Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization

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1 8 Nture Pulishing Group rystls nd luminum slts tivte the NALP3 inflmmsome through phgosoml destiliztion Veit Hornung 1,, Frnz Buernfeind,, Annett Hlle 1, Eivind O Smstd 1,3, Hjime Kono 4, Kenneth L Rok 4, Ktherine A Fitzgerld 1 & Eike Ltz 1,3 Inhltion of sili rystls uses inflmmtion in the lveolr spe. Prolonged exposure to sili n led to the development of siliosis, n irreversile, firoti pulmonry disese. The mehnisms y whih sili nd other rystls tivte immune ells re not well understood. Here we demonstrte tht sili nd luminum slt rystls tivted inflmmsomes formed y the ytoplsmi reeptor NALP3. NALP3 tivtion required phgoytosis of rystls, nd this uptke susequently led to lysosoml dmge nd rupture. Sterile lysosoml dmge (without rystls) lso indued NALP3 tivtion, nd inhiition of either phgosoml idifition or thepsin B tivity impired NALP3 tivtion. Our results indite tht the NALP3 inflmmsome senses lysosoml dmge s n endogenous dnger signl. Crystlline sili, lso known s silion dioxide (SiO ), is found in nture s snd or qurtz. Although ingestion of sili is hrmless nd nontoxi, inhltion of smll sili rystls n led to ute lung inflmmtion. Chroni, mostly ouptionl, inhltive exposure to sili rystls uses the pneumooniosis siliosis. Siliosis is n inurle, irreversile, progressive lung firosis tht is ssoited with other diseses, inluding rdiopulmonry filure, myoteril infetion, utoimmunity nd lung ner. No effetive tretment is ville for siliosis, nd the mehnisms of sili rystl indued lung inflmmtion nd firosis re not well understood 1. Inhltion of sili rystls leds to their deposition in smll irwys of the lung, where muoilil lerne is not funtionl. Susequent ingestion of sili rystls y resident mrophges eliits n inflmmtory response hrterized y the relese of ytokines, suh s interleukin 1 (IL-1; A3663) nd tumor nerosis ftor, nd ftors tht n indue firosis. Moreover, uptke of sili rystls n initite poptoti ell deth with onomitnt relese of ingested sili prtiles, whih re re-engulfed y other lveolr mrophges, induing yle of sustined inflmmtion. Reports hve shown tht other medilly relevnt rystls, suh s rystls of monosodium urte () or lium pyrophosphte dihydrte, whih trigger inflmmtion in gout or pseudogout, respetively, n tivte the ytoplsmi reeptor NALP3 (lso lled ryopyrin or NLRP3) 3. NALP3 is memer of fmily of ytoplsmi Nod-like reeptors tht ontrol the tivity of inflmmtory spse-1 y forming multimoleulr omplexes lled inflmmsomes 4. After eing tivted, NALP3 reruits the dptor moleule ASC, whih in turn inds to prospse-1, leding to its utotlyti proessing nd tivtion. Ative spse-1 tlyzes levge of the proytokines IL-1 nd IL-18, whih re sereted nd iologilly tive only in their proessed forms 6. The upstrem mehnisms of tivtion of the NALP3 inflmmsome re poorly understood. Severl lignds of diverse physiohemil nture hve een reported to tivte the NALP3 inflmmsome. For exmple, in ddition to rystline nd lium pyrophosphte dihydrte, teril ell-wll omponents, severl teril toxins nd high onentrtions of extrellulr re ll reported to tivte the NALP3 inflmmsome 4. Here we investigte the mehnisms of rystl-indued inflmmtion nd demonstrte tht like, sili rystls nd luminum slt (lum) rystls tivted the NALP3 inflmmsome. We further found tht NALP3 tivtion y rystls required phgoytosis nd tht rystls tken up y phgoytosis indued lysosoml swelling nd dmge. NALP3 tivtion y sili rystls ws dependent on lysosoml idifition nd involved the lysosoml ysteine protese thepsin B. Crystl-independent lysosoml dmge ws suffiient to indue NALP3 inflmmtion. Our results suggest ommon mehnism of rystl-indued tivtion of the NALP3 inflmmsome wherey lyosoml perturtion, not the rystl struture itself, is eing sensed. RESULTS indues IL-1 mturtion nd spse-1 tivtion The NALP3 inflmmsome reognizes rystlline mteril ppering in joint fluids s dnger signl 3. Inhltion of sili rystls is known to indue strong proinflmmtory response, inluding the relese of tive IL-1. We thus hypothesized tht sili rystls ould 1 Deprtment of Infetious Diseses nd Immunology, University of Msshusetts Medil Shool, Worester, Msshusetts 16, USA. Division of Clinil Phrmology, Deprtment of Internl Mediine, Ludwig-Mximilins University of Munih 8336, Germny. 3 Institute of Cner Reserh nd Moleulr Mediine, Norwegin University of Siene nd Tehnology, N-7489 Trondheim, Norwy. 4 Deprtment of Pthology, University of Msshusetts Medil Shool, Worester, Msshusetts 16, USA. These uthors ontriuted eqully to this work. Correspondene should e ddressed to: E.L. (eike.ltz@umssmed.edu). Reeived 1 April; epted 1 June; pulished online 11 July 8; doi:1.138/ni.1631 NATURE IMMUNOLOGY VOLUME 9 NUMBER 8 AUGUST 8 847

2 8 Nture Pulishing Group IB: leved IB: leved IL-1β 17 IL-1β 1 1 IB: leved IL-1β priming priming exert their inflmmtory potentil through the tivtion of similrpthwy.weinutedhumnperipherlloodmononuler ells (PBMCs) from severl donors with ked, lipopolyshride (LPS)-free sili rystls. Pro-IL-1 is not onstitutively expressed nd requires trnsriptionl indution in response to, for exmple, Toll-like reeptor stimulus. Although sili rystls did not indue levge nd relese of IL-1 in humn PBMCs y themselves, LPS-primed PBMCs strongly responded to the ddition of sili rystls in dose-dependent wy (Fig. 1). rystl medited tivtion of humn PBMCs ws s potent s other known tivtors of the NALP3 inflmmsome, suh s rystls, or the NALP3-independent stimulus of trnsfeted doule-strnded DNA 7 (poly(da:dt); Fig. 1). IL-1 prodution, s mesured y enzyme-linked immunosorent ssy (ELISA), orrelted strongly with the detetion of leved IL-1 nd spse-1 y immunolot nlysis, whih indited tht tivted ells relesed mture IL-1. Inhiition of spse-1 y the speifi peptide inhiitor z-yvad lmost ompletely olished the relese of IL-1 in response to tretment with sili rystls (Fig. 1). These dt suggest tht sili rystls tivte IL-1 in spse-1-dependent wy in humn immune ells. indues influx of neutrophils into the lung vi IL-1 After exposed people inhle sili rystl dust, lung-resident immune ells susequently engulf the rystl mteril y phgoytosis nd indue n inflmmtory response. Lrge doses of sili dust led to the linil syndrome of ute siliosis, hrterized y the rpid influx of immune ells into the exposed re nd mssive prodution of hemokines nd proinflmmtory ytokines, inluding IL-1. To investigte the in vivo relevne of the IL-1 response in sili-indued lung inflmmtion, we trnsorlly instilled sili rystls into wildtype nd IL-1 reeptor (IL-1R)-defiient mie nd then monitored the mie for ute inflmmtion fter h. We ounted the ells in ronholveolr lvge fluid nd ssessed their phenotype y flow ytometry. We deteted muh more infiltrtion of neutrophils in lungs of wild-type mie exposed to sili rystls thn in those of mie treted with the rrier fluid; this did not our in IL-1R-defiient IB: leved spse , 1 IL-1β ( fold indution ) mie exposed to sili rystl (Fig. ). Other ell types remined mostly unhnged (dt not shown). We noted lmost omplete rogtion of sili-indued influx of neutrophils in mie douly defiient in the dptor moleules MyD88 (A33) nd TRIF, wheres sene of these moleules hd little or no effet on zymosn-indued inflmmtion (Fig. ). Commeril zymosn preprtions re known to tivte signling pthwys dependent on Toll-like reeptor nd detin 1 (refs. 8,9), whih ould explin the lower zymosn-indued neutrophil reruitment. These results olletively indite tht sili rystls indue IL-1 relese nd tht the IL-1 signl trnsdution moleules IL-1R nd MyD88 re ritil in the development of ute inflmmtion in vivo fter exposure to sili rystl. rystls tivte the NALP3 inflmmsome To investigte whether sili rystls n tivte the NALP3 inflmmsome, we did experiments with mrophges from mie defiient z-yvad-fmk Figure 1 indution of the relese of mture IL 1 nd tivted spse-1 y humn PBMCs is spse-1 dependent. ELISA (ove) nd immunolot nlysis (IB; elow) of the prodution of IL-1 ( ) or spse-1 () y humn PBMCs. () PBMCs primed for 3 h with LPS ( pg/ml; + priming) or left untreted ( priming) nd then stimulted for n dditionl 6 h with sili rystls (). Eh dot ove lne represents one donor; smll horizontl lines indite the men. kd, kilodltons. Dt re from two experiments with four independent donors (ELISA) nd or one representtive donor (immunolot). () LPS-primed PBMCs left unstimulted () or stimulted with sili rystls, rystls () or or trnsfeted with poly(da:dt) () nd then inuted for 6 h. Dt re from two experiments with one representtive donor of three. () LPS-primed PBMCs stimulted with sili rystls in the presene (z-yvad-fmk) or sene () of the spse-1 inhiitor z-yvad (1 mm) nd then inuted for 6 h. Results re the men nd s.d. of two independent donors, presented s the fold inrese reltive to only LPS priming (ELISA), or re from one representtive donor (immunolot). Dt re representtive of two experiments. Neutrophils in lvge ( 1 6 ) PBS n = n = 3 n = n = 4 MyD88-KO TRIF-KO IL1R-KO Neutrophils in lvge ( 1 6 ) n = 4 n = 4 MyD88-KO TRIF-KO IL1R-KO Zymosn n = 4 Figure -medited neutrophil influx in model of ute lung inflmmtion is medited y IL-1. Flow ytometry of neutrophils in lung lvge fluid from wild-type mie (), mie douly defiient in MyD88 nd TRIF (MyD88-KO TRIF-KO) or IL-1R-defiient mie (IL-1R-KO) ssessed h fter orotrhel dministrtion of PBS or sili rystls ( mg per mouse; ) or zymosn ( mg per mouse; ). Numers ove rs indite numer of mie per group. Dt re from one representtive experiment of two (error rs, s.d.). 848 VOLUME 9 NUMBER 8 AUGUST 8 NATURE IMMUNOLOGY

3 8 Nture Pulishing Group Superntnts 1, (C7BL6) in NALP3 or the downstrem dptor moleule ASC. Similr to their response to known inflmmsome tivtors suh s rystls, or trnsfeted poly(da:dt), mrophges from wild-type mie produed lrge mounts of IL-1 fter exposure to sili rystls (Fig. 3, left). In ontrst, mrophges lking NALP3 or ASC filed to relese leved IL-1 in response to sili rystls (Fig. 3), whih indited requirement for NALP3 nd ASC in the proessing of IL-1 fter exposure to sili rystls. Consistent with pulished reports 3,7,1,11, responses to rystls nd were dependent on oth NALP3 nd ASC, wheres the response to trnsfeted poly(da:dt) ws ASC dependent yet NALP3 independent (Fig. 3, middle nd left). As IL-1 proessing nd relese ould oth e influened y tivtion y sili rystls, we next exmined the levge of prospse-1 into tive spse-1. We found tht like the relese of IL-1, spse-1 levge ws indued in dose-dependent wy fter tretment with sili rystls,, or poly(da:dt) (Fig. 3). This response ws ompletely dependent on ASC for ll stimuli, s ASC-defiient mrophges filed to trigger spse-1 levge in response to ny of these lignds. Consistent with the IL-1 relese, spse-1 levge in response to poly(da:dt) ws independent of NALP3. A pulished report hs suggested uri id relesed from ells is hief trigger for IL-1 prodution fter stimultion of ells with other rystl preprtions 1. We ssessed whether urise influened the IL-1 response to or sili rystls nd found tht the response to sili ws unimpired fter inution with urise (Fig. 3). We lso generted immortlized mrophge ell lines from wild-type, NALP3- nd ASC-defiient mie nd exmined their response to inflmmsome lignds. Immortlized mrophges hd responses qulittively similr to those of freshly isolted one mrrow derived mrophges (Supplementry Fig. 1 online). These results olletively suggest tht sili rystls tivte the NALP3-ASC omplex, leding to the tivtion of spse- 1 nd susequent levge of pro-il-1 into mture, sereted IL Lyste Figure 3 -medited relese of mtured IL-1 nd tivted spse-1 is medited y the NALP3 inflmmsome. () ELISA of IL-1 prodution y one mrrow derived wild-type, NALP3- defiient or ASC-defiient mrophges primed for 3 h with LPS nd then left unstimulted or stimulted with sili rystls, rystls or or trnsfeted with poly(da:dt); IL-1 ws nlyzed superntnts 6 h fter stimultion (Superntnts) nd intrellulr pro-il-1 ws ssessed in lystes of primed ut unstimulted ells (Lyste). () Immunolot nlysis of the levge of spse-1 to its tive p1 suunit in ells treted s desried in. () ELISA of IL-1 prodution y LPS-primed Superntnts Lyste Superntnts Lyste , (C7BL6) IB: spse-1 p1 IB: spse-1 p1 ASC-KO IB: spse-1 p1 1, , ASC-KO ( µg/ml) ( µg/ml) Ativtion of the NALP3 inflmmsome requires rystl uptke To delinete the upstrem mehnisms involved in sili rystl indued tivtion of the NALP3 inflmmsome, we first determined if uptke of rystlline inflmmsome tivtors influened ell tivtion. We pretreted humn PBMCs with ytohlsin D, well hrterized inhiitor of phgoytosis tht impirs tin filment ssemly, nd then stimulted the ells with or sili rystls s well s with two nonrystlline NALP3 tivtors, the imidzoquinoline derivtive R-848 nd 13. Cytohlsin D ompletely rogted IL-1 relese fter tretment with or sili rystls, wheres the response to R-848 or ws unffeted (Fig. 4). We otined similr results with mouse mrophges (Supplementry Fig. online). These results indite rystl inding nd uptke y phgoytosis re prerequisites for tivtion of the ytosoli NALP3 inflmmsome. To visulize the uptke of rystlline mteril in living ells, we devised method to imge rystls simultneously with ellulr omponents stined with fluoresent dyes (Supplementry Fig. 3 online). We used this method to study the uptke of rystls into mouse mrophges. After inuting ells with sili rystls for 3 min, we stined the ell surfe with the memrne-inding regent fluoresent holer toxin B-suunit in the presene or sene of ytohlsin D. Mrophges rpidly engulfed rystls into intrellulr omprtments y phgoytosis, wheres ells treted with ytohlsin D filed to engulf sili rystls (Fig. 4) or rystls (Supplementry Fig. ) y phgoytosis. The medin length of sili rystls engulfed y phgoytosis ws mm (Fig. 4). It is well known tht phgoytosis of prtiulte mtter in mrophges results in the genertion of retive oxygen speies. rystls trigger the prodution of retive oxygen speies in mrophges, events reported to e linked to disese pthology in siliindued inflmmtion 14. On the sis of those reports nd our own dt emphsizing the importne of phgoytosis in IL-1β response (% mx) Urise mouse mrophges stimulted with sili or rystls or in the presene or sene of urise, presented s the perent of mximum prodution (% mx). Dt re from one representtive experiment of two (error rs, s.d.). NATURE IMMUNOLOGY VOLUME 9 NUMBER 8 AUGUST 8 849

4 8 Nture Pulishing Group sili rystl indued NALP3 tivtion, we hypothesized tht the phgoyte retive oxygen speies system might e involved in this response. To ddress the involvement of this pthwy diretly, we exmined NALP3 inflmmsome responses in mie lking gp91phox, the 91-kilodlton suunit of the phgosoml NADPH oxidse ytohrome. Mie defiient in this suunit lk phgoyte superoxide prodution nd re reported to e hypersuseptile to vrious pthogens 1. However, mrophges from these mie responded normlly to sili rystls,, nd poly(da:dt) (Fig. 4d,e). These results indite tht the phgosoml respirtory-urst oxidse system is not essentil for tivtion of the NALP3 inflmmsome y these stimuli. Phgoytosis of rystls leds to lysosoml destiliztion We next did imging studies with mrophges undergoing phgoytosis of rystlline mteril to monitor the fte of the rgo engulfed. We used onfol refletion mirosopy omined with fluoresene imging to study the suellulr distriution of sili rystls over time (Supplementry Fig. 3). DQ ovlumin is useful tool for monitoring the endo-lysosoml omprtment in living ells in rel time. The fluoresene of the fluorophore BODIPY-FL d R-848 Cytohlsin D (C7BL/6) 1, Cytohlsin D.6.3 rystls Cytohlsin D 1.3 rystls Cytohlsin D 4. gp91phox-ko , (8-hloromethyl-4,4-difluoro-1,3,,7-tetrmethyl-4-or-3,4-diz-sindene) on DQ ovlumin is normlly quenhed unless the protein is proteolytilly proessed into peptides in endo-lysosoml omprtments. In untreted ells, proessed DQ ovlumin ws lolized to smll vesiulr nd tuulr endosomes or lysosomes, s expeted. In ontrst, we deteted lrge swollen lysosomes in most ells exposed to sili rystls. Mny ells hd ytosoli pttern of fluoresently proessed DQ ovlumin, whih indited lysosoml rupture or lekge of lysosoml ontents into the ytosol of rystl-treted ells (Fig. ). Fluoresent inditor dyes tht stin idi environments onfirmed those findings (dt not shown). In ddition, ingested fluoresent dextrn, whih trffis into the lysosoml pthwy, lso stined swollen lysosoml omprtments nd showed ytosoli trnslotion in most ells (Supplementry Fig. 4 online). Suh swelling of lysosomes ws not exlusive to sili rystl treted ells, s rystls eliited similr morphologil hnges nd rupture of lysosoml omprtments (dt not shown). We lso tested if rystls ould e deteted in ells outside phgosoml omprtments y stining the plsm memrne nd phgosoml memrnes of fixed nd permeilized ells with Ipf-KO + ytohlsin D Refletion (sili) Hoehst 1, e Reltive frequeny (C7BL/6) IB: spse-1 p1 gp91phox-ko IB: spse-1 p1 Ipf-KO IB: spse-1 p > rystl length (µm) 1, Figure 4 Inflmmsome tivtion requires phgosoml uptke of rystls ut is independent of the phgosoml retive oxygen speies system. () ELISA of the relese of IL-1 y humn LPS-primed PBMCs left untreted () or treted with inresing doses of ytohlsin D nd then stimulted for 6 h with R-848,, sili rystls or rystls. () Confol mirosopy of C7BL/6 (wild-type) mrophge ells stimulted for h with sili rystls in the presene (elow) or sene (ove) of ytohlsin D ( mm); ell memrnes were stined with fluoresent holer toxin B-suunit (red) nd nulei were stined with Hoehst dye (lue), followed y nlysis of rystl uptke (green). Sle rs, mm. () Length of sili rystls tken up y phgoytosis in C7BL/6 (wild-type) mrophges (n ¼ 1) stimulted for h with sili rystls. (d,e) ELISA of IL-1 in superntnts (d) nd immunolot nlysis of tivted spse-1 in lystes (e) of one mrrow derived wild-type mrophges (), gp91phox-defiient mrophges (gp91phox-ko) or gp91phoxsuffiient, Ipf-defiient mrophges (Ipf-KO; mixed kground ontrol) primed for 3 h with LPS nd then left unstimulted or stimulted for 6 h with sili rystls, rystls or or trnsfeted with poly(da:dt). Dt (error rs, s.d.) re from one representtive experiment of two (, e) or three (). 8 VOLUME 9 NUMBER 8 AUGUST 8 NATURE IMMUNOLOGY

5 8 Nture Pulishing Group Refletion (sili) DQ ovlumin No rystls d Refletion (sili) Hoehst No rystls Figure Phgoytosis of rystls leds to lysosoml destiliztion. (,) Confol mirosopy of wild-type (C7BL/6) mrophges inuted for 6 min with DQ ovlumin (1 mg/ml; red) lone or together with sili rystls (green), then stined with fluoresent holer toxin B-suunit (lue; ); or left untreted or inuted for 6 min with sili rystls (green), then fixed, mde permele (.1% (vol/vol) sponin) nd stined with fluoresent holer toxin B-suunit (red) nd Hoehst dye (lue; ). Originl mgnifition, 18 (min imges) with enlrgement of () or 1.8 () t fr right for oxed res in djent min imges. () Flow fluoresent holer toxin B-suunit. Most rystls were loted in phgosomes tht hd distint memrne stining; however, some rystls were in regions were not ssoited with memrnes nd thus were trnsloted to the ytosol (Fig. ). We otined similr dt when we exmined rystls (dt not shown). To quntify the degree of phgosoml rupture fter rystl tretment, we used ridine ornge, dye used to mesure lysosoml integrity. Aridine ornge fluoreses green in its monomeri stte nd inds to nuler nd ytosoli DNA nd RNA. Beuse of its tioni nture, ridine ornge eomes highly onentrted in idi omprtments, resulting in the formtion of dimers nd the pperne of red fluoresene (Supplementry Fig. online). The mount of red fluoresene of e Fluoresene intensity ( 1 ) Cells Cells ontrol + filomyin DQ ovlumin No rystls ( µg/ml) ( µg/ml) ( µg/ml) Lysosoml ridine ornge fluoresene f IL-1β (pg/ml) nm nm Bfilomyin ridine ornge in lysosomes diretly orreltes with the mount of idi lysosomes in ells. We took dvntge of this property nd monitored the lysosoml ontent of mrophges efore nd fter tretment with sili rystls. Inresing mounts of sili rystls resulted in loss of lysosomes, s indited y less red fluoresene of Fluoresene intensity ( 1 ) ( µg/ml) LysoSensor Bfilomyin (nm) ( µg/ml) nm nm nm nm nm nm ytometry of wild-type nd NALP3-defiient mrophges stined with ridine ornge nd then treted for 3 h with sili rystls. Numers ove rketed lines indite perent ells with loss of lysosoml stining with ridine ornge (exittion, 488 nm; emission, 6 69 nm). (d,e) Flow ytometry of wildtype mrophges treted with filomyin nd left unstined () or stined with 1 mm LysoSensor Green immeditely efore flow ytometry (d), or inuted for 6 min with DQ ovlumin in the presene (+ filomyin) or sene (+ ontrol (dimethyl sulfoxide)) of nm filomyin (e). (f) ELISA of the relese of IL-1 from LPS-primed wild-type mrophges treted with filomyin or left untreted nd then stimulted for 6 h with or sili rystls. Dt re from one representtive experiment of three (,) or two( f; error rs, s.d.). Figure 6 -medited IL-1 prodution is prtilly dependent on thepsin B. () ELISA of IL-1 in LPS-primed wild-type mrophge ells left untreted () or treted with the thepsin B (Cth B) inhiitor CA-74-Me (1 mm) nd then left unstimulted () or stimulted with sili rystls or or trnsfeted with poly(da:dt). () Immunolot nlysis of thepsin B in superntnts of wild-type or NALP3-defiient one mrrow derived mrophges primed for 3 h with LPS nd then left unstimulted or stimulted for 6 h with sili rystls, rystls or or trnsfeted with poly(da:dt). () Confol mirosopy of wild-type mrophges left untreted (left) or inuted for 3 h with sili rystls (pink; right), then stined for 1 h with fluoresent proes for tivted spse-1 (green) nd tivted thepsin B (red) nd then surfe-stined with fluoresent holer toxin B-suunit (lue). Boxed res in min imges t right re enlrged long the fr right mrgin (, top; 1., ottom). Sle rs, mm. Dt re from one representtive experiment of two ( (error rs, s.d.), ) or three().. Cth B inhiitor.8.4 No rystls Cspse-1 Cthepsin B Refletion (C7BL6) IB: Cth B IB: Cth B Cspse-1 Cthepsin B Refletion (sili rystls) 1, NATURE IMMUNOLOGY VOLUME 9 NUMBER 8 AUGUST 8 81

6 8 Nture Pulishing Group ridine ornge (Fig., top). Furthermore, we found lysosoml rupture due to sili phgoytosis to similr extent in ells defiient in NALP3 nd wild-type ells, whih indited tht lysosoml dmge ws independent of NALP3 (Fig., ottom). Colletively, these dt show tht phgoytosis of rystlline mteril leds to tive swelling of phgosomes, followed y phgosoml destiliztion nd rupture, thus resulting in the relese of phgosoml ontents, whih then gin ess to the ytosoli omprtment. Lysosomes ontin plethor of proteolyti enzymes, mny of whih re tivted y idifition of lysosoml pthwys. To ssess the funtion of lysosoml idifition in sili-medited NALP3 tivtion, we used filomyin A to lok the vuolr H + se system, whih is required for the idifition of lysosoml omprtments. As expeted, filomyin A loked the formtion of idi lysosomes in mrophges, s ssessed with the ph-sensitive dye LysoSensor Green, whih fluoreses only fter umultion in idi environments (Fig. d). In ddition, filomyin A suppressed the rpid, dose-dependent pperne of fluoresent DQ ovlumin (Fig. e), whih indited lower lysosoml proteolyti funtion. Notly, filomyin A ompletely loked silimedited IL-1 relese ut hd no effet on (Fig. f), whih suggested pivotl funtion for lysosomes in rystl-medited NALP3 tivtion IB: leved IL-1β e Aridine ornge DNA nd RNA Lysosomes priming priming Aridine ornge DNA nd RNA Lysosomes f -17 Cspse-1 Cthepsin B Cspse-1 Cthepsin B Lysosoml destiliztion triggers inflmmsome tivtion Beuse one importnt funtion of lysosoml idifition is the ph-dependent tivtion of proenzymes to further degrde lysosoml ontents, we hypothesized tht tivtion of lysosoml proenzymes ould e ritil event in sili-medited inflmmsome tivtion. In prtiulr, the thepsin fmily of proenzymes ws likely ndidte. To ddress their funtion in sili-medited inflmmsome tivtion, we tested severl thepsin inhiitors for their ility to ffet sili-medited IL-1 relese. Among the inhiitors tested, CA-74-Me, thepsin B speifi inhiitor 16, led to muh less spse-1 tivtion fter sili rystl tretment (Fig. 6). We lso deteted mture thepsin B in superntnts of rystl-stimulted ells independently of NALP3 (Fig. 6). To ssess whether relese of thepsin B from lysosomes ws temporlly ssoited with spse-1 tivtion, we inuted mouse mrophges with sili rystls nd dded fluoresent peptides tht indite thepsin B tivity y n inrese in red fluoresene nd spse-1 tivity y green fluoresene. When we inuted resting ells with those two reporter peptides, we found red fluoresene in lysosoml omprtments, inditive of thepsin B tivity, wheres resting ells did not quire green fluoresene euse they lked spse-1 tivity (Fig. 6,left). Cells treted with sili rystls quired either red fluoresene euse of lysosoml ontinment of the thepsin B inditor ( µg/ml) ASC-KO Neutrophils ( 1 6 ) g PBS (1 µg) IL-1R-KO ( µg/ml) Cth B inhiitor Bfilomyin d DQ ovlumin ( µg/ml) Cth B inhiitor Bfilomyin h IL-1β response (% mx) DQ ovlumin ( µg/ml) ( µg/ml) ( µg/ml) ( µg/ml) Urise Figure 7 tivtes the NALP3 inflmmsome through lysosoml destiliztion. () ELISA (ove; superntnts) nd immunolot nlysis (elow) of IL-1 prodution y humn PBMCs left untreted or primed for 3 h with LPS ( pg/ml) nd then stimulted for 6 h with inresing doses of lum. Eh dot ove lne represents one donor; smll horizontl lines indite the men. Results re from four donors (ELISA) or one representtive donor (immunolot). () ELISA of IL-1 in superntnts of wild-type, NALP3-defiient or ASC-defiient one mrrow derived mrophges primed for 3 h with LPS nd then stimulted with lum ( mg/ml). () Flow ytometry of neutrophils in peritonel lvge fluid from wild-type mie (n ¼ ) nd IL1R-defiient mie (n ¼ ) given peritonel injetion of lum (1 ug) or PBS h efore. (d f) Confol mirosopy of wild-type mrophges inuted for 6 min with DQ ovlumin (1 mg/ml; red) lone or together with lum (green) nd then stined with fluoresent holer toxin B-suunit (lue; d); or stined with ridine ornge nd then treted with lum (lue; e); or left untreted (right) or inuted for 3 h with lum (pink; left), then stined for 1 h with fluoresent proes for tivted spse-1 (green) nd tivted thepsin B (red) nd then surfe-stined with fluoresent holer toxin B-suunit (lue; f). Sle rs, mm. (g) ELISA of the relese of IL-1 y LPS-primed one mrrow derived mrophges left untreted or treted with the thepsin B inhiitor CA-74-Me (1 mm) or filomyin ( nm) nd then stimulted with lum. (h) ELISA of IL-1 prodution y LPS-primed one mrrow derived mrophges treted for 6 h with lum or rystls in the presene of inresing doses of urise, presented s the perent of the mximum (1%) otined in the sene of urise. Dt (error rs, s.d.) re from one representtive experiment of two (,d,f h) or three (,e). 8 VOLUME 9 NUMBER 8 AUGUST 8 NATURE IMMUNOLOGY

7 8 Nture Pulishing Group peptide or green ytoplsmi fluoresene inditive of spse-1 tivity (Fig. 6, right). Ativted mrophges otined from spse-1-defiient mie did not stin with the spse-1 inditor fluoresent peptide, whih demonstrted the speifiity of the regent (dt not shown). This notle differene etween stining of either lysosoml thepsin B or tivted spse-1 suggested tht ells tht lose lysosoml thepsin B fter lysosoml rupture (loss of red lysosoml fluoresene) rpidly tivte spse-1. These results olletively suggest tht the NALP3 inflmmsome senses lysosoml ontents relesed into the ytosol of phgoyti mrophges fter rystl-indued lysosoml dmge. triggers NALP3 tivtion through lysosoml destiliztion inum slts (lum) re the most ommonly used vine djuvnts, nd ll lum preprtions ontin rystls. inum hydroxide hs een reported to indue the levge of IL-1 nd IL-18 in spse-1-dependent wy 17. To determine whether lum indues inflmmtion y mehnism similr to tht of sili rystls, we did experiments with mixture of luminum hydroxide nd mgnesium hydroxide (Imjet lum). indued IL-1 mturtion nd relese y humn PBMCs (Fig. 7) in spse-1-dependent wy (dt not shown). In mouse mrophges, the lum-indued relese of Lysosome disruption d Leu-Leu- OMe (mm) Dextrn Dextrn e , Leu-Leu- OMe Lysosome disruption 1 Cth B inhiitor IL-1 ws dependent on NALP3 nd ASC (Fig. 7), whih indited tht lum triggers inflmmtion through tivtion of the NALP3 inflmmsome. Additionlly, the influx of neutrophils into the peritoneum fter intrperitonel dministrtion of lum ws dependent on IL-1 tivity (Fig. 7). Of note, lum dded to ells indued onsiderle morphologil hnges nd led to lysosoml rupture, s shown y the ytosoli trnslotion of DQ ovlumin (Fig. 7d) nd the lysosoml stining pttern of ridine ornge (Fig. 7e). As noted with sili rystls, ells inuted with lum stined positively with thepsin B inditor or the spse-1 inditor (Fig. 7f), whih demonstrted lose orreltion of lysosoml loss nd spse-1 tivtion. In greement with tht ide, lum-indued relese of IL-1 ws prtilly dependent on lysosoml idifition nd thepsin B tivity (Fig. 7g). Urise dded to rystl- or -stimulted ells inhiited the rystl eliited relese of IL-1 in dose-dependent wy ut did not hnge the response to lum (Fig. 7h). NALP3 tivtion y rystl-independent lysosoml dmge To determine if lysosoml rupture lone ws suffiient to tivte the inflmmsome, we turned to model with whih we ould ssess the funtion of lysosoml rupture without the requirement of the ddition of known inflmmsome tivtors. We loded mouse mrophges Leu-Leu- OMe Lysosome disruption 1 Cth B inhiitor ASC-KO 1, 1, Leu-Leu- OMe Aridine ornge DNA nd RNA Lysosomes Dextrn 6 Figure 8 Rupture of sterile lysosomes tivtes the NALP3 inflmmsome. () Mirosopy of wild-type mrophge ells inuted for 3 min in the presene of fluoresent dextrn (red) nd then left untreted or treted with hypertoni nd hypotoni solutions to indue lysosoml rupture; left, fluoresent imge; right, trnsmitted light imge. () Immunolot nlysis of tivted spse-1 in superntnts of wild-type or NALP3- defiient one mrrow derived mrophges treted s desried in for h with or without the thepsin B inhiitor CA-74-Me (1 mm or mm); the ddition of or poly(da:dt) serves s ontrol. () Confol mirosopy of wild-type mrophges leled with ridine ornge (top row) or inuted for 3 h in the presene of fluoresent dextrn (red; ottom row) nd inuted with Leu-Leu-OMe (1, mm). (d) ELISA of the relese of IL-1 y LPS-primed wild-type mrophges inuted for 6 h with Leu-Leu-OMe (1 or nm), sili rystls ( mg/ml), or poly(da:dt). (e) ELISA of the relese of IL-1 y wild-type, NALP3-defiient or ASC-defiient one mrrow derived mrophges primed with LPS nd then stimulted with Leu-Leu-OMe ( or 1, mm) or ASC-KO Leu-Leu-OMe Leu-Leu-OMe Cth B inhiitor Bfilomyin (ove) or stimulted with sili rystls or poly(da:dt) (elow) in the presene or sene of the thepsin B inhiitor CA-74-Me (1 mm) or filomyin ( nm). Originl mgnifition, 16 () or 18 (). Dt (error rs, s.d.) re from one representtive experiment of two (,,e) or three (,d). NATURE IMMUNOLOGY VOLUME 9 NUMBER 8 AUGUST 8 83

8 8 Nture Pulishing Group with hypertoni solution nd then pled them in hypotoni medi, whih resulted in the rupture of endo-lysosoml omprtments. This strtegy suessfully trnsloted endo-lysosoml fluoresent dextrn into the ytosol, s shown y diffuse ytosoli stining of ells fter the tretment (Fig. 8). After this rystl-independent disruption of lysosomes, there ws spse-1 levge (Fig. 8, left) tht ws prtilly inhiited y thepsin B inhiitor. Notly, this rystlindependent lysosoml rupture ws ompletely dependent on NALP3 (Fig. 8, right). These results olletively indite tht rupture of lysosoml omprtments nd lekge of lysosoml ontents into the ytosol even in the sene of rystl mteril ws suffiient to trigger tivtion of the NALP3 inflmmsome in prtilly thepsin B dependent wy. Additionlly, we used THP-1 humn monoytes differentited in the presene of phorol 1-myristte 13-ette (PMA) in this lysosoml rupture ssy; this represents model system tht does not rely on miroil stimuli for pro-il-1 priming. Similr to the findings with mouse mrophges, THP-1 ells were lso tivted to relese leved IL-1 fter lysosoml rupture in prtilly thepsin B dependent wy (Supplementry Fig. 6 online). To onfirm those findings, we used n lterntive method to disrupt lysosomes with the regent Leu-Leu-OMe (L-leuyl-L-leuine methyl ester), whih is known to indue lysosoml dmge 18,19. Leu-Leu-OMe is funtionlized dipeptide tht is onverted into memrne-lysing ompound y the lysosoml enzyme dipeptidyl peptidse I. We inuted mouse mrophges with Leu-Leu-OMe t the reported effetive doses nd noted onsiderle swelling nd rupture of lysosomes in most ells, s indited y lysosoml morphology, loss of red fluoresene of ridine ornge nd diffuse ytoplsmi stining y fluoresent dextrn (Fig. 8). Inhiition of lysosoml idifition y filomyin resulted in muh less ltered lysosoml morphology nd rupture (dt not shown). We next ssessed whether Leu-Leu-OMe tivted Il-1 mturtion. We deteted lrge quntities of IL-1 in ellulr superntnts in response to Leu-Leu-OMe, similr to the onentrtions eliited y known inflmmsome tivtors (Fig. 8d). To test whether the Leu-Leu-OMe-indued lysosoml dmge indued NALP3 tivtion, we nlyzed mrophges from mie defiient in NALP3 nd ASC. As noted with sili rystls, ells defiient in NALP3 nd ASC filed to respond to Leu-Leu-OMe-indued lysosoml dmge nd relesed only smll quntities of IL-1 (Fig. 8e, left). Finlly, inhiition of hnges in lysosoml ph y filomyin or inhiition of thepsin B lso inhiited the relese of IL-1 in response to Leu-Leu-OMe (Fig. 8e, right). DISCUSSION Chemilly nd struturlly diverse stimuli, rnging from the smll moleules nd imidzoquinoline derivtives (suh s R-848 nd R-837) to teril toxins nd vrious rystls, n tivte the NALP3 inflmmsome 4. It hs een reported tht sestos nd sili rystls n tivte the NALP3 inflmmsome nd tht lum n tivte the NALP3 inflmmsome 1. Undoutedly, the list of NALP3 inflmmsome tivtors will grow, yet the mehnisms y whih the NALP3 inflmmsome n sense different tivtors re not yet fully understood. One possiility is tht NALP3 ts s pttern-reognition reeptor tht n diretly ind nd respond to vrious lignds. Indeed, reports suggest tht NALP3 tivtors ould gin ess to the ytoplsmi spe,3. A preedent for suh mehnism is the lrge spetrum of lignds tht n e sensed y ertin Toll-like reeptors 4. Another possiility is tht the NALP3 inflmmsome detets n intermedite moleule generted y ommon mehnism eliited y mny different lignds. It hs een shown tht potssium efflux is required for NALP3 tivtion fter vrious inflmmsome stimuli,6. However, it is unler whether potssium efflux lone represents NALP3-inflmmsome stimulus itself or whether low intrellulr potssium ts s n dditionl requirement for inflmmsome ssemly fter the pperne of n s-yet-undefined primry stimulus. An exmple of this potentil mehnism is found in the formtion of the losely relted poptosome 7,8. The loss of intrellulr potssium during poptosis is n erly, essentil step for suessful ssemly of the poptosome, whih forms fter ytohrome is relesed from mitohondri in stress situtions. It is therefore possile tht formtion of the NALP3 inflmmsome is highly fvored t low intrellulr potssium onentrtions, whih my e required yet not suffiient for tivtion. We shve shown here tht tivtion of the NALP3 inflmmsome y rystls or lterntively indued lysosoml dmge required the uptke nd idifition of lysosomes nd tht the ingested rystls led to lysosoml swelling nd lekge. Notly, inhiition of single lysosoml protese, thepsin B, led to sustntil yet inomplete derese in tivtion of the NALP3 inflmmsome y lysosoml dmge. In this ontext it is noteworthy tht the NALP3 stimulus nigeriin, thought to tivte solely y potssium efflux, indues lysosoml lekge nd susequent spse-1 tivtion through the funtion of thepsin B 9. As it hs lso een shown tht pnnexin 1, whih helps in estlishing lrger lysosoml hnnels, is required for nigeriin-medited NALP3 tivtion 3,31, it is oneivle tht pnnexin 1 not only llows ess of teril lignds to the ytoplsm,3 ut ould lso initite lysosoml destiliztion. Other studies hve lso suggested involvement of thepsin B in the funtion of the NALP3 inflmmsome, yet those dt hve indited involvement of thepsin B downstrem of NALP3 3,33 rther thn upstrem of NALP3, s we hve demonstrted here. It will e useful to define moleulr trgets of thepsin B nd potentilly other ph-tivted proteses, s this my led to the identifition of ommon upstrem NALP3-tivting ftors. It hs een shown tht the relted thepsin K is lso involved in innte immune reognition. Cthepsin K ts upstrem of the innte signling reeptor Toll-like reeptor 9 (ref. 34), whih signls from within endo-lysosoml omprtments 3,36. Colletively, our dt suggest the hypothesis tht lysosoml dmge or lekge is pereived y the immune system s n endogenous dnger signl. The NALP3 inflmmsome n respond to internl memrne perturtions nd therey respond to different stimuli, whih ll hve in ommon the ility to indue lysosoml destiliztion. Phgoytes tht ingest endogenous or exogenous rystllized mteril or possily lso ggregted proteins or peptides re thus le to sense lysosoml dmge indued y the rgo tken up y phgoytosis. After the NALP3 inflmmsome is tivted, highly inflmmtory ytokines re generted, whih leds to the reruitment of other immune ells to the ffeted tissue nd to the indution of inflmmtory mehnisms tht ssist in lering the rystllized mteril. However, it is lso possile tht NALP3 tivtion y this mehnism n led to sustined inflmmtion nd resulting tissue dmge. For exmple, hroni inhltive exposure to sili or sestos n led to hroni inflmmtion, tissue dmge nd rinogenesis. NALP3 tivtion y prtiulte mtter n lso intentionlly e exploited therpeutilly. inum slt preprtions, ommonly used s djuvnts, ontin smll rystlline mterils. We hve shown here tht lum tivted immune ells through the NALP3 inflmmsome y mehnism involving lysosoml destiliztion. It is likely tht other vine ndidtes tht re of prtiulte nture or tht n pertur lysosoml memrnes t y similr NALP3-tivtion mehnism. Our findings should thus id in the development of new 84 VOLUME 9 NUMBER 8 AUGUST 8 NATURE IMMUNOLOGY

9 8 Nture Pulishing Group vines nd n led to new therpeuti ides for the tretment of pthologies relted to the NALP3 inflmmsome. METHODS Mie. NALP3-defiient (Nlrp3 / )mie 13, ASC-defiient (Pyrd / )mie 13 nd mie defiient in the protese-tivting ftor Ipf (Nlr4 / ) 37 were provided y Millennium Phrmeutils. C7BL/6 mie, 19/Sv mie, C7BL/6 19 F 1 mie, IL-1R-defiient (Il1r1 / ) mie nd gp91phox-defiient (Cy / )mie were from Jkson Lortories. Mie douly defiient in MyD88 nd TRIF were generted from MyD88-defiient (Myd88 / ) mie nd TRIF-defiient (Tim1 / ) mie (provided y S. Akir). Mie 7 9 weeks of ge were used in ll experiments. All mouse strins were red nd mintined in speifi pthogen free onditions in the niml filities t the University of Msshusetts Medil Shool. All experiments involving live nimls were in ordne with guidelines set forth y the University of Msshusetts Medil Shool Deprtment of Animl Mediine nd the Institutionl Animl Cre nd Use Committee. Regents. Aridine ornge,, filomyin A1, ytohlsin D, LPS, PMA, poly(da:dt) sodium slt, surose nd zymosn were from Sigm-Aldrih. CA-74-Me nd polyethylene glyol 1 were from Cliohem. DQ ovlumin, Alex Fluor 647 onjugted dextrn, Alex Fluor 647 onjugted holer toxin B-suunit, LysoSensor Green nd Hoehst stin were from Moleulr Proes Invitrogen. (Imjet lum djuvnt; mixture of luminum hydroxide nd mgnesium hydroxide) ws from Piere. Leu-Leu-OMeHCl ws from Chem-Impex Interntionl. Urise (Elitek) ws from Snofi-Aventis. rystls (MIN-U-SIL-1) were provided y US. A poly-dispersed preprtion of sili rystls of up to 1 mm in length ws used in ll experiments. rystls were prepred s desried 38. In vivo sili model. Mie were exposed y diret orotrhel instilltion of 4-ml queous suspensions of mg sili rystls (MIN-U-SIL-1) in PBS or mg rude zymosn; ontrol mie reeived PBS. Mie were killed h fter instilltion nd ronholveolr lvge fluid ws otined y three onseutive instilltions nd withdrwls of 1 ml of 1% (vol/vol) BSA in PBS. Reovered fluid ws pelleted y entrifugtion nd ells were ounted. Susequently, ells were stined for surfe mrkers nd neutrophils were identified s ells doule positive for MCA771B (7/4; Serote) nd Ly6G (1A8; BD Biosienes) y flow ytometry. For lum experiments, mie were injeted intrperitonelly with 1 mg of mixture of luminum hydroxide nd mgnesium hydroxide (Piere) in ml PBS. After hllenge (16 18 h), mie were killed nd their peritonel vities were wshed with 6 ml PBS ontining 3 mm EDTA nd 1 U/ml of heprin. Totl peritonel exudte ells were ounted with hemtoytometer nd neutrophils were ounted s desried ove. Cell isoltion nd ulture. Bone mrrow derived mrophges were generted s desried 39. Humn PBMCs were isolted y from whole lood of helthy volunteers y density-grdient entrifugtion (Institutionl Review Bord protool H-1497). Red lood ells were lysed with red lood ell lysis uffer (Sigm). PBMCs nd mrophges were used t density of 1 6 ells per ml. All primry ells nd ell lines exept THP-1 ells were ultured in DMEM supplemented with L-glutmine, iprofloxin (Cellgro) nd 1% (vol/vol) FCS (Hylone). THP-1 ells were ultured in RPMI medium supplemented with 1% (vol/vol) FCS (Hylone), L-glutmine, sodium pyruvte (Cellgro) nd iprofloxin. At 1 d efore stimultion, THP-1 ells were differentited for 3 h with. mm PMA, were wshed three times nd were plted for stimultion. All experiments for immunolot nlysis used serum-free DMEM medium. Where pproprite, ells were stimulted with mm t 1 h efore superntnts were olleted. Immortlized mrophge ell lines. Immortlized mrophge ell lines were generted with pulished J reominnt retrovirus (rrying v-my nd v-rf(mil) onogenes) 4. Primry one mrrow ells were inuted in L99 mouse firolst onditioned medium for 3-4 d for the indution of mrophge differentition. Susequently, ells were infeted with J reominnt retrovirus. Cells were mintined in ulture for 3 6 months nd were slowly wened off L99 superntnt until they were growing in the sene of onditioned medium. Mrophge phenotype ws verified y surfe expression of the mrkers CD11 (M1/7; BD Phrmingen) nd F4/8 (BM8; ebiosienes) s well s rnge of funtionl prmeters, inluding responsiveness to Toll-like reeptor lignds nd teril uptke. Mrophge ell lines were generted from wild-type (C7BL/6), NALP3-defiient nd ASC-defiient mie. Flow ytometry. For evlution of lysosoml rupture, ells were inuted for 1 min with ridine ornge (1 mg/ml), were wshed three times nd then were stimulted. Lysosoml rupture ws ssessed y flow ytometry s loss of emission t 6 6 nm. An LSRII (BD Biosienes) ws used for ll flow ytometry. Dt were quired y DIVA (BD Biosienes) nd were nlyzed with FlowJo softwre (Tree Str). Confol mirosopy. Confol refletion mirosopy ws omined with fluoresene mirosopy on Lei SP AOBS onfol lser-snning mirosope. Refletion ws ptured y plement of the detetor hnnel diretly over the wvelength of the seleted lser hnnel for refletion light pture nd the AOBS mirosope ws set to llow 1% of lser light into the olletion hnnel. Fluoresene ws simultneously ptured y stndrd onfol imging tehniques. ELISA. Cell ulture superntnts were ssyed for IL-1 with ELISA kits from BD Biosienes ording to the mnufturer s instrutions. For mesurement of intrellulr IL-1, ells were wshed nd sujeted to three yles of freezing nd thwing in ssy diluent. Lysosoml rupture in THP 1 ells. THP-1 ells differentited with PMA were inuted for 1 min t 37 1C in hypertoni DMEM medium ontining 1% (vol/vol) polyethylene glyol 1, M surose, mm HEPES, ph 7., nd % (vol/vol) FCS. Cells were susequently wshed nd were inuted for min in hypotoni DMEM medium (DMEM/H O, 3:) for indution of lysosoml rupture. Cells were then inuted for n dditionl 4 h in serumfree DMEM. Stining of thepsin B nd spse-1. After tretments, ells were inuted for 3 min t 37 1C with the fluoresent thepsin B sustrte Mgi Red (resyl violet isustituted y mide linkge to the dipeptide rgininerginine) together with the fluorohrome inhiitor of spse-1, green fluoresent peptide FAM-YVAD-fmk (-roxyfluoresein Tyr-Vl-Al-Asp fluoromethyl ketone), ording to the mnufturer s reommendtions (Immunohemistry Tehnologies). After eing wshed three times in PBS, ells were visulized y onfol mirosopy. Immunolot nlysis. Cell ulture superntnts were preipitted y the ddition of n equl volume of methnol nd. volumes of hloroform, then were vortexed nd entrifuged for 1 min t,g. The upper phse ws disrded nd ml methnol ws dded to the interphse. This mixture ws entrifuged for 1 min t,g nd the protein pellet ws dried t 1C, resuspended in Lemmli uffer nd oiled for min t 99 1C. Smples were seprted y 1% SDS-PAGE nd were trnsferred onto nitroellulose memrnes. Blots were inuted with rit polylonl ntiody to mouse spse-1 p1 (s-14; Snt Cruz Biotehnology), rit polylonl ntiody to the humn spse-1 suunit p1 (s-1; 1 Snt Cruz Biotehnology), rit polylonl ntiody to humn leved IL-1 (Asp116; Cell Signling) or rit polylonl ntiody to mouse thepsin B (AF96; R&D Systems). Aession odes. UCSD-Nture Signling Gtewy ( wy.org): A3663 nd A33. Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS MyD88-defiient nd TRIF-defiient mie were provided y S. Akir (Osk University). We thnk A. Cerny nd Joseph Boulnger for niml husndry nd genotyping; D. Klvkolnu (University of Mrylnd Shool of Mediine) for providing J reominnt retroviruses; nd J. Lee nd H. Kornfeld for help with the lung inflmmtion model. Supported y the Deutshe Forshungsgemeinshft (Ho783/-1 to V.H. nd GK1 to F.B.) nd the NATURE IMMUNOLOGY VOLUME 9 NUMBER 8 AUGUST 8 8

10 8 Nture Pulishing Group US Ntionl Institutes of Helth (R1 AI-6483 to E.L., RO1 AI to K.A.F. nd RO1 AI4343 to K.L.R.). Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Mossmn, B.T. & Churg, A. Mehnisms in the pthogenesis of sestosis nd siliosis. Am. J. Respir. Crit. Cre Med. 17, (1998).. Huux, F. New developments in the understnding of immunology in siliosis. Curr. Opin. Allergy Clin. Immunol. 7, (7). 3. Mrtinon, F., Petrilli, V., Myor, A., Trdivel, A. & Tshopp, J. Gout-ssoited uri id rystls tivte the NALP3 inflmmsome. Nture 44, (6). 4. Petrilli, V., Dostert, C., Muruve, D.A. & Tshopp, J. The inflmmsome: dnger sensing omplex triggering innte immunity. Curr. Opin. Immunol. 19, 61 6 (7).. Agostini, L. et l. NALP3 forms n IL-1-proessing inflmmsome with inresed tivity in Mukle-Wells utoinflmmtory disorder. Immunity, (4). 6. Dinrello, C.A. Interleukin-1, interleukin-18, nd the interleukin-1 onverting enzyme. Ann. NY Ad. Si. 86, 1 11(1998). 7. Muruve, D.A. et l. The inflmmsome reognizes ytosoli miroil nd host DNA nd triggers n innte immune response. Nture 4, (8). 8. Ozinsky, A. et l. The repertoire for pttern reognition of pthogens y the innte immune system is defined y oopertion etween Toll-like reeptors. Pro. Ntl. Ad. Si. USA 97, (). 9. Brown, G.D. et l. Detin-1 is mjor -glun reeptor on mrophges. J. Exp. Med. 196, (). 1. Mrithsn, S. et l. Cryopyrin tivtes the inflmmsome in response to toxins nd. Nture 44, 8 3 (6). 11. Mrithsn, S. et l. Differentil tivtion of the inflmmsome y spse-1 dptors ASC nd Ipf. Nture 43, (4). 1. Kool, M. et l. djuvnt oosts dptive immunity y induing uri id nd tivting inflmmtory dendriti ells. J. Exp. Med., (8). 13. Knnegnti, T.D. et l. Bteril RNA nd smll ntivirl ompounds tivte spse-1 through ryopyrin/nlp3. Nture 44, (6). 14. Fuini, B. & Hurd, A. Retive oxygen speies (ROS) nd retive nitrogen speies (RNS) genertion y sili in inflmmtion nd firosis. Free Rdi. Biol. Med. 34, (3). 1. 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Innte immune tivtion through Nlp3 inflmmsome sensing of sestos nd sili. Siene 3, (8). 1. Eisenrth, S.C., Colegio, O.R., O Connor, W., Sutterwl, F.S. & Flvell, R.A. Cruil role for the Nlp3 inflmmsome in the immunostimultory properties of luminium djuvnts. Nture 43, (8).. Knnegnti, T.D. et l. Pnnexin-1-medited reognition of teril moleules tivtes the ryopyrin inflmmsome independent of Toll-like reeptor signling. Immunity 6, (7). 3. Mrin-Gri, N. et l. Pnnexin-1-medited intrellulr delivery of murmyl dipeptide indues spse-1 Ativtion vi ryopyrin/nlrp3 independently of Nod. J. Immunol. 18, 4 47 (8). 4. Tked, K., Kisho, T. & Akir, S. Toll-like reeptors. Annu. Rev. Immunol. 1, (3).. Petrilli, V. et l. Ativtion of the NALP3 inflmmsome is triggered y low intrellulr potssium onentrtion. Cell Deth Differ. 14, (7). 6. Frnhi, L., Knnegnti, T.D., Duyk, G.R. & Nunez, G. Differentil requirement of PX7 reeptor nd intrellulr K + for spse-1 tivtion indued y intrellulr nd extrellulr teri. J. Biol. Chem. 8, (7). 7. Cin, K., Brtton, S.B. & Cohen, G.M. The Apf-1 poptosome: lrge spsetivting omplex. Biohimie 84, 3 14 (). 8. Krki, P. et l. Intrellulr K + inhiits poptosis y suppressing the Apf-1 poptosome formtion nd susequent downstrem pthwys ut not ytohrome relese. Cell Deth Differ. 14, 68 7 (7). 9. Hentze, H., Lin, X.Y., Choi, M.S. & Porter, A.G. Critil role for thepsin B in mediting spse-1-dependent interleukin-18 mturtion nd spse-1-independent nerosis triggered y the miroil toxin nigeriin. Cell Deth Differ. 1, (3). 3. Pelegrin, P. & Surprennt, A. Pnnexin-1 medites lrge pore formtion nd interleukin-1 relese y the -gted PX7 reeptor. EMBO J., 71 8 (6). 31. Pelegrin, P. & Surprennt, A. Pnnexin-1 ouples to mitotoxin- nd nigeriin-indued interleukin-1 relese through dye uptke-independent pthwy. J. Biol. Chem. 8, (7). 3. Fujisw, A. et l. Disese-ssoited muttions in CIAS1 indue thepsin B-dependent rpid ell deth of humn THP-1 monoyti ells. Blood 19, (7). 33. Willinghm, S.B. et l. Miroil pthogen-indued neroti ell deth medited y the inflmmsome omponents CIAS1/ryopyrin/NLRP3 nd ASC. Cell Host Miroe, (7). 34. Asgiri, M. et l. Cthepsin K-dependent Toll-like reeptor 9 signling reveled in experimentl rthritis. Siene 319, (8). 3. Ltz, E. et l. TLR9 signls fter trnsloting from the ER to CpG DNA in the lysosome. Nt. Immunol., (4). 36. Ltz, E. et l. Lignd-indued onformtionl hnges llosterilly tivte Toll-like reeptor 9. Nt. Immunol. 8, (7). 37. Frnhi, L. et l. Cytosoli flgellin requires Ipf for tivtion of spse-1 nd interleukin 1 in slmonell-infeted mrophges. Nt. Immunol. 7, 76 8 (6). 38. Shiltz, C. et l. Monosodium urte monohydrte rystl-indued inflmmtion in vivo: quntittive histomorphometri nlysis of ellulr events. Arthritis Rheum. 46, (). 39. Sever, M., Coi, E.M. & Fitzgerld, K.A. Toll-like reeptor-dependent nd -independent viperin gene expression nd ounter-regultion y PRDI-inding ftor-1/blimp1. J. Biol. Chem. 81, (6). 4. Roerson, S.M. & Wlker, W.S. Immortliztion of loned mouse spleni mrophges with retrovirus ontining the v-rf/mil nd v-my onogenes. Cell. Immunol. 116, (1988). 86 VOLUME 9 NUMBER 8 AUGUST 8 NATURE IMMUNOLOGY