Thioredoxin-interacting protein links oxidative stress to inflammasome activation

Transcription

1 A rt i l e s Thioredoxin-interting protein links oxidtive stress to inflmmsome tivtion Rongin Zhou 1, Aury Trdivel 1, Bernrd Thorens 2, Inpyo Choi 3 & Jürg Tshopp 1 29 Nture Ameri, In. All rights reserved. The inflmmsome hs mjor role in regulting innte immunity. Deregulted inflmmsome tivity is ssoited with severl inflmmtory diseses, yet little is known out the signling pthwys tht led to its tivtion. Here we show tht interted with thioredoxin ()-interting protein (), protein linked to insulin resistne. Inflmmsome tivtors suh s uri id rystls indued the dissoition of from thioredoxin in retive oxygen speies (ROS)- sensitive mnner nd llowed it to ind. defiieny impired tivtion of the inflmmsome nd susequent seretion of interleukin 1 (IL-1 ). Akin to Txnip / mie, Nlrp3 / mie showed improved gluose tolerne nd insulin sensitivity. The prtiiption of in the inflmmsome tivtion my provide mehnisti link to the oserved involvement of IL-1 in the pthogenesis of type 2 dietes. Ative, mture interleukin 1β (IL-1β) is produed y levge of the intive pro-il-1β preursor y spse-1, whih is tivted in lrge multiprotein omplex lled the inflmmsome 1,2. The inflmmsome, omposed of the Nod-like reeptor protein (lso lled yopyrin or NALP3), CARDINAL, the dptor protein ASC nd spse-1 (ref. 3), is vitl for the prodution of mture IL-1β in response to vriety of signls. The inflmmsome is tivted y teril toxins 4 or pthogen-ssoited moleulr ptterns, suh s murmyldipeptide 5. n lso detet endogenous stress-ssoited dnger signls, suh s ATP 4, monosodium urte rystls () 6 or β-myloid 7. Although retive oxygen speies (ROS), together with low ytoplsmi onentrtions of potssium (K + ), re known to e prerequisites for tivtion of the inflmmsome 8,9, the signling pthwys tht led to tivtion re still poorly understood. Three distint signling pthwys for inflmmsome tivtion hve een proposed. In one model, tivtors enter the ytoplsm vi pnnexin 1, hnnel protein tht is ssoited with nd opened y the purinergi reeptor P2X7 (ref. 1). In seond model, thepsin B relesed from ruptured lysosomes fter phgoytosis of lrge prtiles suh s sili or β-myloid leves n unidentified sustrte nd triggers inflmmsome tivtion 7,11. In third model, ROS, whih re generted y ll known tivtors, indue onformtionl hnge in n unidentified protein tht susequently tivtes the inflmmsome 1. To lrify the reltive ontriution of these proposed signling pthwys, we serhed for previously unknown inding prtners. One of the proteins we identified ws thioredoxin-interting protein (; lso lled VDUP1 or TBP-2), whih in onditions of oxidtive stress inds NALP3 nd leds to its tivtion. RESULTS is n -inding protein A yest two-hyrid sreen using the leuine-rih repets (LRRs) of s it hs identified 12,13 s potentil inding prtner of. We onfirmed the intertion etween nd in humn emryoni kidney (HEK293) T ells. Overexpressed (Fig. 1) nd endogenous (Fig. 1) ound full-length nd the LRR region of, s expeted. The nuleotideinding NACHT domin of lso interted with. Endogenous did not ind other memers of the NLR fmily or other proteins ontining LRRs (Fig. 1), whih suggested tht the intertion is speifi feture of. ontins two rrestin domins nd roxy (C)-terminl extension with no pprent homology to other proteins (Fig. 1). The C-terminl rrestin domin preferentilly interted with (Fig. 1d). Ativtion of the inflmmsome is dependent on the genertion of ROS 14. In ft, ll known tivtors generte ROS nd, onversely, inhiitors of ROS lok inflmmsome tivtion 14. Consistent with tht, we found tht the ddition of ws suffiient to trigger the proessing nd seretion of IL-1β in n ASC spse-1 dependent mnner in THP-1 humn mrophges (Fig. 2 nd Supplementry Fig. 1). In ontrst, we still oserved IL-1β proessing in the sene of the spse-1 tivtor Ipf (Supplementry Fig. 1), whih indited tht Ipf did not prtiipte in the ROS-medited inflmmsome tivtion. hs een identified s inding prtner of redued thioredoxin () 15. diretly interts with the redox-tive domin of nd is thought to funtion s negtive regultor of the redutse tivity. At high onentrtions of, dissoited from (Fig. 2). We lso oserved dissoition with other 1 Deprtment of Biohemistry nd 2 Center of Integrtive Genomis, University of Lusnne, Eplinges, Switzerlnd. 3 Kore Reserh Institute of Biosiene nd Biotehnology, Yusong, Tejon, Repuli of Kore. Correspondene should e ddressed to J.T. (jurg.tshopp@unil.h). Reeived 27 August; epted 11 Novemer; pulished online 2 Deemer 29; doi:1.138/ni.1831 nture immunology DVANCE ONLINE PUBLICATION

2 A rt i l e s 29 Nture Ameri, In. All rights reserved. NACHT PYD LRR VSV IP: Flg IP: Flg Arrestin-N PYD Arrestin-C NLRP2 Nod1 Nod2 Ipf VSV- VSV- Flg-tgged proteins 381 NACHT SDS22 pp32 Flg-tgged proteins (ROS-generting) tivtors suh s nd 14 nd found tht it ws loked y the ROS inhiitor APDC (Fig. 2). Conversely, ROS ( ) used to intert with (Fig. 2). Furthermore, we deteted the ROS-dependent - ssoition fter treting mrophges with or. The ddition of to THP-1 mrophges resulted in muh more ROS fter 3 min (Fig. 3). Simultneously, egn to dissoite from (Fig. 3). Complete dissoition ws evident 1 h fter stimultion, t whih time the - intertion eme detetle. Consistent with tht tivtion time ourse, proessed, tive IL-1β ws evident in ulture superntnts fter 2 h of stimultion (Fig. 3). Together, these dt suggest model wherey is relesed from fter oxidtion of y ROS, whih llows to ind the inflmmsome. medites inflmmsome tivtion We next investigted whether the oserved intertion etween nd ws essentil for tivtion of the inflmmsome. For this, we downregulted expression in THP-1 mrophges through the use of smll interfering RNA. Although omplete rogtion in expression ws not hieved, seretion mture IL-1β nd tivtion of spse-1 were suppressed fter tivtion of the inflmmsome y, or (Fig. 4). We onfirmed the requirement for in tivtion of the inflmmsome using one mrrow derived mrophges (BMDMs) from Txnip / mie (Fig. 4 nd Supplementry Fig. 2). The ltion of resulted in less IL-1β nd less leved spse-1 in the superntnts of ells stimulted with vriety of moleulrly distnt inflmmsome tivtors, inluding nd prtiultes suh s, lum or sili, s well s ATP, whih tivtes IP: Flg d NACHT PYD LRR LRR Flg IP: Flg Flg-tgged proteins -FL -C -N 136 -FL -C -N -FL -C -N Flg- Figure 1 inds to. (,) Immunopreipittion (IP) nd immunolot nlysis of the intertion of Flg-tgged proteins with VSV-tgged () or endogenous () in lystes of HEK293T ells., empty vetor; NACHT, PYD nd LRR, onstruts of ontining only those individul domins (NACHT, nuleotide-inding oligomeriztion domin; PYD, pyrin domin; LRR, leuine-rih repet domin);, ell extrt without immunopreipittion. Below, domins of nd tht intert with eh other. Arrestin-N, N-terminl rrestin domin; Arrestin- C, C-terminl rrestin domin. () Immunopreipittion nd immunolot nlysis of the intertion of LRR-ontining Flg-tgged NLR proteins (NLRP2,, Nod1, Nod2 nd Ipf) nd ontrol proteins (SDS22 nd pp32) with endogenous in lystes of HEK293T ells. (d) Immunopreipittion nd immunolot nlysis of the intertion of vrious VSV-tgged frgments of with Flg-tgged in lystes of HEK293T ells. -FL, fulllength ; -C, onstrut ontining the C terminus of (mino ids ); -N, onstrut ontining the N terminus of (mino ids 1 157). Dt re representtive of three experiments. the inflmmsome vi the P2X7 reeptor. In ontrst, tivtion of the Ipf inflmmsome y Slmonell typhi nd tivtion of the AIM2 inflmmsome y poly(da:dt) were unffeted y ltion (Fig. 4 nd Supplementry Fig. 2), whih indited tht is dispensle for tivtion of these two inflmmsomes. Intrperitonel injetion of eliits n -dependent inflmmtory response in the mouse peritoneum hrterized y mssive neutrophil influx 6. Akin to Nlrp3 / mie, Txnip / mie hd muh less neutrophil influx nd IL-1β prodution in fter injetion of (Fig. 5,), whih supports the ide tht is ritil regultor of inflmmsome tivtion in vivo. In ontrst, zymosn-indued peritonitis, whih is independent 6, ws not ffeted y defiieny (Supplementry Fig. 3). Additionl support for the ide tht is involved in tivtion of the inflmmsome ws provided y experiments using overexpression, whih resulted in more mture IL-1β nd spse-1 in the superntnts of THP-1 mrophges stimulted with, or (Fig. 5). As inds nd therey loks the ntioxidtive ility of 15, downregultion of y short hirpin RNA hd n effet on the tivtion of IL-1β nd spse-1 similr to tht of overexpression (Fig. 5d). Thus, hs n essentil role in tivting the inflmmsome. nd medite gluose-indued IL-1 seretion We took dvntge of the known funtions sried to to gin insight into the physiologil roles of the inflmmsome. In ddition to its funtion s n inhiitor of nd therefore s promoter of ROS genertion 13, hs lso een linked to gluose metolism nd dietes y welth of evidene 16. In et ells, is downregulted y insulin, nd expression is onsistently higher in humns with type 2 dietes 17. Furthermore, muttions re ssoited with hypertriglyeridemi nd lower plsm gluose in type 2 dietes 18. In onfirmtion of pulished dt 19,2, expression ws potently upregulted in response to high gluose onentrtions in Figure 2 ROS indue the dissoition of from nd inding to. () Immunolot nlysis of tive IL-1β (IL-1β p17) in superntnts (SN) nd pro-il-1β in extrts () of THP-1 ells stly expressing short hirpin RNA (sh) speifi for vrious trget genes (elow lnes) nd stimulted for 6 h with (15 µg/ml) or (1 mm). Srmle, Srmle sh shasc shcsp1 IP: Control APDC IP: APDC + + IP: IP: Control ontrol short hirpin RNA; Csp1, spse-1. (,) Immunopreipittion nd immunolot nlysis of the intertion of with () or () in THP-1 ells without pretretment ( ) or pretreted (+) for 3 min with APDC (1 µm) nd susequently treted for 2 h with (1 mm), (15 µg/ml) or (15 µg/ml). Dt re representtive of t lest three independent experiments. DVANCE ONLINE PUBLICATION nture immunology

3 A rt i l e s 29 Nture Ameri, In. All rights reserved. H2 DCFDA (AU) Time (h) SN Time (h) islet ells, nd this indution ws inhiited y otretment with insulin (Fig. 6). Txnip indution n e explined y the presene in the Txnip promoter of rohydrte-response element reognized y the gluosesensing MondoA:Mlx trnsription ftor 18,19. In ontrst to islet ells, mrophges hd onstitutively high protein onentrtions nd this ws influened only mildly y gluose or tivtors (Fig. 6). The oserved upregultion in expression in et islets ws gluose speifi nd ws not side effet of higher osmolrity due to high surose onentrtions; replement of surose with n identil onentrtion (33 mm) of soritol or xylitol hd no effet on expression (Fig. 6). In ddition to its effets on expression, hyperglyemi is reported to indue the relese of IL-1β from islet ells 21. This prompted us to investigte whether mouse islets express inflmmsome omponents., ASC nd spse-1 were ll detetle, lthough their expression ws low reltive to tht in BMDMs (Fig. 7). The low expression of omponents my explin the modest relese of IL-1β from et islets (Fig. 7). The relese of IL-1β ws speifi to gluose, s we did not detet it fter tretment with high onentrtions of soritol or xylitol. To diretly ddress the involvement of the - inflmmsome in the proessing nd seretion of IL-1β in islet ells, IL-1 p17 Time (h) IP: Figure 3 The kinetis of ROS prodution, the dissoition of from nd the ssoition of with in THP-1 ells. () Cellulr ROS t vrious times fter the ddition of tothp-1 ells, ssessed with the ROS-speifi fluoresent proe H 2 DCFDA. AU, ritrry units. () Immunolot nlysis of the temporl intertion of with or, ssessed (s desried in Fig. 2) y immunopreipittion of in THP-1 ells. () Kinetis of the relese of IL-1β into superntnts of THP-1 ells. Dt re representtive of two experiments. P <.5 P <.5 Neutrophils ( 1 6 ) WT Nlrp3 / Txnip / P <.5 P <.1 WT Nlrp3 / Txnip / d PRDI Srmle PRDI- sh Pro-sp1 Csp1 p1 Pro-sp1 Csp1 p1 Srmle si Csp1 p1 Tuulin we isolted islets from wild-type, Txnip / nd Nlrp3 / mie nd exposed them to high gluose onentrtions. IL-1β onentrtions were muh lower in Txnip / nd Nlrp3 / islets thn in wild-type islets (Fig. 7). This gluose-indued seretion of IL-1β ws not medited y or ATP (Supplementry Fig. 4). High gluose onentrtions trigger the tivtion of NADPH oxidse nd the genertion of ROS, whih indites tht hyperglyemi uses tivtion of the inflmmsome through oxidtive stress Gluose-triggered inflmmsome tivtion nd IL-1β seretion ws indeed muh lower in the presene of the ROS inhiitor APDC or the NADPH oxidse inhiitor DPI (Fig. 7d). Glienlmide is drug widely used for the tretment of type 2 dietes. Glienlmide is thought to inhiit the K + hnnel tivity of the ATP-sensitive K + hnnel Kir6.2 (ref. 25). Vrious muttions in the genes enoding the Kir or Sur suunit of this K + hnnel hve een identified tht use either neontl dietes or ongenitl hyperinsulinism 25. As K + efflux is lso key requirement for inflmmsome tivtion, we investigted whether glienlmide inhiited gluosetriggered tivtion of the inflmmsome in et islets. Gluose-medited seretion of IL-1β ws indeed loked y glienlmide, s hs een shown for other tivtors 26,27 (Fig. 7e), whih indited tht tivtion of the inflmmsome in et ells follows the lssil tivtion route. Similr to glienlmide, nd in greement with the oservtion tht oth glienlmide nd Pro-sp1 S. typhi 1:1 1:1 1:1 1:1 Txnip +/+ Txnip / ATP ATP Txnip +/+ Txnip / Csp1 p2 Pro-sp1 Csp1 p2 Pro-sp1 Figure 4 is essentil for tivtion of the inflmmsome. () Immunolot nlysis (IB) of THP-1 ells trnsfeted with ontrol smll interfering RNA (Srmle) or smll interfering RNA speifi for (si) nd stimulted for 6 h with, or. Csp1 p1, tive form of spse-1. () Immunolot nlysis of the seretion of IL-1β nd spse-1 y lipopolyshride-primed Txnip +/+ nd Txnip / BMDMs stimulted with, ATP or. Csp1 p2, tive form of spse-1. () Immunolot nlysis of the seretion of IL-1β from Txnip +/+ nd Txnip / BMDMs stimulted with vrious onentrtions (ove lnes) of S. typhi. Dt re representtive of four (,) or six () experiments. Figure 5 is essentil for tivtion of the inflmmsome in vivo. (,) Neutrophil influx () nd IL-1β onentrtion () in peritonel lvge fluid from wild-type (WT), Nlrp3 / nd Txnip / mie 6 h fter -dependent peritonitis ws eliited y intrperitonel injetion of PBS supplemented with rystls. P vlues, two-wy ANOVA. Dt re representtive of t lest three independent experiments (men nd s.e.m. of three to five mie per group). (,d) Immunolot nlysis of THP-1 ells expressing lentivirl vetor lone (PRDI) or with stle overexpression of (PRDI-) () or trnsfeted with ontrol or -speifi short hirpin RNA (), nd stimulted for 6 h with, or. Dt re representtive of t lest three independent experiments. nture immunology DVANCE ONLINE PUBLICATION

4 A rt i l e s Figure 6 Expression of is indued y gluose. () Immunolot nlysis of in extrts of isolted islets stimulted for 24 h with gluose (left; 5 mm or 33 mm) in the sene (left) or presene (right) of glienlmide (Gli; 1 µm) or insulin (Ins; 5 nm). () Immunolot nlysis of Gluose (mm) 5 33 Tuulin Ins Gli BMDM ATP Gluose Gluose THP-1 Tuulin Gluose Soritol Xylitol expression in extrts of mouse BMDMs (left) nd THP-1 ells (right) stimulted for 6 h with, or ; for 3 min with ATP; or for 24 h with gluose (33 mm). () Immunolot nlysis of expression in isolted islets stimulted for 24 h with 33 mm gluose, soritol or xylitol. Tuulin serves s loding ontrol. Dt re representtive of three experiments. Tuulin 29 Nture Ameri, In. All rights reserved. insulin inhiited gluose-medited upregultion of, insulin resulted in less IL-1β seretion (Fig. 6). Together, these results demonstrte ruil role for the - inflmmsome omplex in the gluose-medited relese of IL-1β from et ells. hs ruil roles in nturl killer ells nd dendriti ells 28,29, whih suggests tht the sene of or might ffet the seretion of ytokines other thn IL-1β. However, we found tht the expression of tumor nerosis ftor, interferon-β nd IL-6 protein ws unltered in mrophges (Supplementry Fig. 5). Txnip / nd Nlrp3 / mie hve similr phenotypes Mie defiient in Txnip hve een nlyzed extensively. Txnip defiieny profoundly ffets energy metolism, inluding ltertions in lood gluose onentrtions, insulin seretion, insulin sensitivity nd ftty id use On the sis of our results reported ove, we expeted tht some of the iologil effets seen in Txnip / mie would lso e evident in Nlrp3 / mie. We therefore exmined gluose tolerne nd insulin tolerne, whih re oth etter in Txnip / mie 3,31. We fed Nlrp3 / mie high-ft diet for 8 weeks (Fig. 8). Gluose tolerne tests showed tht Nlrp3 / mie were more gluose tolernt thn wild-type mie were (Fig. 8). Nlrp3 / mie lso showed BMDMs Islets ASC Csp1 Tuulin d Gluose Soritol Xylitol P <.1 P <.1 Gluose (mm) APDC + DPI + 5 mm gluose 33 mm gluose P <.5 5 P < e WT Gli Figure 7 nd re linked to the seretion of IL-1β from islet ells. () Immunolot nlysis of the expression of omponents of the inflmmsome in extrts of mouse BMDMs nd islets. () Enzyme-linked immunosorent ssy (ELISA) of the seretion of IL-1β y islets stimulted for 24 h with 33 mm gluose, soritol or xylitol. () ELISA of the relese of IL-1β from isolted wild-type, Nlrp3 / or Txnip / islets treted with for 24 h gluose. (d) ELISA of the relese of IL-1β from islets stimulted with gluose in the presene of APDC (1 nm) or DPI (25 µm). (e) ELISA of the inhiition of gluose-medited relese of IL-1β from islets y glienlmide (1 µm) or insulin (5 nm). P vlues, two-wy ANOVA. Dt re representtive of two (,,d) or three (,e) experiments (men nd s.e.m. in e). Nlrp3 / Ins Txnip / elerted lerne of gluose in response to insulin (Fig. 8). These oservtions re in greement with the ide tht nd re prt of the sme signling pthwy. In ontrst to Txnip / mie, how-fed Nlrp3 / mie tht hd fsted overnight hd n unhnged totl insulin ontent of islet ells, serum insulin nd gluose onentrtions (Supplementry Fig. 6), whih indited tht not ll the tivities of nd overlp. DISCUSSION Mny tivtors of the inflmmsome hve een identified, rnging from prtiultes to endotoxins nd K + hnnel openers, yet none of these tivtors diretly intert with. Experiments using the yest two-hyrid tehnique hve shown tht interts with HSP9 nd SGT1, yet oth proteins hve een found to funtion s stilizers of in its intive form nd not s upstrem tivtors 33. Tht sme pproh hs now llowed us to identify s n dditionl inding prtner., in ontrst to SGT1 nd HSP9, is ruil for tivtion of the inflmmsome, whih provides mehnisti explntion for some similrities in the phenotypes of - nd -defiient mie. The involvement of in inflmmsome tivtion is lso onsistent with the reported requirement for ROS in tivtion of the inflmmsome. Our dt suggest tht in resting ells,, whose expression is indued y vrious stimuli suh s gluose, interts with nd is therefore unville for intertion. After n inrese in ROS used y tivtors, is relesed from oxidized nd in turn inds. In ddition to the ROS- xis, the lowering of ytoplsmi K + is essentil for inflmmsome tivtion nd susequent IL-1β relese. Other signling pthwys, suh s lysosyme rupture nd susequent thepsin B relese, my modulte or omplement this pthwy in ertin onditions 11,34. is protein with mny funtions. Although it ws originlly hrterized s -inding protein tht regultes the ntioxidnt funtion of, its funtion seems to e more diverse. For exmple, overexpression of inhiits ell prolifertion vi ell yle rrest. Txnip / mie re lso more suseptile to rinogenesis thn re wildtype mie, nd they hve hemtopoiesis defets, inluding defiient Blood gluose (mm) Time (min) WT Nlrp3 / Blood gluose (% seline) WT Nlrp3 / Time (min) Figure 8 defiieny ffets gluose homeostsis. Gluose tolerne () nd insulin tolerne () of wild-type nd Nlrp3 / mie fed high-ft diet for 8 weeks. P <.5 (Student s t-test). Dt re representtive of three experiments (men ± s.e.m.). DVANCE ONLINE PUBLICATION nture immunology

5 A rt i l e s 29 Nture Ameri, In. All rights reserved. development nd funtion of nturl killer ells. Furthermore, Txnip / mie show impired gluose homeostsis. Some of these findings my eome mehnistilly more omprehensile given the involvement of in tivting the inflmmsome. For exmple, inflmmsome-generted IL-1β is frequently ssoited with tumor progression nd insulin resistne. In greement with tht, -defiient mie hve greter gluose nd insulin tolerne, whih suggests link etween the inflmmsome nd type 2 dietes. Although this onnetion remins purely speultive, the results presented here fvor suh hypothesis. In linil trils, inhiition of IL-1β hs een shown to meliorte type 2 dietes, whih suggests n importnt role for this proinflmmtory ytokine in disese progression 35. Thus, trgeting individul inflmmsome omponents my e n lterntive pproh for treting type 2 dietes. Notly, we hve shown here tht glienlmide, drug widely used for the tretment of type 2 dietes, is potent inhiitor not only of the indution of y gluose ut lso of inflmmsome tivtion in generl 26. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Immunology wesite. Aknowledgments We thnk P. Vndeneele (Ghent University) for ntiody to mouse spse-1 (p2); V. Dixit (Genenteh) for Ipf / mie; R.V. Bruggen (Free University Amsterdm) for S. typhimurium; R. Solri (Glxo) for ntiody to mouse IL-1β; M. Joffrud for tehnil help; nd K. Shroder, C. Dostert nd O. Gross for reding the mnusript. Supported y the Swiss Ntionl Siene Foundtion, MUGEN nd the Swiss Ntionl Center of Competene in Reserh for Moleulr Onology (A.T.). AUTHOR CONTRIBUTIONS R.Z. nd J.T. designed the study nd wrote the mnusript; R.Z. nd A.T. did experiments; I.C. provided mie; nd B.T. provided tehnil support nd oneptul dvie. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Mrtinon, F., Myor, A. & Tshopp, J. The inflmmsomes: gurdins of the ody. Annu. Rev. Immunol. 27, (29). 2. Chen, G., Shw, M.H., Kim, Y.G. & Nuñez, G. Nod-like reeptors: role in innte immunity nd inflmmtory disese. Ann. Rev. Pthol. 4, (29). 3. Agostini, L. et l. NALP3 forms n IL-1β proessing inflmmsome with inresed tivity in Mukle-Wells uto-inflmmtory disorder. Immunity 2, (24). 4. Mrithsn, S. et l. Differentil tivtion of the inflmmsome y spse-1 dptors ASC nd Ipf. Nture 43, (24). 5. Mrtinon, F., Agostini, L., Meyln, E. & Tshopp, J. Identifition of teril murmyl dipeptide s tivtor of the NALP3/ryopyrin inflmmsome. Curr. Biol. 14, (24). 6. Mrtinon, F., Petrilli, V., Myor, A., Trdivel, A. & Tshopp, J. Gout-ssoited uri id rystls tivte the NALP3 inflmmsome. Nture 44, (26). 7. Hlle, A. et l. The NALP3 inflmmsome is involved in the innte immune response to myloid-β. Nt. Immunol. 9, (28). 8. Petrilli, V. et l. Ativtion of the NALP3 inflmmsome is triggered y low intrellulr potssium onentrtion. Cell Deth Differ. 14, (27). 9. Frnhi, L. et l. Critil role for Ipf in Pseudomons eruginos-indued spse- 1 tivtion. Eur. J. Immunol. 37, (27). 1. Mrin-Gri, N. et l. Pnnexin-1-medited intrellulr delivery of murmyl dipeptide indues spse-1 tivtion vi ryopyrin/ independently of Nod2. J. Immunol. 18, (28). 11. Hornung, V. et l. Sili rystls nd luminum slts tivte the NALP3 inflmmsome through phgosoml destiliztion. Nt Immunol 9, (28). 12. Kim, S.Y., Suh, H.W., Chung, J.W., Yoon, S.R. & Choi, I. Diverse funtions of VDUP1 in ell prolifertion, differentition, nd diseses. Cell. Mol. Immunol. 4, (27). 13. Kimul, A.M., Nkmur, H., Msutni, H. & Yodoi, J. Thioredoxin nd thioredoxininding protein-2 in ner nd metoli syndrome. Free Rdi. Biol. Med. 43, (27). 14. Dostert, C. et l. Innte immune tivtion through Nlp3 inflmmsome sensing of sestos nd sili. Siene 32, (28). 15. Nishiym, A. et l. Identifition of thioredoxin-inding protein-2/vitmin D 3 upregulted protein 1 s negtive regultor of thioredoxin funtion nd expression. J. Biol. Chem. 274, (1999). 16. Chen, J., Sxen, G., Mungrue, I.N., Lusis, A.J. & Shlev, A. Thioredoxin-interting protein: ritil link etween gluose toxiity nd et-ell poptosis. Dietes 57, (28). 17. Prikh, H. et l. regultes peripherl gluose metolism in humns. PLoS Med. 4, e158 (27). 18. vn Greevenroek, M.M. et l. Geneti vrition in thioredoxin interting protein () is ssoited with hypertriglyeridemi nd lood pressure in dietes mellitus. Diet. Med. 24, (27). 19. Minn, A.H., Hfele, C. & Shlev, A. Thioredoxin-interting protein is stimulted y gluose through rohydrte response element nd indues et-ell poptosis. Endorinology 146, (25). 2. Turturro, F., Fridy, E. & Welourne, T. Hyperglyemi regultes thioredoxin-ros tivity through indution of thioredoxin-interting protein () in metstti rest ner-derived ells MDA-MB-231. BMC Cner 7, 96 (27). 21. Medler, K. et l. Gluose- nd interleukin-1et-indued et-ell poptosis requires C 2+ influx nd extrellulr signl-regulted kinse (ERK) 1/2 tivtion nd is prevented y sulfonylure reeptor 1/inwrdly retifying K + hnnel 6.2 (SUR/Kir6.2) seletive potssium hnnel opener in humn islets. Dietes 53, (24). 22. Dsu, M.R., Devrj, S. & Jill, I. High gluose indues IL-1β expression in humn monoytes: mehnisti insights. Am. J. Physiol. Endorinol. Met. 293, E337 E346 (27). 23. Suh, S.W. et l. Gluose nd NADPH oxidse drive neuronl superoxide formtion in stroke. Ann. Neurol. 64, (28). 24. Gokulkrishnn, K., Mohnvlli, K.T., Monikrj, F., Mohn, V. & Blsurmnym, M. Sulinil inflmmtion/oxidtion s reveled y ltered gene expression profiles in sujets with impired gluose tolerne nd type 2 dietes ptients. Mol. Cell. Biohem. 324, (29). 25. Httersley, A.T. & Ashroft, F.M. Ativting muttions in Kir6.2 nd neontl dietes: new linil syndromes, new sientifi insights, nd new therpy. Dietes 54, (25). 26. Muruve, D.A. et l. The inflmmsome reognizes ytosoli miroil nd host DNA nd triggers n innte immune response. Nture 452, (28). 27. Lmknfi, M. et l. Glyuride inhiits the ryopyrin/nlp3 inflmmsome. J. Cell Biol. 187, 61 7 (29). 28. Lee, K.N. et l. VDUP1 is required for the development of nturl killer ells. Immunity 22, (25). 29. Son, A. et l. Dendriti ells derived from TBP-2-defiient mie re defetive in induing T ell responses. Eur. J. Immunol. 38, (28). 3. Chen, J. et l. Thioredoxin-interting protein defiieny indues Akt/Bl-xL signling nd pnreti β-ell mss nd protets ginst dietes. FASEB J. 22, (28). 31. Hui, S.T. et l. Txnip lnes metoli nd growth signling vi PTEN disulfide redution. Pro. Ntl. Ad. Si. USA 15, (28). 32. Ok, S.I. et l. Thioredoxin inding protein-2 (TBP-2)/Txnip is ritil regultor of insulin seretion nd PPAR funtion. Endorinology 155, (28). 33. Myor, A., Mrtinon, F., De Smedt, T., Petrilli, V. & Tshopp, J. A ruil funtion of SGT1 nd HSP9 in inflmmsome tivity links mmmlin nd plnt innte immune responses. Nt. Immunol. 8, (27). 34. Dostert, C. et l. Mlril hemozoin is Nlp3 inflmmsome tivting dnger signl. PLoS One 4, e651 (29). 35. Lrsen, C.M. et l. Interleukin-1-reeptor ntgonist in type 2 dietes mellitus. N. Engl. J. Med. 356, (27). nture immunology DVANCE ONLINE PUBLICATION

6 29 Nture Ameri, In. All rights reserved. ONLINE METHODS Regents. Nigeriin, uri id rystls (), ATP, phorol 12-myristte 13-ette, APDC ((2R,4R)-4-minopyrrolidine-2,4-diroxylte), insulin, urise, glienlmide, sili, poly(da:dt) nd gluose were from Sigm; Imjet Alum (luminum hydroxide djuvnt) ws from Piere; ws from Applihem; DPI ws from Alexis; nd, zymosn nd ultrpure lipopolyshride were from Invivogen. S. typhimurium ws gift from R.V. Bruggen. Antiody to humn pro-il-1β (nti humn pro-il-1β) ws produed in house. Monolonl mouse nti- (Cryo-2) ws from Adipogen. Anti mouse IL-1β ws gift from R. Solri. Anti mouse spse-1 (p2) ws gift from P. Vndeneele. The following ntiodies were lso used: nti humn spse-1 (s-622; Snt Cruz), nti humn IL-1β, leved (D116; Cell Signling), nti-asc (AL177; Alexis), nti-ipf (317; ProSi), nti-flg (F7425; Sigm), nti-vsv (V4888; Sigm), nti-tuulin (T5168; Sigm), rit nti- (4-37; Invitrogen), mouse nti- (JYI; MBL) nd nti- (26928; Am). All tissue ulture regents were from Invitrogen. Mie. Nlrp3 /, Txnip / nd P2rx7 / mie hve een desried 6,28. All mie were on C57BL/6 kground. All niml experiments were pproved y lol ethis ommittee. Cell ulture. Humn THP-1 ells were grown in RPMI-164 medi supplemented with 1% (vol/vol) FBS nd 5 µm 2-merptoethnol. THP-1 ells were differentited for 3 h with 1 nm phorol-12-myristte-13-ette. Bone mrrow mrophges were derived from tii nd femorl one mrrow progenitors s desried elsewhere 36 nd were ultured in DMEM omplemented with 1% (vol/vol) FCS, 1 mm sodium pyruvte nd 2 mm L-glutmine 36. Mrophges were primed for h with ultrpure lipopolyshride (1 ng/ml). For IL-1β indution, mrophges were plted in 12-well pltes overnight. The medium ws hnged to Opti-MEM the next morning nd ells were treted for 6 h with (15 µg/ml), lum (2 µg/ml), sili (2 µg/ml), (15 µg/ml) or (.1 1 mm) or for 3 min with ATP (5 mm). For trnsfetion of poly(da:dt) into ells, Lipofetmine 2 (4 µl/ml) ws used ording to the mnufturer s protool (Invitrogen). Cell extrts nd preipitted superntnts were nlyzed y immunolot. Islets from mle C57BL/6 mie were isolted y ollgense digestion nd Histopque density-grdient purifition nd were suspended in RPMI-164 medium ontining gluose (11.1 mmol/l) supplemented with 1% (vol/vol) FBS nd 2 mm l-glutmine 37. The next morning, the medium ws repled with RPMI-164 medium ontining 2.5 mm gluose nd 1% (vol/vol) FBS, followed y inution for 2 h. Islets (4 islets/ml) were then inuted for 24 h or 48 h in the presene of 5 mm or 33 mm gluose, nd superntnts nd ell extrts were olleted. Enzyme-linked immunosorent ssy. Cell ulture superntnts or tissue lystes were ssyed for mouse IL-1β, tumor nerosis ftor (R&D Systems), IL-6 (ebiosiene) nd interferon-β (PBL InterferonSoure) ording to the mnufturer s instrutions. Trnsfetion nd immunopreipittion. Construts were trnsfeted into HEK293T ells y the lium-phosphte method; fter 18 h, ells were olleted nd resuspended in lysis uffer (5 mm Tris, ph 7.8, 5 mm NCl,.1% (vol/vol) Nonidet-P4, 5 mm EDTA nd 1% (vol/vol) glyerol). Extrts were immunopreipitted with nti-flg grose eds nd then were ssessed y immunolot. THP-1 ells (1 1 7 ) were differentited with phorol 12-myristte 13-ette nd then were treted for 2 h with (15 µg/ml), (15 µg/ml) or (1 mm) with or without 3 min of pretretment with APDC (1 µm). THP-1 ells were then resuspended in lysis uffer nd proteins were immunopreipitted from extrts with nti- (C4-37; Invitrogen) or nti- (2632; Am). Knokdown or overexpression in THP-1 ells. THP-1 ells stly expressing short hirpin RNA speifi for, spse-1, ASC, Ipf or were otined s desried 38. THP-1 ells were trnsfeted with smll interfering RNA y the Amx Nuleofetor system nd the Cell Line Nuleofetor kit V ording to the mnufturer s reommendtions. ws loned into the lentivirl vetor prdi292 for overexpression of in THP-1 ells. Slmonell infetion. S. typhimurium ws preultured with trypti soy roth the dy efore the experiment, then ws diluted 1:1 nd inuted for 2 h. Lipopolyshride-primed BMDMs were infeted for 1 h with vrious dilutions of the S. typhimurium ulture (multipliity of infetion, 1 or 1). Cells were wshed nd then inuted for 1 h in OptiMEM ontining gentmyin (Invitrogen). Peritonitis. - or zymosn-indued peritonitis hs een desried 6 ; this ws indued y intrperitonel injetion of 1 mg rystls or.2 mg zymosn dissolved in.5 ml sterile PBS. After 6 h, mie were killed y exposure to CO 2 nd peritonel vities were wshed with 1 ml old PBS. Peritonel lvge fluid ws ssessed y flow ytometry with the neutrophil mrkers Ly6G nd CD11 (BD Phrmingen) for nlysis of the reruitment of polymorphonuler neutrophils. Metoli studies. Mie 6 weeks of ge were fed norml diet or high-ft diet (D12331; Reserhdiets) for 8 weeks. For gluose-tolerne tests, mie were fsted for 8 h nd then were injeted intrperitonelly with gluose t dose of 1.5 mg per g ody weight. Blood smples were otined t vrious time points for gluose mesurements with Gluometer (Rohe) or for insulin mesurements y enzyme-linked immunosorent ssy. ROS ssy. THP-1 ells were loded for 1 min with the ROS-speifi fluoresent proe H 2 DCFDA (2,7 -dihlorofluoresin diette; 1 mm; BioChemik or Fluk), then were wshed twie with PBS nd exposed to. Fluoresene ws ssessed with Titertek Fluoroskn II mirotiter fluoresene plte reder with fluoresein isothioynte filter (exittion, 485 nm; emission, 538 nm). Sttistil nlysis. Smples were nlyzed y Student s t-test for omprison of two groups nd nlysis of vrine (ANOVA) for omprison of multiple groups. 36. Didierlurent, A. et l. Tollip regultes proinflmmtory responses to interleukin-1 nd lipopolyshride. Mol. Cell. Biol. 26, (26). 37. Gotoh, M. et l. Reproduile high yield of rt islets y sttionry in vitro digestion following pnreti dutl or portl venous ollgense injetion. Trnsplnttion 43, (1987). 38. Ppin, S. et l. The SPRY domin of Pyrin, mutted in fmilil Mediterrnen fever ptients, interts with inflmmsome omponents nd inhiits proil-1β proessing. Cell Deth Differ. 14, (27). nture immunology doi:1.138/ni.1831