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1 Enhned detetion of myeloperoxidse tivity in deep tissues through luminesent exittion of ner-infrred nnoprtiles Ning Zhng 1,3, Kevin P Frnis 1, Arun Prksh 2 & Dniel Ansldi 1 npg 213 Nture Ameri, In. All rights reserved. A previous study reported the use of luminol for the detetion of myeloperoxidse (MPO) tivity using optil imging in infiltrting neutrophils under inflmmtory disese onditions. The detetion is bsed on photon-emitting retion between luminol nd n MPO metbolite. Beuse of tissue bsorption nd sttering, however, luminol-emitted blue light n be effiiently deteted from superfiil inflmmtory foi only. In this study we report hemiluminesene resonne energy trnsfer (CRET) methodology in whih luminol-generted blue light exites nnoprtiles to emit light in the ner-infrred spetrl rnge, resulting in remrkble improvement of MPO detetbility in vivo. CRET used 37-fold inrese in luminesene emission over luminol lone in deteting MPO tivity in lung tissues fter lipopolyshride hllenge. We demonstrted dependene of the hemiluminesent signl on MPO tivity using MPO-defiient mie. In ddition, o-dministrtion of 4-minobenzoi id hydrzide (4-ABAH), n irreversible inhibitor of MPO, signifintly ttenuted luminesent emission from inflmed lungs. Inhibition of nitri oxide synthse with nonspeifi inhibitor, l-name, hd no effet on luminol-medited hemiluminesene prodution. Pretretment of mie with MLN12B, seletive inhibitor of IKK-2, resulted in suppression of neutrophil infiltrtion to the lung tissues nd redution of MPO tivity. We lso demonstrted tht CRET n effetively detet MPO tivity t deep tissue tumor foi due to tumor development ssoited neutrophil infiltrtion. We developed sensitive MPO detetion methodology tht provides mens for visulizing nd quntifying oxidtive stress in deep tissue. This method is menble to rpid evlution of nti-inflmmtory gents in niml models. MPO is heme-ontining enzyme tht is bundnt in neutrophils nd mrophges nd funtions s key meditor of the phgoyti oxidtive burst nd inflmmtory proess 1. MPO tlyzes the onversion of hydrogen peroxide nd hloride ions into hypohlorous id, whih is one of the most retive oxidnts produed in vivo 2. Hypohlorous id oxidizes lipids, proteins nd DNA nd thus promotes tissue injury nd inflmmtion 3 during the pthologil progress of hroni obstrutive pulmonry disese 4 (COPD) nd theroslerosis 5. Cirulting onentrtions of MPO hve been shown to predit risk for mjor dverse rdi events. In ddition, mounts of MPO-derived hlorinted ompounds hve been used s speifi biomrkers for disese progression 6. There hs been onsiderble interest in the development of therpeutilly useful MPO inhibitors. A previous study showed tht 4-ABAH irreversibly inhibits hypohlorous id prodution s suiide substrte of MPO 7. A mens of monitoring MPO tivity under in vivo onditions should help estblish the reltionship between MPO indution nd pthologil progression nd filitte prelinil evlution of MPO inhibitors. Noninvsive imging of MPO enzymti tivity with luminol ws previously reported 8. These reserhers found tht luminol speifilly interts with hypohlorous id to produe n emission of luminesent light tht n be deteted through optil imging. When we pplied luminol to severl inflmmtory disese models, we found tht the detetion ws the most sensitive for superfiilly loted inflmmtory foi but ws muh less sensitive, nd ws undetetble in mny ses, for deep tissue inflmmtion. A lk of sensitivity for the detetion of deep tissue inflmmtion is the result of the poor penetrtion pbility of luminol-emitted short-wvelength light (λ mx = 425 nm) through niml tissues. It is well estblished tht photons in the ner-infrred (NIR) region (prtiulrly 7 9 nm) re more penetrtive through niml tissues beuse of redued bsorption nd sttering 9. We questioned whether NIR nnoprtiles ould be used to red shift luminol-emitted blue light to NIR, thus improving the in vivo detetbility of MPO. Previous work hs estblished tht luiferse or horserdish peroxidse onjugted to quntum dots llows luminesene to be bsorbed nd re-emitted by the nnoprtiles, phenomenon known s bioluminesene resonne energy trnsfer 1 (BRET) or CRET 11. We hypothesized tht luminesene energy trnsfer my lso our when the fluoresene eptor is in nononjugted stte. We tested this hypothesis by dministering, mixture of luminol nd ner-infrred quntum dots, to mie with indued lung inflmmtion or metstti ner. We demonstrte tht luminol-emitted 1 Cliper Life Sienes, Almed, Cliforni, USA. 2 Deprtment of Anesthesi nd Periopertive Cre, Sn Frniso Generl Hospitl, University of Cliforni, Sn Frniso, Cliforni, USA. 3 Present ddress: In Vivo Phrmology nd Onology, Phrmron, Beijing, Chin. Correspondene should be ddressed to N.Z. (ning.zhng@phrmron.om). Reeived 17 Otober 211; epted 2 September 212; published online 3 Mrh 213; doi:1.138/nm VOLUME 19 NUMBER 4 APRIL 213 nture mediine

2 h 3 h luminol Rdine Min = 1.e4 Mx = 1.e5 b Fold hnge luminol (effiieny unit) Time (h) d h luminol Epi-fluoresene 1..8 e L H Fluoresene St Du Sl Sp Lu K Br npg 213 Nture Ameri, In. All rights reserved. 3 h short-wvelength light n be red shifted by quntum dots through CRET, resulting in n inrese in MPO detetbility from deep-seted inflmmtory foi. We show the utility of s luminesent MPO redout nd for evlution of MPO inhibition under both in vitro nd in vivo onditions. RESULTS Enhned detetion of MPO tivity with quntum dots To test whether quntum dots n be used to red shift luminolemitted blue light, we onomitntly dministered luminol nd 8-nm-emitting quntum dots () to mie with indued MPO tivity. We seleted lipopolyshride ()-indued pulmonry inflmmtion model beuse of previous diffiulty in using luminol to detet MPO tivity in lung tissues. We intrvenously (i.v.) injeted into mie t 3 h fter delivery nd then imged the mie for luminesent signls (Fig. 1,b). As ompred to luminol-injeted mie tht hd reltively low-level luminesent signls ( ± (men ± s.e.m) photons m 2 sr 1 s 1 ), mie reeiving showed robust luminesent signls ( ± photons m 2 sr 1 s 1 ) in the lungs. MPO detetion by ws 37-fold more sensitive thn detetion by luminol. Longitudinl imging showed tht emitted luminesene rehed its pek t 2 min nd grdully delined to bsl level by 18 min (Fig. 1). We lso monitored the distribution of quntum dots with fluoresent imging (Fig. 1d). Quntum dot-medited fluoresene ws distributed throughout the body. Ex vivo imging deteted fluoresene in the liver, hert, stomh, duodenum, smll intestine, spleen, lungs nd kidney tissues but not in brin tissue, inditing tht quntum.2 Rdint effiieny p/se/m 2 /sr µw/m 2 Min = 6.e7 Mx = 1.e9 Figure 1 Detetion of pulmonry inflmmtion with. ( d) Luminesent nd fluoresent signls deteted in BALB/J mie intrtrhelly delivered with or PBS (ontrol) nd then i.v. injeted with or luminol 3 h lter. Shown re luminesent () nd fluoresent (d) imges deteted with n IVIS Spetrum instrument, s well s quntifitions of the luminesent signls from the lungs (b,). Dt re presented s the men ± s.e.m. (n = 4 per group). (e) Ex vivo nlysis of liver (L), hert (H), stomh (St), duodenum (Du), smll intestine (SI), spleen (Sp), lungs (Lu), kidney (K) nd brin (Br) performed with fluoresent imging (exittion t 745 nm nd emission t 8 nm) nd luminesent imging. dots did not ross the blood-brin brrier. -emitted luminesene ws only detetble in the lungs (Fig. 1e). CRET improved in vivo detetbility of MPO tivity To determine whether CRET ourred with quntum dots in nononjugted stte, we set up in vitro MPO retions with nd without quntum dots nd spetrlly nlyzed the retions. The MPO retion with luminol lone produed luminesene emission pek t 425 nm (Fig. 2). In the presene of quntum dots 8, new luminesene pek t 8 nm ws detetble in quntum dot onentrtion dependent fshion, supporting our hypothesis tht luminesene energy trnsfer ours when the fluoresene eptor is in nononjugted stte. CRET lso ourred under in vivo onditions. We observed three emission peks t 445 nm, 625 nm nd 8 nm in freshly reseted lungs tken from hllenged mie (Fig. 2b). The 8-nm pek, whih omprised 13% of the totl luminesent signls emitted from, is the expeted emission spetrum of 8-nm-emitting quntum dots. The 625-nm pek ws used by tissue bsorption nd re-emission of the luminol signl, s it ws missing from the in vitro MPO spetrl nlysis ssy. We lso nlyzed the emission spetrum under in vivo onditions with n IVIS Spetrum imging system, whih showed tht the CRET signl omprised 81% of the totl detetble luminesene (Fig. 2). When ompred with ex vivo imging tht deteted strong luminesent signls, in vivo imging deteted only ~7% of the luminesene mesured ex vivo (Fig. 2d). These results indite tht in vivo detetion of the luminol-emitted light is dependent predominntly on the red-shifted CRET signl. nture mediine VOLUME 19 NUMBER 4 APRIL

3 npg 213 Nture Ameri, In. All rights reserved. Luminol b d e (rbitrry light unit) 1,4 Luminol +.5 µm QD8 Luminol +.2 µm QD8 1.2 Luminol +.8 µm QD8 1,2 1, Emission spetrum (nm) 9 (rbitrry light unit) Emission spetrum (nm) Peroxidse nd hlorintion tivities of MPO It is well known tht MPO shows hlorintion nd peroxidtion tivity s result of tivtion by hydrogen peroxide. We designed n experiment to disset the ontribution of these tivities with respet to luminesent prodution by MPO. We found muh stronger luminesent signls in MPO retions tht ontined both sodium hloride nd hydrogen peroxide in mnner tht ws dependent on the onentrtion of hydrogen peroxide (Fig. 2e). At hydrogen peroxide onentrtions of 5 µm nd 5 µm, the luminesent emissions of the MPO retions without sodium hloride were 35% nd 49%, respetively, ompred to the identil retions with sodium hloride, inditing tht hlorintion dominted over peroxidtion under these onditions (Fig. 2f). We further ompred the hlorintion nd peroxidse tivities of MPO with those of ltoperoxidse (LPO). from the LPO ssy ws substntilly lower thn tht from the MPO ssy (Fig. 2g). In the presene of sodium hloride, the LPO retion produed only 3% of the luminesent signls s ompred to the MPO retion. medited luminesene is dependent on MPO tivity We exmined the effet of MPO inhibition on -medited luminesent light emission under both in vitro nd in vivo onditions. An inhibition ssy with the irreversible MPO inhibitor 4-ABAH showed tht 4-ABAH dose-dependently inhibited MPO tivity t 1,5 nm with hlf-mximl inhibitory onentrtion (IC 5 ) of 41 nm (Fig. 3,b). At high onentrtions (>1, nm), 4-ABAH inhibited more thn 9% of the MPO tivity. Under in vivo onditions, we found tht pretretment with 4-ABAH ttenuted -medited luminesene in the lungs (Fig. 3). Quntifition of luminesent signls showed tht 4-ABAH tretment before dministrtion suppressed the -indued pulmonry MPO tivity by 74% (Fig. 3d). This inhibition ws sttistilly signifint (P <.5). Anlysis of the lung tissue homogente showed tht 4-ABAH treted lungs hd redued 9 (p/s/m 2 /sr) nm 78 nm 8 nm 82 nm 84 nm CRET Figure 2 Confirmtion tht CRET substntilly improves the in vivo detetbility of MPO tivity. () MPO nd ssy mix nlyzed with TRIAX 32 spetrometer. QD8, quntum dots emitting t 8 nm. (b,) Ex vivo spetrl nlyses of freshly reseted lungs from -hllenged mie with TRIAX 32 spetrometer (b) nd n IVIS Spetrum instrument (). CRET, the umultive red-shifted luminesent signls from the lungs; totl Lum, ll the luminesent signls olleted with n open filter. (d) Comprison of the luminesent signls from the lungs mesured in vivo nd ex vivo. (e,f) The effet of MPO ssy, set up s bove but with hydrogen peroxide dded to the indited finl onentrtions, nlyzed with n IVIS Spetrum instrument. BLI, bioluminesene imging. (g) MPO nd LPO ssys nlyzed Totl Lum with n IVIS Spetrum instrument. Dt re shown s the men luminesent signls ± s.e.m. (,d,f,g). n = 3 per group. f Totl flux (BLI) (p/s/m 2 /sr) In vivo Ex vivo H 2 O 2 MPO in NCI MPO in H 2 O (µm) MPO in NCI MPO in H 2 O H 2 O 2 onentrtion (µm) MPO enzymti tivity ompred to -treted positive ontrols (Fig. 3e), verifying tht the inhibition ws irreversible. The dependene of the medited luminesent signl on MPO tivity ws further supported in n experiment with MPO-defiient mie. In syngenei wild-type C57BL/6 (Mpo +/+ ) mie, intrtrhel hllenge remrkbly indued MPO tivity in the lungs. In ontrst, the MPO tivity ws signifintly (P <.5) less in -hllenged Mpo / mie (Fig. 3f,g). We then investigted the possible role of nitri oxide on luminesene prodution. Pretretment with nonspeifi nitri oxide synthse inhibitor, l-name, hd no impt on the luminesent signl in the lungs of -hllenged mie (Fig. 3h). Effet of MLN12B on MPO indution We previously showed tht -indued lung injury involves nuler ftor κb (NF-κB) tivtion nd tht this indution n be suppressed by MLN12B 12. Here we investigted whether inhibition of the NF-κB signling pthwy by MLN12B ffets MPO tivity nd hypohlorous id prodution during pulmonry inflmmtion. We pretreted mie with MLN12B (3 mg per kg body weight) nd then hllenged them intrtrhelly with. Imging nlysis showed tht MLN12B pretretment mrkedly ttenuted the luminesent signl in the lungs s ompred to -treted positive ontrol mie (Fig. 4). The inhibition ws s muh s 7% (Fig. 4b) nd ws sttistilly signifint (P <.1). We then immunohistohemilly nlyzed the lung tissues to hek whether the inhibition by MLN12B ws ting on neutrophil infiltrtion or inhibition of MPO. Using neutrophilspeifi ntibody, Ly-6G, we observed notieble neutrophil infiltrtion in -treted lung tissues ompred to untreted lungs. MLN12B tretment signifintly redued the infiltrtion of neutrophils to the lung tissues, resulting in derese in MPO tivity (Fig. 4). These results provide evidene of tht the NF-κB signling pthwy is ruil in regulting pulmonry inflmmtion nd MPO prodution. g Totl flux (BLI) Rdine Min = 4.6e3 Mx = 9.6e4 MPO + NCI MPO + H 2 O LPO + NCI LPO + H 2 O 52 VOLUME 19 NUMBER 4 APRIL 213 nture mediine

4 npg 213 Nture Ameri, In. All rights reserved. d (p/s/m 2 /sr) ABAH (nm) No ontrol vehile ABAH * * h 4 h Time fter , Detetion of MPO tivity from tumor metstses It hs been shown tht some mlignnt tumors ttrt neutrophil infiltrtion during their progression 13. We investigted whether we ould use to detet MPO tivity in deep tissue tumor metstses. We estblished systemi MDA-MB-231-lu2 tumor metstsis through intrrdi injetion of tumor ells into NU/NU mie. Bioluminesent imging of the mie, enbled using the lu2 e (p/s/m 2 /sr) b Totl flux (BLI, %) * 6.25 MPO tivity 12.5 vehile 4-ABAH 25 5 MPO tivity (%) 4-ABAH onentrtion (nm) f h 4 h , 1,5 h 4 h Mpo +/+ Mpo / Negtive ontrol vehile 4-ABAH Rdine Min = 1.e4 Mx = 1.e5 Figure 3 -emitted luminesent signl is dependent on MPO tivity. () Anlysis of the MPO ssy with the ddition of 4-ABAH to the indited onentrtions using n IVIS Spetrum instrument. (b) Quntifition of the luminesent signls s perentge of MPO tivity hnge. Dt re shown s the men ± s.e.m. (n = 3 per group). () Imges tken t the indited times fter hllenge of BALB/J mie divided into three groups nd hllenged with with or without 4-ABAH inhibition (8 mg per kg body weight 4-ABAH). Negtive ontrol mie reeived no nd no 4-ABAH. (d) Quntifition of luminesent signls from the lungs. *P <.5 determined by Mnn-Whitney U test. Dt re shown s the men ± s.e.m. (e) MPO tivity of homogented lung tissue determined by the ddition of 8 µl of 2 mm luminol, 1 µl of 8 µm QD8 nd 8 µl of 1,5 µm hydrogen peroxide solution to 8 µl of tissue suspension followed by imging nlysis with n IVIS Spetrum instrument. Dt re shown s the men ± s.e.m. (n = 4 per group). *P <.5 determined by Mnn-Whitney U test. (f) Imging for hemiluminesent signls t 4 h fter i.v. delivery of in mle Mpo / nd (p/s/m 2 /sr) (p/s/m 2 /sr) Mpo +/+ Mpo / luiferse gene engineered into these tumor ells, showed the development of multiple metstses on the limbs nd mndible nd inside the bdominl vity t 3 weeks fter tumor-ell injetion (Fig. 5). At the sites of tumor metstses, in lose proximity to the humerus, tibi nd femur, osteolyti lesions were evident by miro-omputed tomogrphy (miroct) imging (Fig. 5b d). After i.v. delivery of to the mie, we were ble to detet luminesent signls t ll of the h g Rdine Min = 3.e4 Mx = 2.e5 * h 4 h Time fter vehile L-NAME h 4 h Time fter ontrol C57BL/6 mie hllenged with. (g) Quntifition of the luminesent signls from the lungs. Dt re shown s the men ± s.e.m. *P <.5 determined by Mnn-Whitney U test. (h) Quntifition of the luminesent signls from the lungs of -hllenged BALB/J mie injeted with l-name (1 mg per kg body weight intrperitonelly (i.p.)) t 6 min before in vivo imging with. Dt re shown s the men ± s.e.m. (n = 3 per group). 1. b MLN12B + MLN12B + MLN12B Rdine Min = 8.e3 Mx = 1.e5 (p/s/m 2 /sr) ** ** Neutrophil MPO Thori region 1 µm 1 µm 1 µm 1 µm 1 µm 1 µm Figure 4 Effet of MLN12B tretment on MPO tivity. () Indution of MPO tivity monitored with in BALB/J mie divided into three groups nd dosed with MLN12B (3 mg per kg body weight orlly) or ontrol vehile before intrtrhel hllenge. Negtive ontrol mie reeived no nd no MLN12B. (b) Quntifition of the luminesent signls from the lungs. Dt re shown s the men ± s.e.m. (n = 4 per group). **P <.1 determined by Mnn-Whitney U test. () Fluoresene mirosopy performed with Nune multispetrl hrge-oupled devie (CCD) mer to visulize neutrophils (green) nd MPO (red) in the lung tissues. Nulei were stined with DAPI (blue). Originl mgnifition, 4. nture mediine VOLUME 19 NUMBER 4 APRIL

5 npg 213 Nture Ameri, In. All rights reserved. Figure 5 Imging MPO tivity in MDA-MB- 231-lu2 tumor metstses. () Bioluminesent imging visulizing tumor metstses t 3 weeks in NU/NU mie intrrdilly injeted with MDA-MB-321-lu2 tumor ells. (b d) High-mgnifition miroct imges obtined using Quntum FX µct system of the shoulders nd knee joints showing osteolyti lesions used by tumor metstses. Arrows indite regions of osteolyti lesions. (e) MPO tivity in these lesions nlyzed with. (f i) Immunohistologil nlysis of the tumor metstses (f,g) or helthy tissues from similr re in ontrol mouse (h,i) nlyzed for neutrophils (green; f,h) nd MPO (red; g,i) with Nune multispetrl CCD mer. Nulei re shown in blue. Originl mgnifition, 4. (j) Co-registrtion of multimodlity threedimensionl bioluminesent nd miroct imges obtined using n IVIS Spetrum instrument nd Quntum FX µct system, respetively. The DLIT-mesured depth (mm) of the tumor metstses is shown bove eh of the metstti nodules. metstti foi (Fig. 5e), inditing the presene of MPO tivity in these lesions. Immunohistohemil nlysis showed inresed stining of neutrophils nd MPO in MDA-MB-231-lu2 tumor metstses (Fig. 5f,g) s ompred with helthy tissues from ontrol NU/NU mie in the sme ntomil lotions (Fig. 5h,i). We further performed three-dimensionl bioluminesent imging of the mie nd estimted the depth of eh metstti lesion with reonstrution lgorithm (DLIT) progrm. We o-registered the three-dimensionl bioluminesent imges with three-dimensionl miroct imges to disply ll metstti lesions in referened ntomil setting (Fig. 5j). The mesured tumor depths rnged from 1.2 mm to 6.8 mm, onfirming tht medited CRET is suitble for monitoring MPO in deep tissue inflmmtory foi. DISCUSSION Our nnoprtile-bsed luminesent detetion pproh relies on similr energy trnsfer mehnism s do BRET nd CRET, whih use onjugtion of luminesent enzymes to quntum dots, llowing luminesene to be bsorbed nd re-emitted by the nnoprtiles t longer wvelength. Reently, fluoresent nnoprtiles suh s quntum dots hve been used s in vivo imging gents beuse of their brod bsorption spetr, high quntum yields nd photostbilities 14,15. Their blue-inresing extintion oeffiieny llows fluoresent nnoprtiles to be exited more effiiently by shorter-wvelength blue light 16, suh s tht emitted by luminol, with re-emission of this light in the NIR rnge. The mjor finding in this study is tht nnoprtiles in nononjugted form n lso effetively onvert luminesene to NIR photons. By enbling this photo shift, our nnoprtile-bsed luminesent detetion method llows in vivo ssying of endogenous enzymes suh s MPO. Chroni exposure to lrge mounts of is reported to be ssoited with the development nd progression of mny types of pulmonry diseses, inluding sthm nd COPD 17. The pthogenesis of COPD is hrterized by neutrophil infiltrtion of pulmonry tissues nd elevted onentrtions of MPO in bronholveolr lvge fluids 18. MPO is the only enzyme in the body tht is known to produe hypohlorous id, whih oxidizes lipids, proteins nd DNA 3,4. e b f h 1 µm 1 µm 1 µm d g i 1 µm j Hypohlorous id n be deteted with luminol beuse of the light-emitting retion of these two omponents. However, the short wvelength of luminol-emitted luminesene limits its pplition for the detetion of deep tissue MPO tivity. Using n -indued lung injury model, we demonstrted tht quntum dots were ble to onvert luminol-emitted blue light to NIR photons tht were pble of penetrting tissue t depth. Although the effiieny of energy onversion ws only ~13%, the detetbility of MPO tivity in the lung tissues ws improved by 37-fold in -injeted mie ompred to luminol-injeted mie. Our study demonstrted tht onversion to NIR luminesene is the unequivol ftor for sensitive MPO detetion in vivo. MPO prodution in the lungs during -indued injury orrelted with neutrophil infiltrtion in the lung tissues. In ddition, we found diret evidene of elevted mounts of MPO protein in the lung tissues. Using Mpo / mie, we demonstrted tht the -medited luminesent signl is dependent on MPO tivity. We lso ruled out ny involvement of nitri oxide in luminesene genertion using l-name, nonspeifi nitri oxide synthse inhibitor 19. We then investigted the bility of to monitor the phrmologil inhibition of MPO. 4-ABAH is the most potent MPO inhibitor tht funtions s suiide MPO substrte 6. We demonstrted tht 4-ABAH pretretment signifintly ttenuted MPO tivity in the lungs. MPO gene expression is under the regultion of number of trnsription ftors, inluding myeloid nuler ftors 1α nd 1β 2 nd grnuloyte olony-stimulting ftor 13. However, there is no evidene of diret involvement of NF-κB in MPO gene regultion. Nevertheless, evidene showed tht under inflmmtory disese onditions, NF-κB tivtion is ompnied by n elevtion of MPO tivity in inflmed tissues. In previous study of -indued lung injury in Sprgue-Dwley rts, pulmonry tissues showed NF-κB tivtion nd elevtion of MPO tivity 21. Tretment with penehylidine hydrohloride resulted in inhibition of the NF-κB pthwy, redution of MPO tivity nd suppression of inflmmtory ell infiltrtion to the lung tissues. An inrese in MPO tivity hs lso been observed under other disese onditions, suh s olitis 22. These studies demonstrted tht inhibition of NF-κB tivtion is ssoited with H-Heb Phgoyti soure intensity 54 VOLUME 19 NUMBER 4 APRIL 213 nture mediine

6 npg 213 Nture Ameri, In. All rights reserved. the meliortion of disese symptoms nd n ttenution of MPO tivity. Our previous study demonstrted tht MLN12B suppressed NF-κB tivtion during -indued lung injury 12. MLN12B is potent nd seletive smll-moleule inhibitor of IKK-2 (ref. 23). Here we found tht pretretment with MLN12B ttenuted neutrophil infiltrtion to the lung tissues. MPO omprises between 2% nd 5% of the totl ellulr protein nd is the most bundnt protein in neutrophils 24. Humn monoytes lso ontin MPO-positive grnules. However, they re fewer in number thn neutrophils. In ddition, MPO expression is lost during differentition into tissue mrophges 25. Thus, substntil ontribution of monoytes, mrophges or both to MPO tivity during lung injury is unlikely. Previous studies hve shown tht most mlignnt tumors ontin substntil number of leukoyti infiltrtes tht re linked to poor ptient prognosis, with inresing evidene showing tht neutrophils tively prtiipte in this proess 26. Using systemi MDA-MB-231-lu2 metstti model, we showed tht suessfully visulized tumor metstses of vrious depths, thus estblishing tht the nnoprtile-bsed luminesent detetion pproh n be used to ssess MPO tivity in lesions beyond superfiil levels. In ddition to monitoring pulmonry inflmmtion nd the tivity of neutrophils in onogenesis, it is possible tht ould be used for the dignosti detetion of other neutrophil-ssoited inflmmtory onditions, suh s theroslerosis, peritonitis, rthritis nd sepsis. Clinil pplition of the nnoprtile-bsed MPO detetion method my beome possible if is proven sfe for use in humns. Of the two gents used in the formultion, luminol hs been used to tret humns with lopei ret 27. It n be rpidly lered from the system without using toxiity 28. The development of fluoresent quntum dots for prelinil nd linil pplitions involves sfety onsidertions. In prelinil tumor-trgeting studies, there hve been no observtions of short-term toxiity of dmium-ontining quntum dots in mie 29,3. The long-term toxiity of these prtiles to both nimls nd humns remins unknown. Nevertheless, onerns regrding dmium toxiity hve led to the reent development of dmium-free quntum dots 31 nd gold-bsed nnomterils 32. The prospet of pplying the nnoprtile-bsed luminesent detetion method in the lini my drw more reserh to this re. Methods Methods nd ny ssoited referenes re vilble in the online version of the pper. Aknowledgments We thnk H. Xu for spetrometer nlysis, E. Lim nd K. Wong for tehnil ssistne nd S. Ry nd R. Singh for vluble disussion. AUTHOR CONTRIBUTIONS Experimentl design nd onepts were devised by N.Z. nd K.P.F., D.A. onduted ll the experiments exept the Mpo / study, whih ws performed by D.A., A.P. nd K.P.F., D.A. nd N.Z. performed the dt nlysis. N.Z. wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is vilble online t reprints/index.html. 1. Hegens, A., Vernooy, J.H., Heering, P., Mossmn, B.T. & Wouters, E.F. Myeloperoxidse modultes lung epithelil responses to pro-inflmmtory gents. Eur. Respir. J. 31, (28). 2. Winterbourn, C.C. Reoniling the hemistry nd biology of retive oxygen speies. Nt. Chem. Biol. 4, (28). 3. Dvies, M.J., Hwkins, C.L., Pttison, D.I. & Rees, M.D. Mmmlin heme peroxidses: from moleulr mehnisms to helth implitions. Antioxid. Redox Signl. 1, (28). 4. O Donnell, C. et l. 3-hlorotyrosine in sputum of COPD ptients: reltionship with irwy inflmmtion. COPD 7, (21). 5. Hzen, S.L. & Heineke, J.W. 3-hlorotyrosine, speifi mrker of myeloperoxidsetlyzed oxidtion, is mrkedly elevted in low density lipoprotein isolted from humn therosleroti intim. J. Clin. Invest. 99, (1997). 6. Mlle, E., Furtmüller, P.G., Sttler, W. & Obinger, C. Myeloperoxidse: trget for new drug development? Br. J. Phrmol. 152, (27). 7. Kettle, A.J., Gedye, C.A. & Winterbourn, C.C. Mehnism of intivtion of myeloperoxidse by 4-minobenzoi id hydrzide. Biohem. J. 321, (1997). 8. Gross, S. et l. Bioluminesene imging of myeloperoxidse tivity in vivo. Nt. Med. 15, (29). 9. Altinoğlu, E.I. & Adir, J.H. Ner infrred imging with nnoprtiles. Wiley Interdisip. Rev. Nnomed. Nnobiotehnol. 2, (21). 1. So, M.-K., Xu, C., Loening, A.M., Gmbhir, S.S. & Ro, J. Self-illuminting quntum dot onjugtes for in vivo imging. Nt. Biotehnol. 24, (26). 11. Wng, H.Q. et l. Influene of quntum dot s quntum yield to hemiluminesent resonne energy trnsfer. Anl. Chim. At 61, (28). 12. Ansldi, D. et l. Imging pulmonry NF-κB tivtion nd therpeuti effets of MLN12B nd TDZD-8. PLoS ONE 6, e2593 (211). 13. Orit, T., Shimozki, K., Murkmi, H. & Ngt, S. Binding of NF-Y trnsription ftor to one of the is-elements in the myeloperoxidse gene promoter tht responds to grnuloyte olony-stimulting ftor. J. Biol. Chem. 272, (1997). 14. Wu, X. et l. Immunofluoresent lbeling of ner mrker Her2 nd other ellulr trgets with semiondutor quntum dots. Nt. Biotehnol. 21, (23). 15. Medintz, I.L., Uyed, H.T., Goldmn, E.R. & Mttoussi, H. Quntum dot bioonjugtes for imging, lbeling nd sensing. Nt. Mter. 4, (25). 16. Chn, W.C. & Nie, S. Quntum dot bioonjugtes for ultrsensitive nonisotopi detetion. Siene 281, (1998). 17. Mihel, O. et l. Severity of sthm is relted to endotoxin in house dust. Am. J. Respir. Crit. Cre Med. 154, (1996). 18. Linden, M. et l. Airwy inflmmtion in smokers with nonobstrutive nd obstrutive hroni bronhitis. Am. Rev. Respir. Dis. 148, (1993). 19. Homyoun, H., Khvndgr, S. & Dehpour, A.R. The involvement of endogenous opioids nd nitrioxidergi pthwy in the ntionvulsnt effets of foot-shok stress in mie. Epilepsy Res. 49, (22). 2. Nuhpryoon, I. et l. PEBP2/CBF, the murine homolog of the humn myeloid AML1 nd PEBP2β/CBFβ proto-onoproteins, regultes the murine myeloperoxidse nd neutrophil elstse genes in immture myeloid ells. Mol. Cell Biol. 14, (1994). 21. Shen, W., Gn, J., Xu, S., Jing, G. & Wu, H. Penehylidine hydrohloride ttenutes -indued ute lung injury involvement of NF-κB pthwy. Phrmol. Res. 6, (29). 22. Islm, M.S. et l. Anti-inflmmtory effets of phytosteryl ferultes in olitis indued by dextrn sulphte sodium in mie. Br. J. Phrmol. 154, (28). 23. Ngshim, K. et l. Rpid TNFR1-dependent lymphoyte depletion in vivo with seletive hemil inhibitor of IKKβ. Blood 17, (26). 24. Shultz, J. & Kminker, K. Myeloperoxidse of the leuoyte of norml humn blood. I. Content nd loliztion. Arh. Biohem. Biophys. 96, (1962). 25. Bos, A., Wever, R. & Roos, D. Chrteriztion nd quntifition of the peroxidse in humn monoytes. Biohim. Biophys. At 525, (1978). 26. Gregory, A.D. & Houghton, A.M. Tumor-ssoited neutrophils: new trgets for ner therpy. Cner Res. 71, (211). 27. Irie, S. The tretment of lopei ret with 3-minophthl-hydrzide. Curr. Ther. Res. Clin. Exp. 2, (196). 28. Snders, J.M., Chen, L.J., Burk, L.T. & Mtthews, H.B. Metbolism nd disposition of luminol in the rt. Xenobioti 3, (2). 29. Lrson, D.R. et l. Wter-soluble quntum dots for multiphoton fluoresene imging in vivo. Siene 3, (23). 3. Stroh, M. et l. Quntum dots spetrlly distinguish multiple speies within the tumor milieu in vivo. Nt. Med. 11, (25). 31. Go, J. et l. In vivo tumor-trgeted fluoresene imging using ner-infrred nondmium quntum dots. Bioonjug. Chem. 21, (21). 32. Hhn, M.A., Singh, A.K., Shrm, P., Brown, S.C. & Moudgil, B.M. Nnoprtiles s ontrst gents for in-vivo bioimging: urrent sttus nd future perspetives. Anl. Bionl. Chem. 399, 3 27 (211). nture mediine VOLUME 19 NUMBER 4 APRIL

7 npg 213 Nture Ameri, In. All rights reserved. ONLINE METHODS Regents. We obtined bteril, (from Slmonell bortus-equi), 4-ABAH, l-name nd LPO from Sigm-Aldrih Chemil Co. (St. Louis, MO), luminol from Biosynth Chemistry & Biology (Its, Illinois), QD8 from Life Sienes (Grnd Islnd, NY) nd mouse MPO from R&D Systems (Minnepolis, MN). Animls. We obtined 8- to 1-week-old femle BALB/J, C57BL/6 nd Mpo / mie from the Jkson Lbortory (Br Hrbor, Mine) nd 8- to 1-week-old femle NU/NU mie from Chrles River (Wilmington, MA). All niml studies were pproved by the Institutionl Animl Cre nd Use Committee t Cliper, Almed, CA. Imging nlysis of MPO tivity. Aute lung injury ws eliited with intrtrhel delivery of 5 µl of 1 mg/ml solution to BALB/J mie using 22-guge intubtor. After 3 4 h, mie were i.v. injeted with 125 µl of luminol (4 mg/ml) or µl of (125 µl of 4 mg/ml luminol mixed with 12.5 µl QD8 (1 pmol)) nd imged in n IVIS Spetrum instrument (Cliper, Almed, CA) for 5 min with n open filter. Three-dimensionl bioluminesene imging nd mct o-registrtion. Mie were i.p. injeted with 15 mg per kg body weight luiferin, nesthetized nd imged with nrrowbnd light emission filters (58 64 nm). After optil imging, the mie were imged with Quntum FX µct system. Threedimensionl reonstrution of the optil nd µct imges nd o-registrtion of the two were performed with the Living Imge4.2 Softwre. In vitro MPO ssy with. In 96-well blk plte, n MPO tivity ssy ws set up s follows: 8 µl of.5 µg/ml MPO in 15 mm NCl (ph 7.4) ws dded to eh well nd then 8 µl of 2 mm luminol nd 1 µl of 8 µm QD8 were dded. Eighty miroliters of 15 µm hydrogen peroxide solution ws dded to initite the retion. The plte ws immeditely ssyed with n IVIS Spetrum instrument with 5-min exposure time. In nother experiment, the retion mixture ws set up with either 2 nm of LPO or 2 nm of MPO in finl volume of 24 µl. The retions were ssyed s desribed bove. To study the effet of MPO inhibition by 4-ABAH, 1 µl of the 4-ABAH stok solutions were dded to the retion mix before the ddition of hydrogen peroxide to obtin finl 4-ABAH onentrtions of, 6.25, 12.5, 25,.5, 1, 15, 2, 25, 5, 1, nd 1,5 nm. Luminesent signls from eh well were quntified using the Living Imge softwre. Anlysis of CRET by spetrometry. In 1.5-ml ependorff tube, 5 µl of MPO solution (.6 µm in 15 mm NCl, ph 7.4) ws mixed with 5 µl of luminol (2, µm) nd vrious quntities of 8-nm emitting quntum dots to hieve finl onentrtions of,.5,.2 nd.8 µm. Then 5 µl of hydrogen peroxide solution (2, µm) ws dded to the retion mix, nd the smples were nlyzed with TRIAX 32 spetrometer (Jobin Yvon Instruments, NJ). Freshly reseted lung tissues from injeted mie were lso pplied to spetrl nlysis with the TRIAX 32 spetrometer. Dependene of medited luminesene on MPO tivity. To test the effet of MPO inhibition on medited light emission, the MPO inhibitor 4-ABAH (8 mg per kg body weight i.p.) ws injeted into hllenged BALB/J mie 15 min before in vivo imging with. The effet of nitri oxide inhibition ws studied by o-treting the -hllenged mie with l-name (1 mg per kg body weight i.p.) t 6 min before in vivo imging. To study the effet of NF-κB pthwy inhibition on -indued luminesent signls, BALB/J mie were pre-dosed with MLN12B (in.5% methylellulose, 3 mg per kg body weight orlly) t 16 h nd 1 h before hllenge. Mie were pplied to imging nlysis fter i.v. injetion of. To study the impt of MPO defiieny on deteted luminesene, 8-week-old mle Mpo / mie were hllenged with, s bove, nd pplied to imging detetion of the hemiluminesent signl fter i.v. delivery of. Age-mthed C57BL/6 ontrol mie were used for omprison. Histology nd fluoresene mirosopy with Nune. Histologil nlysis ws performed with Penthrome stining kit following the mnufturer s instrutions (Ameri Mster Teh Sientifi, In., Lodi, CA). Reseted lungs were fixed overnight with 4% prformldehyde nd embedded in prffin. Conseutive 5-µm-thik setions were prepred nd seprtely stined with neutrophil-speifi ntibody, Ly6G fluoresein isothioynte (FITC) (1:1 dilution, BD Phrmingen, Sn Diego, CA, tlog number , lone number RB6-8C5) nd n MPO rbbit polylonl ntibody (1:1, dilution, Abm, Cmbridge, MA, tlog number b45977). A seondry Cy3 donkey rbbit-speifi ntibody (Jkson ImmunoReserh Lbortories In., West Grove, PA, tlog number ) ws used for visulizing MPO-positive ells. The nulei were stined with DAPIontining mounting medi (Vetor lbs, Burlingme, CA). Fluoresene mirosopy ws performed with Nune multispetrl CCD mer (Cliper, Hopkinton, MA). Sttistil nlyses. Signifine between two mens ws lulted using two-tiled Mnn-Whitney U test. P <.5 ws onsidered signifint. Sttistil nlyses were performed using Prism 5 (GrphPd Softwre, In., L Joll, CA). nture mediine doi:1.138/nm.311