Polymer-Coated Metal-Oxide Nanoparticles Inhibit IgE Receptor Binding, Cellular Signaling, and Degranulation in a Mast Cell-like Cell Line

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1 Polymer-Coted Metl-Oxide Nnoprticles Inhiit IgE Receptor inding, Cellulr Signling, nd Degrnultion in Mst Cell-like Cell Line Vn. Orteg, Jmes D. Ede, Dvid oyle, Jmes L. Stfford, nd Greg G. Goss * FULL PPER Previous reports hve shown tht nnoprticles (NPs) cn oth enhnce nd suppress immune effector functions; however the mechnisms tht dictte these responses re still uncler. Here, the effects of polycrylic cid (P) functionlized metl-oxide NP re investigted on RL-2H3 (representtive mmmlin grnulocyte-like cell line) cell viility, cellulr degrnultion, immunogloulin E (IgE) receptor inding, nd cell signling pthwys relted to immune function. The incresing development of P-NPs s pesticide dispersnts nd s drug crriers in therpeutics necessittes their investigtion for sfe production. Using two in vitro experimentl pproches, this study demonstrtes tht pre-exposing RL-2H3 cells, or IgE ntiodies, to P-NPs (TiO 2, CeO 2, ZnO, Fe 2 O 3, nd P-Cpsules (NP coting control) over 24 h, significntly decrese the inding cpcity of IgE for Fcε receptors, inhiit the phosphoryltion of intrcellulr signling proteins (e.g., MPK ERK) tht medite degrnultion, nd inhiited RL-2H3 cell degrnultion. In ddition, nd unlike the other NPs tested, P-TiO 2 significntly reduced RL-2H3 viility, in time (4 24 h) nd dose-dependent mnner (>5 µg ml 1 ). Together, these dt demonstrte tht P-NPs t su-lethl doses cn interct with cell surfce structures, such s receptors, to suppress vrious stges of the RL-2H3 degrnultory response to externl stimuli, nd modify immune cell functions tht cn impct host-immunity. 1. Introduction The unique physico-chemicl properties of nnoprticles (NPs) hve cptured the interest of the consumer industry nd the iomedicl community for their potentil to improve product V.. Orteg, J. D. Ede, Dr. D. oyle, Dr. J. L. Stfford, Dr. G. G. Goss Deprtment of iologicl Sciences University of lert Edmonton, lert, Cnd T6G 2E9 E-mil: greg.goss@ulert.c Dr. G. G. Goss Ntionl Reserch Council (Cnd) Ntionl Institute for Nnotechnology Edmonton, lert, Cnd T6G 2M9 This is n open ccess rticle under the terms of the Cretive Commons ttriution License, which permits use, distriution nd reproduction in ny medium, provided the originl work is properly cited. DOI: 1.12/dvs qulity nd ugment prctices for the tretment of disese, respectively. When compred to their lrger ulk equivlents, mterils in the nnoscle hve incresed chrge, lrger surfce re to volume rtios nd cn exhiit quntum fluorescence. [1 ] s such, NPs re eing developed s fluorescent gents for cell imging, s cncer therpeutics [2 ] nd s dditives in pints nd cosmetics, nd for use in industril pplictions. [1 ] This incresed use hs spurred the rpid development of NPs of vrying sizes, mteril types, nd surfce functionliztions, ech with their own unique chrcteristics nd potentil for toxicity (e.g., poptosis, [3 ] cell stress, [4 ] nd ltering ctlytic enzymes. [5 7 ] ). For exmple, re metl NPs ggregte redily, precipitte in solution nd often relese free metl ions, which cn increse the vriility of oserved effects nd oscure the source of NP toxicity. [8 ] y contrst, functionlizing metl NPs with polymers such s polycrylic cid (P), increses dispersiveness in solutions [9 ] nd reduces dissolution of the metl core. [1 ] ccordingly, P-NP-medited toxicity could e driven y different mechnisms when compred to core mterils lone. The need to study P-NP toxicity is prticulrly relevnt given the incresing development of these prticles s oth pesticide dispersnts for the griculturl industry, [11 ] nd s drug delivery crriers in nno-medicines. [12 ] The common hypothesis is tht NPs with identicl cotings will interct with iologicl tissues in similr mnner since the coting will dominte the interction nd potentilly lessen toxic effects. [3 ] However, the use of highly monodispersed NPs my lso lter trnsloction nd deposition cross tissues nd interctions with cells, when compred to effects from re NPs. Thus, the sfe dministrtion of NPs requires comprehensive ssessment of their iocomptiility with oth trget nd non-trget tissues. One key considertion in the development of NP-enled products is the response of the host immune system to exposure. Indeed, while some NPs hve een specificlly designed to ctivte the immune response (e.g., vccine djuvnts [13 ] ), others hve lso een shown to either indvertently suppress [14 ] or ctivte [15 ] vrious immune responses. For exmple, dv. Sci. 215, 2, The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim wileyonlinelirry.com (1 of 12) 1514

2 FULL PPER in rts, intrvenous dministrtion of polymeric NPs resulted in decresed serum histmine levels,[14] while intrperitonel dministrtion of re TiO2 NPs promoted llergic sensitiztion nd incresed the numer of inflmmtory cells in mice.[15] However, the mechnisms y which NPs modulte immune responses, nd the wider repercussions for immune functioning, re not well estlished. ssessing the effects of NPs on innte immunity is prticulrly importnt ecuse innte immune cells form the first line of cellulr defense ginst invding pthogens, nd s such, re lso one of the first immune cell types tht re likely to encounter nd respond to NPs.[14] Mst cells re innte cells locted throughout the ody ut especilly in mucosl linings of the respirtory nd gstrointestinl trct where they ct s erly detectors nd responders to ntigenic prticles.[16] Mst cells detect foreign invders using high ffinity surfce memrne Fcε receptors (FcεRI), which, when engged nd oligomerized y ntigen-ound immunogloulin E (IgE) ntiodies, medite cell ctivtion. This oligomeriztion ctivtes complex cscde of intrcellulr signls tht termintes in the extrcellulr ε relese of vesicle-stored ntimicroil nd inflmmtory grnules (e.g., β-hexosminidse (β-hex), histmines, nd others) in process known s degrnultion (see review y Gilfilln nd Tkczyk[17] for detils). In short, the extrcellulr region of the FcεRI α lph chin inds the Fc region of IgE, while the F region of IgE contins the ntigen. Once two FcεRI re crosslinked, memrne-ound nd receptor-ssocited Src-fmily kinses (e.g., LYN, SYK) initite signl trnsduction y shifting FcεRI to phosphorylted stte, y dding phosphte groups to the tyrosine residues of the immunoreceptor tyrosine-sed ctivtion motif (ITM) on the intrcellulr β nd γ-chin of the FcεRI til. Signl propgtion is further enhnced y downstrem ctivtion of clcium chnnels nd other signl kinses, such s mitogen-ctivted protein kinses (MPK) nd relted extrcellulr signl-relted kinses (ERK), to medite degrnultion of the cell (see Figure 1). The immortlized rt sophilic leukemi (RL-2H3) cell line is commonly used s model for in vitro studies of mst cell-like immune functions,[18, 19] including degrnultion. RL cells express endogenous FcεRI nd effectively ind IgE on their surfce in process clled sensitiztion. ε Figure 1. Schemtic representtion of the degrnultory signling cscde of RL-2H3 (dpted from refs.[17] nd[34]). Degrnultion occurs when FcεRI re cross-linked y ntigen-ound IgE, resulting in the recruitment of tyrosine kinses, tyrosine-protein kinse (LYN) nd spleen tyrosine kinse (SYK) to the trnsmemrne immunoreceptor tyrosine-sed ctivtion motif (ITMs) of the receptor. This results in the phosphoryltion of tyrosines within the trnsmemrne dptor molecule Linker for ctivtion of T cells (LT) tht coordintes the downstrem signling of multiple cytosolic dptor molecules, including, GR2-relted dptor protein (GDS), SH2-domin-contining leukocyte protein of 76 kd (SLP76), mong others not shown for simplicity. s well, the exchnge fctor, VV nd the signling enzyme, Phospholipse Cγ1 (PLCγ), which regultes clcium moiliztion nd ctivtion of Protein Kinse C (PKC), medite the exocytosis of degrnultory products, such s β-hexosminidse. VV lso ctivtes the RS-MPK (ERK) pthwy, which stimultes the production of trnscription fctors tht generte cytokines nd contriutes to degrnultion. Two experiments were designed to test the effects of NP exposures on vrious components of the degrnultory pthwy. Experiment 1 ws designed to test the effects of NP-exposed RL-2H3 cells degrnultion y mesuring: 1. IgE inding FcεRI (Figure 2,c,e), 2. MPK ERK phosphoryltion (Figure 3,c,e), nd 3. Exocytosis of degrnultory products (i.e., β-hexosminidse) (Figure 4,c,e). Experiment 2 tested the effects of NP-exposed IgE ntiodies on identicl endpoints of the degrnultory pthwy s were mesured in Experiment 1 (i.e., Figures 2 4,d,f) (2 of 12) wileyonlinelirry.com 215 The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim dv. Sci. 215, 2, 1514

3 Once sensitized, RL-2H3 cn e ctivted y dinitrophenyl (DNP)-humn serum lumin (HS), lignd tht stimultes RL-2H3 degrnultion y cross-linking IgE-primed FcεRI. [17 ] The propensity for NPs to ind nd lter the conformtionl structure nd iologicl function of proteins, [5,7 ] suggests tht they my lso lter the function of surfce memrne receptors, including FcεRIs. Hung et l. [2 ] showed tht DNP-lumin leled gold NPs directly stimulted FcεRI-medited responses y providing cross-linking of receptors. However, re gold prticles themselves did not ind to FcεRI, nor did they lter degrnultory ehvior. Intrcellulr signling (e.g., MPK pthwy) hs een demonstrted to e n effective mens to monitor FcεRI-medited interctions nd cellulr effector function in RL-2H3 cells. [21 ] Thus, the lignd-receptor-signl trnsduction xis cn e used to understnd how NPs lter immune function. In this study, we investigted the impcts of vrious commercilly ville P functionlized metl-oxide NPs (TiO 2, ZnO, CeO 2, nd Fe 2 O 3 ) on the degrnultory response of RL- 2H3 cells. Specificlly, our im ws to ssess the impct of P- NPs on the interction etween FcεRI nd IgE, nd on ctivted intrcellulr signls tht determine cell effector functions. further im ws to provide comprtive dt on cell function nd viility for cells exposed to NPs with distinct metl cores, ut possessing identicl primry prticle size nd P functionliztion. This llows the testing of our underlying hypothesis tht similr sized core NPs, functionlized with common coting, will provide similr cellulr response. 2. Results 2.1. Chrcteriztion of P-NP Spectrl Optics The opticl properties were mesured for ech P-NP to chrcterize their intrinsic sornce nd fluorescence. sornce for ech P-NP occurred t round 25 nm, with P-TiO 2 soring the highest t nerly 1..u. (Supplementry Figure 1, Supporting Informtion). None of the P-NPs displyed fluorescent properties, side from P-Fe 2 O 3, which emitted fluorescence (9 RFU) t round 35 nm, when excited t 25 nm (Supplementry Figure 1, Supporting Informtion) RL-2H3 Viility ws Reduced when Cells were Preexposed to P-TiO 2 fter 4 h, RL-2H3 viility ws significntly decresed to 88 ± 2.1% of control (1 ± 1.%), when exposed to 2 µg ml 1 P-TiO 2, which ws further decresed to 67 ± 8.8% of control (1.3 ±.4%) fter 24 h (Supplementry Figure 2, Supporting Informtion). t this sme time point, viility ws lso reduced to 87 ± 5.5% from exposures to 1 µg ml 1 P-TiO2. In contrst, exposure to P-ZnO, P-Fe 2 O 3, P-CeO2, nd P-Cp NPs hd no significnt effects on cell viility t concentrtions 2 µg ml 1 nd for exposures up to 24 h (Supplementry Figure 2 e, Supporting Informtion). No chnges in viility were oserved for concentrtions less thn 5 µg ml 1 over 24 h for ny of the P-NPs (dt not shown) P-TiO 2 -Exposed RL-2H3 Cells hd Reduced inding of IgE to FcεRI IgE inding to RL-2H3 FcεRI ws not significntly different thn controls when RL-2H3 cells were pre-exposed for 2 h to 2 µg ml 1 P-Cps nd P-CeO 2, followed y sensitiztion with IgE t 5, 1, 2 ng ml 1 (Figure 2,e). However, IgE inding ws significntly decresed (i.e., reltive shift in MFI) (35.33 ± 2.67%, ± 1.67%, nd 76 ± 1.%) t ll IgE concentrtions (5, 1, 2 ng ml 1, respectively) when preexposed to 2 µg ml 1 P-TiO2, compred to controls (44 ± 1.73%, ± 2.3%, 94 ±.%, respectively) (Figure 2 c). No differences in receptor inding were oserved for IgE concentrtions elow 5 ng ml 1 for ll P-NPs (dt not shown) inding of IgE to FcεRI ws Reduced when Pre-exposed to P-NPs When pre-exposed to single concentrtion (2 µg ml 1 ) of P-Cp or P-CeO 2 for 1 h, IgE ntiody inding (t 2 ng ml 1 ) to FcεRI significntly decresed (P-Cp: 77 ± 4.81%, P-CeO 2 : 8.25 ± 3.99%, respectively) reltive to pired controls (P-Cp: 88 ± 2.59%, P-CeO 2 : 9 ± 2.21%, respectively), while NP-exposed IgE t 5 nd 1 ng ml 1 were not different (Figure 2,f). y contrst, pre-exposure of IgE to single concentrtion of P-TiO 2 (2 µg ml 1 ), resulted in significntly reduced IgE inding t the lowest IgE concentrtion (5 ng ml 1 ) (13.76 ± 4.88%), reltive to unexposed pired control (32.29 ±.31%) (Figure 2 d). Exposure of 1 nd 2 ng ml 1 IgE to P-TiO 2 did not significntly chnge the percent of IgE ound to FcεRI. No differences in inding were oserved for IgE concentrtions elow 5 ng ml 1 for ll P- NPs (dt not shown). When single concentrtion of IgE (2 ng ml 1 ) ws pre-exposed for 1 h to 5, 1, nd 2 µg ml 1 P-Cps nd P-CeO 2, no significnt chnges in reltive IgE inding were oserved (Supplementry Figure 3, Supporting Informtion). However, the IgE inding decresed in dose-dependent mnner (68.33 ± 12.35%, ± 3.84%, nd 17 ±.58%, respectively), compred to unexposed control cells (1.3 ± 6.98%), when single concentrtion of IgE (2 ng ml 1 ) ws pre-exposed for 1 h to 5, 1, 2 µg ml 1 P-TiO2, (Supplementry Figure 3, Supporting Informtion). Het denturing of IgE (negtive control) resulted in negligile IgE inding (3. ± 3.%), indicting tht fluorescence from secondry IgG-PE ntiody required functionl IgE to e mesured y flow cytometry MPK ERK Phosphoryltion ws Reduced when Cells or IgE were Exposed to P-NPs Untreted control cells express reltively low levels of phosphorylted MPK ERK. The ddition of IgE t 5, 1 or 2 µg ml 1 induced dose-dependent phosphoryltion in vehicle-exposed cells for oth the NP-exposed cell nd NPexposed IgE experiments ( Figure 3 2 ). Cells tht were not sensitized with IgE ( ng ml 1 ) ut were exposed to NPs (2 µg FULL PPER dv. Sci. 215, 2, The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim wileyonlinelirry.com (3 of 12) 1514

4 FULL PPER % IgE inding 1 Cpsule - 2 µg/ml % IgE inding Vehicle contol Cpsule - 2 µg/ml * FL2 Fluorescence FL2 Fluorescence % IgE inding 1 TiO 2-2 µg/ml 8 6 * 4 * 2 * % IgE inding 1 Vehicle contol TiO 2-2 µg/ml * FL2 Fluorescence FL2 Fluorescence % IgE inding 1 CeO 2-2 µg/ml % IgE inding 1 CeO 2-2 µg/ml * FL2 Fluorescence FL2 Fluorescence Figure 2. Reltive percent expression of IgE ound on RL-2H3 cells. Pnels,c,e: RL-2H3 cells were pre-exposed to 2 µg ml 1 P-NPs (() cpsule control; (c) TiO 2 ; (e) CeO 2 ) for 2 h followed y IgE sensitiztion (5, 1, 2 ng ml 1 ). Pnels,d,f: IgE (5, 1, 2 ng ml 1 ) ws pre-exposed to P-NPs for 1 h prior to sensitiztion of unexposed RL-2H3 cells. s (cells or IgE; white rs in histogrm; lue plots in flow cytometry outputs) were exposed to ddh 2 O in plce of P-NPs (lck rs in histogrm; red plots in flow cytometry outputs). Secondry got-nti mouse IgG ntiody conjugted to phycoerythrin (PE) ws dded to sensitized cells nd fluorescence mesured y flow cytometry. Vlues from unexposed (NPs or IgE) controls were sutrcted from ll experimentl vlues nd dt re presented normlized to positive controls. Dt re mens ± SEM n = 5 6 independent experiments. * denote signifi cnt differences (NOV, p <.5) etween tretments t ech IgE concentrtion. ml 1 ) lso hd incresed phosphorylted MPK ERK for P- TiO 2 nd P-CeO 2 in oth experimentl pproches. This increse ws less pronounced when the P-Cps were used. Cells tht were pre-exposed to P-TiO 2 for 2 h prior to sensitizing with IgE hd significntly decresed phosphorylted MPK ERK t oth mid (1 ng ml 1 ) nd high (2 ng ml 1 ) IgE concentrtions (3.18 ±.8%, 3.25 ±.53%, respectively), reltive to vehicle-exposed cells t the sme IgE concentrtions (4.73 ±.45%, 5.27 ±.24%, respectively) (Figure 3 c). IgE t 5 ng ml 1 lso hd reduced MPK ERK phosphoryltion (3.12 ±.28%), lthough not significntly different thn pired vehicle control (4.26 ±.35%) (Figure 3 c). y contrst, cells tht were pre-exposed to P-CeO 2 nd P-Cps for 2 h did not hve significntly decresed phosphorylted MPK ERK t ech 1514 (4 of 12) wileyonlinelirry.com 215 The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim dv. Sci. 215, 2, 1514

5 Fold Induction of p44/42 ERK1/ Vehicle Cpsule - 2 µg/ml IgE concentrtion (ng/ml) Fold Induction of p44/42 ERK1/ Cpsule - 2 µg/ml IgE concentrtion (ng/ml) FULL PPER NP NP Fold Induction of p44/42 ERK1/ TiO 2-2 µg/ml IgE concentrtion (ng/ml) * * Fold Induction of p44/42 ERK1/ TiO 2-2 µg/ml * IgE concentrtion (ng/ml) NP NP Fold Induction of p44/42 ERK1/ CeO 2-2 µg/ml IgE concentrtion (ng/ml) Fold Induction of p44/42 ERK1/ CeO 2-2 µg/ml IgE concentrtion (ng/ml) NP NP Figure 3. Reltive percent induction of ERK1/2 p44/42 MP Kinse (MPK) pthwy in RL-2H3 cells. Pnels,c,e: RL-2H3 cells were pre-exposed to 2 µg ml 1 P-NPs (() cpsule control; (c) TiO 2 ; (e) CeO 2 ) for 2 h followed y IgE sensitiztion (5, 1, 2 ng ml 1 ). Pnels,d,f: IgE (5, 1, 2 ng ml 1 ) ws pre-exposed to 2 µg ml 1 P-NPs for 1 h prior to sensitiztion of unexposed RL-2H3 cells. s (cells or IgE;, white rs) were exposed to ddh 2 O in plce of P-NPs (+, lck rs). RL-2H3 cells were ctivted with M DNP-HS. Reltive percent chnge in MPK ERK phosphoryltion ws determined y correcting for the expression of endo-erk1/2 p44/42, then normlizing to unexposed negtive control cells. Dt re mens ± SEM n = 4 independent experiments. Different upper-cse letters denote signifi cnt differences (one-wy NOV, p <.5) etween IgE concentrtions for ech tretment group, followed y pirwise Tukey multiple comprison test. * denote signifi cnt differences (two-wy NOV, p <.5) within given IgE concentrtion, followed y onferroni multiple comprison test. IgE concentrtion (5, 1, 2 ng ml 1 ) (CeO 2 : 2.97 ±.33%, 3.87 ±.59%, 4.4 ±.76%; Cp: 3.17 ±.48%, 2.99 ±.38%, 3.86 ±.8%, respectively) reltive to vehicle-exposed cells t the sme IgE concentrtions (5, 1, 2 ng ml 1 ) (CeO 2 : 2.88 ±.53%, 3.27 ±.39, 4.26 ±.88%; Cp: 3.22 ±.36%, 3.77 ±.27%, 4.73 ±.52%, respectively) (Figure 3,e). When cells were sensitized with IgE pre-exposed to P- TiO 2, significnt decrese in phosphorylted MPK ERK ws oserved for 5 ng ml 1 IgE (1.74 ±.1%), with trend towrd decresed phosphoryltion for 1 ng ml 1 (2.57 ±.22%,) nd 2 ng ml 1 (2.26 ±.21%) IgE, reltive to vehicle-exposed cells t the sme IgE concentrtions (2.34 ± dv. Sci. 215, 2, The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim wileyonlinelirry.com (5 of 12) 1514

6 FULL PPER.18%, 2.54 ±.17%, 2.76 ±.18%, respectively) (Figure 3 d). Cells sensitized with P-Cp-exposed IgE lso hd decresed phosphorylted MPK ERK t ech IgE concentrtion (5, 1, 2 ng ml 1 ) (2.29 ±.38%, 2.78 ±.41%, 2.97 ±.59%, respectively), ut this decrese ws not significntly different thn vehicle-exposed IgE t the sme IgE concentrtions (2.53 ±.47%, 3.19 ±.53%, 3.68 ±.58%, respectively) (Figure 3). Similrly, the phosphorylted MPK ERK for cells sensitized with P-CeO 2 -exposed IgE ws lso slightly decresed (2.92 ±.1% 3.29 ±.24%, 3.26 ±.64%, respectively), reltive to vehicle-exposed IgE (3.6 ±.62%, 3.8 ±.7%, 4.6 ± 1.21%, respectively), t IgE concentrtions of 5, 1, 2 µg ml 1, respectively, ut this decrese ws lso not significntly different (Figure 3 f) RL-2H3 Cellulr Degrnultion ws Reduced when Cells were Pre-exposed to P-NPs Degrnultion decresed following P-NP exposures to RL- 2H3 cells, with the mgnitude dependent on NP type, concentrtion nd length of exposure ( Figure 4 ). For exmple, t 1 h, 2 µg ml 1 P-TiO 2 significntly decresed RL-2H3 degrnultion (51.1 ± 7.6%) (Figure 4 c), reltive to controls (94.4 ± 2.4%), however, exposures to P-Cps nd P- CeO 2 t the sme concentrtions did not (Figure 4,e). fter 4 h, degrnultion levels decresed to 7.3 ± 1.77% of control (95.1 ± 2.2%) from exposures to 2 µg ml 1 P-TiO2 (Figure 4 c). However, fter 24 h, the degrnultion vlues for P-TiO 2 were comprle s those oserved t 1 h. Similr dose- nd time-dependent reductions in degrnultion were oserved for P-CeO 2 over time (Figure 4 e). However, unlike P-TiO 2, the reduction continued to decrese with longer P-CeO 2 exposure periods up to 24 h, nd this occurred t doses tht did not ffect cellulr viility. Degrnultion lso significntly decresed from exposure to P-Cp, y pproximtely two fold for ll tested doses t 4 h (5 µg ml 1 : 54.3 ± 12.%, 1 µg ml 1 : 5.7 ± 14.%, 2 µg ml 1 : 63.4 ± 12.1%) (Figure 4 ). fter 24 h, degrnultion remined inhiited. Finlly, P-ZnO nd P-Fe 2 O 3 -exposed cells lso hd reduced degrnultion vlues (Supplementry Figure 4, Supporting Informtion), despite lso not eliciting reduction in viility t ny dose or exposure period. Exposures less thn 1 µg ml 1 did not significntly ffect degrnultion for ll NPs nd exposure periods (dt not shown) RL-2H3 Degrnultion ws rogted when IgE ws Pre-exposed to P-NPs Degrnultion ws rogted in RL-2H3 cells tht were sensitized with IgE (2 ng ml 1 ) pre-exposed to 5, 1, nd 2 µg ml 1 P-TiO 2 (5 µg ml 1 : ± 5.45, 1 µg ml 1 : ± 2.18, nd 2 µg ml 1 : 15. ± 1.23%), when compred to cells sensitized with unexposed IgE (1.8 ± 1.73%), (Figure 4d). s well, P-TiO 2 lowered the reltive relese of β-hex elow unsensitized negtive control cells (33 ± 3.48%). Cells sensitized with IgE pre-exposed to P-CeO 2, lso displyed significntly lower degrnultion t ech NP concentrtion (5 µg ml 1 : 7.25 ± 5.66%, 1 µg ml 1 : ± 5.66% nd 2 µg ml 1 : 79. ± 6.55%), ut to lesser extent thn P-TiO 2 (Figure 4 f). On the contrry, cells sensitized IgE pre-exposed to P-Cp (5, 1, 2 µg ml 1 ), did not hve decresed degrnultory responses (5 µg ml 1 : ± 5.66%, 1 µg ml 1 : 93.5 ± 5.24% nd 2 µg ml 1 : 97.5 ± 6.93%), reltive to unexposed cells (93. ± 9.23%) (Figure 4) RL-2H3 Degrnultion Recovers when P-TiO2 is Removed from Exposure Solution Despite significnt decrese in degrnultion fter 2 h exposure to 1 nd 2 µg ml 1 P-TiO 2 (41.33 ± 8.67% nd ± 1.93%, respectively), replcing P-TiO 2 with fresh MEM culture medi resulted in time-dependent return to control degrnultion levels for oth tested doses over 24 h ( Figure 5 ). The 24 h time point lso resulted in significntly more degrnultion for the 2 µg ml 1 dose (116.8 ± 14.1%), reltive to control cells (1.3 ± 1.97%). 3. Discussion 3.1. Generl Summry of Results This study demonstrtes tht P-NPs cn ffect the viility of mmmlin grnulocytes nd these effects were dependent on the P-NP core mteril, concentrtion, nd durtion of exposure. Our dt lso indicted tht degrnultion ws suppressed, to vrying extents, y ll P-NPs t sulethl doses, nd especilly y P-TiO 2. This my e due to decresed IgE inding y FcεRI nd/or decresed ctivtion of MPK ERK when the RL-2H3 cells were exposed to P-NPs. Together, these dt suggest tht P-NP exposure significntly impcts the cellulr degrnultion response nd y extension, grnulocyte-medited innte immune responses Effects of P-NPs on Cell Viility We hve demonstrted tht P-TiO 2 significntly ffected the viility of RL-2H3 cells (Supplementry Figure 2, Supporting Informtion), which ws similr response oserved in primry goldfish ( Crssius urtus ) neutrophils. [22 ] lthough P-NPs were more toxic to neutrophil viility thn to RL- 2H3 cells, similr trends ssocited with concentrtion, exposure durtion, nd prticle type were oserved. Results from previous studies show tht P-NPs hve stronger negtive chrge (rnging from 2 to 5 mv), smller primry prticle size (5 1 nm), higher dispersiveness nd lower ggregtion ehviors [1,22 ] thn their unfunctionlized metl-oxide counterprts in low ionic strength solutions. [23 ] These physico-chemicl properties hve een credited with lessening toxicity to firolst [3 ] nd humn neutrophils [24 ] tht is typiclly oserved when cells re exposed to unfunctionlized NPs using similr dosing (<1 µg ml 1 ) nd exposure (<48 h) conditions. [3 ] In prticulr, the strong negtive chrge of P-NPs is elieved to mitigte the negtive effects on cell viility, s there 1514 (6 of 12) wileyonlinelirry.com 215 The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim dv. Sci. 215, 2, 1514

7 % degrnultion g Cpsule exposure period (hours) 5 µg/ml 1 µg/ml 2 µg/ml % degrnultion Cells lone Cpsule concentrtion (µg/ml) FULL PPER % degrnultion TiO 2 exposure period (hours) 5 µg/ml 1 µg/ml 2 µg/ml % degrnultion Cells lone TiO 2 concentrtion (µg/ml) % degrnultion CeO 2 exposure period (hours) 5 µg/ml 1 µg/ml 2 µg/ml % degrnultion 12 1 c c c Cells lone CeO 2 concentrtion (µg/ml) Figure 4. Reltive percent degrnultion (β-hex relese) of RL-2H3 cells. Pnels,c,e: RL-2H3 cells were pre-exposed to 5, 1, 2 µg ml 1 P- NPs (() cpsule control; (c) TiO 2 ; (e) CeO 2 ) for 1, 2, 4 or 24 h followed y IgE sensitiztion (2 ng ml 1 ) for 1 h. Pnels,d,f: IgE (2 ng ml 1 ) ws pre-exposed to 5, 1, 2 µg ml 1 P-NPs for 1 h prior to sensitiztion of unexposed RL-2H3 cells. s (cells or IgE) were exposed to ddh 2 O in plce of P-NPs. Degrnultion ssy (excittion/emission: 36 nd 45 nm, respectively) regents were dded to experimentl groups following exposures. Vlues from unexposed controls were sutrcted from ll experimentl vlues nd dt re presented s normlized to positive controls (.1 ng ml 1 DNP-HS). Dt re mens ± SEM n = 5 6 independent experiments. Different lower-cse letters denote signifi cnt differences (two-wy NOV, p <.5) within n RL-2H3 exposure period for ech P-NP (pnels,c,e), followed y onferroni multiple comprison test. Different lower-cse letters denote signifi cnt differences (one-wy NOV, p <.5) etween exposed IgE (pnels,d,f), followed y pirwise Tukey multiple comprison test. would e less electrosttic interction nd disruption of negtively chrged phosphtidylcholine nd phosphtidylserine vesicles of the plsm memrne when exposed to ctionic NPs. [25 ] However, nionic P-NPs could still medite cell toxicity y intercting with lood proteins, creting protein corons (i.e., the protein dsorption lyer tht forms round NPs [26 ] ) tht influence the fte nd trnsport of NPs within circultion nd into the cells of nimls. Surprisingly, cell viility ws distinctly ffected y ech P-NPs, despite hving identicl surfce cotings. The most significnt decreses were oserved with P-TiO 2, which my e relted to the smller sized ggregtes tht form when in dv. Sci. 215, 2, The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim wileyonlinelirry.com (7 of 12) 1514

8 FULL PPER % degrnultion TiO 2-1 µg/ml TiO 2-2 µg/ml solution fter 24 h when compred to the other P-NPs. [22 ] This would increse the numer of single P-TiO 2 NPs in suspension nd the ville surfce re to dversely interct with cells. Other considertions my include the mount of P polymer formed on the surfce of the NP. During synthesis, the interction etween the inorgnic metl ions nd the P polymers is chrge-dependent process. Thus, ZnO my ttrct more P thn TiO 2, for exmple, nd form thicker polymer cots tht could msk dverse core mteril-medited effects such s toxicity. The effects on viility were likely not relted to the relese of core metl ions s the dissolution of P-NPs hve not een demonstrted in ultrpure wter, [1 ] or in simulted iologicl solutions. [23 ] s well, viility did not decrese over 24 h when RL-2H3 cells were exposed to Ti, Zn, Fe, nd Ce ions (dt not shown) t dilyzed concentrtions previously determined for P-NPs. [1 ] 3.3. Effects of P-NPs on Cellulr Degrnultion P-NPs (P-CeO 2, P-Fe 2O 3, P-Cps) lso significntly inhiited cellulr degrnultion t concentrtions tht did not ffect viility t ny P-NP dose or exposure durtion when RL-2H3 cells were pre-exposed. The pttern of inhiition ws lso NP-specific. For exmple, P-TiO 2 ws the only P-NP tht exhiited dose nd time-dependent effect when cells were exposed, while P-ZnO, P-Cps, nd P-Fe 2 O 3 hd more sustined inhiitory effects over 24 h. Exposure to P-CeO 2 resulted in delyed effect, with inhiition occurring fter 4 nd 24 h. These differences re likely relted to chnges in their physico-chemicl properties over time, when in suspension. [22 ] Recovery time (h) fter 2h TiO 2 exposure Figure 5. Reltive percent degrnultion (β-hex relese) of RL-2H3 cells exposed to 1 µg ml 1 or 2 µg ml 1 P-TiO2 for 2 h. Exposure solutions were replced with fresh cell medi for 2, 4, nd 24 h recovery periods, followed y sensitiztion with IgE (2 ng ml 1 ). s were exposed to ddh 2 O in plce of P-NPs. Degrnultion ssy (excittion/emission spectr: 36 nd 45 nm, respectively) regents were dded to experimentl groups following ech recovery time point. Vlues from unexposed controls were sutrcted from ll experimentl vlues nd normlized to positive controls (.1 ng ml 1 DNP-HS). Dt re mens ± SEM n = 5 6 independent experiments. Different lower cse letter denote signifi cnt differences (two-wy NOV, p <.5) for ech recovery durtion period, followed y onferroni multiple comprison test. Different upper-cse letters denote signifi cnt differences (one-wy NOV, p <.5) etween recovery periods for ech exposure concentrtion, followed y pirwise Tukey multiple comprison test. For exmple, over 24 h period, discrete NP ggregtes (>15 nm) formed for P-CeO 2 nd P-ZnO, [22 ] signifying tht NP surfce res tht were ville to interct with memrne receptors lso decresed over time. We elieve tht this my hve een responsile for the mitigtion of the effect on degrnultion in these mterils. P-TiO 2 on the other hnd ws highly monodispersed NP tht hd the smllest-sized ggregtes s mesured y DLS (see Supplementry Tle 1, Supporting Informtion). P-TiO 2 lso demonstrted the most immedite effects on degrnultion suggesting tht the incresed surfce re to volume rtio ville for P-TiO 2 NPs my hve incresed the numer of individul prticles tht could interct with FcεRI, without indvertently cross-linking nd ctivting receptor-medited degrnultion, which hs een previously shown with DNP-coted gold NPs. [2 ] With n estimted FcεRI per cell, the distnce etween djcent receptors is pproximtely 32.6 nm. [2 ] s such, lrger NPs (>2 nm) tht possess ound cell-ctivting lignd, such s DNP, could indvertently increse degrnultion vi incresed receptor oligomeriztion, [2 ] while smller NPs, such the Pcoted ones (<1 nm) used in this study, would e less likely to do so. Thus, P-NP ggregtion sttes likely ply n importnt role in regulting RL-2H3 degrnultion, which is influenced y core mteril properties, despite hving identicl surfce functionliztion. Further experimenttion would e required to decipher the precise detils of these interctions ut our results support this notion. Our findings re in greement with other studies of innte grnulr cells following NP exposures to primry humn mst cells, [ 27 ] primry murine peritonel mst cells, [ 28 ] primry fish neutrophils, [ 29 ] nd RL-2H3 cells. [ 14,2 ] Conversely, other studies hve reported tht some NPs (re TiO 2 ) ugment degrnultion y directly ctivting L -type voltgegted C 2+ chnnels y stimulting the influx of extrcellulr C 2+, triggering signling cscdes tht ctivte histmine relese. [ 18 ] re TiO 2 NPs cn lso ct s n djuvnt in the genertion of ovlumin-specific IgE nd IgG ntiodies, [ 15 ] which in turn could sensitize mst cells to ugment future llergic rections Plusile Explntions for Suppressed Degrnultion There re reports tht cron-sed fullerenes suppress mst cell degrnultion y scvenging free rdicls, therey reducing cellulr rective oxygen species (ROS) levels required to ctivte degrnultion. [27 ] It hs lso een proposed tht C 6 fullerenes inhiit degrnultion in mst cells y competitively preventing grnule relese from SNRE-ound secretory vesicles, [14 ] whilst Murer-Jones et l., [28 ] hve suggested tht cell sored SiO 2 NPs (25 nm) sufficiently interfere with cytoskeletl nd memrne-fusion mchinery to prevent the exocytosis of cellulr grnules. We cnnot discount the possiility tht internliztion of P-NPs through memrne recycling events results in disruption of the degrnultion responses. Internliztion of NPs in immune cells hs een shown to occur through clthrin nd nonclthrin medited endocytosis. [3 ] However, given tht pre-exposing IgE to P-NPs prior to sensitizing unexposed cells reduces memrne inding nd degrnultion, 1514 (8 of 12) wileyonlinelirry.com 215 The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim dv. Sci. 215, 2, 1514

9 suggests tht cell responses cn lso e inhiited t the plsm memrne level. Future studies will focus on the potentil for internliztion of these P-NPs nd the possiility tht disruption of intrcellulr processes involved in degrnultion lso contriute to the oserved inhiitory effects. In our study, we elieve tht suppression of the degrnultion response occurs when receptor-medited events re ostructed. The size rchitecture of NPs hs een reported to ply significnt role in the ctivtion or suppression of degrnultion in mst cells where DNP-coted gold NPs lrger thn 19.8 nm, cross-linked FcεRI resulting in incresed β-hex secretion. However, when these NPs were less thn 19.8 nm, cross-linking did not occur nd less β-hex ws relesed. [2 ] Given tht the primry prticle dimeters of the P- NPs tested in this study re etween 3 nd 1 nm [1,22 ] nd hve high negtive chrge, [22 ] we investigted the hypothesis tht these smller mterils my still ind FcεRI nd stericlly ostruct IgE from properly ttching to the receptor nd therefore inhiit the degrnultory response. Our dt showed tht cells pre-exposed to P-TiO 2 hd significnt reductions in IgE inding, reltive to control, suggesting tht P-TiO 2 ltered the ttchment of IgE to FcεRI. These findings confirm the results of Hung et. l., [2 ] in demonstrting tht NPs cn interct directly with FcεRI nd lso extend these results y reporting tht even smller prticles (3 1 nm) cn inhiit cell effector functions Effects of P-NPs on Cellulr MPK ERK Phosphoryltion In support of our findings, ntigen-induced phosphoryltion of the receptor signling trget, MPK ERK, ws significntly reduced when cells were pre-exposed to P-NPs implying tht ltertions in IgE inding occurred. MPK ERK re receptor signling kinses tht direct the regultion nd ctivtion of diverse rry of cellulr processes, including prolifertion, differentition, nd poptosis, s well s severl pro-inflmmtory functions (cytokine nd eicosnoid production, phgocytosis, degrnultion) in mst cells. [31 ] MPKs, such s ERK, re ctlyticlly inctive t resting stte, ut when phosphorylted from upstrem signling pthwys initited from FcεRI suunits such s LYN, they ecome ctivted to regulte cell functions. [17 ] Thus, our results support the hypothesis tht prtil reductions in MPK ERK phosphoryltion contriute to decresed RL- 2H3 degrnultion when RL-2H3 cells were pre-exposed to P-NPs. Similrly, when IgE ws pre-exposed to P-TiO 2 nd P- CeO 2, our results demonstrted tht the efficcy of IgE to sensitize RL-2H3 cells nd medite degrnultion vi MPK ERK signling pthwys ws reduced. This ws prticulrly true for P-TiO 2 exposures t lower IgE concentrtions (5 ng ml 1 ), where distinct seprtion in the inding of IgE for FcεRI ws oserved (Figure 2d). When IgE ws first pre-exposed to P- TiO 2, this tretment rogted degrnultion t ll tested doses (Figure 4 d) nd lso elow control levels in non-treted cells, indicting tht P-TiO 2 lso inhiited sl relese of β-hex. Interestingly, the mgnitude of decresed MPK ERK phosphoryltion ws similr regrdless of whether RL-2H3 cells or IgE were pre-exposed to P-TiO 2 suggesting tht the modifiction of RL-2H3 degrnultion y P-NPs is likely medited vi the FcεRI. Tken together, our results demonstrte tht P-TiO 2 - exposed IgE reduced receptor inding cpcity, therey decresing intrcellulr signling (MPK ERK) nd ultimtely reducing or rogting the relese of grnulr products (degrnultion) tht help promote pro-inflmmtory responses in vertertes Implictions of P-NP Immune Modultion The recovery of the degrnultion response following the replcement of NP-exposure medi with NP-free medi demonstrtes tht the suppressive effects of P-NPs re not permnent. P-NPs were likely loosely ound to FcεRI nd the dynmic interction etween the receptor, other proteins in solution, nd the P-NPs needs further investigtion. We hypothesize tht P-NPs detched from the receptor following removl of NP-exposure medi, therey llowing for IgE to ind FcεRI nd the degrnultory process to e restored. This hypothesis is consistent with the concept of soft nd hrd protein corons in which proteins tht hve lower ffinities for NPs initilly ind the NP, ut re replced y proteins with higher ffinity for the NPs, creting the hrd coron. [26,32 ] The implictions could e significnt if vitl circultory proteins, such s immunogloulins (e.g., IgE, IgG), form prt of the NP-protein coron nd re prevented from executing their iologicl roles. For exmple, we hve previously demonstrted tht lctte dehydrogense (LDH) ctivity decreses when exposed to metl NPs y ltering the conformtionl structure of the protein. [5,7 ] If nimls re exposed to P-sed mterils either directly s therpeutic or indvertently through environmentl exposure, this my result in diminution of the functionl ctivity of IgE medited responses, nd therey impede the ctivtion of immune function nd ltertion of their ntimicroil ctivity. Future reserch should focus on determining which lood proteins re ound y vrious NP formultions nd whether this will impct the immune response of cells nd orgnisms. 4. Conclusion P-NPs inhiited ntigen-induced RL-2H3 degrnultion t sulethl doses. The decresed degrnultion ws medited y ffecting the ttchment of IgE for its endogenous FcεRI, resulting in the diminished phosphoryltion of MPK ERK, nd diminished RL-2H3 degrnultion. Furthermore, ech P-NPs hve een shown to distinctly modulte oth viility nd degrnultion, despite hving the sme surfce functionliztion nd primry prticle sizes. These distinct differences in responses my e relted to oserved differences in their chrge nd gglomerting properties, prticulrly over time. [22 ] These results further demonstrte tht understnding physicochemicl properties of NPs ply importnt roles in determining effector functionl responses of the innte immune system. Future studies should focus on the physico-chemicl properties of P-NPs tht modulte specific cellulr toxicity nd degrnultion in immune cells. FULL PPER dv. Sci. 215, 2, The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim wileyonlinelirry.com (9 of 12) 1514

10 FULL PPER 5. Experimentl Section Polymer-Coted Metl-Oxide Nnoprticle nd Chrcteriztion Informtion : The functionlized derivtives used in this study consisted of P-coted metl oxide NPs, including TiO 2 (rutile), ZnO, CeO 2, Fe 2 O 3 tht were mnufctured y Vive Crop Protection Inc. (formerly Vive Nno Inc.) (Toronto, ON, Cnd) nd were kindly donted for this study. lso included in the study were P nnocpsules (Cps) tht do not hve metl core. These were used s coting control for oserved effects of the metl-oxide NPs. Working concentrtions were mde from primry stock NPs (1 g L 1 in ddh 2 O) y diluting with ddh 2 O. ll stocks were stored t 4 C nd protected from light. The methods used to synthesize re descried y Felix et l. [1 ] We hd conducted extensive chrcteriztion of stock P-NPs s previously reported. [6,1,22 ] These included hydrodynmic dimeter, polydispersity index, nd zet potentil t nd 24 h for working stock solutions t concentrtions etween 1 nd 2 µg ml 1 in ddh 2 O, plus trnsmission electron microscopy (TEM) imges for ech P-NP. These dt re reported previously, [22 ] nd is lso presented in Supplementry Tle 1. dditionlly, primry prticle size, ph, metl purity, nd percent free metl relesed t.5 nd 72 h s reported y Felix et l. [1 ] is summrized in Supplementry Tle 2, Supporting Informtion. The reported NP core sizes were mesured etween 3 nd 9 nm using TEM (Supplementry Figure 5, Supporting Informtion) nd the hydrodynmic sizes in suspension were pproximtely etween 5 nd 1 nm. [22 ] Only Fe 2 O 3 hd lrger ggregtes t pproximtely 14 nm, likely due to their mgnetic properties. [22 ] Importntly, P-NPs with ll the cores remin highly monodispersed over 24 h nd relese only trce mounts of free metl ions over 72 h, with the mjority of ions relesed in the fi rst 3 min. [1 ] Metl purity is ove 98% for ll P-NP, side from CeO 2, with the remining contminting metl eing the N + stilizer used during the synthesis process. To determine the intrinsic sornce nd fluorescence of ech P-NP, sornce nd fluorescence spectrum mesurements were performed nd plotted (Supplementry Figure 1,, Supporting Informtion). Ech P-NP were diluted in ddh 2 O to 5 µg ml 1 nd loded into qurtz cuvette. n sornce spectrum from 19 to 82 nm (Hewlett Pckrd 8452 diode rry spectrophotometer) nd fluorescence emission spectrum up to 11 nm (excittion 25 nm, Cry Eclipse photoluminescence spectrometer) were then recorded. RL-2H3 Cell Line : The rt sophil RL-2H3 cell line ws grown t 37 C with 5% CO 2 in culture medi consisting of miniml essentil medium (MEM) with Erle s lnced slt solution (Sigm-ldrich, Cnd) supplemented with 1% M L -glutmine, 1% penicillin/ streptomycin, nd 1% het-inctivted FS s descried y Cortes et l. [21 ] Cells were pssed every third dy y hrvesting cells in n RL-2H3 hrvest uffer ( M EDT, M NCl, M HEPES, M KCl, ph 7.4) t 37 C with 5% CO 2 for 1 min, followed y pipetting to detch cells from cell culture plte (D iosciences, Mississug, Cnd). Cells were seeded into new flsks t su-cultivtion rtio of 1:1. Exmintion of P-NP Exposure on RL-2H3 Cell Viility Using Flow Cytometry : Flow cytometry (eckmn Coulter Qunt SC, Mississug, Cnd) ws used to mesure the effects of NP exposure on cell viility, with propidium iodide (PI) used s fluorescent mrker for cell deth. PI penetrtes dmged memrnes of necrotic nd/or lte poptotic cells nd intercltes with nucleic cids to enhnce its fluorescence 2 3 times. [33 ] The ssy ws performed y growing cells to confluence over 3 d in RL-2H3 culture medi. Cells were hrvested s descried ove nd enumerted with Trypn lue stining solution (Sigm- ldrich, Cnd) on hemocytometer to ensure cultures hd >95% vile cells nd to determine the numer of cells in the culture. Following enumertion, cells were resuspended in fresh culture medi nd seeded in 96-well flt-ottom culture pltes (Corning Costr, US) t cells per well for NP exposure periods less thn 24 h. Cells were then incuted for 1 h t 37 C to llow for cell ttchment to wells. For viility experiments, RL-2H3 cells were exposed to one of fi ve P-NPs (TiO 2, CeO 2, ZnO, Fe 2 O 3, Cps) t concentrtions of 1, 1, 5, 1, 2 µg ml 1 for exposure periods of 1, 2, 4, nd 24 h t 37 C. cells received n equl volume of sterile doule deionized (dd) wter, while no dditionl solutions were provided to unexposed control cells. Following ech exposure, cell culture medi ws spirted from wells nd hrvest uffer ws dded to ech well. Cells were hrvested s ove nd trnsferred to 1.5 ml centrifuge tues nd wshed twice in phosphte uffered sline (PS) y centrifugtion t 4 g for 7 min to remove cell medi nd NPs. The cell pellets were gently disrupted nd resuspended in PS/PI (1 µg ml 1 ) mixture nd nlyzed y flow cytometry (FL2 fi lter) for indictions of cell deth y monitoring for increses in PI fluorescence, nd for chnges in cell profi le outputs, reltive to unexposed control cells. Cell viility ws determined y clculting the percentge of living cells tht did not fluoresce for PI (i.e., not ded or dying) to the unexposed control popultion using the following eqution [(percent vile of experimentl tretment/percent vile of unexposed control cells) 1]. Experimentl pproches to Exmine P-NP Effects on the Degrnultory Signling Cscde of ctivted RL-2H3 Cells : Figure 1 depicts the two experimentl pproches used in this study to exmine the effects of NPs on RL-2H3 degrnultion. The fi rst pproch preexposed RL-2H3 cells to P-NPs, while the second pproch preexposed IgE ntiodies to P-NPs. For oth pproches, mesured endpoints included: cellulr degrnultion, IgE receptor inding, nd MPK signling. The following sections descrie the mterils nd methods for ech mesured endpoint. RL-2H3 Degrnultion Experiments: Exmintion of P-NP Exposures on RL-2H3 Degrnultion : Cellulr ctivtion nd ws ssessed vi the β-hexosminidse relese ssy. [19 ] The ssy ws performed y seeding RL-2H3 cells per well, suspended in cell culture medi, into 96-well flt-ottom plte (Costr). Cells were then incuted for 1 h t 37 C to llow for cell ttchment, followed y triplicte dosing of ech P-NP (TiO 2, CeO 2, ZnO, Fe 2 O 3, Cps) t concentrtions of 1, 5, 1, 2 µg ml 1 for 1, 2, 4, nd 24 h t 37 C. Tretment groups for these experiments included:, which received n equl volume of sterile ddh 2 O; Positive control, which included IgE-sensitized nd DNP-HS stimulted group, nd received n equl volume of cell culture medi; nd, Negtive control, which lso received n equl volume of cell culture medi lone. Following tretments, exposure medi were removed y spirtion nd tretment groups were wshed in Tyrode s uffer ( M HEPES, M NCl, M CCl 2, M D-glucose, M NHCO 3, M NH2PO 4, MgCl2, S.1%, ph 7.4). 2 ng ml 1 of mouse nti-dnp IgE m (Sigm-ldrich, Cnd) ws then dded to P-NP exposed, vehicle control nd the positive control groups for 1 h t 37 C, while Tyrode s uffer lone ws dded to negtive control groups. Following incution, ll solutions were removed nd cells were gin wshed with Tyrode s uffer, nd Tyrode s uffer contining.1 ng ml 1 DNP-HS (ioserch Technologies Inc. US) ws dded to P-NP exposed, vehicle control, nd positive control groups, while Tyrode s uffer lone to negtive control group. ll groups were incuted for 1 h t 37 C. Following incution, the levels of β-hex relesed y the RL- 2H3 cells were determined y incuting the cellulr superntnts with M of 4-methylumelliferyl N-cetyl-β- D -glucosminide s descried y Nl. [19 ] Perkin-Elmer microplte reder (36 nm excittion; 45 nm emission) nlyzed smple fluorescence. Reltive fluorescence units (RFU), signifying the cellulr relese of β-hex, were represented s normlized vlues y setting positive control cells to 1%. The reltive relese for ll smples ws then clculted using the following eqution [(RFU of experimentl tretment RFU of negtive control)/rfu of IgE positive control cells RFU of negtive control) 1]. Exmintion of RL-2H3 Degrnultory Recovery : RL-2H3 cells were exposed s descried ove to 1 nd 2 µg ml 1 P-TiO2 for 2 h. The P-TiO 2 exposure medi ws then replced with fresh culture medi nd degrnultion mesured t, 2, 4, nd 24 h postreplcement, to test if the P-NPs hd permnent or temporry effect on RL-2H3 degrnultion. The degrnultion ssy ws conducted s descried ove. For ech recovery time point, cells tht did not hve the P-TiO 2 exposure medi removed were mesured for degrnultion 1514 (1 of 12) wileyonlinelirry.com 215 The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim dv. Sci. 215, 2, 1514

11 to ensure tht the responses were consistent with the degrnultion experiments descried in Section 2.5. Exmintion of P-NPs on IgE-medited Degrnultory Properties : Degrnultion ws ssessed on RL-2H3 cells using IgE ntiodies tht were first pre-exposed to P-TiO 2, P-CeO 2, nd P-Cps. IgE ntiodies (2 ng ml 1 ) were pre-exposed on ice for 1 h with vrying P-NP concentrtions (5, 1, 2 µg ml 1 ) prior to sensitizing cells for 1 h t 37 C. n equl volume of sterile ddh 2 O ws used s vehicle control. The degrnultion ssy ws then conducted s descried ove. IgE ntiody inding Experiments: Exmintion of P-NP Exposures on IgE inding to the Surfce of RL-2H3 Cells : Three seprte experiments were conducted to determine whether IgE receptor inding chnged in the presence of P-NPs. In the fi rst experiment, RL-2H3 cells were hrvested nd enumerted s descried ove nd seeded in 96-well culture pltes t densities of cells per well nd llowed to dhere. Cells were then exposed for 2 h t 37 C to P-Cps, P-TiO 2 or P-CeO 2 t 2 µg ml 1, nd pired to ddh 2 O s vehicle controls. Following exposure, cells were wshed with PS nd sensitized with IgE suspended in MEM cell culture medi t fi ve concentrtions (12.5, 25, 5 1, 2 ng ml 1 ), or with MEM lone s negtive control. Cells were then incuted for 1 h t 37 C. Following sensitiztion, cells were wshed with ice-cold PS to remove unound IgE, nd 1. µg of phycoerythrin (PE)- conjugted secondry ntiody (got nti-mouse IgG (H+L) PE, Sigm-ldrich, Cnd) ws dded to ech tretment group nd incuted on ice, in the drk for 3 min. n IgG secondry lone control group received 1. µg IgG-PE ntiody. Following incution, cells were wshed with PS nd hrvested s descried ove nd resuspended in Tyrodes uffer for nlysis of PE fluorescence using flow cytometry. Men fluorescence intensity (MFI) vlues were ssigned to ech tretment group to quntify differences. The second nd third experiments tested the inding of IgE to the FcεRI with nd without prior exposure to P-NPs. The second experiment exposed single IgE concentrtion (2 ng ml 1 ) for 1 h to P-TiO 2, P-CeO 2, nd P-Cp t three concentrtions (5, 1, 2 µg ml 1 ), to determine whether IgE inding would chnge with incresing concentrtions of P-NPs. n equl volume of ddh 2 O ws used s vehicle control nd dentured IgE (9 C for 2 min) ws used s negtive control. Conversely, the third experiment exposed vrying IgE concentrtions (12.5, 25, 5, 1, 2 ng ml 1 ) to single concentrtion (2 µg ml 1 ) of P-TiO 2, P-CeO2, nd P-Cp, or ddh 2 O (vehicle control) to determine the effects on IgE inding t lower sensitizing IgE concentrtions, reltive to unexposed IgE. For these experiments, RL-2H3 cells were seeded s descried ove nd sensitized with one of the P-NP-exposed IgE tretments for 1 h t 37 C. Following sensitiztion, cells were wshed with ice-cold PS uffer to remove unound IgE, nd 1. µg of PE-conjugted IgG secondry ntiody ws dded to ech tretment group nd incuted on ice, in the drk for 3 min. IgG secondry lone control groups received 1. µg IgG-PE. Following incution, PE fluorescence ws nlyzed using flow cytometry s descried ove. ssessment of P-NP Exposures on RL-2H3 MPK ERK1(p44)/ ERK2(p42) Phosphoryltion : RL-2H3 MPK ERK ws used s signl trnsduction iomrker in two experiments to ssess chnges in cell ctivtion when exposed to P-NPs. In the first experiment, cells were seeded t RL-2H3 cells per well into flt-ottom 6-well pltes (Corning Costr, US) nd incuted for 2 d t 37 C in culture medi to llow for confluent growth ( cells per well). Cells were then incuted with or without P-TiO 2, P-CeO 2 or P-Cp t 2 µg ml 1 in culture medi for 2 h t 37 C. n equl volume of sterile ddh 2 O ws used for vehicle controls. Negtive control cells were left undistured. Tretments were then removed nd cells were wshed twice in PS prior to sensitiztion for 1 h in culture medi to 5, 1 or 2 ng ml 1 IgE for oth NP-exposed nd vehicle control cells. Negtive control cells did not receive IgE. DNP-HS (.1 ng ml 1 ) ws then dded to IgEsensitized cells for 8 minutes to cross-link the receptors nd ctivte the cells. The second experiment ws designed identiclly s ove, however IgE ntiodies (2 ng ml 1 ), insted of cells, were pre-exposed with or without P-TiO 2, P-CeO 2, or to P-Cps (2 µg ml 1 ) for 1 h nd then used to sensitize the cells. For vehicle control tretments, IgE were pre-exposed to n equl smll volume of sterile ddh 2 O. In oth experiments, cells ctivted with DNP-HS were wshed twice with PS nd lysed with 2 µl of ice-cold lysis uffer (1% Triton X-1, M NCl, M Tris, phosphtse, nd protese inhiitors (Roche from Moleculr iology Services Unit, ph 8.) using mechnicl disruption. Cell lysttes were then prepred for Western lotting techniques y diluting 1:1 with 2 Lemmli reducing uffer (.5 M Tris, 1% glycerol, 1% SDS. 1% romophenol lue, 5% β-mercptoethnol), oiled t 95 C for 1 min, nd then seprted on SDS-PGE gel (1% crylmide, 1% SDS, 1.5 M Tris seprting uffer). Proteins were then trnsferred to nitrocellulose memrnes for 1 h t 1 V nd lotted with nti-erk1(p44)/erk2(p42) MPK (L34F12) nd nti-phospho-p44/ p42 MPK (Erk1/2) XP mouse m purchsed from Cell Signling Technologies (US) t finl dilution of 1:4 (v/v). Immunorective protein nds were detected using got nti-rit IgG (H+L) HRPconjugted p (io-rd) t finl dilution of 1:5 (v/v) nd visulized on uthordiogrphy film (GE Helthcre) using the SuperSignl West Pico Chemiluminescent Sustrte kit (Pierce iotechnology). nlysis of nd intensity ws performed using ImgeJ v1.47, which ws downloded from Sttistics : Sttisticl nlyses were performed using the GrphPd 6. sttisticl softwre progrm. To investigte effects of NPs on cell viility, IgE receptor inding, nd degrnultion from NP-exposed cells, two-wy nlysis of vrince (NOV) with onferroni multiple comprison test ws performed. To investigte effects of NPs on cellulr MPK ERK phosphoryltion nd degrnultion from NP-exposed IgE, one-wy NOV with pirwise Tukey multiple comprison test ws performed. proility of p <.5 ws considered signifi cnt. Dt vlues re presented s men ± stndrd error on the men (SEM). Supporting Informtion Supporting Informtion is ville from the Wiley Online Lirry or from the uthor. cknowledgements This work ws supported y grnts from Nturl Sciences nd Engineering Reserch Council (NSERC) of Cnd to J.L.S. nd G.G.G. nd the NNNI Environment Cnd, nd NRC Nnotechnology Inititive (Grnt No. RES2319) to J.L.S. nd G.G.G. s well s the lert Innovtes Technology Futures nnoworks collortive reserch greement (CR Reference No. 2965) to G.G.G. V..O. ws supported y NSERC post-grdute doctorl scholrship, Queen Elizeth II Grdute Scholrship nd lert Innovtes Helth Solutions Grdute Studentship. J.D.E. ws supported y n NSERC Vnier Cndin Grdute Scholrship nd n lert Innovtes Technology Futures Grdute Student Scholrship. uthors thnk Dn Snell, Dr. Dnut Chmot, Dr. enjmin Montgomery, nd Christin Lwford for technicl nd/or editoril ssistnce. uthors lso thnk Vive Crop Protection nd Dr. Drren nderson for their generous provision of P-NPs. Received: Mrch 25, 215 Revised: June 7, 215 Pulished online: July 14, 215 [1] D. Y. Li, Wiley Interdiscip. Rev.: Nnomed. Nnoiotechnol. 212, 4, 1. [2] J. P. M. lmeid, E. R. Figuero, R.. Drezek, Nnomedicine 213, 1, 53. [3] M. Hmzeh, G. I. Sunhr, Toxicol. In Vitro 212, 27, 864. FULL PPER dv. Sci. 215, 2, The uthors. Pulished y WILEY-VCH Verlg GmH & Co. KG, Weinheim wileyonlinelirry.com (11 of 12) 1514