Biological effects of double-walled carbon nanotubes on the innate immune system: An in vitro study on THP-1 human monocytes

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Biological effects of double-walled carbon nanotubes on

the innate immune system: An in vitro study on THP-1

human monocytes

Samir Dekali, Christine Bachelet, Séverine Maunoir-Regimbal, Emmanuel

Flahaut, Jean-Claude Debouzy, David Crouzier

To cite this version:

Samir Dekali, Christine Bachelet, Séverine Maunoir-Regimbal, Emmanuel Flahaut, Jean-Claude

De-bouzy, et al.. Biological effects of double-walled carbon nanotubes on the innate immune system:

An in vitro study on THP-1 human monocytes. Toxicology, Elsevier, 2016, vol. 365, pp. 1-8.

�10.1016/j.tox.2016.07.019�. �hal-01486943�

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To cite this version :

Dekali, Samir and Bachelet, Christine and

Maunoir-Regimbal, Séverine and Flahaut, Emmanuel and Debouzy,

Jean-Claude and Crouzier, David Biological effects of double-walled

carbon nanotubes on the innate immune system: An in vitro study on

THP-1 human monocytes. (2016) Toxicology, vol. 365. pp. 1-8.

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Biological

effects

of

double-walled

carbon

nanotubes

on

the

innate

immune

system:

An

in

vitro

study

on

THP-1

human

monocytes

Samir

Dekali

a,

_*

_,

_Christine

_Bachelet

a

_,

_Séverine

_{Maunoir-Regimbal}

a

_,

_Emmanuel

_Flahaut

b

_,

Jean-Claude

Debouzy

a

,

David

Crouzier

a

a_{InstitutdeRechercheBiomédicaledesArmées(IRBA),DépartementEBR/UnitédesRisquesTechnologiquesEmergents,1,placeduGénéralValérieAndré,} BP73,91223Brétigny-sur-OrgeCedex,France

b_{CIRIMAT,UniversitédeToulouse,CNRS,INPT,UPS,UMRCNRS-UPS-INPNo.5085,UniversitéToulouse3PaulSabatier,Bât.CIRIMAT,118,routedeNarbonne,} 31062ToulouseCedex9,France

Keywords: THP-1monocytes

Double-walledcarbonnanotubes Inflammation

Innateimmunity LPS

ABSTRACT

DWCNTshavenumerousindustrialandbiomedicalapplicationsandseveralstudiesreportedthatthey couldactasimmunomodulatorsystems.Theimmunesystemisthefirstlineofdefenceofthehuman bodywhenexposedtoparticulatematter.InordertoinvestigateDWCNTs’roleoninnateimmunity,we usedTHP-1monocyticcellsforthepurposeofthisstudy.

WeshowedthatDWCNTswerenotcytotoxicuntil6h,24h,48hand72hofincubationwithTHP-1 monocyticcells(concentrationstestedfrom10to50mg/mL).From6hto72hofincubationofTHP-1cells with DWCNTs,we measured a significantincreaseof the baseline cell index usingxCELLigence1

technologyshowingcelladhesion.After24hofexposure,DWCNTsagglomerateswerelocalizedinTHP-1 monocytecytoplasmandcelladhesionwasobservedsimultaneouslywithasignificantincreaseinthe expressionofCD11bandCD14cellsurfaceproteins.Pro-inflammatorycytokinesecretion(IL-1b,IL-6, IL-8,TNF-aandIL-10)wasalsomeasuredinsupernatantsafter6hor24hofexposuretoDWCNTs.This pro-inflammatoryresponsewasincreasedinTHP-1monocyticcellspre-treatedwithLPS.

Altogether,ourdataindicatethatDWCNTsinduceanincreasedpro-inflammatoryresponseofTHP-1 monocytesandseemtomodulatecellsurfaceproteinexpressionconfirmingthatDWCNTscouldactas stimulatorsofinnateimmunity.

1.Introduction

Nano-objectscanbedefinedashavingatleastonedimension lessthan 100nanometers (nm)in length.Amongnano-objects, nanotubesaredefinedashavingatleasttwodimensionsinthe nanoscale(lessthan100nm)(ISO,2008).Carbonnanotubes(CNTs) arecylindricalcarbonmolecules,whichcanbedividedintotwo majorgroups:single-walledcarbonnanotubes(SWCNTs),which aremadeupofasinglegraphitesheet,wrappedintoacylindrical tube,and multi-walledcarbon nanotubes(MWCNTs)which are multiple SWCNTs, concentrically nested. Due to their unique physico-chemical properties such as electrical conductivity, mechanical strength and thermal conductivity, MWCNTs could be or are being used in various industrial sectors such as

electronics,aerospaceornanomedicine(Baughmanetal.,2002; Mundraetal.,2014).

However,theirintrinsicphysico-chemicalpropertiesalsoraise the question of their effects on human health. Interestingly, a recent study showed that anthropogenic carbon nanotubesare foundintheairwaysofasthmaticParisianchildren,indicatinga dailyenvironmental exposure tothesenano-objects ( Kolosnjaj-Tabietal.,2015).TheInternationalAgencyforResearchonCancer (IARC) recently classified one type of MWCNTs (MWCNT-7) as “possiblycarcinogenictohumans”(Group2B);andSWCNTsand MWCNTs(excludingMWCNT-7)werecategorisedasnot classifi-ableastotheircarcinogenicitytohumans(Group3)(Grosseetal., 2014).SeveralstudiesreportpotentiallytoxiceffectsofSWCNTsor MWCNTstohumanhealthbutthesebiologicaleffectsaremerely beingdiscussedandstillnotverywellunderstood(Ongetal.,2014; Liuetal.,2013;Miozzietal.,2016).Theimmunesystemisthefirst lineofdefenceof thehumanbodywhenexposedtoparticulate matterlikeCNTs.Severalstudieshavepointedouttheimplications oftheimmuneresponseafterexposuretoMWCNTs.Arecentstudy

* Correspondingauthor.

E-mailaddress:samir.dekali@gmail.com(S.Dekali).

showedthatICR(TheInstituteforCancerResearch)miceexposed to MWCNT-7 (single intraperitoneal administration) exhibit sustained stimulating effects on immune and inflammatory responses (Yamaguchi et al.,2012).Otherauthorsshowed that MWCNTsintratrachealinstillationmightinduceallergicresponses inmicethroughBcellactivationbyTh2-typecytokines(IL-4,IL-5 and IL-10), and IgE production (Park et al., 2009). Moreover, Lavernyandcolleaguesshowed,usingperipheralblood mononu-clear cells (PBMCs), that exposure toMWCNTs canpromote or suppressimmuneresponsedependingonthecell’sorigin:from healthysubjectsandfrompatientswithallergyagainsthousedust mites, respectively (Laverny et al., 2013). Interestingly, it has recentlybeenshownthatMWCNTexposureincreasesadhesionof THP-1monocytestoendothelialcellsinaninvitromodelofthe alveolo-capillary barrier (constituted of A549 epithelial and HUVECs endothelial cell lines) simultaneously with oxidative stress,indicatinganincreasedexpressionofTHP-1monocyticcell surfaceadhesionproteins(Caoetal.,2015).Ithasbeenshownthat monocytesinresponsetoenvironmentalstimulicandifferentiate intoinflammatoryoranti-inflammatorysubsets(Yangetal.,2014). Pondman etal. showed that complement opsonisation ofCNTs significantly increase their internalization with concomitant down-regulationofpro-inflammatorycytokinesandup-regulation of anti-inflammatory cytokines in both U937 cells and human monocytes(Pondmanetal.,2014).It wasnotablyreportedthat CNTsinjectedintomiceinducedinflammationandactivation of innate cells such as monocytes/macrophages, resulting in pro-inflammatorycytokinesecretion,whichinturnstimulatesThcells (Grecco et al., 2011; Yamaguchiet al., 2012). Hamiltonand his colleaguesinvestigatedhowMWCNTs’physico-chemical charac-teristics mayinfluencebioactivityonTHP-1 macrophages.They foundthatincreasedMWCNTlengthordiameterraised inflam-masomeactivationandpro-inflammatoryresponseinthesecells, similarly to alveolar macrophages isolated from C57BL/6 mice (Hamiltonetal.,2013).

In the present study, we tested CNTs (approximately 80% double-walledCNTs,20%SWCNTsandafewtriple-walledCNTs)to explore theeffects onTHP-1 monocytephenotype,CNTcellular internalization and pro-inflammatory response, using flow cytometry,transmissionelectronmicroscopy(TEM)andmultiplex ELISA.Ouraimwas todeterminetheroleofCNTsonmonocyte function.Weshowed,after24hofexposure,thatCNTswerenot cytotoxicbutwereinternalizedbyTHP-1monocyticcells.THP-1 cellsbecameadherentandsignificantincreasesin pro-inflamma-torycytokinesand chemokinesweremeasuredin supernatants (IL-1

b

,IL-6,IL-8,TNF-

a

andIL-10)comparedtocontrols.Moreover, we measured an increasedexpression of CD11b and CD14 cell surfaceproteinscomparedtocontrols.CD11bisthe

a

subunitof the integrin cell surface receptor complement receptor 3 and functionsin a heterodimerwithCD18toallowrecognition and phagocytosisofiC3bopsonizedparticles(Pahletal.,1993).CD11b is selectively expressed on the surface of mature monocytes, macrophages,granulocytesandnaturalkillercells.CD14,onthe otherhand,functionsinconcertwithLPSbindingproteinand TLR-4inthedetectionandbindingoflipopolysaccharide(Metheetal., 2005).

Because DWCNTs areat the interfacebetweenSWCNTs and MWCNTs witha largernumber of walls,we believethat these resultscanbeextrapolatedtoallkindofCNTs.

2.Methods 2.1.Cellculture

THP-1 human monocytic cell line was obtained from the EuropeanCollectionofCellCultures(ECACC,Reference88081201),

and grownin RPMI 1640medium containing GlutamaxTM_(Life Technologies,Reference61870)supplementedwith10%(v/v) heat-inactivatedfetalbovineserum(FBS;Lonza,ReferenceDE14-801) andantibiotics100U/mLpenicillinand100

m

g/mLstreptomycin (LifeTechnologies,Reference15140).Inordertoavoidanycellular stress,cellsweremaintainedinexponentialgrowthbetween2and 9!105_cells/mLina

humidifiedatmosphereat37"_Cand5%CO

2.

2.2.DWCNTssynthesis,preparationandcharacterization

DWCNTswereproducedbycatalyticchemicalvapour decom-position(CCVD)ofCH4overMgC1xCoxOsolidsolutioncontaininga smalladditionofMolybdenum(Flahautetal.,2003).AfterCCVD, thecatalystandsupportwereremovedbytreatmentofthesample with a concentrated aqueous hydrochloricacid solution. High-resolutiontransmissionelectronmicroscopy(TEM)showedthata typicalsampleconsistsof77%DWCNTs, 18%SWCNTsand5% triple-walledCNTs.DiameterdistributionofDWCNTsrangedfrom0.5to 2.5nmforinnertubes(medianinnerdiameter=2.0nm)andfrom 1.2to3.2nmforoutertubes(medianouterdiameter=1.3nm).The lengthofindividualDWCNTsusuallyrangesbetween1and10

m

m, althoughbundlesmaybemuchlonger(upto100

m

m,atleast).Due tothesynthesisandcatalyst-eliminationprocess,thewallsofthe DWCNTsare not expectedtobe functionalized (particularlyby oxygen-containing functional groups). Brunauer Emmet Teller (BET)specificsurfaceareawas985m2_/g.

DWCNTsweresterilizedandthendispersedinPBScontaining 25%(v/v)FBSataconcentrationof5mg/mLbyvortexingfor30s and sonicating for 5min in anultrasonic bath (AdvantageLab, CataloguenumberAl04-02)atroomtemperature.Thisprocedure was repeated_fivetimes. A“sham stocksolution” ofsterilePBS containing25%(v/v)FBSwaspreparedandsonicatedinparallelfor useascontrol.Thesestocksolutionswerekeptat4"_C.Beforecell

exposure,theDWCNTs’stocksolutionwas_firstlyvortexedfor30s andthensonicatedfor5mininanultrasonicbath.Suspensions werethen diluted in the complete cell culturemedium to the requiredconcentration.

DWCNTsweresuspendedataconcentrationof200

m

g/mLin thecompleteculturemediumandthenparticlesizedistribution was measured using laser diffraction granulometry (Horiba, ParticaLA-950V2).

2.3.Trypanblueexclusionassay

THP-1 cells were seededin 12-well plates and immediately incubatedwithDWCNTsatdifferentconcentrations(0,10,25and 50

m

g/mL)inRPMI1640mediumsupplementedwith10%(v/v)FCS for 6h, 24h, 48hor 72hat 37"_{C. Then, cell suspensions were}

dilutedtohalfwith100

m

LofamixcontainingRPMI1640medium supplementedwith10%(v/v)FCS(50%v/v)andTrypanblue(Life Technologies, Catalogue number 15250-061) (50% v/v). After 1min,viableanddeadcellswerecountedunderalightmicroscope (LeitzLaborluxK).

2.4.LIVE/DEAD1_{cytotoxicityassay}

Cell suspensions were exposed to DWCNTs at different concentrations(0,10,25and50

m

g/mL)in24-wellcultureplates for48hor 72h. Cytotoxicitywas thenassessed usingtheLIVE/ DEAD1 _{viability/cytotoxicity kit for mammalian cells (Life} Technologies,ReferenceL-3224)accordingtothemanufacturer’s instructionsand analysedusing aflow cytometer(Becton Dick-insonBiosciences,LSRII).Thisassayallowssimultaneous determi-nationofliveanddeadcellsusingtwoprobes:ontheonehand, non fluorescent cell-permeant calcein AM is cleaved by cyto-plasmic esterasesproducing intensely green _fluorescentcalcein

well retained within live cells, on the other hand, ethidium homodimer(EthD-1)enterscellswithdamagedmembranesand, uponbindingtonucleicacids,producesabrightred_{fluorescence.} EthD-1isexcludedbytheintactplasmamembraneoflivecells. CountBrightTM_{AbsoluteCountingBeads(LifeTechnologies,} Refer-enceC36950) wereused toquantifycellpopulations,and were addedpriortodataacquisitionona BDLSRIITM

flowcytometer withBDFACSDiva1_{software.Fluorescenceemissionofcellswas} analyzedusing488nmexcitationwavelength. Foreach sample, 3!103 _{beads events were collected to ensure statistically} significantdeterminationofsamplevolumes,andmean fluores-cenceof about5.5! 104 cells was determined.A gating witha combinationofparametersseparatedallevents.CountingBeads were brightly fluorescent at the two emission wavelengths (

l

excitation=488nm;

l

emission for calcein=530nm/bandpass 30nm and

l

emission for ethidium homodimer–1=610nm/ 20nm bandpass) while DWCNTs were not. No interference betweenbeadandDWCNTfluorescencewasrecorded(datanot shown).DataanalysiswasrealizedusingFCSExpress1_Software (DeNovoSoftware).

2.5.xCELLigencereal-timecellanalysis(RTCA):celladhesionassay RealtimemonitoringofTHP-1livingcellswasperformedusing thexCELLigence RTCA DP instrument (ACEA Biosciences) which wasplacedinahumidifiedincubatorat37"_Cand5%CO

2.

Cell adhesion experiments were carried out using 16-well plates (ACEA Biosciences, Reference 00300600880). Initially, 100

m

L of culture medium was added to each well and a backgroundmeasurementstepwas performedasa background signalgeneratedbycell-freemedium.Toinitiatetheexperiment, THP-1 cells were counted and seeded at concentrations of 3!104_{viablecells/wellanddirectlyexposedtoDWCNTs(10and} 50

m

g/mL) or 15ng/mL phorbol 12-myristate13 acetate (PMA; Sigma–Aldrich,ReferenceP8139).Controlcellswereonly incubat-edwithcellculturemedium. Eachconditionwas performedin quadruplicatewithaprogrammedsignaldetectionscheduleonce everyminuteduring72hofincubation.

2.6.Surfaceantigenexpression

After48hor72hofincubationofDWCNTswith7.5! 105THP-1 monocytes,cellswerecentrifuged,washedwithPBSandincubated with200

m

L of a mixsolution of dilutedFITC-conjugated anti-human CD11b (BeckmanCoulter, Reference IMO530)and RD1-conjugatedanti-humanMY4(CD14,BeckmanCoulter,Reference 6603262) monoclonal antibodies (1:10 and 1:40 dilutions respectively)in PBS for40minat4"_{C. Cellswerethenwashed}

twicewith2mLof PBS.Cell pelletwas thenresuspended with 0.5mL of PBS containing 1

m

g/mL Hoechst 33258 (Invitrogen, ReferenceH3569)andkeptoniceprotectedfromlightuntilflow cytometryanalysis. Individualcompensation settingswere per-formed using antibody-capture beads (Beckton Dickinson Bio-sciences, CompBeads). Appropriate isotypic controls (Beckman Coulter;IgG1-FITC,ReferenceA07795;andMsIgG2B-RD1, Refer-ence6603038) wereusedtodeterminenon-specificstainingof monoclonal antibodies, based on the _{fluorescence} minus one (FMO) controls. 5! 104 events were recorded. Gating on the Forward Scatter (FSC) area and FSC width signals performed doublets discrimination. Staining with Hoechst 33258 and UV excitation of the dye gated out dead cells. Mean fluorescence intensity(MFI)foreachlabellingwasquantifiedandtherelative fluorescenceintensity(RFI)wascalculatedasfollows:

RFI=MFI/MFIofcorrespondingFMOcontrol

2.7.Inflammatorycytokinereleasedetection

THP-1monocyteswereincubatedwithdifferentconcentrations ofDWCNTs(0,10,25and50

m

g/mL)in24-wellcultureplatesfor 6h or 24h. Pre-treatments with 100ng/mL LPS were also performed to investigate THP-1 pro-inflammatory response of conditionedcells.Aftertheseincubations,cellswerecentrifugedat 2000!g for 10min. Then, supernatants were collected and an additional centrifugation at 2000!g for 10min was realized beforestorageat#80"_{Cuntilcytokinereleaseanalysis.}

Quantifi-cationofIL-1

b

,IL-6,IL-8,IL-10,IL-12,andTNF-

a

wasperformed simultaneouslyinsupernatants(50

m

L)usingtheCytometricBead Arraykit(CBA;BecktonDickinsonBiosciences)accordingtothe manufacturer’sinstructions.SampleswereacquiredonaLSRIITM flow cytometer and concentrations of each cytokine were determined in pg/mL on the basis of standard curves and regression analysis. Limits of detection for each protein using theCBAkitwere:IL-1

b

(7.2pg/mL),IL-6(2.5pg/mL),IL-8(3.6pg/ mL),IL-10(3.3pg/mL),IL-12(1.9pg/mL)andTNF-

a

(3.7pg/mL). 2.8.Electronmicroscopy

Cellular suspensions were fixed in a solution of 4% (v/v) glutaraldehyde in 0.1Mcacodylate bufferat pH7.4. Cells were post-fixedina1%(v/v)osmiumtetroxidesolutionduring2hat roomtemperature.THP-1monocyteswerethenmixedinaliquid lowmeltingpointgelatinesolutionandquicklybroughtbackto roomtemperature.Samplesofsolidgelatinewerecut,dehydrated andembeddedinSpurrresin(Sigma-Aldrich,ReferenceEM0300). Semi-thinsectionsof1

m

mstainedwithToluidinebluewereused for histological analysis (data not shown). Ultrathin sections stainedwithuranylacetateandpoststainedwithleadcitratewere observed on a JEOL 1010 transmission electron microscope. PhotographsweretakenwithaGatanES500camera.

2.9.Statisticalanalysis

Data are representedas mean of three independent experi-ments$standarddeviation(SD).Datawereanalysedbyone-way analysis of variance (ANOVA) followed by Dunnett’s t-test to comparethedifferentgroupstreatedtothecontrol(

a

risk=0.05). Toexaminedifferencesbetweenpairsofgroups,Student’st-tests (

a

risk=0.05)wereused.

3.Results

3.1.CharacterizationofsizedistributionofDWCNTsintherelevant biologicalmedia

To analyze size distribution of DWCNTagglomerates in cell culture media,we usedlaser diffraction. Sizeanalysis revealed micronic agglomerates with sizes comprised between 3.9 and 44.9

m

m(Fig.1).Themeasuredmediandiameteris6.9$1.7

m

m. 3.2.DWCNTsdonotinducecytotoxicityonTHP-1monocyticcells

TomeasurecytotoxicityofDWCNTsonTHP-1monocyticcells avoidinginterferenceswiththeassay,weperformed,ontheone hand,Trypanblueassay.After6h,24h,48hor72hofincubation, DWCNTsdidnotinducesignificantcytotoxicityonTHP-1 mono-cytes(Fig.2).

On the other hand, we used the LIVE/DEAD1 _viability/ cytotoxicityassaykit.Nosignificantdecreaseinthecellviability was observed after48hor72hof incubationwithTHP-1 cells (Fig.S1).

3.3.DWCNTsinducecelladhesion

To determineifDWCNTshaveaneffectoncelladhesion,we usedxCELLigence1_{technology.Realtimemonitoringoflivingcells} using xCELLigence1 _{technology is a useful high throughput} screening methodallowingtomeasurecelladhesion.Themore cellsareattachedtotheelectrodesoftheE-plate,thelargerthe increaseinelectrodeimpedance.

We showed that baseline cell index was immediately, significantly increased after incubation with 15ng/mL PMA

(positive control) indicating that THP-1 monocytes became adherents (Fig. 3). After 6h of exposure to 10 or 50

m

g/mL DWCNTs,baselinecellindexesweresignificantlyincreased.From 20h to 72h, after 50

m

g/mL DWCNT incubation, baseline cell indexesweresignificantlymoreincreasedthan10

m

g/mLDWCNTs. Consequently, increase in baseline cell index seems to be concentration-dependent.

Toensurethatincreasesinbaselinecellindexeswerenotdueto sedimentation of DWCNT agglomerates, we used control wells containingonlyDWCNTs.Nosignificantincreaseinthebaseline cellindexwasmeasured(datanotshown).

3.4.DWCNTsareinternalizedbyTHP-1monocyticcellsandinduce CD11bandCD14expression

InordertodetermineifTHP-1monocytesareabletointernalize DWCNTs, we performed observations by TEM. After 24h of incubation of 10

m

g/mL DWCNTs,THP-1 cells became adherent and we observed nanoparticle agglomerates in the cytoplasm (Fig.4AandB).Theseagglomerateslooklikecarbonencapsulated Conanoparticlesdescribedtobepresentintherawmaterial.This maybeanindirectevidenceofthepresenceofassociatedCNTs. Size of internalizedagglomerates was in the micronrange. No DWCNTswereobservedincellnucleinorinthenucleolus.

BecauseweobservedDWCNTinternalizationandcelladhesion, weinvestigated ifexpressionofdifferentiation surfacemarkers wasinduced.Oncedifferentiated,THP-1cellsexpressCD11band CD14ontheirplasmamembrane(Moeenrezakhanlouetal.,2008). CD11bexpressionwassignificantlyincreasedafter48hand72h DWCNTexposureuptoapproximately36%and60%respectively foreachtestedconcentration,comparedtocorrespondingcontrols (Fig.4C).SignificantamountsofCD14werealsomeasuredafter 48hand72hDWCNTexposureuptoapproximately22%and24% respectively for each tested concentration compared to corre-spondingcontrols(Fig.4D).

3.5.DWCNTsinducepro-inflammatorycytokineandchemokine releasebyTHP-1monocytes

ToexploretheDWCNTs’pro-inflammatoryeffectswemeasured cytokineandchemokinesecretion(IL-1

b

,IL-6,IL-8,TNF-

a

,IL-10 andIL-12)inTHP-1cellsupernatants,after6hor24hofexposure. Todetermineifdifferentialeffectswereobservedonstimulated THP-1monocytes,thesameexperimentswereperformedonLPS pre-treatedcells.

EffectsofDWCNTsonTHP-1cellsafter6hofincubation

Significant amounts of IL-1

b

and IL-8 were detected after incubationwith25and50

m

g/mLDWCNTscomparedtocontrols (Fig.5AandC),whilesignificantreleasesofIL-6,TNF-

a

andIL-10 wereonlymeasuredinresponse to50

m

g/mLDWCNTexposure (Fig.5B,D,E).SignificantsecretionofIL-12wasnotdetectedinany ofthecellsupernatantsinourconditions(Fig.5F).

IncaseofLPSpre-treatedTHP-1cells,significantamountsof IL-1

b

weredetected at25and 50

m

g/mL(Fig.5A),and significant releasesofTNF-

a

andIL-10weremeasuredinresponseto50

m

g/ mLDWCNTincubation(Fig.5DandE)comparedtocontrols.

Statistical comparisons revealed significant differences be-tweencellswithorwithoutLPSpre-treatmentforIL-6(10

m

g/mL;

Fig.3B),IL-8(0,10and25

m

g/mL;Fig.5C)andIL-10(25and50

m

g/ mL;Fig.5E).

EffectsofDWCNTsonTHP-1cellsafter24hofincubation

Significant increase in IL-1

b

secretion was measured after incubationwith25and50

m

g/mLDWCNTscomparedtocontrols (Fig.5A).SignificantsecretionsofIL-6,IL-8,TNF-

a

andIL-10were

Fig.1.SizedistributionofDWCNTsintheculturemedium.Size analysiswas performedusinglaserdiffraction.DWCNTsweresuspendedinRPMI1640cell culturemediumsupplementedwith10%(v/v)FBS.ThecurverepresentsDWCNTs sizedistributionofonerepresentativeexperiment.

Fig.2. CytotoxicityofDWCNTsonTHP-1cells.THP-1cellswereincubatedfor6h, 24h,48hor72htoDWCNTs.CellviabilitywasmeasuredusingTrypanblue.Data representthemean(SD)ofthreeindependentexperiments.

Fig.3. RealtimecellmonitoringofTHP-1monocytesexposedtoDWCNTs.Curves representthebaselinecellindexacrosstime.Controlcells(bluecurve,baseline)are onlyincubatedincellculturemedium.THP-1monocyteswereincubatedwith 15ng/mL phorbol 12-myristate 13-acetate(PMA; red curve),10 or 50mg/mL DWCNTs(orangeandredcurves,respectively).Datarepresentonerepresentative experimentofquadruplicatesduring72h(Forinterpretationofthereferencesto colourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.).

Fig.4. DWCNTsinternalizationanddifferentiationsurfacemarkersexpressionbyTHP-1cells.Afterexposureto10mg/mLDWCNTsfor24h,THP-1cellswereobservedby transmissionelectronmicroscopy(A;scalebar2mm).DWCNTsweredetectedinthecytoplasmofthesemonocytes(B;scalebar1mm).Afterexposureto10mg/mLDWCNTs for48h(CandD,greybars)or72h(CandDblackbars),CD11b(C)andCD14(D)differentiationsurfacemarkerexpressionbyTHP-1cellswasdeterminedbyflowcytometry. Meanfluorescenceintensityforeachlabelingwasquantifiedandexpressedastherelativefluorescenceintensity(RFI)reportedinpercentageofthecorrespondingcontrol. Datarepresentthemean(SD)ofthreeindependentexperiments.One-wayANOVAandDunett’spost-test(comparisonsvs.controlcellsnotexposedtoDWCNTs)were performed(*p<0.1;**p<0.01).

Fig.5.Pro-inflammatorypotentialofDWCNTsonTHP-1cells.Cellswereexposedforhor24hto0,10,25,50mg/mLDWCNTswithoutorwith100ng/mLLPSpre-treatment (hatchedorgreybarsrespectively).IL-1b,IL-6,IL-8,TNF-a,IL-10andIL-12werethenmeasuredbyELISAmultiplexincellsupernatants(A,B,C,D,EandFrespectively).Data representthemean(SD)ofthreeindependentexperiments.One-way ANOVAand Dunett’spost-testwereperformedtocompareeachDWCNTsexposedcellsto correspondingcontrols(*p<0.1;**p<0.01;***p<0.001).One-wayANOVAandTukey’spost-testwerealsoperformedtocompareforeachDWCNTsconcentrationnotLPS pre-treatedcellsvs.pre-treatedcells(#p<0.1;##p<0.01;###p<0.001).

detectedinresponsetothehighestdoseofDWCNTs(50

m

g/mL;

Fig.5B–D).

ForLPSpre-treatedTHP-1monocyticcells,significantamounts of IL-1

b

, IL-6 and IL-8 were secreted at 10, 25 and 50

m

g/mL compared tocontrols(Fig.5A–C).SignificantincreasesinTNF-

a

andIL-10wererespectivelydetectedat25and50

m

g/mLDWCNTs (Fig.5D),and50

m

g/mLDWCNTs(Fig.5E).

Statistical comparisons revealed significant differences be-tweencellswithorwithoutLPSpre-treatmentforIL-1

b

(10,25, 50

m

g/mL;Fig.3A),IL-6(10,25,50

m

g/mL;Fig.3B),IL-8(0,10,25 and50

m

g/mL;Fig.5C)andIL-10(25

m

g/mL;Fig.5E).

4.Discussion

Theobjectiveofthisstudywastoassessinvolvementofinnate immunityandinflammatoryresponseafterDWCNTexposure.For thispurpose,monocyticTHP-1cellswereincubated6h,24h,48h or72hwithDWCNTs.Wehaveclearlyshownthatsecretionof pro-inflammatorycytokines(IL-1

b

,IL-6,IL-8,TNF-

a

andIL-10)after exposuretoDWCNTswasconcomitanttocellularadhesionandto CD11b/CD14increasedexpression.

The stateof agglomerationof DWCNTswas characterized in exposuremediacontaining10%FCS.Giventhattheprimarylength ofDWCNTsusuallyrangesbetween1and10

m

m,wefoundthat DWCNTswereagglomeratedinthecellculturemediumwithasize distributionbetween3.9and 44.9

m

m. Interestingly,Saueretal. haverecentlyshownthatbovineserumalbumin(BSA)containing mediainducedstrongeragglomerationthanporcinelung surfac-tant(Saueretal.,2015).Consequently,wehypothesizedthatthe agglomerationcouldbeduetothelargeamountofFCS(containing BSA)inthecellculturemediumusedinthisstudy.

WefoundthatDWCNTsdidnotinducecytotoxicityafter6h, 24h, 48h and 72h of incubation with THP-1 monocytes. In literature,THP-1cellsaremainlyuseddifferentiatedin “macro-phage-like”cellsafterincubationwithinducerssuchasphorbol 12-myristate13-acetate(PMA)or 12-O-tetrade-canoylphorbol-13-acetate(TPA)(Tsuchiyaetal.,1982;Lanoneetal.,2009).Onlyafew studies were focused on DWCNT toxicity on monocytic cells. Recently,PescatoriandcolleaguesincubatedaseriesofMWCNTs differing in termsof bothfunctional groups anddiameterwith THP-1undifferentiatedcells(Pescatorietal.,2013).Todetectcells undergoingapoptosisandnecrosistheyanalysedannexin-VFITC and propidium iodidestainingby flowcytometry. Their results were consistent with our _findings, as they did not report cytotoxicityuntil100

m

g/mLafter24hofexposure.Lavernyand colleaguesalsopublishedresultsconsistentwithourstudy,asthey didnot reportcellviabilitydecreaseafter 48hofincubation of humanperipheralbloodmononuclearcells(PBMC)withMWCNTs (Laverny et al., 2013). However, Haniu and colleagues found cytotoxicityafter24hofincubationofundifferentiatedTHP-1cells to10or100

m

g/mLMWCNTs(Haniuetal.,2011).Thisresultcould be due to the smallest length of MWCNTs (size distribution between7and10

m

m).Interestingly,otherauthorsexposedDHD/ K12/Trbratcellsduring6hand24htothesamebatchofDWCNTs usedinourstudyandtheyshowedasignificantreductionincell viability(Fioritoetal.,2014).Wehypothesizedthattheseratcolon adenocarcinomacellsaremoresensitivetoDWCNTsthanTHP-1 monocyticcells.

As innate immunity adherence to endothelia or to the extracellularmatrixis aprerequisiteforextravasationof mono-cytes into injured tissues; we have investigated the ability of DWCNTstoinducemonocyticadhesion.Recently,Caoetal.found that exposure of HUVECs to MWCNTs significantly increased oxidative stress and THP-1 monocyte adhesion, while another studydidnotrevealanycytotoxicityofCNTsonthesamecellline (Cao et al., 2015; Flahaut et al., 2006). Using xCELLigence1

technology,wefoundthatdirectincubationwithDWCNTs,THP-1 became adherentto E-plates indicating a direct role on innate immunity. Thisresultis consistent withtheonly study(to our knowledge) showing that CNTs stimulate adhesion of THP-1 monocytes(DeNicolaetal.,2013).However,contrarytoourstudy theyfoundthatmonocyteswerenotabletointernalizeCNTs.This could be attributed to different physico-chemical properties betweentheCNTsused.

In addition to DWCNT agglomerate phagocytosis, we also showedmaturationofTHP-1monocyteswithincreasedCD11band CD14 expression. Normally, CD14 is downregulated during monocyte differentiation in macrophages (Daigneault et al., 2010;SteinbachandThiele,1994).Consequently,wesupposethat cellsobtainedafterDWCNTincubationarenotfullydifferentiated macrophages.However,we didnot findany otherstudyin the literature investigating effects of DWCNT exposure on CD14 expression by human monocytes. Some authors have recently shown that CD14 and CD11b were up-regulated after MWCNT incubationwithRAW264.7macrophageswhileitdown-regulated acomplexoftoll-likereceptor4andmyeloiddifferentiationfactor 2(TLR4/MD2)(Mengetal.,2015).Thus,theyhypothesizedthat up-regulation of CD14 was related to TLR4 endocytosis and that significant increases of CD11b acting as pattern recognition receptorislinkedtotheMWCNTphagocytosisprocess.Another computational study also suggested that CNTs might also be recognizedaspathogensbytoll-likereceptors(Turabekovaetal., 2014).Consequently,ontheonehand,increasedCD14expression couldalsobeassociatedtoTLR4endocytosisand/orrecognitionof DWCNTsbytoll-likereceptors,butfurtherexperimentsneedtobe carried out to confirm this mechanism. On the other hand, phagocytosis has been described as the main mechanism of internalizationpathwayforCNTagglomerates,bundles,clustersor single dispersed nanotubes 1

m

m or more in length, while endocytosisordiffusionwereattributedtosmallestobjects(Raffa etal.,2010).Thus,increasedCD11bexpressionisverylikelyrelated to the observed phagocytosis of CNT aggregates by THP-1 monocytes.AsElgrablietal.haverecentlyshownthatoxidative stressisimplicatedinMWCNTdegradationbyTHP-1 macrophage-likecells, it would beinterestingtoperform additional experi-mentsinordertoinvestigateifthispathwayisalsoinvolvedafter THP-1monocyticcellsexposure(Elgrablietal.,2015).

Yamaguchi and colleagues demonstrated that MWCNTs in-ducedsustainedstimulatingeffectsonimmuneandinflammatory responsesafterintraperitonealadministration inICR micewith increasednumbersofleukocytes,monocytesandgranulocytesin peripheralblood (Yamaguchiet al.,2012).Consequently, it was relevanttoinvestigatethepro-inflammatoryeffectsofDWCNTson monocytesandweshowed astrongpro-inflammatoryresponse after6hor24hofincubation.Concomitantlywithcelladhesion and DWCNT phagocytosis, a significant release of IL-1

b

was measured. This result corroborates the study of Meunier and colleaguesdemonstratingthatthesamebatchofDWCNTsinduced IL-1

b

releasebyprimaryhumanmonocytesrequiringpotassium effluxandphagocytosis(Meunieretal.,2012).Thestrongincrease inIL-1

b

couldbeinagreatpartduetothefactthattheseDWCNTs arenotsurfacefunctionalized.Itwasshowninvitro,aswellasin vivo,thatCNTchemicalsurfacefunctionalizationinduced signifi-cantdecreasesofIL-1

b

secretionandrecruitmentofneutrophils and monocytes (Yang et al., 2013). Interestingly, Kanno et al. recentlyinvestigatedroleRhokinases(ROCK1,and2)knowntobe involvedinmanycellularfunctionssuchasadhesion,regulationof cytoskeleton,andphagocytosis(Kannoetal.,2015).They demon-strateROCKsinvolvementininflammatoryresponseafterTHP-1 differentiatedmacrophagesexposuretoMWCNTs.Asweobserved adhesion,phagocytosisandsignificantIL-1

b

releasebyunprimed

andLPS-primedTHP-1monocytes,wesuggestthatROCKsarealso involvedinthisinflammatoryresponse.

ExposureofTHP-1monocytestoDWCNTsalsomainlyinduced asignificantincreaseofIL-6secretioninLPSprimedTHP-1whilea significantincreasewasonlymeasuredafter50

m

g/mLexposurein unprimedTHP-1cellsupernatants.Someauthorshavepreviously shown that IFN-

g

and TNF-

a

are implicated in IL-6 and IL-6 receptor modulation (Sanceau et al., 1991). IL-1

b

may have inducedTNF-

a

secretioninTHP-1monocytesasitwaspreviously shown in human mononuclear cells in vitro by Ikejima and colleagues(Ikejimaetal.,1990).TheobservedIL-1

b

andTNF-

a

secretionafter6hofexposureofTHP-1cellstoDWCNTsmayhave induced a significant increase of IL-6 secretion after 24h of exposure.IL-1

b

andTNF-

a

secretionmayhaveactivatedtheB-cell specifictranscriptionfactorNF-

k

Bandconsequently pro-inflam-matorysecretionofIL-6andIL-8aspreviouslydescribed(Bonizzi and Karin, 2004). IL-6 is known to play an important role in monocyticcelldifferentiation(Chomaratetal.,2000).Moreover, wealsomeasuredverysignificantamountsofIL-10atthehighest DWCNTconcentration.IL-10mayalsopromotemonocyte differ-entiationinmacrophage-likecellsaspreviouslyshownbyAllavena andcolleagues(Allavenaetal.,1998).Wethereforehypothesized that DWCNTs, as observed after exposure to the highest concentration,mimicIFN-

g

effectsandactas monocyte-macro-phageactivators.

5.Conclusions

In conclusion, ourexperiments showed that DWCNTs could modifymonocytephenotypealsoinducingastronginflammatory responsedespitenodecreaseincellviability.Thus,theseDWCNTs could also potentially have a significant impact on the innate immunesystem.Thesepropertiesmustbecautiouslytakeninto accountespeciallyinpublichealthornanomedicineissues.Dueto thespecificnatureoftheDWCNTsusedinthiswork,theseresults maybeextrapolatedtobothSWCNTsandMWCNTsingeneral. Declarationofintereststatement

ThisworkwassupportedbyagrantoftheFrenchMinistryof DefencefortheresearchprogramNANO-HF.Allauthorsdeclare thattheyhavenocompetinginterests,andaresolelyresponsible forthecontentandwritingofthispublication.

Acknowledgement

The authors thank Edgar Gentilhomme for his technical assistance.

AppendixA.Supplementarydata

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