SUPPLEMENTARY INFORMATION doi:1.138/nture1188 1mM CCl 2 (min) 3 4 6 CCl 2 (mm) for 4min.1. 1 (mm) Pro- d WT GdCl 3 R-68 -/- P2x7r -/- -/- Csp1 -/- WT -/- P2x7r -/- -/- Csp1 -/- Csp1 (p2) (p17) Pro-Csp1 Pro- Nigericin dsdna Flgellin WT -/- P2x7r -/- -/- Csp1 -/- WT -/- P2x7r -/- -/- Csp1 -/- WT -/- P2x7r -/- -/- Csp1 -/- WT -/- P2x7r -/- -/- Csp1 -/- c sirna N.C. Aim2 Nlrc4 Pro- Pro- Pellet Csp1 (p2) Pro-Csp1 oligomer dimer monomer CCl 2 dsdna Flgellin Pro- Pro- Pro- Supplementry Figure 1. Extrcellulr clcium induces secretion independent of the AIM2 or NLRC4 inflmmsomes., (1 mg ml -1 for 3 h)-primed BMDMs were treted with CCl 2 (1 mm) for the indicted time durtion (left) or with the indicted concentrtions (right) of CCl 2 for 4 min., -primed BMDMs from WT,, P2x7r,, or Cspse-1 (Csp1)-deficient mice were cultured without dditionl tretment () or treted with nigericin (2 mm) for 3 min, dsdna (1 mg ml - 1 dsdna with 2. ml ml -1 Lipofectmine 2) for 3 min, or flgellin (. µg ml -1 flgellin with 2 µl ml -1 DOTAP) for 4 min. For detection of oligomers, pellets from whole-cell lystes were cross-linked with disuccinimidyl suerte (DSS) nd nlyzed y immunolot for. c, BMDMs trnsiently trnsfected with scrmled (negtive control, N.C.), Aim2, or Nlrc4 sirnas were primed with nd then treted with ( mm) for 4 min, CCl 2 (1 mm) for min, dsdna for 4min, or flgellin for min. d, -primed BMDMs were treted with GdCl 3 (1 mm, gonist of the CSR) or R-68 (1 mm, llosteric gonist of the CSR) for min. Cell culture superntnts () nd cell lystes () were nlyzed y immunolotting s indicted. All immunolot dt shown re representtive of more thn three independent experiments. WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION c d CPPD Nigericin dsdna Flgellin CCl 2 CCl 2 CCl 2 CCl 2 CCl 2 GdCl 3 MgCl 2 Pro- e CCl 2 (1 mm) (mm) f (2 mm) CCl 2 (mm).7 1.3 2...3.7 1.3 2.. Pro- Pro- Pellet oligomer oligomer dimer dimer monomer monomer Conc. (mm) 4 3 2 1 : Free C 2+ Free 4-.7 1.3 2.. CCl 2 (1 mm) (mm) Conc. (mm) 4 3 2 1 CCl 2 : Free C 2+ Free 4-.3.7 1.3 2.. (2 mm) (mm) Supplementry Figure 2. Extrcellulr clcium inhiits -driven NLRP3 inflmmsome ctivtion y formtion of C 2+ - complexes, ut does not inhiit CPPD or nigericininduced NLRP3 inflmmsome ctivtion or AIM2 or NLRC4 inflmmsome ctivtion., -primed BMDMs were treted without or with CPPD (2 mg/ml) in the sence or presence of dded CCl 2 (1 mm)., -primed BMDMs were treted without or with nigericin (2 mm) in the sence or presence of dded CCl 2 (1 mm). c. -primed BMDMs were treted with dsdna (1 mg ml -1 dsdna with 2. ml ml -1 Lipofectmine 2) or flgellin (. mg ml - 1 flgellin with 2 ml ml -1 DOTAP) in the sence or presence of dded CCl 2 (1 mm) s indicted. d, -primed BMDMs were treted with (2 mm) in the sence or presence of dded CCl 2 (1 mm), GdCl 3 (1 mm), or MgCl 2 (1 mm). e, -primed BMDMs were treted with 1 mm CCl 2 in the presence of vrious concentrtions ( to mm) of. f, -primed BMDMs were treted with 2 mm in the presence of vrious concentrtions ( to mm) of CCl 2. Cell culture superntnts () nd cell lystes () were nlyzed y immunolotting for nd oligomeriztion. Estimted concentrtion of free C 2+ nd 4- (ottom) were clculted y C/Mg//EGTA Clcultor v1 constnts from Theo Schoenmkers' Cheltor (http://www.stnford.edu/~cptton/cmgegta-ts.htm), sed on RPMI (ph nd concentrtions of MgCl 2 nd monovlent ions) t 37 C with the stndrd.42 mm C 2+ efore dded CCl 2. 2 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH CCl 2 (1 mm) N.C. sirna 4 6 8 Csr sirna #1 4 6 8 Csr sirna #2 4 6 8 Csr sirna #3 4 6 8 (pmole) Pro- CCl 2 (1 mm) N.C. sirna sirna sirna Csr sirna #3 4 6 8 4 6 8 4 6 8 4 6 8 (pmole) Pro- Csr Supplementry Figure 3. Optimiztion of experimentl conditions for gene knockdowns., Three sirna oligonucleotides were tested for Csr knockdown experiments. BMDMs were trnsiently trnsfected with indicted mount of scrmled (negtive control, N.C.) or three independent Csr sirnas. Then cells were stimulted with CCl 2 (1 mm) for min fter priming with. Cell culture superntnts () nd cell lystes () were nlyzed y immunolotting s indicted. Csr si RNA #3 ws effective t inhiiting relese into the superntnt with miniml effects on pro- nd ctin., BMDMs were trnsiently trnsfected with the indicted mount of scrmled (negtive control, N.C.) or selected sirnas for knockdown of,, or Csr. Then cells were stimulted with CCl 2 (1 mm) for min fter priming with. Cell culture superntnts () nd cell lystes () were nlyzed y immunolotting s indicted. WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION sirna N.C. Csr Csr sirna N.C. Csr sirna N.C. Csr Pro- Csp1 (p2) Pro-Csp1 Nigericin Pro- Csp1 (p2) Pro-Csp1 MSU Pro- Csp1 (p2) Pro-Csp1 CPPD Pro- Csp1 (p2) Pro-Csp1 dsdna Pro- Csp1 (p2) Pro-Csp1 Flgellin Pro- Csp1 (p2) Pro-Csp1 Supplementry Figure 4. The CSR is essentil for the ctivtion of the NLRP3 inflmmsome ut not the AIM2 or NLRC4 inflmmsomes. BMDMs were trnsiently trnsfected with scrmled sirna (negtive control, N.C.) or Csr sirna nd then either not sujected to further tretment (), or treted with nigericin (2 mm for 3 min), MSU (2 mg ml -1 for 1h), CPPD (2 mg ml -1 for 1h), dsdna (1 mg ml -1 dsdna with 2. ml ml -1 Lipofectmine 2 for 3 min), or flgellin (. mg ml -1 flgellin with 2 ml ml -1 DOTAP for 4 min) fter priming with. Cell culture superntnts nd cell lystes were nlyzed y immunolotting for nd cspse-1. 4 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH PLC inhiitor Edelfosine (mm) 4 2 1 Pro- dsdna U73122 Flgellin U73122 Pro- c PKC inhiitor GF1923X C-1 Pro- Supplementry Figure. PLC-IP 3 medited intrcellulr clcium ccumultion triggers NLRP3 ut not AIM2 or NLRC4 inflmmsome ctivtion., (1 mg ml -1 for 3 h)-primed BMDMs were co-treted with ( mm) nd PLC inhiitor (edelfosine), nd secretion ws nlyzed., -primed BMDMs were treted with dsdna (1 mg ml -1 dsdna with 2. ml ml -1 Lipofectmine 2 for 3 min), or flgellin (. mg ml -1 flgellin with 2 ml ml -1 DOTAP for 4 min) in the presence of U73122 (PLC inhiitor, 1 mm). Cell culture superntnts nd cell lystes were nlyzed y immunolotting for. c, -primed BMDMs were co-treted with ( mm) nd PKC inhiitors ( mm GF1923X nd mm C-1), nd secretion ws nlyzed y immunolot. WWW.NATURE.COM/NATURE
RESEARCH SUPPLEMENTARY INFORMATION.2 1 1 2 3 Reltive Intensity (fold increse) Time (min): 7 + APB mm + BAPTA-AM 1 mm 6 4 3 + 2-APB Ionomycin 1 12 + BAPTA-AM 9 6 3 1 1 2 Time (min) 2 3 3 3 3 3 3.2 1 1 2 3 Reltive Intensity (fold increse) Time (min): 4 CCl2 CCl2 CCl2 + APB mm CCl2 + BAPTA-AM 1 mm 3 1 1 8 6 4 2 CCl2 + 2-APB CCl2 + BAPTA-AM Ionomycin CCl2 1 1 2 Time (min) 2 c.2 1 1 2 3 Reltive Intensity (fold increse) Time (min): 4 GdCl3 GdCl3 GdCl3 + APB mm GdCl3 + BAPTA-AM 1 mm 3 1 GdCl3 + 2-APB GdCl3 + BAPTA-AM 6 Reltive intensity Ionomycin 6 GdCl3 4 2 1 1 2 Time (min) 2 26 Supplementry Figure 6. nd CSR gonists induce intrcellulr clcium influx. BMDMs were plted on 4-chmered coverglss dishes nd stined with fluo-4/am. Cells were treted with or without the IP3 receptor locker 2-APB ( mm) or intrcellulr clcium cheltor BAPTA-AM (1 mm), followed y tretment with 1 mm (), 1 mm CCl2 () or 1 mm GdCl3 (c). The estimted free [C2+]o in () is.22 mm, when djusted for C2+- complexes. Imges of untreted cells were cquired (t=), then, CCl2, or GdCl3 were dded nd cells were imged for 3 min with cquisition t 1 sec intervls. Sustined C2+ trces in () re likely due to the vilility of extrcellulr C2+ through the P2X7 receptor, which is necessry for -induced (ut not CCl2-induced) NLRP3 inflmmsome ctivtion (Fig. 1). After 3 min, ionomycin ws dded to the medium to finl concentrtion of mm. Imges were cquired on Leic SP Confocl Imging System, nd nlyzed using Imris 7.3.1 imges from three independent experiments re shown in left W W W. N softwre. A T U R E. C O M Representtive / NAT U R E pnels, nd the fold increse in solute intensity of ll cells in field reltive to time (2-
SUPPLEMENTARY INFORMATION RESEARCH Imges were cquired on Leic SP Confocl Imging System, nd nlyzed using Imris 7.3.1 softwre. Representtive imges from three independent experiments re shown in left pnels, nd the fold increse in solute intensity of ll cells in field reltive to time (2-2 cells per field) is shown in right pnels. For ll comprisons ( vs + 2-APB, vs + BAPTA-AM, GdCl 3 vs GdCl 3 + 2-APB, GdCl 3 vs GdCl 3 + BAPTA-AM, CCl 2 vs CCl 2 + 2-APB, CCl 2 vs CCl 2 + BAPTA-AM), p-vlues from n unpired t-test were <.1. Cytokine (pg/ml) 1 8 6 4 IL-6 BAPTA-AM TNF- BAPTA-AM TPEN 2 Pro- CCl 2 CCl 2 CCl 2 c Pellet Thpsigrgin Pro- oligomer dimer monomer d Reltive Csp1 ctivity (fold) 1. 1....64.32 1.6 8 4 2 CCl 2 (nm) 1 Z-VAD -FMK Supplementry Figure 7. Increse of intrcellulr clcium triggers NLRP3 inflmmsome ctivtion, ut not IL-6 or TNF- production., -primed BMDMs were treted with CCl 2 (1 mm) lone or in the presence of BAPTA-AM (1 mm). Cell culture medi were collected nd, IL-6, nd TNF- levels were mesured y cytokine multiplex ssy., -primed BMDMs were treted with (2 mm) in the presence of BAPTA-AM (1 mm) or TPEN (1 mm). c, -primed BMDMs were treted with or without thpsigrgin ( nm) for 3 min, nd were nlyzed for secretion nd pyroptosome y immunolot. d, The ctivity of recominnt humn ctive cspse-1 ws mesured in the presence of the indicted concentrtion of CCl 2 using colorimetric cspse-1 ctivity ssy. Dt represent the men s.e.m from three or four independent experiments. WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION Sup Adcy ctivtor NKH477 (mm) 1 1 Sup Adcy ctivtor 1 1 2 c PDE4 inhiitors (mm) Ro-2-1724 (R)-(-)-Roliprm Zrdverine 1 2 1 2 1 2 Sup Pro dsdna Pro- dsdna Pro- Sup Sup Sup CCl 2 Pro Flgellin Pro- Flgellin Pro- d (2 mm) Ro-2-1724 (R)-(-)-Roliprm Zrdverine Forskolin CCl 2 (1 mm) Ro-2-1724 (R)-(-)-Roliprm Zrdverine Forskolin e pg/ml 7 6 4 3 2 1 IL-6 TNF- Pellet Pro- oligomer Unt only +Ro 2-1724 +Roliprm +Forskolin +NKH477 CCl2 CCl2+Ro 2-1724 CCl2+Roliprm CCl2+Forskolin CCl2+NKH477 KH7 dimer g sirna: RPMI N.C. Csr N.C. Csr m-3m3fbs KH7 PLC ctivtor Adcy inhiitor N.C. Csr N.C. Csr Pro- monomer f KH7 (mm): 1 8 6 4 2 KH7 (mm): 1 camp (pmol/1x1 6 cells) Pro- h Reltive gene expression (x-fold vs. Adcy3 gene) 24 22 2 18 2. 1. 1...1.8.6.4.2. N.D. N.D. N.D. N.D. N.D. N.D. 1 2 3 4 6 7 8 9 Adcy suptypes i Pull-down: Bed only Pull-down: Bed only Pull-down: -ed (mm) 1. 3.. 7. 1 Pull-down: -ed camp (mm) 1. 3.. 7. 1 te te WB: WB: Supplementry Figure 8. camp directly suppresses NLRP3 inflmmsome ctivtion., (1 mg ml -1 for 3 h)-primed BMDMs were treted with the indicted doses of NKH477 (Adcy ctivtor), in the presence of ( mm) or CCl 2 (1 mm), nd then were nlyzed for secretion y immunolots., -primed BMDMs were treted with the indicted 8 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH doses of forskolin in the presence of dsdna (1 mg ml -1 dsdna with 2. ml ml -1 Lipofectmine 2 for 3 min) or flgellin (. mg ml -1 flgellin with 2 ml ml -1 DOTAP for 4 min). Cell culture superntnts nd cell lystes were nlyzed y immunolotting for. c, primed BMDMs were treted with the indicted doses of PDE4 inhiitors (Ro 2-1724, (R)-(- )-Roliprm, or zrdverine) in the presence of dsdna (1 mg ml -1 dsdna with 2. ml ml -1 Lipofectmine 2 for 3 min) or flgellin (. mg ml -1 flgellin with 2 ml ml -1 DOTAP for 4 min). Cell culture superntnts nd cell lystes were nlyzed y immunolotting for. d, -primed BMDMs were co-treted with (2 mm) for 3 min or CCl 2 (1 mm) for 4 min either lone, or in the presence of Ro 2-1724, (R)-(-)-Roliprm, zrdverine, or forskolin. Cell culture superntnts nd lystes were nlyzed for secretion nd oligomeriztion. e, -primed BMDMs were treted with (2 mm) or CCl 2 (1 mm) lone or in the presence of PDE4 inhiitors, n Adcy ctivtor, or n Adcy inhiitor. Cell culture medi were collected nd IL-6 nd TNF- levels were mesured y ELISA. Dt represent the men s.e.m from three independent experiments. f, -primed BMDMs were treted with the indicted doses of KH7, n inhiitor of solule Adcy, nd nlyzed for secretion. g, The function of is unffected y Csr knockdown. BMDMs were trnsiently trnsfected with scrmled (negtive control, N.C.),, or Csr sirnas. After priming with, cells were treted with nothing (RPMI),, m-3m3fbs (PLC ctivtor), or KH7 (Adcy inhiitor). Cell culture superntnts () nd cell lystes () were nlyzed y immunolotting s indicted. h, Reltive gene expression profile of Adcy sutypes in BMDMs ssyed y RT-QPCR. i, Cyclic AMP hs no effect on the interction of NLRP3 with. The lystes of -primed BMDMs were incuted with -conjugted eds or unconjugted eds in the presence of or camp (with vrious concentrtion s indicted) for 3 min t RT. After wshing, ound proteins were eluted from the eds nd nlyzed y immunolot for. The gel running nd Western lots for the two ssys were done simultneously, nd chemiluminescent detection ws done on the sme X-ry film from which oth immunolot imges were tken. WWW.NATURE.COM/NATURE 9
RESEARCH SUPPLEMENTARY INFORMATION PKA inhiitor: KT72(mM).1. 1.. 1 1 2 (2mM) sirna N.C. Prkc Pk Pro- Pro- PKA inhiitor: H89 (mm).1. 1.. 1 1 2 (2mM) Pro- dsdna CCl 2 Pro- Pro- Pro- Flgellin Pro- Supplementry Figure 9. NLRP3 inflmmsome ctivtion is not suppressed y protein kinse A (PKA)., -primed BMDMs were treted with the indicted doses of PKA inhiitors, KT72 or H89 for 4 min, nd then nlyzed for secretion., BMDMs were trnsiently trnsfected with scrmled sirna (N.C.) or Prkc sirna nd then treted without () or with ( mm) for 4 min, or CCl 2 (1 mm) for min, dsdna for 3 min, or flgellin for min fter priming with. secretion ws nlyzed y immunolotting. 1 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Helthy control priming priming Forskolin Z-VAD- FMK Ro 2-1724 (R)-(-)- Roliprm Pro- MW2 1 1 2 MW3 1 1 2 Pro- MW4 1 1 2 MW 1 1 2 MW6 1 1 2 Pro- c MW2 Ro2-1724 (mm) 1 2 MW3 Ro2-1724 (mm) 1 1 2 MW4 Ro2-1724 (mm) 1 1 2 MW Ro2-1724 (mm) 1 2 MW6 Ro2-1724 (mm) 1 2 Pro- d MW2 (R)-(-)-Roliprm (mm) 1 2 MW3 (R)-(-)-Roliprm (mm) 1 1 2 MW4 (R)-(-)-Roliprm (mm) 1 1 2 MW (R)-(-)-Roliprm (mm) 1 2 MW6 (R)-(-)-Roliprm (mm) 1 2 Pro- WWW.NATURE.COM/NATURE 11
RESEARCH SUPPLEMENTARY INFORMATION e FMF1 M68I/V726A 1 2 FMF2 V726A/+ 1 2 Z-VAD-FMK FMF3 M694V/M694V 1 2 Z-VAD-FMK FMF4 M694V/V726A Ro 2-1724 (R)-(-)- Roliprm Pro- f MW2 2-APB (mm) 1 1 MW3 2-APB (mm) 1 1 MW4 2-APB (mm) 1 1 MW 2-APB (mm) 1 1 MW6 2-APB (mm) 1 1 Pro- g MW2 BAPTA-AM (mm) 1 2 MW3 BAPTA-AM (mm) 1 2 MW4 BAPTA-AM (mm) 1 2 MW BAPTA-AM (mm) 1 2 MW6 BAPTA-AM (mm) 1 2 Pro- Supplementry Figure 1. The role of camp nd clcium signling in the pthogenesis of CAPS., Helthy control PBMCs were non-primed or primed with (1 mg ml -1 ) for 3h nd then treted with forskolin ( mm), Z-VAD-FMK (2 mg ml -1 ), Ro 2-1724 (2 mm), or (R)-(-)- Roliprm (2 mm) for 4 min. Cell lystes nd cell culture superntnts were nlyzed for secretion y immunolot., PBMCs from five MWS ptients with F23C muttion in NLRP3 were non-primed or primed with (1 mg ml -1 ) for 3h nd then treted with the indicted dose of forskolin. Cell lystes nd cell culture superntnts were nlyzed for secretion y immunolot. c,d, -primed or -primed PBMCs from five MWS ptients were treted with the indicted dose of Ro 2-1724 (c) or (R)-(-)-Roliprm (d) Cell lystes nd cell culture superntnts were nlyzed for secretion y immunolot. e, primed or -primed PBMCs from FMF ptients (with MEFV muttions s indicted) were treted with the indicted dose of forskolin (first three pnels) or treted with 2 mm Ro 2-1724 or 2 mm (R)-(-)-Roliprm for 4 min (fourth pnel). Cell lystes nd cell culture superntnts were nlyzed for secretion y immunolot. f,g, -primed or primed PBMCs from five MWS ptients were treted with the indicted dose of 2-APB (f) or BAPTA-AM (g). Cell lystes nd cell culture superntnts were nlyzed for secretion y immunolot. 12 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH K +? C 2+ CSR P2X7R K + PLC Gq Gi Adcy camp IP 3 C 2+ NLRP3 camp IP 3 R ASC Inctive NLRP3 Pro-Csp1 Endoplsmic Reticulum Pro- Active Csp1 : LRR : NBD : PYD : CARD Supplementry Figure 11. Proposed moleculr mechnism of NLRP3 inflmmsome ctivtion medited y the CSR. In mcrophges stimulted y PAMPs, such s, the NLRP3 inflmmsome is ctivted in response to extrcellulr dnger signls,, or C 2+, through CSRmedited signl trnsduction pthwys. The CSR, G protein-coupled receptor, ctivtes PLC, which induces intrcellulr C 2+ relese from the ER following the interction of IP 3 with its receptor. The CSR lso inhiits denylte cyclse nd susequently reduces camp levels. An increse in intrcellulr C 2+ nd/or decrese in camp ctivtes the NLRP3 inflmmsome, which my e regulted y coordintion of intrcellulr C 2+ nd camp levels. WWW.NATURE.COM/NATURE 13
RESEARCH SUPPLEMENTARY INFORMATION Activtion Suppression CSR gonist : C 2+ : camp : Triggering : Inctive NLRP3 inflmmsome : NLRP3 inflmmsome ctivtion Supplementry Figure 12. Proposed model of NLRP3 inflmmsome ctivtion regulted y the lnce of C 2+ nd camp. In the unstimulted stte, the NLRP3 inflmmsome is not ctivted due to lnce etween the levels of intrcellulr C 2+ nd camp (left). Upon stimultion of -primed mcrophges with CSR gonists, incresed intrcellulr C 2+ nd decresed camp coordintely lter the lnce to ctivte the NLRP3 inflmmsome (middle). This ctivtion cn e locked y deliertely incresing camp with n Adcy ctivtor or PDE4 inhiitor (right, top) or lterntively y decresing intrcellulr C 2+ with PLC inhiitor or IP 3 R locker (right, ottom). In ddition, the NLRP3 inflmmsome cn e ctivted y incresing intrcellulr C 2+ without chnging camp (middle, top) or decresing camp without chnging intrcellulr C 2+ (middle, ottom) in the sence of known NLRP3 inflmmsome stimuli. Autoinflmmtory disese muttions of NLRP3 reduce its inding ffinity for camp, pushing the lnce towrd inflmmsome ctivtion. 14 WWW.NATURE.COM/NATURE