Minor compounds from virgin olive oil attenuate LPS-induced inflammation via visfatin-related gene modulation on primary human monocytes

J Food Biochem. 2019;43:e12941. wileyonlinelibrary.com/journal/jfbc  | 1 of 10 https://doi.org/10.1111/jfbc.12941 © 2019 Wiley Periodicals, Inc. Received:20February2019 | Revised:7May2019 | Accepted:13May2019 DOI: 10.1111/jfbc.12941 FULL ARTICLE Minor compounds from virgin olive oil attenuate LPS‐induced inflammation via visfatin‐related gene modulation on primary human monocytes Maria E. Martin1 | Maria C. Millan‐Linares2,3 | Maria C. Naranjo3 | Rocío Toscano3,4 | Rocio Abia3 | Francisco J. G. Muriana3 | Beatriz Bermudez1 | Sergio Montserrat‐de la Paz4 Abbreviations: AMPK,AMP‐activatedproteinkinase;CCR2,Chemokinereceptortype2;COX, Cyclooxygenase; GAPDH,Glyceraldehyde‐3‐phosphatedehydrogenase;HTyr, Hydroxytyrosol; LPS, Lipopolysaccharide; MCP‐1,Monocytechemoattractantprotein‐1;MUFA,Monounsaturatedfattyacid;NAD, Nicotinamide adenine dinucleotide; NAMPT, Nicotinamide phosphoribosyltransferase; NF‐κB,Nuclearfactor‐kappaB;NMNAT, Nicotinamide mononucleotide adenyltransferase; NOS, Nitric oxide synthase; PPARγ,Peroxisome proliferator‐activatedreceptorgamma;SIRT, Sirtuin; SQ, Squalene; TNF‐α, Tumor necrosis factor α; UF, Unsaponifiable fraction. 1FacultyofBiology,DepartmentofCell Biology,UniversidaddeSevilla,Seville,Spain 2CellBiologyUnit,InstitutodelaGrasa, CSIC, Seville, Spain 3Instituto de la Grasa, CSIC, Seville, Spain 4DepartmentofMedicalBiochemistry, MolecularBiologyandImmunology,School ofMedicine,UniversidaddeSevilla,Seville, Spain Correspondence SergioMontserrat‐delaPaz,Departmentof MedicalBiochemistry,MolecularBiology, andImmunology,SchoolofMedicine, UniversidaddeSevilla,Av.Dr.Fedriani3, 41071Seville,Spain. Email:[email protected] Abstract Wehaveanalyzedtheeffectsofminorcompoundsfoundintheunsaponifiablefraction(UF)andinthephenolicfraction(PF)ofvirginoliveoil(VOO)onLPS‐induced inflammatory response via visfatin modulation in human monocytes. For this purpose, monocyteswereincubatedwith UF and PFatdifferentconcentrationsand the pro‐inflammatory stimulus LPS for 24 hr; squalene (SQ) and hydroxytyrosol (HTyr),themaincomponentsinUFandPF,respectively,werealsoused.Therelative expression of both pro‐inflammatory and anti‐inflammatory genes, as well as othergenesrelatedtotheNAD+‐biosyntheticpathwaywasevaluatedbyRT‐qPCR; andthesecretionofsomeofthesemarkerswasassessedbyELISAprocedures.We found that UF,SQ,PF,andHTyrpreventedfrom LPS‐induceddysfunctional gene expressionandsecretionviavisfatin‐relatedgenemodulationinhumanmonocytes. ThesefindingsunveilapotentialbeneficialroleforminorcompoundsofVOOinthe preventionofinflammatory‐disorders. Practical application Inthisproject,potentialhealthbenefitsofVOOmicronutrients(unsaponifiableand phenoliccompounds)wereconfirmedthroughanti‐inflammatoryassays.Ourresults reveal new interesting researching goals concerning nutrition by considering the role ofbioactiveVOOcompoundsinthepreventionandprogressofdiseasesrelatedto inflammation. KEYWORDS NAMPT,oliveoil,phenols,unsaponifiable,visfatin 2 of 10 | MARTIN eT Al. 1 | INTRODUCTION Traditional Mediterranean includes the intake of virgin olive oil (VOO).Moreover,VOOisresponsibleoftheremarkablehealthy benefitsofthisdiet(Montserrat‐delaPaz,Bermudez,etal.,2016). Regarding its composition, virgin olive oil contains large amounts ofmonounsaturatedfattyacids(MUFAs)aswellasseveralminor phenolics and other bioactive compounds included in the unsaponifiablefraction(UF)(Lopezetal.,2014).Ithasbeenpreviously shown that both the phenolic fraction (PF) and UF from VOO modulate gene expression involved in oxidative stress and inflammation(Aparicio‐Sotoetal.,2018;Cardeno,Sanchez‐Hidalgo,& Alarcon‐delaLastra,2013;Cardeno,Sanchez‐Hidalgo,Aparicio‐ Soto,&Alarcon‐delaLastra,2014).Additionally,itisknownthat PFandUFfromVOOdecreasetheexpressionofbothcyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) involving nuclear factor kappa B (NF‐κB)‐mitogen‐activated protein kinase(MAPK)signalingpathwayinlipopolysaccharide(LPS)‐ac‐ tivated murine macrophages (Cardeno et al., 2014; Rosignoli, Fuccelli, Fabiani, Servili, & Morozzi, 2013; Scoditti et al., 2014). It has also been established the anti‐atherogenic properties of PFandUFfromVOOnotonlyinvivobutalsoinvitroconditions (Meza‐Mirandaetal.,2016;Montserrat‐delaPaz,Naranjo,etal., 2016).PFandUFfromVOOactthroughdifferentinflammatory pathwaysmodulatingimmunesystemcells.VOOdietprevented theactivationoftheJanuskinase/signaltransducersandactivatorsoftranscription(JAK/STAT),MAPKssignalingpathwaysand upregulated nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and haem oxygenase (HO‐1) protein expressions (Aparicio‐Soto, Sanchez‐Hidalgo,Rosillo,Castejon,&Alarcon‐delaLastra,2016). It is known that visfatin or nicotinamide phosphoribosyltransferase (NAMPT) is secreted by activated leukocytes as a pre‐B‐ cellcolony‐enhancing factor inbonemarrowstroma(Samalet al., 1994). In the rescue route of nicotinamide adenine dinucleotide (NAD+)generation,intracellularNAMPT(iNAMPT) appears to be the rate‐limiting enzyme (Montserrat‐de la Paz, Naranjo, et al., 2017). Previous studies have proved the significance of NAMPT‐medi‐ atedNAD+recycling,incooperationwithNAD+‐dependentprotein deacetylases (sirtuins), directly on the expression of peroxisome proliferator‐activated receptors (PPARs), NF‐κB, and endothelial NOS (eNOS); many data also provide evidence of an indirect role in metabolism, aging, inflammation, cell proliferation, and differentiation (Bermudez et al., 2017). Cells also produce NAMPT to create anextracellularsupply(eNAMPT)withcytokine‐likeactivity(Dahl, Holm,Aukrust,&Halvorsen,2012).Ourpreviousdatarevealedthat within atherosclerotic carotid plaques, the expression of NAMPT is increasedandeNAMPTcausesinflammatoryandmatrixdegrading responses (Dahl et al., 2012, 2007). Considering useful effects of VOOsecondarycompounds,thisstudyfocussesontheirpotential tomodulateLPS‐mediatedactivationofprimaryhumanmonocytes via visfatin/NAMPT‐relatedgenemodulation,whichisrelatedtoseveral diseases, such as atherosclerosis. 2 | MATERIAL AND METHODS 2.1 | Preparation of UF and squalene (SQ) from VOO For UF extraction, we used VOO of Picual variety. According to IUPAC2401,usingstandardmethods,UFwasisolated(Cardenoet al.,2014;Paquot,1992).Briefly,atemperatureof80°Csaponified 5gofVOOrefluxingwith avolumeof50 mlofa2N solutionof potassiumhydroxidepreparedinethanol(Panreac,Barcelona,Spain), with gently boiling and 20 min after the suspension turned clear. The addition of 50 ml of distillate water through the top of the condenser stopped heating and then the mixture started rotating movement. The solution was washed many times with water when cooled to 30–35°C,andUFwasfinallyextractedbydistillationusingarotary evaporator(B‐480model,BüchiLabortechnik,EssenGermany)and the solvent diethyl ether (Panreac) for evaporation at 30°C under vacuum conditions. In oven, the fraction extracted was dried for 15 min at 103 ± 2°C. Sample production was between 1.3% and 1.4%.The quantitative evaluation of the aliphatic alcohols, sterols, and triterpenic dialcohols contained in the UF was determined in accordancewith European Regulation EEC/2568/91 for olive oil.As described previously, the unsaponifiable matter was extracted and thenfractionedusingTLC(thinlayerchromatography)witha65:35 mixtureofhexaneandethyletheronasilicagelplate(Kieselgel60 F254,Merck,Germany).Componentssuchastriterpenicdialcohols, aliphaticalcohols,andsterols,werecharacterizedbytheirretention factor. Then the triterpenic dialcohols recovered from the plate were converted from erythrodiol and uvaol to trimethylsilyl ethers; first of all adding a volume of 200 μlofa9:3:1v/v/vmixtureofpyridinehexamethyldisilazane‐trimethylchlorosilane and then analyzing by GC(gaschromatography).TheHP5890seriesIIgaschromatograph includeda30m,0.32mmi.d.,TracsilTRB‐5(95%ofdimethylpolysiloxane,5%ofdiphenyl,filmthickness0.25μm) capillary column and aflameionizationdetector(Teknokroma,Barcelona,Spain).Settings for the gaschromatographwerebothinjectorand detector300°C and isothermal column 275°C; split ratio 1:50 and hydrogen flow rate1.0ml/minand130Kpa.Foralcoholchromatographicmeasurement, the protocol was similar to that for sterols and triterpene diols, buttheoventemperaturewasasfollows:215°Cduring5min,then 3°C/minraisingto290°Candkept2minmore.Therelativeretentiontimeletusidentifythepeakareastoquantify.Fortocopherols, weusedanR‐tocopherolstandardcurvetoperformquantification comparingpeakareasandidentificationbyHPLC(high‐performance liquid chromatography) (Montserrat‐de la Paz, Fernandez‐Arche, Angel‐Martin,&Garcia‐Gimenez,2014).AnHPLCsystem(Hewlett‐ Packard,Minnesota,US1050)wasusedtoexaminethesolution10% weightin hexane (Panreac)test sample.The equipmentconditions were: 290 nm excitation and 330 nm emission wavelengths with a fluorescence detector (Shimadzu RF‐535), as well as an analytical column silica (250 mm × 4 mm i.d. × 5 μm)(Merck,SuperspherSi60) at30°Cand1ml/min,400barflowrate.Table1includesresultsas mg/100 mg UF calculating total concentrations as the sum of individual concentrations. | 3 of 10 MARTIN eT Al. Forsqualene(SQ)quantification,weusedanexternalstandard curvetocomparethepeakareasandcarriedoutaGCidentification.Briefly,1mlofinternalSQstandard(Sigma‐Aldrich,StLouis, MO,USA)(5mg/ml)wasmixedwith0.5gofVOO.Thenbothavolume of 400 ml of 2 N of potassium hydroxide in methanol and 2 ml of heptane were added and the whole mixture was agitated vigorously.Afterbecomingclear,theuppersolution,1μl was injected intoanHP5890(Hewlett‐Packard)GCtoperformSQquantification. The equipment included a split injection system (split ratio 1:50) and a flame ionization detector. Components were separatedonafusedsilicacapillarycolumnSPB‐5(30m×0.25mm i.d.) coated with 5% of phenyl‐95% methylsilicone, 0.25 μm of thickness (Supelco). The equipment conditions were: 1 ml/min hydrogenflowrate,130Kpa;280°Cinjectortemperature,300°C detectortemperature;oventemperatureswere240°Cfor6min initiallyandthenrisingat5°C/minto280°C.Resultsareexpressed as mg/100 mg UF and total concentration is calculated as the sum of individual concentrations. 2.2 | Preparation of PF and hydroxytyrosol (HTyr) from VOO PF was obtained using the method described byVazquez‐Roncero, JanerdelValle,andJanerdelValle(1973).Anamountof50gofVOO wasextractedwith125mlofmethanol:water(80:20,v/v).Firstthe resultant was mixed using a vortex during 1 min at a speed of 5,000 g followed by sonication during 15 min. Methanolic phase was concentrated after decantation in vacuum conditions under a stream of nitrogenatatemperaturelowerthan35°Cuntilitreachedasyrupy consistency.PFwasthenlyophilizedandstoredat–80°C.BothquantitativeandqualitativeanalysisofPFwerecarriedoutaccordingtothe COI/T20/29doc. This protocol is based on direct extraction and quantification of the phenolic minor polar compounds using a methanol solutionandHPLC.Onceextracted,analiquotofthesupernatantphase wastaken,filtered,andinjectedintoaHPLCsystemwhichincluded C18reverse‐phasecolumn(25cm×4.6mm),typeSpherisorbODS‐2 (5 mm) and a spectrophotometric UV detector at 280 nm. Phenols content was quantified after measurement of areas of the related chromatographicpeaksandexpressedinµMofphenolsin50µgPF/ ml. Table 2 includes detailed data concerning the composition of the isolatedPF.Toinvitrotreatments,bothHTyrandSQwerepurchased bySigma‐Aldrich(Madrid,Spain). 2.3 | Blood collection and isolation of human monocytes To perform this study, we proceeded according to Good Clinical Practice Guidelines and in line with the principles outlined in the HelsinkiDeclarationoftheWorldMedicalAssociation.Healthymale blooddonorsaged<35declaredtobenon‐smokersandnottaking anymedication.Participantsagreedtogiveinformedconsentatthe UniversityHospitalVirgendelRocio,Seville.Fromalargeantecubitalvein,peripheralbloodsamplesweretakenandthencollectedinto K3EDTA‐containingtubes(BectonDickinson,NJ,USA).Centrifugation over a Ficoll‐Histopaque (Sigma) gradient isolated peripheral blood mononuclearcells(PBMCs)fromperipheralbloodsamples.Thenusing CD14microbeadsandLScolumnsonamidiMACSsystem(Miltenyi Biotec, Madrid, Spain) according to the manufacturer's instructions,monocyteswerefinallyisolatedfromPBMCs.Flowcytometry (FACScanto II flow cytometer and FACSDiva software, BD) showed apurityoftheCD14monocytesisolations>90%routinely.Isolated monocyteswerefinallyresuspendedinRPMI1640cell‐culturemediumsupplementedwith10%heat‐inactivatedfetalbovineserum,L‐ glutamine, penicillin, and streptomycin. 2.4 | Cell treatments Monocytes(5×105cells/ml)wereincubatedwith LPS (100 ng/ml) followedornotof24hrtreatmentin24‐wellplateswithUFat25and 50 μg/ml,SQat50μM,PFat25and50μg/ml, or HTyr at 41 μMfrom TABLE 1 MaincompositionofUFfromEVOO Component mg/kg (ppm) Squalene 5,681 Total sterols 1,289 Campesterol 40.7 β‐sitosterol 7.8 Δ5‐Avenasterol 69.7 Total methylsterols 69.8 Total triterpenic alcohols 568 Total aliphatic alcohols 163 C24 40.8 C26 71.9 α‐tocopherol 87 TABLE 2 CompositionofPFfromEVOOusingCOI/T20/29doc Component µM Phenol (50 µM PF/ml) Hydroxytyrosol 41.07 Tyrosol 43.09 Vanillicacid 5.09 p‐Coumaricacid 3.42 Decarboxymethyl oleuropein aglycone (dialdehyde) 10.27 Tyrosyl acetate 4.97 Decarboxymethyl ligstroside aglycone (dialdehyde) 11.33 Pinoresinol 6.21 Cinnamic acid 6.89 Acetoxy‐pinoresinol 6.22 Oleuropein aglycone, aldehyde form 39.18 Ligstroside aglycone, dialdehyde form 26.20 Luteolin 4.18 Apigenin 0.88 4 of 10 | MARTIN eT Al. VOO.SQandHTyrconcentrationandHTyrconcentrationinhuman monocytes was selected based on previous studies and these concentrationareequivalenttotheconcentrationofSQinUFat50µg/ml andofHTyrinPFat50µg/ml.CellsincubatedonlywithLPSwere considered as positive control and untreated cells were considered as negative control. Supernatants were removed after the treatments, andthenstoredat−20°C.CellswereusedtoRNAextraction. 2.5 | RNA isolation and quantitative real‐time PCR analysis Trisure Reagent (Bioline) was used to extract total RNA. In a NanoDropND‐1000Spectrophotometer(Thermo Scientific),RNA qualitywasevaluatedbyA260/A280 ratio. Reverse transcription was performed using 1 μg RNA (iScript, BioRad). About 20 ng of the cDNAobtainedwasusedastemplateforreal‐timepolymerasechain reactionamplifications.Amplificationofeachspecificgeneproduct wasperformedusingaCFX96system(BioRad).EveryPCRreaction containedbrilliantSYBRgreenQPCRSupermix(BioRad),theprimer pairs for the corresponding gene and cDNA template. As housekeepinggenesglyceraldehyde3‐phosphatedehydrogenase(GAPDH) was used. Table 3 shows both the sequence and the information for the primers. Every reaction was done in triplicate. To estimate the relativemRNAexpressionofanalyzedgenes,theaveragethreshold cycle (Ct) values of the triplicates were used. With the standard 2‐ (ΔΔCt)method,themagnitudeofchangeofmRNAexpressionforcandidategeneswasassessed.ResultswerenormalizedusingGAPDH expression and showed as percentage of control samples. 2.6 | Measurement of cytokine release Todeterminelevelsofvisfatin,IL‐1β,IL‐6,TNF‐α,andIL‐10inculturesupernatantstheenzyme‐linkedimmunosorbentassay(ELISA)wasused, accordingtotheinstructionsofthemanufacturer(Diaclone,Besancon, France).Thelevelsofvisfatin,IL‐1β,IL‐6,TNF‐α,andIL‐10inculture supernatantsweremeasuredbyELISA,followingtheindicationsofthe manufacturer (Diaclone, Besancon, France). Using calibration curves from serial dilution of human recombinant standards in each assay, the concentrationsofcytokinewereestimatedandexpressedinpg/ml. 2.7 | Statistical analysis Data were expressed as arithmetic mean ± SD (standard deviations). StatisticalanalysesweredoneusingGraphPadPrismVersion5.01 Target No. GenBank Direction Sequence (5ʹ ‐‐> 3ʹ) Visfatin/NAMPT NM_005746.2 Forward GCCAGCAGGGAATTTTGTTA Reverse TGATGTGCTGCTTCCAGTTC NMNAT‐1 NR_022787.3 Forward TCTCCTTGCTTGTGGTTCATTC Reverse TGACAACTGTGTACCTTCCTGT NMNAT‐2 NM_015039.3 Forward GTAAAACGACGGCCAGT Reverse TAATACGACTCACTATAGG NMNAT‐3 NM_001200047.2 Forward GTAAAACGACGGCCAGT Reverse TAATACGACTCACTATAGG SIRT‐1 NM_012238.4 Forward CAGACCCTCAAGCCATGTTT Reverse TAATACGACTCACTATAGG PPARγNM_001330615.1 Forward CTGAATGTGAAGCCCATTGAA Reverse GATCCTTTGGATTCCTGAAA MCP‐1 NM_011333.3 Forward ACTGAAGCTCGTACTCTC Reverse CTTGGGTTGTGGAGTGAG CCR2 NM_001123041.2 Forward AGAGGCATAGGGCAGTGAGA Reverse GCAGTGAGTCATCCCAAGAG TNF‐αNM_000594 Forward TCCTTCAGACACCCTCAACC Reverse AGGCCCCAGTTTGAATTCTT IL‐1βNM_000576 Forward GGGCCTCAAGGAAAAGAATC Reverse TTCTGCTTGAGAGGTGCTGA IL‐6 NM_000600 Forward TACCCCCAGGAGAAGATTCC Reverse TTTTCTGCCAGTGCCTCTTT IL‐10 NM_001289746 Forward ATGCACAGCTCAGCACTGC Reverse GTTTCGTATCTTCATTGTCATGTAGG GAPDH NM_002982.3 Forward CACATGGCCTCCAAGGAGTAAG Reverse CCAGCAGTGAGGGTCTCTCT TABLE 3 SequencesofRT‐qPCR primers for gene expression analysis | 5 of 10 MARTIN eT Al. software(SanDiego,CA,USA).Significanceofparametervariations wasevaluatedbyone‐wayanalysisofvariance(ANOVA),following Tukey multiple comparisons test as post hoc test. Those P values fewer 0.05 were determined statistically significant. 3 | RESULTS 3.1 | Effects of minor compounds from VOO on visfatin/eNAMPT secretion in LPS‐treated human monocytes TheeffectsofUF,SQ,PF,andHTyrofVOOonsecretionofvisfatin (eNAMPT) in human primary monocytes are showed in Figure 1. LPScausedanincreaseintheconcentrationofvisfatin(eNAMPT) in the medium of human primary monocytes compared to untreated controlcells.Interestingly,thisLPS‐inducedsecretionofvisfatinwas prevented by UF at 50 μg/ml,PFat25and50μg/ml and HTyr at 50 μMbutnotbyUFat25μg/mlorSQat41μM.Inthecaseof PF,theeffectwasdependentonconcentration(50μg/ml > 25 μg/ ml, p < 0.05). These observations suggest that minor compounds in VOO,probablyphenolics,suchasHTyr,mayhaveabeneficialrolein regulating visfatin secretion in activated human monocytes. 3.2 | Effects of minor compounds from VOO on expression of NAMPT‐ and inflammatory‐related genes in LPS‐treated human monocytes GenesinvolvedintheNAD+ biosynthetic salvage pathway, including NAMPT(Figure2A),NMNAT‐1(Figure2B),andNMNAT‐3(Figure2D), weredownregulatedbyLPSinhumanprimarymonocytes;however, NMNAT‐2genewasunaffected(Figure2C).WhenLPS‐treatedcells wereincubatedwithUF,PF,HTyr,andSQnotonlytheeffectofLPS ontheabovegeneswasabrogated(e.g.,NAMPTgenewithUFat 25 μg/ml and 50 μg/mlandSQat50μM)butalsotheirtranscriptional activity was upregulated. This was particularly prominent withPFandHTyr,andconcentration‐dependent(50μg/ml > 25 μg/ ml, p<0.05)withPFonNMNAT‐1 and NMNAT‐2 genes. In addition, the expression of genes encoding NAD+‐dependent deacetylase SIRT‐1(Figure2E)anditsdownstreamtargetPPARγ (Figure 2F) was repressedbyLPSbutupregulatedinthepresenceofVOO‐derived fractionsorcompounds,withnotableeffectsofPFandHTyrand withtheexceptionofSQthatonlyretainedmRNAofPPARγ gene to a level similar to that of untreated control cells. We also observed thatLPS‐inducedupregulationofMCP‐ 1 gene (Figure 2G) (which encodes an important factor for the recruitment of cells within foci of inflammation) and CCR2 gene (Figure 2H) (which encodes the receptor for MCP‐1) was abolished by either UF, PF, HTyr, or SQ, reachingmRNAlevelslowerthanthosefoundintheuntreatedcontrolcellsbyPFat50μg/ml or HTyr at 41 μM.Theseobservations suggestthatminorcompoundsinVOO,probablySQandphenolics suchasHTyr,mayhaveabeneficialroleinregulatingNAD+ biosynthetic pathway and the inflammatory response in activated human monocytes. 3.3 | Effects of minor compounds from VOO on gene expression and of pro‐ and anti‐inflammatory cytokines in LPS‐treated human monocytes TogaininsightintotheeffectsofVOO‐derivedfractionsandcompounds in human primary monocytes upon LPS treatment, we analyzed secretion and gene expression of both pro‐inflamma‐ tory (IL‐1β, IL‐6, and TNF‐α)andanti‐inflammatory(IL‐10)cytokines. UF,PF,HTyr,andSQdiminishedLPS‐inducedreleaseofIL‐1β (Figure3A),IL‐6(Figure3B),andTNF‐α (Figure 3C) as well as expressionofgenesencodingthesecytokines(Figure3E–G).Inthecase ofUFandPF,theireffectswereconcentration‐dependent(50μg/ ml > 25 μg/ml, p<0.05).TheIL‐10anti‐inflammatorycytokinesecretion was increased by UF at 50 μg/ml,PFat25and50μg/ml, and HTyr at 50 μM(Figure3D)whilealltestedVOO‐derivedfractions and compounds increased the transcriptional activity of IL‐10 gene (Figure 3H) above the level of untreated control cells. The effects ofUFandPFonIL‐10secretionandofPFonIL‐10 gene expression were concentration‐dependent (50 μg/ml > 25 μg/ml, p < 0.05). These observations reinforce the notion that minor compounds of VOO,probablySQandphenolicssuchasHTyr,mayhavearolein reducing the inflammatory response in activated human monocytes. 4 | DISCUSSION “Mediterraneandiet”isdescribedasprotectivepreventingcardiovasculardiseases,notonlybyimprovingclassicalriskfactors,but alsobypromotingananti‐inflammatoryeffect,whileotherslike “meat‐based”or“Westernized”dietsareconsideredasinductive ofinflammatoryconditions(Naranjoetal.,2016).Theactivation FIGURE 1 EffectsofUF,SQ,PF,andHTyrfromVOOon visfatin/eNAMPTsecretioninLPS‐treatedhumanmonocytes.Cells wereuntreated(C)ortreatedwithLPS(100ng/ml)intheabsence (LPS)orpresenceofUF(at25and50μg/ml),SQ(at50μM),PF(at 25 and 50 μg/ml), or HTyr (at 41 μM)for24hr.Theconcentration ofvisfatininculturesupernatantswasmeasuredbyELISA.Values shown are the mean ± SD (n=3)andthosemarkedwithdifferent lowercase letters are significantly different (p < 0.05) 6 of 10 | MARTIN eT Al. FIGURE 2 EffectsofUF,SQ,PF,andHTyrfromVOOonexpressionofNAMPT‐andinflammatory‐relatedgenesinLPS‐treatedhuman monocytes.Cellswereuntreated(C)ortreatedwithLPS(100ng/ml)intheabsence(LPS)orpresenceofUF(at25and50μg/ml),SQ (at 50 μM),PF(at25and50μg/ml), or HTyr (at 41 μM)for24hr.TheexpressionofNAMPT(A),NMNAT‐1(B),NMNAT‐2 (C), NMNAT‐3 (D), SIRT‐1 (E), PPARγ (F), MCP‐1 (G), and CCR2(H)geneswasmeasuredbyqPCR.Valuesshownarethemean±SD (n=3)andthosemarkedwith different lowercase letters are significantly different (p < 0.05) | 7 of 10 MARTIN eT Al. FIGURE 3 EffectsofUF,SQ,PF,andHTyrfromVOOonsecretionandgeneexpressionofpro‐andanti‐inflammatorycytokinesinLPS‐ treatedhumanmonocytes.Cellswereuntreated(C)ortreatedwithLPS(100ng/ml)intheabsence(LPS)orpresenceofUF(at25and 50 μg/ml),SQ(at50μM),PF(at25and50μg/ml), or HTyr (at 41 μM)for24hr.TheconcentrationofIL‐1β(A),IL‐6(B),TNF‐α(C),andIL‐10(D) inculturesupernatantswasmeasuredbyELISA,whereastheexpressionofIL‐1β (E), IL‐6 (F), TNF‐α (G), and IL‐10 (H) genes was measured by qPCR.Valuesshownarethemean±SD (n=3)andthosemarkedwithdifferentlowercaselettersaresignificantlydifferent(p < 0.05) 8 of 10 | MARTIN eT Al. ofcirculatingleukocytesisknowntobearemarkableprocessduringinflammation(Montserrat‐delaPaz,Rodriguez,etal., 2017). Amongtheleukocytes,wheninjuryoccurs,theearliestandquickest immune cells to react are the monocytes, and its activation causes the increase and perpetuation of the inflammatory state (Libby,Nahrendorf,&Swirski,2016).Herein,weaimedtoaddress the relevance of UF from VOO in modulating LPS‐mediated inflammatory response in human primary monocytes. Our study shows thatUFand PF, as wellasHTyr fromVOO couldreduce the inflammatory response via downregulation of visfatin. This protein has a potential involvement in inflammation and several other disorders, including myocardial failure, atherosclerosis, metabolic syndrome, malignancies, neurodegeneration, and aging (Montserrat‐delaPaz,Lopez,etal.,2014).Thereisrecentevidence ofLPStargetingvisfatinasabiomarkerofinflammation(Iwasaet al.,2016).However,therole ofvisfatin/NAMPT is controversial. RecentstudieshavebeenfocusedoneNAMPT,whichisthought to act as a pro‐inflammatory cytokine (Laiguillon et al., 2014). However, Lin et al. (2015) and our research group also reported that iNAMPT may have beneficial anti‐inflammatory properties (Bermudez et al., 2017). In the present study, this dual role of eNAMPT (visfatin) and iNAMPThasbeenexploredbyanalyzing theconcentrationofvisfatin/eNAMPTinthemediumofcellsand therelativemRNAlevelsofNAMPT/iNAMPT. Other compounds in the unsaponifiable of olives, such as oleanolic acid and related triterpenoids, have been shown to inhibit visfatin secretion in murine 3T3‐L1 adipocytes (Rodriguez‐Rodriguez, 2015). The effects on visfatin secretion that we observed were dependent on the concentrationofUFandPFfromVOO,suggestingthatthesefractions, probablyduetoHTyr,haveanti‐inflammatorypropertiesthrough regulation of visfatin exit from cells. The results or our study agree withapreviousstudyinwhichUFofVOOexhibitedantioxidant andinhibitoryactivityofNF‐κB‐mediatedsignalinginmurineperitonealmacrophagestreatedwithLPS(Cardenoetal.,2014).Itis noteworthy that a nutritional intervention demonstrated that the ingestionwithVOO(25ml/day,577mgofphenoliccompounds/ kg)for4weeksreducedserumlevelsofvisfatin/eNAMPTin30 patientswithtype‐2diabetes(Santangeloetal.,2016).Thiseffect was accompanied with an improvement in the metabolic control of glucoseandintheprofileofcirculatinginflammatoryadipokines. Previously,Gesingandcols(2017)showthatcirculatinglevelsof eNAMPT and iNAMPT protein expression in PBMC were selectively increased in patients with acute infections and symptomatic chronic inflammatory conditions compared to healthy controls and also compared to patients with asymptomatic chronic inflammatory diseases, allergic manifestations, or obesity. OurstudyisthefirstinshowingaroleforUF,PF,SQ,andHTyr from virgin olive oil in the regulation of genes involved in NAD+ biosynthesis, such as NAMPT (iNAMPT), NMNAT‐1, NMNAT‐2, and NMNAT‐3. NMNAT‐1 has been recently reported to exhibit protection of neuronal function against tau‐induced inflammation and atrophy in a mouse model of Alzheimer´s disease (Musiek et al., 2016).ApossibleroleofNMNAT family members in the biology of innate immune cells and in the inflammatory response mediated bymonocytesremainstobeestablished.ThefactthatLPSdownregulated the gene encoding NAMPT suggests that iNAMPT is not involved in the inflammatory response induced by LPS in human monocytes. These findings could be indicative of contrary activities ofvisfatin/eNAMPTandiNAMPTininflamedtissues(Benito‐Martin et al., 2014). BecauseNAMPTisakeyNAD+biosyntheticenzymeandSIRT1 requiresNAD+foritsenzymaticactivitythatincludestheabolishment of inflammatory mediator production and cell recruitment (Busch, Mobasheri, Shayan, Stahlmann, & Shakibaei, 2012; Dong etal.,2014),wealsoanalyzedtheimpactoftheUF,PF,SQ,andHTyr fromVOOinthegeneregulationofSIRT1 and its downstream target PPARγ(Quetal.,2016).Inarecentstudy,SIRT1 has been described as a critical immune regulator whose expression can be downregulated in human macrophages following the release of inflammatory cytokinesuponcellstimulationwithLPS(Jiaetal.,2016).Incontrast, the deficiency of SIRT1 in human monocytes and mononuclear cells has been associated to systemic and possibly vascular wall inflammation,leadingtoatherogenesis(Kitada,Ogura,&Koya,2016).In human monocytes, we found that all tested fractions or compounds ofVOOreversedthereductionofSIRT1 gene expression caused by LPS.OurresultsmatchwiththosereferredtothepolyphenolresveratrolinTNF‐αtreatedhumanendothelialcells(Liuetal.,2017). Furthermore, we observed that the gene expression profile for PPARγ was similar as to SIRT1;theUF,PF,andHTyrnotonlyrestoring but also potentiating PPARγgeneexpressioninLPS‐treatedhuman monocytes.PPARγ agonists were reported to suppress inflammation byinhibitingNF‐κBsignalingpathwaysinendothelialcells(Marcone, Haughton, Simpson, Belton, & Fitzgerald, 2015). Therefore, our studysuggeststhatcompoundsintheUFofVOOmaycounteract LPS‐inducedpro‐inflammatoryresponsebyrestoringmRNAlevels of SIRT1 and PPARγ genes in human monocytes. Toevaluatemoredeeplyintotheanti‐inflammatoryactionofUF, PF, SQ, and HTyr from VOO in human monocytes, we investigated whether these fractions or compounds played a role in the regulation of MCP‐1 and CCR2. MCP‐1,alsoknownasCCL2, is mainly produced by monocytes/macrophages in response to many stimuli and conditions forexamplecytokines,oxidativestressorgrowthfactors(Deshmane, Kremlev,Amini,&Sawaya,2009).Afterbindingthischemokinetothe receptor CCR2, both migration and infiltration of many cell systems including monocytes, natural killer cells, T lymphocytes, and memorycells,arefacilitated(Vakilian,Khorramdelazad,Heidari,Rezaei,& Hassanshahi,2017).Wefoundthatalltestedfractionsorcompounds ofVOOavoidedLPS‐inducedup‐regulationofMCP‐1 and CCR2 genes in human monocytes. Sanchez‐Fidalgo, Cardeno, Sanchez‐Hidalgo, Aparicio‐Soto,anddelaLastra(2013)andSanchez‐Fidalgo,Cardeno, Sanchez‐Hidalgo,Aparicio‐Soto,Villegas,etal.(2013)demonstrated that dietary VOO polyphenols and unsaponifiable supplementation reduced MCP‐1 in a chronic colitis in mice. Other natural polyphenols such as curcumin have been shown to exert similar effects on MCP‐1geneexpressioninseveralcelllines(Karimian,Pirro,Majeed,& Sahebkar,2016).OurfindingswereconsistentwiththenotionthatUF | 9 of 10 MARTIN eT Al. andPF,andnotablyHTyrofVOOmaybeusefulforpreventingrecruitment and chemotaxis of monocytes across the vascular endothelium. These observations were consistent with the decrease in the medium and prevention of LPS‐induced gene expression of pro‐ inflammatorycytokines(IL‐1β, IL‐6, and TNF‐α) in human monocytes. Allthefractions(inadose‐dependentmanner),SQ,andHTyrexerted these beneficial effects. Of note, they also promoted the release and gene expression of the anti‐inflammatory cytokine IL‐10. Our study is complementary to a previous one in which squalene was shown to reducemRNAlevelsofpro‐inflammatorycytokinesinhumanmonocytes and neutrophils (Cardeno et al., 2015). This is in line with evidenceofanti‐inflammatorypropertiesofUFofvirginoliveoil(Amiot, 2014;Cardoso,Catarino,Semiao,&Pereira,2014). 5 | CONCLUSIONS ThisstudyunveilsnewbeneficialeffectsofUF,includingSQ,PF,and HTyr,fromVOOandestablishesthepotentialuseofthesefractions/ compounds in the prevention and treatment of inflammatory disorders.Withinthiscontext,theconsumptionofVOOanditsbioactive components have shown a broad range of promising activities in different inflammatory and autoimmune diseases and it may be a relevant approach in the prevention and management of these disorders. ACKNOWLEDGMENTS AuthorsthankCellBiologyUnitofInstitutodelaGrasaforitsassistance duringthefulfilmentofthiswork.B.B.andS.M.acknowledgefinancial supportfrom“VOwnResearchPlan”(UniversityofSeville). CONFLICTS OF INTEREST The authors state no conflict of interest. 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