Role of scavenger receptors in silica nanoparticle-induced cytokine responses in bronchial epithelial cells

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Role of scavenger receptors in silica nanoparticle-induced cytokine responses in bronchial epithelial cells

Magne Refsnes*, Tonje Skuland, Johan Øvrevik, Marit Låg

SectionofAirQualityandNoise,DepartmentofEnvironmentalHealth,NorwegianInstituteofPublicHealth,POBox222Skøyen,N-0213,Oslo,Norway

HIGHLIGHTS

Silicananoparticles(SiNP)induceIL-6,CXCL8,IL-1αandIL-1βandTGF-αresponsesinepitheliallungcells.

Thescavenger-receptorsSR-B1,LOX-1andCXCL16areinvolvedintheSiNP-inducedcytokineandTGF-αresponses.

TheSR-B1-inducedresponsesaremediatedviaMAP-kinase-andNF-kB-independentmechanisms.

ARTICLE INFO

Articlehistory:

Received20May2021

Receivedinrevisedform15September2021 Accepted8October2021

Editor:Dr.AngelaMally Availableonline12October2021

Keywords:

Silicananoparticles Scavengerreceptors Bronchialepithelialcells Cytokines

TGF-α

Signallingpathways

ABSTRACT

Amajorchallengeinnanoparticle(NP)researchistoelucidatehowNPsactivateinitialtargetsincells, leadingtocytotoxicityandinflammation.Wehavepreviouslyshownthatsilica(Si)NPsinducepro- inflammatoryresponsesinbronchialepithelialcells(BEAS-2B)viamechanismsinvolvingtransforming growthfactor(TGF)-αrelease,andactivationofMAP-kinasep38andJNKbesidesNF-k^B^(p65).În^the presentstudy,therolesofscavengerreceptors(SRs)inSiNP-inducedcytokineresponsesinBEAS-2Bcells wereexaminedbysiRNAsilencing.CellsexposedtoSi10andSi50(nominalsizes10and50nm)showed marked interleukin(IL)-6,CXCL8, IL-1α,IL-1βresponses.TransientknockdownofSR-B1,LOX-1and CXCL16reducedtheSi10-andSi50-inducedcytokineresponses,toadifferentmagnitudedependenton the particle size, SR and cytokine. Si10-inducedTGF-α responses were also markedly reducedby knockdownofSR-B1andCXCL16.Furthermore,theroleofSR-B1inSi10-inducedphosphorylationsof p65andMAP-kinasesp38andJNKwereexamined,andnosignificantreductionswereobservedupon knockdownofSR-B1.Inconclusion,LOX-1andCXCL16andespeciallySR-B1seemtohaveimportantroles inmediatingcytokineresponsesandTGF-αreleaseduetoSiNPexposureinBEAS-2Bcells,withouta down-streamroleofMAP-kinaseandNF-k^B.

1.Introduction

Theuseofengineerednanoparticles(NPs)inthedevelopment ofnewmaterials,giving advantageousproperties forconsumer productsandtherapeutics,israpidlyincreasingword-wide.With theemerginguse,itisofuttermostimportancetoavoidthatthese new NP/nanomaterials (NM) possess properties that induce adverseacuteor chronichealtheffects in humansoraffectthe

plethoraoforganismsintheenvironment.Alotofresearchhas focusedonhazardcharacterizationofNPs(Neletal.,2013)andalso onexposureassessment,toidentifywhichNPsmayrepresenta potential health problem for humans, either in occupational settings, upon drug therapy or for the general population/

consumers. To better predict NP toxicity and develop adverse outcomepathways(AOPs)fordifferentclassesofNPs,itisessential to elucidate the molecular initiating events ultimately causing effectsobservedfromNPexposure.

Non-crystalline (amorphous) silica nanoparticles (SiNPs) are knowntoinducecytotoxic,genotoxicandimmunotoxicresponses (Napierskaetal.,2010;Murugadossetal.,2017;Pavanetal.,2019).

A large range of invitro studies has shown that SiNPs induce inﬂammation-relatedresponses,includingreleaseofthecytokines interleukin(IL)-6andCXCL8.Furthermore,smallSiNPsappearto befarmorepotentonamassbasisthansimilarsubmicro-sized Abbreviations:NPs,Nanoparticles;SiNPs,Silicananoparticles;Si10,SiNPswith

nominalsize10nm;BEAS-2B,bronchial epithelial cells2B; SR-B1, scavenger receptor B1; LOX-1, lectin-like oxidized LDL receptor-1; CXCL16, C-X-C motif chemokineligand16;IL,interleukin;TGF-α,transforminggrowthfactor-α;TACE, tumournecrosisfactor-αconvertingenzyme.

*Correspondingauthor.

E-mailaddress:[email protected](M.Refsnes).

http://dx.doi.org/10.1016/j.toxlet.2021.10.004

ContentslistsavailableatScienceDirect

Toxicology Letters

j o u r n a l h o m e p a g e : w w w . e l s e vi e r . c o m / l o c a t e/ t o x l e t

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particles(Linetal.,2006;Napierskaetal.,2009;Napierskaetal., 2012;Lågetal.,2018;Sandbergetal.,2012;Skulandetal.,2014a).

WehavepreviouslyshownthattheSiNP-inducedIL-6andCXCL8 responsesinBEAS-2Bcellsinvolvep38mitogen-activatedprotein kinase (MAP-kinase) and c-Jun N-terminal kinases (JNK), and nuclear factor-

k

^B ^(NF-

k

^B) signalling, as well as cleavage and releaseofmembrane-boundtransforminggrowthfactor-α(TGF-α) throughtumournecrosisfactor-αconvertingenzyme(TACE)(Låg etal., 2018;Skuland etal., 2014b).Furthermore,SiNP-induced cytokine responses partially seem to involve reactive oxygen species (ROS)-dependent mechanisms in the BEAS-2B cells, at high,butnotlowconcentrationsofSiNPswithnominalsizeof50 nm(Si50)(Refsnesetal.,2019).

Scavengerreceptors(SRs)areasuperfamilyofreceptorsthat mightmediatetheeffectofNPs.SRswereinitiallyshowntobind andinternalizeoxidizedlipoproteins,butarecurrentlyknownto bind a variety of ligands including exogenous pathogens, and negativelycharged particles.SRs andtheirinherent properties havebeenidentiﬁedandclassiﬁedinto10eukaryotefamilies(A- J). These receptors are expressed in a variety of cell types, includingmacrophages,dendritic-,endothelial-and epithelial cells. Binding of ligands to SRs facilitates cellular uptake and ligand removal/clearance. SR members are known to regulate pathophysiologicalstatesincludingairway diseasesassilicosis, atherosclerosis, pathogen infections, immunesurveillance, and cancer. In general, studies have focused on Class A scavenger receptors(SR-A),however,researchhasalsobeendirectedagainst theroleofClassBscavengerreceptors(SR-B)(Gowdyetal.,2015;

Zani et al., 2015; Shannahan et al., 2015a; Hoekstra, 2017).

DifferentSRshave beenshown tobe involvedinparticle- and nanoparticle recognition, uptake, inﬂammatory (cytokine) and /or cytotoxic responses, as revealed in different cell types (Shannahan et al., 2015a; Nakayama, 2018). SR-A1 and also anothermemberoftheSR-Aclass(MARCO),wereearlyreported to mediate the uptake, cytotoxicity and/or inﬂammatory responses (cytokines) of larger crystalline silica particles in alveolarmacrophagesandmastcellsuponinhalationorinstilla- tion in mice (Hamilton et al., 2006; Brown et al., 2007).

Subsequently,ithasbeenreportedthat SR-A1andMARCOalso mightmediatesuchresponsesinmacrophageswhenexposedto variousNPs,includingSiNPs(Orretal.,2011),carbonnanotubes (Wang etal.,2020),gold-NPs (Françaet al.,2011)and polysty- rene-NPs(Kannoetal.,2007).

ThescavengerreceptorSR-B1isexpressedinmultipletissues and cell types across the body, although expressed at varying levels. The SR-B1 is able to bind to a wide variety of ligands, including anionic phospholipids and native and modiﬁed lip- oproteins.Interestingly,SR-B1andalsoSR-B2(CD36),havebeen reportedtomediatetheeffectofsilicaandothernano-sizedand micro-sizedparticles(Nakayama,2018;Shannahanetal.,2015b).

Evidence also suggests that SR-B1 is involved in canonical inﬂammasome activation and subsequent pro-inﬂammatory responsesin mouse macrophages and humanperipheralblood monocytes, after exposure to crystalline silica and amorphous SiNPs(Tsugitaetal.,2017).IncontrasttoSR-A,SR-B1alsoseemsto playamoreprominentroleinepithelialcells(Hoekstraetal.,2010;

Sharmaetal.,2015),butitsrolewithrespecttoNPshasbeenless elucidated.IthasbeenreportedthatSR-B1mediatestheuptakeof silver-NPsinratlungepithelialcells,leadingtocelltoxicityandan IL-6response(Shannahanetal.,2015b).BothSR-B1andtheclass-E SR,LOX-1(lectin-likeoxidizedLDLreceptor-1),hasbeenreported tobehighlyexpressedinhumanbronchialepithelialcells.Others, suchasCXCL16(chemokine(C-X-Cmotif)ligand16)achemokine witha dualroleasclassGSR,and SREC-1(scavengerreceptors expressedontheendothelialcells),appeartobemoremoderately expressedinbronchialepithelialcells(Dieudonnéetal.,2012).

Inthepresentstudy,weexaminetheroleofthreeSRs,SR-B1, LOX-1 and CXCL16, in regulationof 10nm SiNP (Si10)-induced cytokine and growth factor responses in BEAS-2B cells. The involvementoftheseSRswasalsoexaminedafterexposurewith largerSi50toverifytheroleofSRsineffectsfromNPsofdifferent sizes. Finally, therolesof MAPK- and NF-

k

^B-pathwaysⁱⁿ ^Si10-

inducedSR-B1-mediatedresponseswereinvestigated.

2.Methods 2.1.Materials

LHC-9 and Dulbecco's Modified Eagle Medium: Nutrient MixtureF-12(DMEM/F12)mediumwerepurchasedfromGibco, Life technologies, NY, USA.PureColTM collagenfrom Advanced BioMatrix,Inc,CA,USA.Thecellcultureflaskswereobtainedfrom Nunc A/S, Roskilde, Denmarkand the 6-wellplates and 10cm culture discs from Corning, Lowell, MA 01851 USA. Silica nanoparticle with nominal size of 10 nm (Si10) and 50 nm (Si50)werefromKiskerBiotech,Steinfurt,Germany.ELISAcytosets for IL-6 and CXCL8 were purchased from Invitrogen Life TechnologiesLtd,UK,whileIL-1α,IL-1βand TGF-αDuoSetwere fromR&DSystems,Inc,UK.Thespecificantibodiesagainstp-p38, p-JNKandp-p65wereboughtfromCellSignalingTechnology,Inc., Danvers,MA,USA,β-actinwasdeliveredfromSigma-Aldrich,St.

Louis,MO,USA.SupersignalWestDurawasobtainedfromThermo FisherScientiﬁcInc,Rockford,IL,USA,whiletheRe-blotstripping solutionwas fromChemicon, Millipore,Billerica, MA,USA.DC- Proteinassay,30%acrylamid-BIS,TEMED,APSusedforWestern analysis,werepurchasedfromBioRadLaboratoriesLtd,UKandthe nitrocellulosemembranefromWhatmanGmbH,Dassel,Germany.

siRNAagainstSR-B1, LOX-1andCXCL16,and controlsiRNAwere boughtfromCellSignalingTechnology,Inc.,Danvers,MA01923, USAandHiPerfecttransfectionreagentfromQiagenGmbH,Hilden, Germany.PerfectPureRNAisolationkitwasboughtfrom5PRIME, Gaithersburg,MD20878,USA.High CapacitycDNAArchiveKit, SYBR^TMGreenPCRMasterMix,TaqManUniversalPCRMastermix and TaqMan Gene Expression Assays were obtained from Life Technologies(ThermoFisherScientiﬁcInc,Waltham,MA02454, USA).TheprimersforSR-B1,LOX-1,SREC-1andCXCL16weremade forusbyIntegratedDNATechnologies,Inc.,IA,USA.TheLDHkit wasobtainedfromSigma-Aldrich(Merck,Darmstadt,Germany).

Anti-EGFR,neutralizing,wasfromMillipore,Billerica,MA,USA.

2.2.Cellcultureandexposureconditions

BEAS-2Bcells,aSV-40hybrid(Ad12SV40)transformedhuman bronchialepithelialcellline(EuropeanCollectionofCellCultures, ECACC, Salisbury, UK) were maintained in LHC-9 medium in collagen-coatedﬂasks(PureColTM)inahumiﬁedatmosphereat37

Cwith5%CO₂,withrefreshmentofmediumeverysecondday.

Thecells(passages8–50)weresubsequentlyseededin35mm6- wellcultureplates(300000cellsperwell)oron100mmculture dishesinLHC-9mediumandculturedfor24h.Thereafter,thecells werestarvedfor24hinserum-freeDMEM/F12beforeexposureto Si10andSi50.Cytokinerelease(CXCL8,IL-6,IL-1αandIL-1β)was assessedafter20 hexposure, whileTGF-αreleasewas assessed after4hexposure.Analysisofp38-,JNK-andNF-

k

B-phosphory- lationbyWesternblottingwasperformedafter2hexposure.Total exposurevolumewas1.0mLin35mm(six-well)and5mLinthe culturedishes.

2.3.Preparationandcharacterisationofthenanoparticlesolution Stocksolutions(25mg/mL)oftheSi10andSi50nanoparticles were dispersed in sterile water (2.3 mg/mL) and sonicated for

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approximately2minonice(untilspecificultrasoundenergyof420 Jwasgiventothenanoparticles).Bovineserumalbumin(BSA,final concentration 0.15 %) and phosphate buffed saline (PBS, final dilution1x)wereaddedtotheparticlesolution,accordingtothe methodbyBihariandco-workers(Biharietal.,2008).Thisgavea

“particleworkingsolution”withaﬁnalparticleconcentrationof2 mg/mL. Dynamic light scatter (DLS), transmission electron microscopy(TEM),surfaceareaandchemicalelementcomposition ofSi10andSi50werepreviouslyreported(Lågetal.,2018).The Si10andSi50sizesasmeasuredbyTEManalysiswere10.81.6 and 34.7 1.7 nm, respectively, whereas the surface areas as measuredbyBrunauer-Emmett-Teller(BET)analysiswere244and 86.2m²/g,respectively.

2.4.Quantiﬁcationofscavengerreceptorexpressions

Inthepresentstudy,themRNAexpressionlevelsofSR-B1,LOX- 1,SREC-1andCXCL16wereinvestigatedinBEAS-2Bcellsbyuseof qPCR. RNA was isolated and cDNA synthesised as previously described(Lågetal.,2018).qPCRwasperformedwithforwardand reverseprimersforSR-B1(forward:5⁰-TTTGAAGGCATCCCCACCTA, reverse:5⁰-TGAATTCCAGACTCCAGGCAC),LOX-1(forward:5⁰-TTG TCCGCAAGACTGGATCTG,reverse:5⁰-TGGCATCCAAAGACAAGC ACT),SREC-1(forward:5⁰-GACTCCTTCTCATCCGATCCreverse:5⁰- GGCGCGGAGGCTTAGGGATGG)andCXCL16(forward:5⁰-CATCAA TTCCTGAACCCATGG,reverse:5⁰-GAATCGTCTCCGGAAACACCT)in SYBR^TMGreenPCRMasterMixandforβ-actinwithapre-designed TaqManGene Expression Assays (Hs01060665_g1)in TaqMan¹ Real-Time Universal PCR Master Mix. The expression of the differentgeneswasnormalizedagainstβ-actin,ahouse-keeping gene,and expressed asfold changecompared totheuntreated controlascalculatedbythe

DD

^Ct-method^(Låg^et^al.,^2018).

2.5.Suppressionofscavengerreceptors

TheBEAS-2BcellsweretransfectedwithHiPerfect(6

m

^L/well)

andsiRNAagainstSR-B1,LOX-1andCXCL16(10nM)immediately afterseedingthecells(200000cellsin2.3mLmedium/wellina6- wellcultureplate).After2daysinculturethecellswereexposedto Si10andSi50.ThetransfectionefﬁciencywasveriﬁedbyqPCRof thegene-expressionat48and72hafterthetransfectionprocess.

Toensurethatthecytokine-inhibitionwasduetoknock-downof the mRNA of the respective scavenger receptors, and not an unspeciﬁcreaction,thecellswerealsotransfectedwithnon-sense siRNA(10nM).

2.6.CytokineanalysisofIL-6,CXCL8,IL-1αandIL-1β

Cell culture media were collected 20 h after exposure and centrifugedat300xgtoremovecelldebrisandat10,000xgto removeﬂoatingSiNPs.IL-6,CXCL8,IL-1αandIL-1βproteinlevels weredeterminedbysandwichELISAaccordingtothemanufac- turer'sguidelines.Absorbancewasmeasuredandquantiﬁedbya platereader(TECANSunrise)equippedwithadedicatedsoftware (MagellanV1.10).

2.7.AnalysisofTGF-αrelease

ThereleaseofTGF-αwasdetermined4hafterexposurewitha 30minpre-treatmentofthecellculturestoaneutralizationEGFR antibody(anti-EGFR5

m

^g/mL).^After^exposure^to^Si10^particles^the

mediawerecollectedandcentrifugedat300xgand10,000xgto removecelldebrisandﬂoatingsilicaparticles.Theproteinlevelsof TGF-α were determined by sandwich ELISA, according to the manufacturer'sguidelines.

2.8.Cytotoxicity

Thecytotoxicitywasevaluatedbymeasuringlactatedehydro- genase (LDH) release in the cell culture media after 20 h of exposureandcalculatedasdescribedinpreviousstudies(Lågetal., 2018).Themeasurementsweredoneaccordingtothemanufac- ture’sprocedures.

2.9.Westernanalysis

Phosphorylations of the MAPKs p38 and JNK, and the transcriptionfactor p65in BEAS-2Bcells afterexposuretoSi10 have previously been investigated by Western analysis, with characterizationofthetime-courserelationships(Lågetal.,2018).

Basedonthesetime-courses,theBEAS-2Bcellswereexposedto Si10particlesfor2h,andthecelllysateanalysedforp-p38,p-JNK and p-p65. The cell lysates were prepared and analysed as described inthe previousstudy withβ-actin asanendogenous control(Lågetal.,2018).

2.10.Statisticalanalysis

Statistical analyses of 4–6 independent biological replicates wereperformedbyusingone-wayANOVAwithDunnetsMultiple comparisontest(usingGraphPadPrismsoftware(version8.0Inc., SanDiego,CA).

3.Results

3.1.SiNP-inducedcytokineresponses

Inlinewithapreviousstudy(Lågetal.,2018)Si10andSi50 exposure(050/0200

m

^g/mL)^induced^aconcentration-dependent and progressive increase in IL-6, CXCL8, IL-1α and IL-1β releasesinBEAS-2Bcells(Fig.1).Somereductionincellviability wasobservedat50

m

^g/mL^Si10^and²⁰⁰

m

^g/mL^Si50ⁱⁿ^accordance

with our previous study (data not shown). Thus, in further experiments 25

m

^g/mL ^Si10 ^was ^chosen ^to ^minimize^that ^NP-

inducedcytotoxicitycouldinﬂuencethecytokineresponses.

3.2.Involvementofscavenger-receptorsinSiNP-inducedcytokine responses

InitiallyanalysisofSRexpressionsbyqPCRshowedrelatively highlevelsofSR-B1,LOX-1andCXCL16(Ct20–25)inunexposed BEAS-2Bcells,buta minimallevel(Ct>35)ofSREC-1.Thus,to address the potential role of SRs in SiNP-induced cytokine responsesinBEAS-2Bcells,thecellsweretransfectedwithsiRNA against SR-B1, LOX-1 and CXCL16, 48 h before exposure. The transient knockdown of SR-B1 significantly reduced the Si10- inducedCXCL8,IL-6,IL-1αandIL-1βresponses(Fig.2A).ThesiRNA againstLOX-1andCXCL16alsosignificantlyreducedtheCXCL8,IL- 6andIL-1βresponses.However,Si10-inducedIL-1αresponsewas onlyreducedbythesiRNAagainstCXCL16,notLOX-1.TheSi10- inducedIL-1βresponseswerereducedtoapproximatelythesame magnitudebysiRNAsagainstSR-B1,LOX-1andCXCL16,whereas theothercytokinesseemedtobelessreducedbysiRNAsagainst CXCL16andLOX-1thanSR-B1,althoughnotsignificantly.Next,we exploredwhetherSRscouldbeinvolvedineffectsfromSiNPsof othersizes.Thus,theeffectsofSR-B1,LOX-1andCXCL16silencing by siRNA were examined for Si50-induced cytokine responses.

SimilarpatternsasforSi10wereobserved,butwithlesssignificant reductionsinthecytokineresponses(Fig.2B).Thus,forCXCL8no significantreductionwasobservedaftersilencingofLOX-1.ForIL- 1αnoreductionswereobservedneitherforLOX-1norCXCL16,and forIL-1βnotforanyoftheSR-receptors.TheefficiencyofsiRNA-

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mediatedsilencingofSRswerevalidatedbyqPCR.SiRNAtrans- fections against SR-B1, LOX-1 and CXCL16, almost completely knockeddown(approximately80–90%)theirrespectivereceptor mRNAlevels(Fig.2C).TheLOX-1andCXCL16siRNAsdidnotaffect theSR-B1levels.However,siRNAagainstSR-B1appearedtocausea slight, but not statisticallysigniﬁcantreductions in LOX-1 and CXCL16expressions(Fig.2C).

3.3.TheinvolvementofscavengerreceptorsinSi10-inducedTGF-α release

PreviouslywehaveshownthatSiNPsinduceTGF-αviaaTACE- mediatedmechanism,andthatTGF-αcontributedtoSiNP-induced

cytokines(Lågetal.,2018; Skulandetal.,2014b).Wetherefore investigatedtheinvolvementofthedifferentSRsinSi10-induced TGF-αreleasebytransfectingwiththesiRNAagainstSR-B1,LOX-1 and CXCL16. In linewith previous observations, Si10 exposure induced a marked TGF-α release in BEAS-2B cells after 4 h exposure,andsigniﬁcantreductionsbysiRNAsagainstSR-B1and CXCL16wereobserved,butnotbysiRNAagainstLOX-1(Fig.3).

3.4.Scavengerreceptor-B1andintracellularsignalling

Previous studiessuggest that activationof MAP-kinasesp38 andJNKandthetranscriptionfactorNF-

k

^B^(p65)^partly^mediate

SiNP-inducedIL-6andCXCL8formation(Lågetal.,2018;Skuland Fig.1.Concentration-dependenteffectsofSi10andSi50exposureoncytokinerelease.TheBEAS-2BcellswereexposedtoSi10(0-50m^g/mL)^and^Si50^(0-200m^g/mL)^for²⁰^h beforeanalysisforIL-6(A,B),CXCL8(C,D),IL-1α(E,F)andIL-1β(G,H)byELISA.TheresultsarepresentedbythemeanSEMof4independentexperiments.*Signiﬁcantly differentfromcontrol,p0.05.

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et al., 2014b). We therefore explored whether SRs could be involvedinSiNP-inducedp38,JNKorNF-

k

^B^activationⁱⁿ^BEAS-2B

cells,bytransientknockdownofSR-B1.However,silencingofSR- B1didnotaffecttheSi10-inducedphosphorylationofp38andJNK (2h), indicating that thesesignalling pathways wereactivated through a SR-B1-independent mechanism, at least at early timepoints(Fig.4).AlthoughSi10-inducedp65-phosphorylation seemedpartiallyreducedbysiRNAagainst SR-B1,thereduction wasnotsigniﬁcant,indicatingnoorlittledown-streamroleforNF-

k

^Bⁱⁿ^mediating^regulation^of^cytokine^responsesⁱⁿ^BEAS-2B^cells

(Fig.4).

4.Discussion

Thepresentresultssuggestthatthescavenger-receptorsSR-B1, LOX-1andCXCL16haveimportantrolesinmediatingSiNPs(Si10 and Si50)-induced activation of IL-6, CXCL8, IL-1α and IL-1β responses, in bronchial epithelial cells (BEAS-2B). SR-B1 and CXCL16 were also involved in SiNP-induced TGF-α release, as demonstratedforSi10.Furthermore,SR-B1seemstomediateSiNP- inducedcytokineresponsesthroughMAPK-andNF-

k

^B-indepen-

dentmechanisms,aspresentlyshownforSi10.

AnimportantﬁndinginthepresentstudywithBEAS-2Bwas thatsilencingofSR-B1bytransientknockdownreducedtheSiNP- induced increase of IL-6 and CXCL8, supporting an up-stream involvement of SR-B1 for the induction of these cytokines.

Furthermore, SiNP-induced IL-1α and IL-1β responses, were similarlyaffectedasIL-6andCXCL8upontransfectionwithsiRNA againstSR-B1.ThismaysuggestthatSiNP-inducedIL-1αandIL-1β formationviaSR-B1isaninitialcriticaleventinvolvedinSiNP- induced IL-6 and CXCL8 responses. However, it should not be excluded that IL-6 and CXCL8 formation also involves IL-1- independentpathways.Previously,manystudieshaveshownthat IL-1, in addition to TNF-α, is important in triggering of pro- inﬂammatoryresponses,includingcytokinesasCXCL8andIL-6, andultimatelyinexacerbationanddevelopmentofinﬂammatory diseases (Dinarello, 2000). Interestingly, we have shown that larger-sizedcrystallinesilica(Min-U-Sil)andSi10arepromoting theinductionofIL-6andCXCL8releaseatleastpartlyviaIL-1,but notTNF-α,intri-culture modelswithhumantypeIIlikealveolar epithelialA549cellsculturedincombinationwithTHP-1mono- cytes/macrophages and endothelial cells (Herseth et al., 2009;

Skulandetal.,2020a).

Fig.3.TheroleofdifferentscavengerreceptorsinmediatingSi10-inducedTGF-α release.TheBEAS-2BcellsweretransfectedwithsiRNAsagainstSR-B1,LOX-1and CXCL16orwithnon-targetingsiRNA(siNT)twodayspriortotheexposureofSi10.

OnehbeforeexposingthecellstoSi10(25m^g/mL)^for⁴^h,^the^cells^werepre-treated withaneutralizationEGFRantibody(anti-EGFR,5m^g/mL).^The^cells^were^analysed forTGF-αbyELISA.Theresultsarepresentedas%ofSi10-inducedresponsesbythe mean SEM of 4 experiments, *signiﬁcantly different from control (siNT),

#signiﬁcantlydifferentfromSi10(siNT),p0.05.ThemeanSEMcontrolvalue was6.52pg/mLandthemeanSEMSi10(NT)responsewas20.68.5pg/mL.

Fig.2. TheroleofdifferentscavengerreceptorsinmediatingSi10–andSi50-inducedcytokineresponses.TheBEAS-2BcellsweretransfectedwithsiRNAsagainstSR-B1,LOX-1 andCXCL16orwithsi-nontargetingRNA(siNT)twodayspriortotheexposureofSi10(25m^g/mL)ând^Si50⁽²⁰⁰m^g/mL)^for²⁰^h.Â)Si10-inducedcytokineresponsesB)Si50- inducedcytokineresponses.C)GeneexpressionlevelsofSR-B1,LOX-1andCXCL16beforeandaftertreatmentwithsiRNAsagainstthedifferentSRsasanalysedbyqPCR.InA) andB)cellsupernatantswereanalysedforCXCL8,IL-6,IL-1αandIL-1βbyELISA.Theresultsarepresentedas%ofSiNP-inducedresponsesandrepresentthemeanSEMof4- 6independentexperiments,*significantlydifferentfromcontrol,p0.05;#significantlydifferentfromSi10-andSi50-exposedcells,p0.05.ThemeanSEMcontrolvalues were580146,590130,162.8and3.02.5pg/mLforCXCL8,IL-6,IL-1αandIL-1β,respectively.ThemeanofSi10-responseswas25800pg/mLforCXCL8,28000pg/mL forIL-6,390pg/mLforIL-1αand96pg/mLforIL-1β.ThemeanoftheSi50-inducedresponseswas5900pg/mLforCXCL8,10000pg/mLforIL-6,170pg/mLforIL-1αand17pg/

mLforIL-1β.Thegeneexpression(C)wasperformedbyqPCR.

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ItshouldbenotedthattheSi10-inducedcytokineresponsesin thebronchialepithelialcellsdoesnotsolelyseemtobemediated bySR-B1.OurdatasuggestthatLOX-1andCXCL16,alsoreportedto belocalizedonepitheliallungcells(Dieudonnéetal.,2012),may beimportantinmediatingSiNP-inducedcytokineregulation.Thus, transfectionswithsiRNAsagainstLOX-1andCXCL16alsoshowed signiﬁcant,reductionsofSi10-inducedIL-6,CXCL8,IL-1αandIL-1β responsesintheBEAS-2Bcells.Althoughnotsigniﬁcantlydifferent fromtheeffectofSR-B1siRNA,theimpactofLOX-1andCXCL16 silencingonSiNP-inducedcytokineresponses,seemedsomewhat lower.Notably,theinvolvementofSR-B1,LOX-1andCXCL16was observedfordifferentsizesofSiNP,supportingamoregeneralrole ofSRsinSiNP-inducedcytokineresponses.

The pathogenesisof silica-induced airway disease hasbeen extensivelyexamined.Thesilanolgroupsareregardedascritical for the silica-induced responses (Pavan and Fubini, 2017).

Furthermore,themajorfocushasbeenoninflammasome(NLRP3) activationandIL-1βformationinmacrophages.Theseeventsare regarded as essential in the adverse outcome pathways for inflammation in silicosis. Anumber of studieshas shownthat phagocytosis/internalizationofcrystallineandamorphoussilica in macrophages is crucial for the inflammatory responses via induction of lysosomal rupture and activation of the NLRP3 inflammasome.These processessubsequentlyinducecaspase-1 activationandcleavageofpro-IL-1βandenhanceIL-1βsecretion (Dostertetal.,2008;Hornungetal.,2008;Kusakaetal.,2014) that is crucial for lung disease development (Schroder and Tschopp,2010).Notably,ithasalsobeendemonstratedacritical role for scavenger receptors as an initial event in triggering internalizationofsilicaparticlesandsubsequentinflammasome activation.ThishasespeciallybeenshownforSR-A1andMarco (Hamiltonetal.,2006;Brownetal.,2007).However,theSR-B1 hasalsobeenreportedtobeimportantforinternalizationofsilica particlesinmacrophages.Thus,silicaparticlesinteractwithSR- B1viaa binding siteinthe extracellularhelix,thus leadingto internalizationofsilicaparticlesandsubsequentinflammasome activation(andIL-1βformation)andpro-inflammatoryresponses asextensively characterizedby Tsugita and coworkers(Tsugita etal.,2017).Inthepresentstudy,SR-B1wasshowntobeinvolved inSi10-inducedcytokineresponsesinhumanbronchialepithelial cells(BEAS-2B).Ingeneral,theroleofscavengerreceptorsinlung epithelial cells has been much less examined than in macrophages. However, SR-B1 has been reported to be localized in human bronchial epithelial lung cells, and to internalize exogenous agents such as double-stranded RNA (Dieudonné etal.,2012).InothercelltypesasHEK293cells,SR-B1hasbeen reportedtomediate fattyaciduptake(Wang etal., 2020).The partial reduction of the SiNP-induced cytokine responses

obtained by the siRNAs against SR-B1, LOX-1 and CXCL16, respectively,maypossiblybeexplainedbythereservecapacity forinternalizationofSiNPviatheseSRs.Althoughitmayappear thatSR-B1andotherSRsprimarilyareinvolvedinSiNPbinding, internalizationandsubsequentinﬂammasomeactivation,more indirect effects involving uptake of SiNP-induced mediators shouldnotbeexcluded.

Overall,thepresentstudyextendsourpreviouscharacteriza- tion of cellular signaling pathways for SiNP-induced IL-6 and CXCL8responses(Lågetal.,2018;Skulandetal.,2014b)andalsoon IL-1αandIL-1βresponses(Skulandetal.,2020b),andsuggeststhat severalindependentpathwaysmaybeinvolved.Ourcurrentdata suggestthatSiNPsinducecytokineresponsesviaSR-B1,indepen- dentoftheshort-termactivationoftheMAP-kinasesp38andJNK, andprobablytheNF-

k

^B^pathway.^The^role^ofMAP-kinasesandNF-

k

^BⁱⁿSR-mediated responsesremains unclear.Withfewexcep- tions SRs have only short cytosolic tails that lack discernible signalling motifs (Canton et al., 2013). However, it has been reportedthatSRs,asespeciallyexaminedforCD36,interactwith various co-receptors/signalling proteins in hetero-multimeric signalling complexes known as signalosomes. These have also beenreportedtoincludeMAPK.However,thesecomplexeshave shown a large context-dependent variability, with differences dependingontheexogenousagents,celltypesandotherfactors (Cantonetal.,2013).Notably,wehavepreviouslyshownthatlarger micron-sized crystalline silica particles trigger a rapid MAPK- phosphorylation withinminutes ofcontact withthe cellmem- brane of A549 cells. These early signalling responses were triggered prior to particleuptake, which occurredfrom 14 h after exposure (Ovrevik et al., 2006). Thus, it is tempting to speculatethatMAPKsignallingcouldderivefromearlysignalling inducedthroughinteractionsbetweenSiNPsandcellmembrane components(Kettigeretal.,2016;Alkhammashetal.,2015),while SRs,maybeinvolvedin thelater bindingand internalisationof SiNPs,possiblyleadingtolysosomedamageandinﬂammasome activation(Gómezetal.,2017;Peetersetal.,2013).Interestingly,it has been reported that JNK- and MEK inhibition reduce SR- mediatedinternalization(Sulahianetal.,2008).MAPK-activation could therefore be an upstream event. Such an upstream or independentrolewouldexplainwhySR-B1silencingdidnotaffect MAPKresponsesinSiNP-exposedBEAS-2Bcells,despiteitseffect oncytokineregulation.AnupstreamroleofMAPKwouldcomply with a more indirect activation of SRs through SiNP-induced mediators.However,thepotentialmechanismsbehindthisneedto beclariﬁed.Inaddition,therolesofMAPKsandNF-

k

^Bⁱⁿ^LOX-1-

and CXCL16-mediatedcytokine responsesinSiNP-exposed cells remain to be addressed, as well as the role of these SRs and mechanismsinothercelltypes.

Fig.4. TheroleofSR-B1inSi10–inducedphosphorylationofMAPKp38andJNK,andNF-kB(p65).TheBEAS-2Bcellswerepre-treatedwithsiRNAsagainstSR-B1,twodays priortotheexposuretoSi10(25mg/mL)for2h.Thecontrolsweretreatedwithnon-sensesiNT.Thecellswereanalysedforp38-,JNK-andp65-phosphorylation,andβ-actin( ACTB,loadingcontrol)byWesternanalysis.TheresultsarepresentedbyrepresentiveblotsaswellasthemeanSEMof4-5independentexperimentsafterquantiﬁcation.

(7)

5.Conclusions

Thescavenger-receptorSR-B1,aswellasLOX-1andCXCL16,are showntobeinvolvedininductionofIL-6,CXCL8,IL-1αandIL-1β responses,aswell asTGF-αformation, duetoSiNP exposurein BEAS-2Bcells.LackofeffectfromSR-B1silencingonSiNP-induced phosphorylation of MAPKs and NF-

k

^B ^is ^compatible ^with ^an

independentorupstreamroleofthesepathways.

DeclarationofCompetingInterest

The authors declare that they have no known competing ﬁnancial interests or personal relationships that could have appearedtoinﬂuencetheworkreportedinthispaper.

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