMed] 6. Balgoma, D.; Astudillo, A.M.; Pérez-Chacón, G.; Montero, O.; Balboa, M.A.; Balsinde, J. Markers of monocyte activation revealed by lipidomic proﬁling of arachidonic acid-containing phospholipids. J. Immunol. 2010, 184, 3857–3865. [CrossRef][PubMed] 7. Gil-de-Gómez, L.; Astudillo, A.M.; Meana, C.; Rubio, J.M.; Guijas, C.; Balboa, M.A.; Balsinde, J. A phosphatidylinositol species acutely generated by activated macrophages regulates innate immune responses. J. Immunol. 2013, 190, 5169–5177. [CrossRef] 8. Gil-de-Gómez, L.; Astudillo, A.M.; Guijas, C.; Magrioti, V.; Kokotos, G.; Balboa, M.A.; Balsinde, J. Cytosolic

group IVA and calcium-independent group VIA phospholipase A2s act on distinct phospholipid pools in zymosan-stimulated mouse peritoneal macrophages. J. Immunol. 2014, 192, 752–762. [CrossRef] 9. Sugiura, T.; Nakajima, M.; Sekiguchi, N.; Nakagawa, Y.; Waku, K. Diﬀerent fatty chain compositions of alkenylacyl, alkylacyl and diacyl phospholipids in rabbit alveolar macrophages: High amounts of arachidonic acid in ether phospholipids. Lipids 1983, 18, 125–129. [CrossRef] 10. Blank, M.L.; Smith, Z.L.; Cress, E.A.; Snyder, F. Molecular species of ethanolamine plasmalogens and transacylase activity in rat tissues are altered by fish oil diets. Biochim. Biophys. Acta 1994, 1214, 295–302. [CrossRef] 11. Chilton, F.H.; Fonteh, A.N.; Surette, M.E.; Triggiani, M.; Winkler, J.D. Control of arachidonate levels within inﬂammatory cells. Biochim. Biophys. Acta 1996, 1299, 1–15. [CrossRef] 12. Chilton, F.H. Potential phospholipid source(s) of arachidonate used for the synthesis of leukotrienes by the human neutrophil. Biochem. J. 1989, 258, 327–333. [CrossRef][PubMed] 13. Yamashita, A.; Hayashi, Y.; Nemoto-Sasaki, Y.; Ito, M.; Oka, S.; Tanikawa, T.; Waku, K.; Sugiura, T. Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms. Prog. Lipid Res. 2014, 53, 18–81. [CrossRef][PubMed] 14. Yamashita, A.; Hayashi, Y.; Matsumoto, N.; Nemoto-Sasaki, Y.; Koizumi, T.; Inagaki, Y.; Oka, S.; Tanikawa, T.;

Sugiura, T. Coenzyme-A-independent transacylation system; possible involvement of phospholipase A2 in transacylation. Biology 2017, 6, 23. [CrossRef][PubMed] 15. Murphy, R.C.; Folco, G. Lysophospholipid acyltransferases and leukotriene biosynthesis: Intersection of the Lands cycle and the arachidonate PI cycle. J. Lipid Res. 2019, 60, 219–226. [CrossRef][PubMed]

16. Dennis, E.A.; Cao, J.; Hsu, Y.H.; Magrioti, V.; Kokotos, G. Phospholipase A2 enzymes: Physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem. Rev. 2011, 111, 6130–6185.

17. Balboa, M.A.; Sáez, Y.; Balsinde, J. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J. Immunol. 2003, 170, 5276–5280. [CrossRef][PubMed]

18. Pérez, R.; Melero, R.; Balboa, M.A.; Balsinde, J. Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide. J. Biol. Chem. 2004, 279, 40385–40391. [CrossRef][PubMed]

19. Pérez, R.; Balboa, M.A.; Balsinde, J. Involvement of group VIA calcium-independent phospholipase A2 in macrophage engulfment of hydrogen peroxide-treated U937 cells. J. Immunol. 2006, 176, 2555–2561. [CrossRef] Cells 2019, 8, 799 17 of 20

20. Balsinde, J.; Balboa, M.A. Cellular regulation and proposed biological functions of group

VIA calcium-independent phospholipase A2 in activated cells. Cell. Signal. 2005, 17, 1052–1062. [CrossRef] 21. Winkler, J.D.; Sung, C.M.; Bennett, C.F.; Chilton, F.H. Characterization of CoA-independent transacylase activity in U937 cells. Biochim. Biophys. Acta 1991, 1081, 339–346. [CrossRef] 22. Winkler, J.D.; Sung, C.M.; Chabot-Fletcher, M.; Griswold, D.E.; Marshall, L.A.; Chilton, F.H.; Bondinell, W.; Mayer, R.J. β-lactams SB 212047 and SB 216754 are irreversible, time-dependent inhibitors of coenzyme A-independent transacylase. Mol. Pharmacol. 1998, 53, 322–329. [CrossRef][PubMed] 23. Winkler, J.D.; Fonteh, A.N.; Sung, C.M.; Heravi, J.D.; Nixon, A.B.; Chabot-Fletcher, M.; Griswold, D.E.; Marshall, L.A.; Chilton, F.H. Eﬀects of CoA-independent transacylase inhibitors on the production of lipid inﬂammatory mediators. J. Pharmacol. Exp. Ther. 1995, 274, 1338–1347. [PubMed] 24. Asai, K.; Hirabayashi, T.; Houjou, T.; Uozumi, N.; Taguchi, R.; Shimizu, T. Human group IVC phospholipase

A2 (cPLA2γ). Roles in the membrane remodeling and activation induced by oxidative stress. J. Biol. Chem. 2003, 278, 8809–8814. [CrossRef][PubMed] 25. Astudillo, A.M.; Pérez-Chacón, G.; Meana, C.; Balgoma, D.; Pol, A.; del Pozo, M.A.; Balboa, M.A.; Balsinde, J. Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis. J. Biol. Chem. 2011, 286, 35299–35307. [CrossRef][PubMed] 26. Valdearcos, M.; Esquinas, E.; Meana, C.; Gil-de-Gómez, L.; Guijas, C.; Balsinde, J.; Balboa, M.A. Subcellular localization and role of lipin-1 in human macrophages. J. Immunol. 2011, 186, 6004–6013. [CrossRef] [PubMed] 27. Rubio, J.M.; Rodríguez, J.P.; Gil-de-Gómez, L.; Guijas, C.; Balboa, M.A.; Balsinde, J. Group V secreted

phospholipase A2 is up-regulated by interleukin-4 in human macrophages and mediates phagocytosis via hydrolysis of ethanolamine phospholipids. J. Immunol. 2015, 194, 3327–3339. [CrossRef] 28. Guijas, C.; Astudillo, A.M.; Gil-de-Gómez, L.; Rubio, J.M.; Balboa, M.A.; Balsinde, J. 2012. Phospholipid sources for adrenic acid mobilization in RAW 264.7 macrophages: Comparison with arachidonic acid. Biochim. Biophys. Acta 2012, 1821, 1386–1393. [CrossRef] 29. Gil-de-Gómez, L.; Astudillo, A.M.; Lebrero, P.; Balboa, M.A.; Balsinde, J. Essential role for ethanolamine plasmalogen hydrolysis in bacterial lipopolysaccharide priming of macrophages for enhanced arachidonic acid release. Front. Immunol. 2017, 8, 1251. [CrossRef] 30. Zoeller, R.A.; Rangaswamy, S.; Herscovitz, H.; Rizzo, W.B.; Hajra, A.K.; Das, A.K.; Moser, H.W.; Moser, A.; Lazarow, P.W.; Santos, M.J. Mutants in a macrophage-like cell line are defective in plasmalogen biosynthesis, but contain functional peroxisomes. J. Biol. Chem. 1992, 267, 8299–8306. 31. Gaposchkin, D.P.; Zoeller, R.A. Plasmalogen status inﬂuences docosahexaenoic acid levels in a macrophage cell line: Insights using ether lipid-deﬁcient variants. J. Lipid Res. 1999, 40, 495–503. [PubMed] 32. Gaposchkin, D.P.; Farber, H.W.; Zoeller, R.A. On the importance of plasmalogen status in stimulated arachidonic acid release in the macrophage cell line RAW 264.7. Biochim. Biophys. Acta 2008, 1781, 213–219. [CrossRef][PubMed] 33. Rubio, J.M.; Astudillo, A.M.; Casas, J.; Balboa, M.A.; Balsinde, J. Regulation of phagocytosis in macrophages by membrane ethanolamine plasmalogens. Front. Immunol. 2018, 9, 1723. [CrossRef][PubMed] 34. Ono, T.; Yamada, K.; Chikazawa, Y.; Ueno, M.; Nakamoto, S.; Okuno, T.; Seno, K. Characterization of a novel

inhibitor of cytosolic phospholipase A2α, pyrrophenone. Biochem. J. 2002, 363, 727–735. [CrossRef][PubMed] 35. Kokotos, G.; Hsu, Y.H.; Burke, J.E.; Baskakis, C.; Kokotos, C.G.; Magrioti, V.; Dennis, E.A. Potent and selective

ﬂuoroketone inhibitors of group VIA calcium-independent phospholipase A2. J. Med. Chem. 2010, 53, 3602–3610. [CrossRef][PubMed] 36. Dedaki, C.; Kokotou, M.G.; Mouchlis, V.D.; Limnios, D.; Lei, X.; Mu, C.T.; Ramanadham, S.; Magrioti, V.; 2+ Dennis, E.A.; Kokotos, G. β-Lactones: A novel class of Ca -independent phospholipase A2 (group VIA iPLA2) inhibitors with the ability to inhibit β-cell apoptosis. J. Med. Chem. 2019, 62, 2916–2927. [CrossRef][PubMed] 37. Vasilakaki, S.; Barbayianni, E.; Leonis, G.; Papadopoulos, M.G.; Mavromoustakos, T.; Gelb, M.H.; Kokotos, G.

Development of a potent 2-oxoamide inhibitor of secreted phospholipase A2 guided by molecular docking calculations and molecular dynamics simulations. Bioorg. Med. Chem. 2016, 24, 1683–1695. [CrossRef] [PubMed] 38. Shirai, Y.; Balsinde, J.; Dennis, E.A. Localization and functional interrelationships among cytosolic group 2+ IV, secreted group V, and Ca -independent group VI phospholipase A2s in P388D1 macrophages using GFP/RFP constructs. Biochim. Biophys. Acta 2005, 1735, 119–129. [CrossRef][PubMed] Cells 2019, 8, 799 18 of 20

39. Balboa, M.A.; Balsinde, J.; Dillon, D.A.; Carman, G.M.; Dennis, E.A. Proinﬂammatory macrophage-activating properties of the novel phospholipid diacylglycerol pyrophosphate. J. Biol. Chem. 1999, 274, 522–526. [CrossRef][PubMed]

40. Balsinde, J.; Balboa, M.A.; Yedgar, S.; Dennis, E.A. Group V phospholipase A2-mediated oleic acid mobilization in lipopolysaccharide-stimulated P388D1 macrophages. J. Biol. Chem. 2000, 275, 4783–4786. [CrossRef] 41. Pindado, J.; Balsinde, J.; Balboa, M.A. TLR3-dependent induction of nitric oxide synthase in RAW 264.7

macrophage-like cells via a cytosolic phospholipase A2/cyclooxygenase-2 pathway. J. Immunol. 2007, 179, 4821–4828. [CrossRef][PubMed] 42. Ruipérez, V.; Astudillo, M.A.; Balboa, M.A.; Balsinde, J. Coordinate regulation of TLR-mediated arachidonic

acid mobilization in macrophages by group IVA and group V phospholipase A2s. J. Immunol. 2009, 182, 3877–3883. 43. Balsinde, J.; Fernández, B.; Diez, E. Regulation of arachidonic acid release in mouse peritoneal macrophages. The role of extracellular calcium and protein kinase C. J. Immunol. 1990, 144, 4298–4304. [PubMed] 44. Balsinde, J.; Fernández, B.; Solís-Herruzo, J.A.; Diez, E. Pathways for arachidonic acid mobilization in zymosan-stimulated mouse peritoneal macrophages. Biochim. Biophys. Acta 1992, 1136, 75–82. [CrossRef]

45. Balboa, M.A.; Balsinde, J. Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells. J. Biol. Chem. 2002, 277, 40384–40389. [CrossRef] 46. Balboa, M.A.; Pérez, R.; Balsinde, J. Ampliﬁcation mechanisms of inﬂammation: Paracrine stimulation

of arachidonic acid mobilization by secreted phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydroperoxyeicosatetraenoic acid. J. Immunol. 2003, 171, 989–994. [CrossRef][PubMed] 47. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef] 48. Rodríguez-Prados, J.C.; Través, P.G.; Cuenca, J.; Rico, D.; Aragonés, J.; Martín-Sanz, P.; Cascante, M.; Boscá, L. Substrate fate in activated macrophages: A comparison between innate, classic, and alternative activation. J. Immunol. 2010, 185, 605–614. [CrossRef] 49. Balboa, M.A.; Balsinde, J.; Dennis, E.A. Involvement of phosphatidate phosphohydrolase in arachidonic acid mobilization in human amnionic WISH cells. J. Biol. Chem. 1998, 273, 7684–7690. [CrossRef] 50. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆ Ct method. Methods 2001, 25, 402–408. [CrossRef] 51. Valdearcos, M.; Esquinas, E.; Meana, C.; Peña, L.; Gil-de-Gómez, L.; Balsinde, J.; Balboa, M.A. Lipin-2 reduces proinflammatory signaling induced by saturated fatty acids in macrophages. J. Biol. Chem. 2012, 287, 10894–10904. [CrossRef][PubMed] 52. Bligh, E.G.; Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911–917. [CrossRef][PubMed] 53. Diez, E.; Balsinde, J.; Aracil, M.; Schüller, A. Ethanol induces release of arachidonic acid but not synthesis of eicosanoids in mouse peritoneal macrophages. Biochim. Biophys. Acta 1987, 921, 82–89. [CrossRef] 54. Fine, J.B.; Sprecher, H. Unidimensional thin-layer chromatography of phospholipids on boric acid-impregnated plates. J. Lipid Res. 1982, 23, 660–663. [PubMed] 55. Astudillo, A.M.; Pérez-Chacón, G.; Balgoma, D.; Gil-de-Gómez, L.; Ruipérez, V.; Guijas, C.; Balboa, M.A.; Balsinde, J. Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells. Biochim. Biophys. Acta 2011, 1811, 97–103. [CrossRef][PubMed] 56. Guijas, C.; Pérez-Chacón, G.; Astudillo, A.M.; Rubio, J.M.; Gil-de-Gómez, L.; Balboa, M.A.; Balsinde, J. Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase

A2-dependent synthesis of lipid droplets in human monocytes. J. Lipid Res. 2012, 53, 2343–2354. [CrossRef] 57. Guijas, C.; Meana, C.; Astudillo, A.M.; Balboa, M.A.; Balsinde, J. Foamy monocytes are enriched in cis-7-hexadecenoic fatty acid (16:1n-9), a possible biomarker for early detection of cardiovascular disease. Cell Chem. Biol. 2016, 23, 689–699. [CrossRef] 58. Astudillo, A.M.; Meana, C.; Guijas, C.; Pereira, L.; Lebrero, R.; Balboa, M.A.; Balsinde, J. Occurrence and biological activity of palmitoleic acid isomers in phagocytic cells. J. Lipid Res. 2018, 59, 237–249. [CrossRef] Cells 2019, 8, 799 19 of 20

59. Rodríguez, J.P.; Guijas, C.; Astudillo, A.M.; Rubio, J.M.; Balboa, M.A.; Balsinde, J. Sequestration of 9-hydroxystearic acid in FAHFA (fatty acid esters of hydroxy fatty acids) as a protective mechanism for colon carcinoma cells to avoid apoptotic cell death. Cancers 2019, 11, 524. [CrossRef] 60. Pérez, R.; Matabosch, X.; Llebaria, A.; Balboa, M.A.; Balsinde, J. Blockade of arachidonic acid incorporation into phospholipids induces apoptosis in U937 promonocytic cells. J. Lipid Res. 2006, 47, 484–491. [CrossRef] 61. Fahy, E.; Subramaniam, S.; Brown, H.A.; Glass, C.K.; Merrill, A.H., Jr.; Murphy, R.C.; Raetz, C.R.; Russell, D.W.; Seyama, Y.; Shaw, W.; et al. A comprehensive classiﬁcation system for lipids. J. Lipid Res. 2005, 46, 839–861. [CrossRef][PubMed] 62. Chilton, F.H.; Fonteh, A.N.; Sung, C.M.; Hickey, D.M.; Torphy, T.J.; Mayer, R.J.; Marshall, L.A.; Heravi, J.D.; Winkler, J.D. Inhibitors of CoA-independent transacylase block the movement of arachidonate into 1-ether-linked phospholipids of human neutrophils. Biochemistry 1995, 34, 5403–5410. [CrossRef][PubMed]

63. Balsinde, J.; Barbour, S.E.; Bianco, I.D.; Dennis, E.A. Arachidonic acid mobilization in P388D1 macrophages 2+ is controlled by two distinct Ca -dependent phospholipase A2 enzymes. Proc. Natl. Acad. Sci. USA 1994, 91, 11060–11064. [CrossRef][PubMed] 64. Balsinde, J.; Balboa, M.A.; Insel, P.A.; Dennis, E.A. Diﬀerential regulation of phospholipase D

and phospholipase A2 by protein kinase C in P388D1 macrophages. Biochem. J. 1997, 321, 805–809. [CrossRef][PubMed] 65. Balsinde, J.; Fernández, B.; Solís-Herruzo, J.A. Increased incorporation of arachidonic acid into phospholipids in zymosan-stimulated mouse peritoneal macrophages. Eur. J. Biochem. 1994, 221, 1013–1018. [CrossRef] [PubMed] 66. Buczynski, M.W.; Stephens, D.L.; Bowers-Gentry, R.C.; Grkovich, A.; Deems, R.A.; Dennis, E.A. TLR-4 and sustained calcium agonists synergistically produce eicosanoids independent of protein synthesis in RAW 264.7 cells. J. Biol. Chem. 2007, 282, 22834–22847. [CrossRef][PubMed] 67. Rouzer, C.A.; Ivanova, P.T.; Byrne, M.O.; Milne, S.B.; Marnett, L.J.; Brown, H.A. Lipid profiling reveals arachidonate deficiency in RAW264.7 cells: Structural and functional implications. Biochemistry 2006, 45, 14795–14808. [CrossRef][PubMed] 68. Okuno, T.; Gijón, M.A.; Zarini, S.; Martin, S.A.; Barkley, R.M.; Johnson, C.A.; Ohba, M.; Yokomizo, T.; Murphy, R.C. Altered eicosanoid production and phospholipid remodeling during cell culture. J. Lipid Res. 2018, 59, 542–549. [CrossRef][PubMed] 69. Emilsson, A.; Sundler, R. Differential activation of phosphatidylinositol deacylation and a pathway via diphosphoinositide in macrophages responding to zymosan and ionophore A23187. J. Biol. Chem. 1984, 259, 3111–3116. 70. Casas, J.; Gijón, M.A.; Vigo, A.G.; Crespo, M.S.; Balsinde, J.; Balboa, M.A. Phosphatidylinositol

4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells. Mol. Biol. Cell 2006, 17, 155–162. [CrossRef]

71. Balsinde, J. Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipid incorporation and remodelling. Biochem. J. 2002, 364, 695–702. [CrossRef][PubMed] 72. Boilard, E.; Surette, M.E. Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. J. Biol. Chem. 2001, 276, 17568–17575. [CrossRef][PubMed] 73. Nikolaou, A.; Kokotou, M.G.; Vasilakaki, S.; Kokotos, G. Small-molecule inhibitors as potential therapeutics

and as tools to understand the role of phospholipases A2. Biochim. Biophys. Acta 2019, 1864, 941–956. [CrossRef][PubMed] 74. Balsinde, J.; Balboa, M.A.; Dennis, E.A. Identification of a third pathway for arachidonic acid mobilization

and prostaglandin production in activated P388D1 macrophage-like cells. J. Biol. Chem. 2000, 275, 22544–22549. [CrossRef] [PubMed] 75. Marshall, J.; Krump, E.; Lindsay, T.; Downey, G.; Ford, D.A.; Zhu, P.; Walker, P.; Rubin, B. Involvement

of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils. J. Immunol. 2000, 164, 2084–2091. [CrossRef][PubMed] 76. Degousee, N.; Ghomashchi, F.; Stefanski, E.; Singer, A.G.; Smart, B.P.; Borregaard, N.; Reithmeier, R.;

Lindsay, T.F.; Lichtenberger, C.; Reinisch, W.; et al. Groups IV, V, and X phospholipases A2s in human neutrophils: Role in eicosanoid production and gram-negative bacterial phospholipid hydrolysis. J. Biol. Chem. 2002, 277, 5061–5073. [CrossRef][PubMed] Cells 2019, 8, 799 20 of 20

77. Pérez-Chacón, G.; Astudillo, A.M.; Ruipérez, V.; Balboa, M.A.; Balsinde, J. Signaling role for lysophosphatidylcholine acyltransferase 3 in receptor-regulated arachidonic acid reacylation reactions in human monocytes. J. Immunol. 2010, 184, 1071–1078. [CrossRef][PubMed] 78. Ramanadham, S.; Ali, T.; Ashley, J.W.; Bone, R.N.; Hancock, W.D.; Lei, X. Calcium-independent

phospholipases A2 and their roles in biological processes and diseases. J. Lipid Res. 2015, 56, 1643–1668. [CrossRef][PubMed] 79. Jenkins, C.M.; Mancuso, D.J.; Yan, W.; Sims, H.F.; Gibson, B.; Gross, R.W. Identiﬁcation, cloning, expression

and puriﬁcation of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J. Biol. Chem. 2004, 279, 48968–48975. [CrossRef] [PubMed]

80. Balsinde, J.; Pérez, R.; Balboa, M.A. Calcium-independent phospholipase A2 and apoptosis. Biochim. Biophys. Acta 2006, 1761, 1344–1350. [CrossRef][PubMed] 81. Fuentes, L.; Pérez, R.; Nieto, M.L.; Balsinde, J.; Balboa, M.A. Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase

A2. J. Biol. Chem. 2003, 278, 44683–44690. [CrossRef][PubMed] 82. Brown, H.A.; Murphy, R.C. Working towards an exegesis for lipids in biology. Nat. Chem. Biol. 2009, 5, 602–606. [CrossRef] [PubMed] 83. Dean, J.M.; Lodhi, I.J. Structural and functional roles of ether lipids. Protein Cell 2018, 9, 196–206. [CrossRef] [PubMed] 84. Koivuniemi, A. The biophysical properties of plasmalogens originating from their unique molecular architecture. FEBS Lett. 2017, 591, 2700–2713. [CrossRef][PubMed] 85. Honsho, M.; Fujiki, Y.Plasmalogen homeostasis. Regulation of plasmalogen biosynthesis and its physiological consequence in mammals. FEBS Lett. 2017, 591, 2720–2729. [CrossRef][PubMed] 86. Yamashita, A.; Kamata, R.; Kawagishi, N.; Nakanishi, H.; Suzuki, H.; Sugiura, T.; Waku, K. Roles of C-terminal

processing, and involvement in transacylation reaction of human group IVC phospholipase A2 (cPLA2γ). J. Biochem. 2005, 137, 557–567. [CrossRef][PubMed] 87. Yamashita, A.; Tanaka, K.; Kamata, R.; Kumazawa, T.; Suzuki, N.; Koga, H.; Waku, K.; Sugiura, T. Subcellular

localization and lysophospholipase/transacylation activities of human group IVC phospholipase A2 (cPLA2γ). Biochim. Biophys. Acta 2009, 1791, 1011–1022. [CrossRef][PubMed] 88. Stewart, A.; Ghosh, M.; Spencer, D.M.; Leslie, C.C. Enzymatic properties of human cytosolic phospholipase

A2γ. J. Biol. Chem. 2002, 277, 29526–29536. [CrossRef] 89. Breton, M.; Colard, O. Protein kinase C promotes arachidonate mobilization through enhancement of CoA-independent transacylase activity in platelets. Biochem. J. 1991, 280, 93–98. [CrossRef][PubMed] 90. Winkler, J.D.; Sung, C.M.; Huang, L.; Chilton, F.H. CoA-independent transacylase activity is increased in human neutrophils after treatment with tumor necrosis factor-α. Biochim. Biophys. Acta 1994, 1215, 133–140. [CrossRef] 91. Fonteh, A.N.; Chilton, F.H. Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation. J. Immunol. 1992, 148, 1784–1791. [PubMed] 92. Baker, P.R.; Owen, J.S.; Nixon, A.B.; Thomas, L.; Wooten, R.; Daniel, L.W.; O’Flaherty,J.T.; Wykle, R.L. Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases. Biochim. Biophys. Acta 2002, 1592, 175–184. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).