Macrophage sensing of single-walled carbon nanotubes via Toll-like receptors

1 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 www.na u e.com/scien i ic epo s Mac ophage sensing o single- walled ca bon nano ubes ia Toll- like ecep o s Sou a P. Mukhe jee1, Olesja Bonda enko1,2, Pekka Kohonen 1, Fe nando T. Andón1,3, Táňa B zico á1,4, Isabel Gessne 5, Sanjay Ma hu 5, Massimo Bo ini6,7, Paolo Calliga i8, Lo enzo S ella 8, Elena Kisin9, Anna Sh edo a9,10, Reija Au io11, Heli Salminen-Mankonen12, Rii a Lahesmaa12 & Beng Fadeel1 Ca bon-based nanoma e ials including ca bon nano ubes (CNTs) ha e been shown o igge in lamma ion. Howe e , how hese ma e ials a e ‘sensed’ by immune cells is no known. He e we compa ed he e ec s o wo ca bon-based nanoma e ials, single-walled CNTs (SWCNTs) and g aphene oxide (GO), on p ima y human monocy e-de i ed mac ophages. Genome-wide ansc ip omics assessmen was pe o med a sub-cy o oxic doses. Pa hway analysis o he mic oa ay da a e ealed p onounced e ec s on chemokine-encoding genes in mac ophages exposed o SWCNTs, bu no in esponse o GO, and hese esul s we e alida ed by mul iplex a ay-based cy okine and chemokine p o iling. Condi ioned medium om SWCNT-exposed cells ac ed as a chemoa ac an o dend i ic cells. Chemokine sec e ion was educed upon inhibi ion o NF-κB, as p edic ed by ups eam egula o analysis o he ansc ip omics da a, and Toll-like ecep o s (TLRs) and hei adap o molecule, MyD88 we e shown o be impo an o CCL5 sec e ion. Mo eo e , a speci ic ole o TLR2/4 was con i med by using epo e cell lines. Compu a ional s udies o elucida e how SWCNTs may in e ac wi h TLR4 in he absence o a p o ein co ona sugges ed ha binding is guided mainly by hyd ophobic in e ac ions. Taken oge he , hese esul s imply ha CNTs may be ‘sensed’ as pa hogens by immune cells. Ca bon-based nanoma e ials including ca bon nano ubes (CNTs) and g aphene oxide (GO) a e po en ial candi- da es o a ious applica ions in medicine such as d ug deli e y and imaging1. Howe e , he success ul ansla ion o nanoma e ials o biomedical applica ions equi es a de ailed unde s anding o he biological in e ac ions o he ma e ials. In pa icula , in e ac ions o nanoma e ials wi h he immune sys em, he i s line o de ense agains o eign in usion, a e o key impo ance2. The inna e immune sys em is deployed in de ense agains mic oo ganisms and in ol es he ecogni ion o pa hogen-associa ed molecula pa e ns (PAMPs) by (PRRs) on he su ace o phagocy ic cells. The immune sys em also esponds o issue damage, a p ocess ha is igge ed by so-called dange o damage-associa ed molecula pa e ns (DAMPs)2. We p e iously hypo hesized ha nan- opa icles migh be ecognized di ec ly as nanopa icle-associa ed molecula pa e ns o NAMPs by cells o he immune sys em3. Howe e , expe imen al e idence o suppo his idea was la gely lacking. Hence, while he e 1Nanosa e y & Nanomedicine Labo a o y, Di ision o Molecula Toxicology, Ins i u e o En i onmen al Medicine, Ka olinska Ins i u e , 17177, S ockholm, Sweden. 2Labo a o y o En i onmen al Toxicology, Na ional Ins i u e o Chemical Physics and Biophysics, Tallinn, 12618, Es onia. 3Labo a o y o Cellula Immunology, Humani as Clinical and Resea ch Ins i u e, 20089, Rozzano-Milano, I aly. 4Depa men o Gene ic Toxicology and Nano oxicology, Ins i u e o Expe imen al Medicine AS CR, 14220, P ague, Czech Republic. 5Ino ganic and Ma e ials Chemis y, Uni e si y o Cologne, 50939, Cologne, Ge many. 6Depa men o Expe imen al Medicine and Su ge y, Uni e si y o Rome To Ve ga a, Rome, 00173, I aly. 7San o d Bu nham P ebys Medical Disco e y Ins i u e, La Jolla, CA, 92037, USA. 8Depa men o Chemical Sciences and Technologies, Uni e si y o Rome To Ve ga a, Rome, 00133, I aly. 9Exposu e Assessmen B anch, Na ional Ins i u e o Occupa ional Sa e y and Heal h, Mo gan own, WV, 26505, USA. 10Depa men Pha macology & Physiology, Wes Vi ginia Uni e si y, Mo gan own, WV, 26505, USA. 11Facul y o Social Sciences, Uni e si y o Tampe e, 33014, Tampe e, Finland. 12Tu ku Cen e o Bio echnology, Uni e si y o Tu ku, 20520 Tu ku, and Åbo Akademi Uni e si y, 20500, Tu ku, Finland. Sou a P. Mukhe jee and Olesja Bonda enko con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be add essed o B.F. (email: [email p o ec ed]) Recei ed: 10 May 2017 Accep ed: 3 Janua y 2018 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 is an eme ging body o li e a u e on nanoma e ial e ec s on he immune sys em, he e a e ew i any s udies in which e idence o speci ic ‘sensing’ o nanopa icles by immune-compe en cells has been p o ided. P e ious wo k has shown ha p o eins adso bed on o he su ace o nanopa icles can ac i a e mac ophages ia su ace ecep o s, esul ing in he sec e ion o p o-in lamma o y cy okines4, bu whe he nanoma e ials hemsel es a e ecognized by immune cells h ough speci ic ecep o s is no known. Single-walled and mul i-walled CNTs as well as ca bon nano ibe s ha e been shown o induce p o-in lamma o y and p o- ib o ic esponses, especially ollowing pulmona y exposu e5. Speci ically, exposu e o SWCNTs was shown o elici acu e in lamma ion and ea ly-onse ib osis in he lungs o mice, wi h neu ophil accumula ion, ollowed by mac ophage in lux, and an ea ly ele a ion o p o-in lamma o y cy okines ollowed by p oduc ion o p o- ib o ic cy okines6. Mo eo e , ele a ed numbe s o dend i ic cells (DCs) a e ound in he lungs o mice ollowing pha yngeal aspi a ion o SWCNTs7. Fu he mo e, based on he li e a u e a ailable a he ime, ce ain igid and ‘needle-like’ MWCNTs we e classi ied by he In e na ional Agency o Resea ch on Cance (IARC) as being po en ially ca cinogenic o humans8, and a mo e ecen , in-dep h examina ion o in i o and in i o s udies has a i med he o iginal e alua ion ha some MWCNTs a e po en ially ca cinogenic, while he da a a e inconclusi e o o he s9. Fo g aphene-based ma e ials, a consensus on oxici y o heal h isks has ye o eme ge, al hough conside able e o s a e being in es ed in o de o add ess his ques ion in a sys ema ic ashion10,11. In a ecen s udy, so-called g aphene nanopla ele s we e shown o induce pulmona y oxici y in mice a high doses, bu no lung ib osis12. In ano he ecen inhala ion s udy in a s, g aphene nanopla ele s showed low oxici y, wi h no dis inc pa hology o in lamma ion; he ma e ials we e inges ed by lung mac ophages13. In a s udy on single-laye GO shee s wi h la e al dimensions below 500 nm, no signi ican cy o oxic esponses we e no ed using A549 lung ca cinoma cells, and no in lamma ion o g anuloma o ma ion was obse ed in i o ollowing in ape i oneal injec ion14, while a mo e ecen s udy sugges ed ha he impac o GO on human pe iphe al blood-de i ed cells was dependen on he la e al dimensions o GO15. He e we p esen de ailed mechanis ic in i o s udies o add ess he impac o well-cha ac e ized and endo oxin- ee SWCNTs and GO on p ima y human mac ophages. Guided by global ansc ip omics analysis o mac ophages exposed o hese ma e ials, we ocused ou s udies on chemokine signaling and could show ha SWCNTs, bu no GO, induced chemokine sec e ion in mac ophages. Ou expe imen al esul s sugges ed ha SWCNTs we e sensed by Toll-like ecep o s (TLRs), PRRs on he su ace o phagocy ic cells16. Cellula up ake o SWCNTs was no equi ed o chemokine signaling in exposed mac ophages. Molecula docking sugges ed ha SWCNTs may in e ac wi h TLR4 bo h ia he ip and he side-walls. These s udies indica e ha immune cells a e able o ‘sense’ SWCNTs h ough speci ic immune ecep o s and as such a e ele an o ou unde s anding o he impac o hese ma e ials on human heal h. Resul s Cha ac e iza ion o ca bon-based nanoma e ials. SWCNTs, p oduced by he high p essu e CO dis- p opo iona ion p ocess (HiPco) echnique, and GO, syn hesized by a modi ied Humme ’s me hod, we e cha ac- e ized using an a ay o analy ical echniques. T ansmission elec on mic oscopy (TEM) e ealed ha SWCNTs had an a e age diame e o 1–4 nm and an a e age leng h o 0.5–2 µm, whe eas GO had an a e age diame e (la e al size) o 1.1 ± 0.3 µm. The su ace cha ge (ζ-po en ial) was −42.3 ± 0.9 mV and −42.0 ± 1.2 mV o SWCNTs and GO, espec i ely. The samples we e also cha ac e ized ollowing dispe sion in cell cul u e medium (i.e., DMEM) wi h and wi hou 10% FBS (Fig.S3a–e). The ζ-po en ial emained nega i e in DMEM + FBS, hough less nega i ely cha ged when compa ed o samples dispe sed in wa e . DLS measu emen s sugges ed ha SWCNT and GO we e less agglome a ed in DMEM + FBS han in medium wi hou FBS, hough DLS esul s o non-sphe ical objec s should be in e p e ed wi h cau ion. Nanoma e ials a e equen ly con amina ed wi h lipopolysaccha ide (LPS) o endo oxin, he cell wall com- ponen o G am-nega i e bac e ia17. The e o e, bo h ma e ials we e es ed by using he con en ional limulus amoebocy e lysa e (LAL) assay and ound o be endo oxin- ee (da a no shown). Mo eo e , in o de o exclude po en ial a e ac s due o in e e ence wi h he assay, which could skew he in e p e a ion o he biological da a, we also pe o med a mac ophage ac i a ion es based on he e alua ion o TNF-α sec e ion by p ima y human monocy e-de i ed mac ophages (HMDM) in he p esence and absence o a speci ic LPS inhibi o 18. LPS (100 ng/ mL) was included as a posi i e con ol. Using his app oach, he CNTs we e con i med o be endo oxin- ee, as mac ophage sec e ion o TNF-α in esponse o CNTs was e y low and no a ec ed by polymyxin B (Fig.S1a–c). Cy o oxici y assessmen and cellula up ake. We hen assessed bo h nanoma e ials o cy o ox- ici y using he lac a e dehyd ogenase (LDH) elease assay. ZnO nanopa icles (100 µg/mL) which a e known o be cy o oxic o mac ophages19, we e included as a posi i e con ol. SWCNT exposu e esul ed in a sligh , dose-dependen inc ease in LDH elease a 24 h (14–16% mo e LDH elease compa ed o he con ol a con- cen a ions 30 and 100 µg/mL, espec i ely; p < 0.05), whe eas GO did no yield any cy o oxici y in HMDMs (Fig.S1d). Nex , we assessed o cellula up ake o he nanoma e ials. TEM imaging con i med ha bo h nano- ma e ials we e in e nalized by HMDM a 24 h (Fig.S2). SWCNTs and GO we e mos ly ound wi hin memb ane enclosed acuoles, sugges ing ha he up ake had occu ed h ough an ac i e, mos likely endocy ic p ocess. Engul men o GO changed i s appea ance om a 2D s uc u e o a densely packed s uc u e (Fig.S2). Mic oa ay analysis o mac ophage esponses. To u he e alua e he impac o he wo nanoma e ials on mac ophages, we pe o med ansc ip omics analyses ollowing exposu e o 6 h o 24 h o 10 o 30 µg/ml (i.e., sub-cy o oxic doses) o SWCNT o GO. A yme ix® GeneChip® Human Genome U219 a ays we e employed o global assessmen o gene exp ession. The mic oa ay da a ha e been submi ed o he Gene Exp ession Omnibus Da abase (GEO accession no. GSE83516). Few di e en ially exp essed genes we e no ed a 6 h (da a no shown) and we he e o e ocused ou analysis on he 24 h exposu e ime-poin . The nanoma e ial exposu e a he www.na u e.com/scien i ic epo s/ 3 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 la e ime-poin esul ed in a speci ic ansc ip ional esponse wi h 52 di e en ially exp essed genes in esponse o SWCNTs, while 7 genes we e di e en ially exp essed in esponse o GO (mul ig oup analysis by wo-way ANOVA, p < 0.05, > 1.5- old change compa ed o con ols) (Fig.1a, and Supplemen a y Table1). In e es ingly, he di e en ially exp essed genes showed no o e lap be ween he wo nanoma e ials. Subsequen canonical pa h- way en ichmen analysis o di e en ially exp essed genes using he Molecula Signa u es Da abase showed ha he h ee biological pa hways wi h he mos signi ican en ichmen we e hose in ol ed in cy okine-cy okine ecep- o in e ac ion, chemokine signaling pa hway and chemokine ecep o binding pa hway (mul iple es ing co ec ed p- alues lowe han 10−25) (Fig.1b, and Supplemen a y Table2). In addi ion, ou analysis showed en ichmen o he NF-κB pa hway (comp ising 7 genes, q- alue 10−5.7). Indeed, analysis o ansc ip ional egula ion ne - wo ks showed ha NF-κB was a cen al ne wo k connec ing up egula ed chemokines, and, mo eo e , ha se - e al membe s and a ge s o he NF-κB pa hway we e signi ican ly modula ed in esponse o SWCNTs (Fig.2a). Fu he mo e, ups eam egula o analysis using he Ingenui y Pa hway Analysis (IPA) so wa e showed ha he mos signi ican ly modula ed NF-κB pa hway ne wo k membe s we e RELA (p65), IRF7 and NFKBIA (IκBα) (Z-sco es 3.4, 2.9 and 2.4, espec i ely) (Fig.2b). No ably, acco ding o a ecen bioin o ma ics s udy, RELA and NFKBIA a e bo h among he i e key genes media ing NF-κB pa hway- ela ed in lamma o y esponses and mac- ophage ac i a ion20. Taken oge he , he ansc ip omics analysis sugges ed ha chemokine signaling pa hways a e p ominen ly de egula ed by SWCNTs, bu no by GO, and ha NF-κB is a po en ial ups eam egula o o he ansc ip ional esponses o SWCNTs. Valida ing he ansc ip omics esul s. To alida e hese esul s, we assessed o mac ophage p oduc- ion o chemokines. To his end, a mul iplexed immunoassay o he de ec ion o a de ined se o cy okines/ chemokines ollowing exposu e o SWCNTs o GO (10–100 µg/mL) was applied. We ocused he analysis on ou chemokines (CCL3/MIP-1α, CCL5/RANTES, CXCL9 and CXCL10) based on he signi ican up egula ion o he co esponding genes acco ding o ou ansc ip omics analysis (abo e). As shown in Fig.3a–d, SWCNT-exposed cells p oduced high le els o all ou chemokines, while GO-exposed cells ailed o sec e e hese chemokines, which is hus in acco dance wi h he mic oa ay esul s. The sec e ion o CCL3 and CCL5 in SWCNT-exposed HMDM was dose-dependen (Fig.3e–h). These da a hus co obo a ed he ansc ip omics esul s. To u he con ol o any po en ial endo oxin con amina ion, he CNTs we e subjec ed o calcina ion a 250 °C o emo e Figu e 1. T ansc ip omics analysis o mac ophages exposed o ca bon-based nanoma e ials. Global gene exp ession p o iling o human monocy e-de i ed mac ophages (HMDM) was conduc ed a e 24 h exposu e o single-walled ca bon nano ubes (SWCNTs) o g aphene oxide (GO) (see Supplemen a y Table1). (a) Hea map o di e en ly exp essed genes in esponse o 10 and 30 µg/mL GO (line 1 and 2, espec i ely) o 10 and 30 µg/ mL SWCNTs (line 3 and 4, espec i ely). Up egula ed ansc ip s a e p esen ed in ed and down egula ed ansc ip s in g een (log2 old change > 0.75). The genes a ec ed upon exposu e o SWCNTs (n = 52) and GO (n = 7) did no o e lap. (b) Canonical pa hways modula ed in HMDM a e exposu e o SWCNTs (see Supplemen a y Table2); anking was pe o med acco ding o mul iple es ing co ec ed p- alue. www.na u e.com/scien i ic epo s/ 4 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 any esidual endo oxins. The samples we e echa ac e ized (in cell cul u e medium) and shown o display simila ζ-po en ial alues while DLS measu emen s showed no signi ican changes (i.e., no agglome a ion) (Fig.S4a,b). We hen moni o ed he p oduc ion o CCL5 in mac ophages ollowing exposu e o 12 h o calcined SWCNTs (30 µg/mL) and ound ha he SWCNTs we e s ill capable o igge ing CCL5; mo eo e , his was no a ec ed by polymyxin B, indica ing ha he obse ed e ec is in insic o he SWCNTs and no a esul o mic obial con am- ina ion (Fig.S5a). Dissec ing he signaling pa hway. As no ed abo e, ups eam egula o analysis o he mic oa ay da a indica ed ha he NF-κB signaling pa hway was in ol ed in he ansc ip ional egula ion o chemokine exp es- sion by SWCNTs. To alida e his in silico p edic ion, HMDM we e p e ea ed wi h he NF-κB inhibi o , Bay 11-7082, and chemokine sec e ion in esponse o SWCNT exposu e was de e mined. SWCNT-induced p oduc- ion o CCL3 (Fig.4a) was signi ican ly educed upon p eincuba ion wi h Bay 11-7082 (10 µM) and he sec e ion o CCL5 was comple ely blocked by he inhibi o (Fig.4b), hus con i ming a ole o NF-κB. Nex we aimed o add ess how NF-κB is ac i a ed by SWCNTs. Figu e 2. Ups eam egula o analysis o he ansc ip omics esul s. (a) The NF-κB ne wo k was iden i ied as a po en ial ups eam egula o o SWCNT- igge ed esponses in HMDM acco ding o ups eam egula o analysis (p < 0.01; Z-sco e > 2 S.D.). (b) Ups eam egula o analysis73 o he da a iden i ied he modula ion o NF-κB ne wo k membe s in HMDM exposed o SWCNTs o 24 h. Da a we e analyzed h ough he use o IPA (QIAGEN Inc., www.qiagenbioin o ma ics.com/p oduc s/ingenui y-pa hway-analysis). www.na u e.com/scien i ic epo s/ 5 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 Figu e 3. SWCNTs, bu no GO, igge mac ophage sec e ion o chemokines. Sec e ion o chemokines by p ima y human mac ophages (HMDM) a e a 24 h exposu e o SWCNTs o GO as measu ed by a mul i-plex immunoassay. (a–d) Exposu e o HMDM o 30 µg/mL SWCNTs showed a signi ican inc ease in CXCL9, CXCL10, CCL3/MIP-1α, and CCL5/RANTES, while he e was no esponse in cells exposed o GO a he same concen a ion. (e,g) Dose-dependen sec e ion o CCL3/MIP-1α, and CCL5/RANTES in cells exposed o SWCNTs, while he e was no esponse o GO a any o he concen a ions es ed ( ,h). Da a a e shown as mean alues ± S.D. o h ee independen expe imen s using cells om di e en dono s; p- alues by S uden ’s - es , * < 0.05; *** < 0.001. www.na u e.com/scien i ic epo s/ 6 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 The ecogni ion o so-called PAMPs by di e en amilies o e olu iona ily conse ed PRRs (PRRs) ini ia es a signaling cascade ha leads o he ansc ip ion o in lamma o y cy okines and chemokines o elimina e pa ho- gens and a ac o he immune cells o he si e o in ec ion21. In pa icula , Toll-like ecep o s (TLRs) play a key ole in inna e immuni y16. TLRs ac i a e mul iple signaling pa hways by ec ui ing adap o p o eins, such as myeloid di e en ia ion ac o 88 (MyD88), which ini ia e signal ansduc ion pa hways ha culmina e in he ac i- a ion o ansc ip ion ac o s, eg., NF-κB, wi h ensuing cy okine/chemokine p oduc ion. TLR4 is a ecep o o bac e ial LPS while TLR2 has speci ici y o mul iple mic obial componen s de i ed om bac e ia, ungi, i uses, and mycoplasma21. The oxidized phospholipid, 1-palmi oyl-2-a achidonyl-sn-glyce o-3-phospho ylcholine (oxPAPC) is known o inhibi LPS signaling ia TLR2 and TLR422. To assess whe he SWCNTs a e capable o ac i a ing NF-κB ia TLRs, we p eincuba ed HMDM wi h oxPAPC (30 o 60 µg/mL) p io o exposu e o SWCNTs (30 µg/mL). LPS (100 ng/mL) was included as a posi i e con ol. As shown in Fig.5a, oxPAPC signi i- can ly educed LPS-induced sec e ion o CCL5. Mo eo e , SWCNT-induced p oduc ion o CCL5 was comple ely blocked. Nex , we es ed whe he inhibi ion o he adap o p o ein, MyD88 would supp ess NF-κB ac i a ion. To his end, cells we e p eincuba ed wi h Pepinh-MYD, a 26 aa pep ide ha blocks MyD88 signaling by inhibi ing i s homodime iza ion23. Pepinh-Con ol, a con ol pep ide, was included as a nega i e con ol. Pepinh-MYD (25 µM) signi ican ly educed LPS-induced NF-κB ac i a ion, as de e mined by he quan i ica ion o p65 phospho yla- ion, and SWCNT-induced ac i a ion o NF-κB was also blocked by Pepinh-MYD, bu no by Pepinh-Con ol (25 µM) (Fig.5b). Mo eo e , inhibi ion o MyD88 impeded chemokine p oduc ion in cells exposed o SWCNTs. Thus, Pepinh-MYD (25 µM) signi ican ly educed LPS-induced CCL5 sec e ion, and SWCNT- igge ed elease o CCL5 was also educed by Pepinh-MYD, bu no by he con ol pep ide (Fig.5c). These da a hus p o ided e i- dence o TLR2/4-MyD88-NF-κB signaling in SWCNT-induced chemokine p oduc ion in human mac ophages. SWCNTs a e in e nalized by HMDM a 24 h (Fig.S2) and we ecen ly p o ided e idence ha mac ophage up ake o SWCNTs may occu al eady a e a ew hou s24. To assess whe he cellula up ake o SWCNTs is equi ed o chemokine esponses, we de e mined he sec e ion o CCL5 in HMDM exposed o SWCNTs (30 µg/ mL) ollowing p eincuba ion wi h o wi hou cy ochalasin D (10 µM), an inhibi o o ac in polyme iza ion ha blocks endocy osis24. As shown in Fig.5d, cy ochalasin D did no a ec LPS-induced o SWCNT-induced p o- duc ion o CCL5, sugges ing ha his e en is elayed ia cell su ace signaling. We also cul i a ed mac ophages in medium wi h o wi hou 10% FBS in o de o es whe he he e ec o SWCNTs on chemokine sec e ion was in luenced by he p esence o se um p o eins. As shown in Fig.S5b, SWCNT- igge ed p oduc ion o CCL5 was compa able in he p esence o absence o se um. LPS was included as a posi i e con ol. To con i m he indings ob ained in monocy e-de i ed mac ophages, and in o de o add ess he ole, i any, o speci ic TLRs o he ‘sensing’ o SWCNTs, we used HEK293 cells s ably ans ec ed wi h human TLR2 o TLR4 and an NF-κB-inducible epo e gene25. Fu he mo e, in o de o asce ain whe he SWCNTs a e capable o TLR ac i a ion pe se, o whe he he in e ac ion is due o se um p o eins adso bed on he su ace o he nanoma e- ials26, we pe o med he expe imen s in epo e cell lines cul u ed in medium supplemen ed o no wi h 10% e al bo ine se um (FBS). LPS (100 ng/mL) was used as a posi i e con ol. LPS igge ed p onounced ac i a ion o TLR4 and a signi ican ac i a ion o TLR2 (Fig.6a). In e es ingly, SWCNTs also ac i a ed TLR4 and, o a lesse deg ee, TLR2, and he le el o ac i a ion in he p esence and absence o FBS was indis inguishable. The la e inding sugges ed ha SWCNTs a e sensed di ec ly by TLRs (Fig.6b). Func ional ole o chemokine sec e ion. Chemokines (G eek: kinos, mo emen ) a e named o hei ole in inducing di ec ed mig a ion o chemo axis in neighbo ing esponsi e cells. We he e o e add essed whe he he condi ioned medium o mac ophages exposed o SWCNT e sus GO would ac as a chemoa ac an o o he immune cells. Conside ing ha CCL3 and CCL5 a e he mos e ec i e chemoa ac an s o imma u e DCs27, we s udied cell mig a ion using p ima y human monocy e-de i ed DCs e sus non-di e en ia ed p ima y human monocy es. Su ace exp ession o he chemokine ecep o , CCR5 was highe o DCs han monocy es (Fig.7a), Figu e 4. SWCNT- igge ed chemokine sec e ion is NF-κB-dependen . P e ea men wi h he NF-κB inhibi o , Bay 11-7082 (10 µM) o HMDM exposed o 30 µg/mL SWCNTs o medium alone educed he sec e ion o CCL3 (a) and comple ely blocked he sec e ion o CCL5 (b), by mul i-plex assay. Da a a e mean alues ± S.D. o h ee independen expe imen s using cells om di e en dono s; p- alues by S uden ’s - es , * < 0.05; *** < 0.001. www.na u e.com/scien i ic epo s/ 7 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 in line wi h published esul s28. Fu he mo e, condi ioned medium o SWCNT-exposed HMDM was a weak chemoa ac an o monocy es, bu p omo ed he mig a ion o DCs (p < 0.05) (Fig.7b). Thus, he condi ioned medium se ed as a chemoa ac an o cells exp essing CCR5 and did no ema kably a ec he mig a ion o cells ha exp essed low le els o CCR5. Howe e , condi ioned medium o GO-exposed HMDM did no p omo e cell mig a ion (Fig.7b), in line wi h he obse a ion ha only SWCNTs igge ed chemokine sec e ion. Molecula docking s udies. P e ious heo e ical s udies ha e sugges ed ha C60 ulle enes and CNTs may block K+ channels29,30. In addi ion, ecen compu a ional s udies sugges ed ha he in e nal hyd ophobic pocke s o some TLRs migh be capable o binding ca bon-based nanos uc u es31. We pe o med molecula modelling o he TLR4:CNT complex o u he elucida e how TLRs can in e ac wi h he CNTs; he s udies we e done in he absence o a p o ein co ona. To be e ep oduce he expe imen al condi ions, bo h p is ine and ca boxyla ed CNTs we e modelled. The O:C a om a io co esponding o he expe imen al ze a po en ial was se o 0.1532, which esul ed in 84 ca boxyl g oups. Docking simula ions o p is ine CNTs ga e ise o a unique clus e o e y simila poses wi hin he p e-de ined ene gy ange (10 kcal/mol). Con e sely, ca boxyla ed CNTs showed di e se binding modes, wi h in e ac ion ee ene gies di e ing only sligh ly (~1 kcal/mol). The bes sco ing bind- ing mode, obse ed in bo h p is ine and ca boxyla ed CNTs, e ealed wo egions in TLR4, one in e ac ing wi h he ip o he CNT and ano he in con ac wi h i s side-walls (Fig.8a,b). While he i s egion is localized in a highly hyd ophobic a ea, which encompasses esidues om Ile108 o Asn265, he second is ound in he loops a ound Ile412 and Leu434 (Fig.9a,b). In oxidized CNTs, His159 and A g264 a e also wi hin a dis ance o he ca boxyl g oups ha is compa ible wi h sal b idge o ma ion. Al e na i ely, in he second egion, A g382, His431 and His458 a e close enough o o m ion-pai in e ac ions wi h ca boxyl g oups on he CNT side-walls. O e all, he bes binding mode is essen ially guided by hyd ophobic con ac s be ween TLR4 and CNTs, bu in he case o ca boxyla ed CNTs he in e molecula in e ac ion is s eng hened by sho - ange elec os a ics. The second and hi d op binding modes o ca boxyla ed CNTs co espond o a comple ely di e en con igu a ion, in which side-walls a e in close con ac wi h a la ge po ion ( om esidue 87 o 289) o he TLR pa allel be a-shee pa e n Figu e 5. TLR2/4- and MyD88-dependen sec e ion o chemokines. (a) Signi ican educ ion o CCL5 sec e ion in HMDM a e 12 h exposu e o SWCNT (30 µg/mL) in he p esence o he TLR2/4 inhibi o , oxPAPC (30 o 60 µg/mL). oxPAPC also blocked CCL5 sec e ion igge ed by LPS (0.1 µg/mL) in a dose- dependen manne . Fu he mo e, he MyD88 inhibi o , Pephinh-MYD (25 µM), bu no Pepinh-Con ol (25 µM), educed NF-kB p65 phospho yla ion (b) and CCL5 exp ession (c) in cells exposed o SWCNT (30 µg/ mL) o 12 h. Pephinh-MYD (25 µM) also educed NF-kB ac i a ion and CCL5 sec e ion by LPS (0.1 µg/mL). NF-kB p65 phospho yla ion and CCL5 exp ession was de e mined by ELISA. (d) Cy ochalasin D (10 μM), an inhibi o o ac in polyme iza ion, does no a ec CCL5 sec e ion in HMDM exposed o 12 h o SWCNT (30 µg/ mL). LPS (0.1 µg/mL) was included as a con ol. CCL5 le els we e de e mined by ELISA. Da a shown in panels a o d a e epo ed as mean alues ± S.D. o a leas h ee independen expe imen s using cells om di e en dono s. p* < 0.05; ** < 0.01; *** < 0.001 (one-way ANOVA wi h pos -hoc ukey’s es ). www.na u e.com/scien i ic epo s/ 8 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 in he inne pa o he p o ein (Fig.8c– , Fig.9a,b). In e es ingly, he a ea o in e ac ion o hese poses in e sec s one o he wo egions al eady obse ed o he bes pose. This inding is consis en wi h he negligible ene gy di e ence o hese binding modes wi h espec o he bes sco ing mode. Ne e heless, as opposed o he la e , hese wo classes o poses a e clea ly domina ed by elec os a ic in e ac ions, as shown by he dis ances be ween he ca boxyl g oups and some cha ged esidues (His179, A g257, A g289, A g355), which a e compa ible wi h he p esence o s abilizing ion-pai in e ac ions. Discussion The inna e immune sys em does no espond o mic obes in a nonspeci ic manne ; in ac , pa hogen ecogni- ion by he inna e immune sys em is speci ic, elying on PRRs ha ha e e ol ed o de ec molecula signa u es known as PAMPs21. Thus, a ela i ely small numbe o immune ecep o s a e employed by mac ophages and o he immune cells o de ec a as a ay o mic oo ganisms; i is in iguing o specula e ha simila p inciples o mechanisms migh be deployed o immune ecogni ion o a ious classes o nanopa icles33. Howe e , o da e, he e a e ew i any examples o speci ic immune sensing o enginee ed nanoma e ials and he p oblem is con ounded by a numbe o ac o s. Fi s , many s udies a e pe o med using nanopa icles ha a e no well Figu e 6. SWCNTs igge TLR2 and TLR4 ac i a ion. (a) HEK 293 cells co- ans ec ed wi h human TLR2 (HEK-Blue™ hTLR2) o TLR4 (HEK-Blue™ hTLR4 cells) and an NF-κB/AP-1-sec e ed emb yonic alkaline phospha ase (SEAP) epo e gene we e exposed o SWCNT (30 µg/mL) o 12 h in he p esence o absence o 10% FBS. LPS (0.1 µg/mL) was included as a posi i e con ol. SWCNTs ac i a ed TLR2/4 independen ly o he p esence o se um in he cul u e medium. Da a a e shown as mean alues ± S.D. o h ee independen expe imen s. (b) Schema ic diag am showing he ‘sensing’ o SWCNTs by HMDMs ia TLR ecep o s esul ing in MyD88-dependen ac i a ion o NF-kB leading o nuclea ansloca ion o NF-kB wi h ansc ip ion and sec e ion o CCL5. The sec e ed chemokine(s) induce chemo axis o immune cells bea ing he co esponding ecep o (s). Figu e 7. Mac ophage sec e ed ac o s p omo e mig a ion o DCs. (a) Exp ession o he chemokine ecep o CCR5 in p ima y human monocy es (Mo) and monocy e-de i ed dend i ic cells (DCs) was de e mined by low cy ome y. The a e age exp ession o CCR5 in cells om h ee di e en dono s is depic ed. (b) Mig a ion o monocy es (Mo) and DCs in esponse o condi ioned medium (CM) o human monocy e-de i ed mac ophages (HMDM) exposed o 100 µg/mL SWCNT (CM-SWCNT) o 100 µg/mL GO (CM-GO). Cell mig a ion (3 h pe iod) was de e mined by using answell chemo axis mic ochambe s. Da a a e shown as mean alues ± S.D. o h ee independen expe imen s; p* < 0.05, S uden ’s - es . www.na u e.com/scien i ic epo s/ 9 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 cha ac e ized, o no uni o m in appea ance wi hin he same sample, making i di icul o d aw conclusions ega ding speci ic p ope ies o nanopa icles and hei biological beha io 34. In addi ion, nanopa icles a e e- quen ly con amina ed wi h bac e ial endo oxin, as may be he case o o he bioma e ials, leading po en ially o e oneous esul s, especially when s udying in e ac ions wi h immune-compe en cells17. Fu he mo e, nanopa - icles a e known o apidly adso b p o eins and o he biomolecules, and his is hough o endow he nanopa i- cles wi h a new, biological ‘iden i y’ such ha hese adso bed biomolecules could dic a e biological in e ac ions: cells may no ‘see’ he p is ine nanopa icle su aces35. In addi ion o hese conside a ions, i has been a gued ha he e a e no nano-speci ic (i.e., size-dependen ) biological e ec s o nanopa icles, and he e o e no no el e ec s a e o be expec ed36. Howe e , i is wo h no ing ha many biological p ocesses anspi e a he nano-scale. Thus, i ollows om his a gumen ha nanopa icles, as a unc ion o hei small size, may in e e e wi h bio- logical p ocesses in a manne no seen o la ge pa icles37. Pa k e al.29 showed ha pu i ied SWCNTs blocked Figu e 8. Molecula docking o CNTs and TLR4. Resul s o docking simula ions o p is ine and ca boxyla ed CNTs and TLR4. (a) The bes binding mode o p is ine CNT. (b) bes binding mode o ca boxyla ed CNT. 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J Compu Chem 30, 2785–2791 (2009). www.na u e.com/scien i ic epo s/ 17 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 Acknowledgemen s This wo k was suppo ed by he Eu opean Commission unded p ojec s, FP7-NANOMMUNE (g an ag eemen no. 214281), FP7-MARINA (g an ag eemen no. 263215), FP7-NANOSOLUTIONS (g an ag eemen no. 309329), Flagship P ojec GRAPHENE (g an ag eemen s no. 604391 and 696656), and COST Ac ion MODENA (TD1204) (schola ship awa ded o T.B.). F.T.A. was suppo ed, in pa , h ough a pos doc ellowship om he Galician Go e nmen , Spain (Resolu ion 21, Ma ch 2013). We hank D . Kjell Hul enby, Elec on Mic oscopy Co e Facili y, Ka olinska Ins i u e , o echnical assis ance. Au ho Con ibu ions S.P.M. and O.B. pe o med in i o expe imen s and analyzed da a; F.T.A. and T.B. con ibu ed o in i o expe imen s and da a analysis; I.G. cha ac e ized he SWCNT and GO samples, supe ised by S.M., E.K., M.B., and A.S. con ibu ed o he ma e ial cha ac e iza ion o he SWCNT samples; P.C. and L.S. pe o med he molecula docking s udies; H.S.M. and R.A. pe o med he mic oa ay expe imen s, supe ised by R.L., and P.K. conduc ed he downs eam da a analysis; B.F. concei ed and coo dina ed he s udy, and w o e he pape wi h S.P.M. and O.B.; all au ho s app o ed he inal e sion o he pape . Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h ps://doi.o g/10.1038/s41598-018-19521-9. Compe ing In e es s: The au ho s decla e ha hey ha e no compe ing in e es s. Disclaime : The indings and conclusions in his epo a e hose o he au ho s and do no ep esen he iews o he Na ional Ins i u e o Occupa ional Sa e y and Heal h o he Uni ed S a es. Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C e- a i e Commons license, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in a c edi line o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons license and you in ended use is no pe - mi ed by s a u o y egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/. © The Au ho (s) 2018