1、IMMUNOLOGYESCRT-dependent membrane repairnegatively regulates pyroptosisdownstream of GSDMD activationSebastian Rhl1,2,Kateryna Shkarina3*,Benjamin Demarco3*,Rosalie Heilig3*,Jos Carlos Santos3*,Petr Broz1,3Pyroptosis is a lytic form of cell death that is induced by inflammatory caspases uponactivat
2、ion of the canonical or noncanonical inflammasome pathways.These caspasescleave gasdermin D(GSDMD)to generate an N-terminal GSDMD fragment,whichexecutes pyroptosis by forming membrane pores.We found that calcium influx throughGSDMD pores serves as a signal for cells to initiate membrane repair by re
3、cruiting theendosomal sorting complexes required for transport(ESCRT)machinery to damagedmembrane areas,such as the plasma membrane.Inhibition of the ESCRT-III machinerystrongly enhances pyroptosis and interleukin-1b release in both human and murinecells after canonical or noncanonical inflammasome
4、activation.These results not onlyattribute an anti-inflammatory role to membrane repair by the ESCRT-III system butalso provide insight into general cellular survival mechanisms during pyroptosis.Gasdermin D(GSDMD)is a pore-formingprotein that induces pyroptosis,a necroticform of cell death that is
5、initiated after in-flammasome activation(13).The detec-tion of pathogen-or host-derived dangersignalsbyinflammasomestriggersthe activationofinflammatorycaspases(caspase-1and-11inmiceand caspase-1 and-4 in humans),which cleaveGSDMDtoreleaseautoinhibitiononitsN-terminaldomain(GSDMDNT)(1,2).The GSDMDNT
6、tar-gets the plasma membrane andorganelles,whereit forms large pores to initiate pyroptosis(47).Damage to the plasma membrane does not neces-sarily result in cell death,as it has been observedthat the influx of Ca2+ions from the extracel-lular milieu triggers repair programs involvingeither the endo
7、cytosis of a damaged membraneor its shedding in the form of ectosomes(811).This latter mechanism relies on components ofendosomalsortingcomplexesrequiredfor trans-port(ESCRT-0 and-III and associated factorsthatcontrolESCRTrecruitmentanddisassembly)and has been specifically implicated in the res-tora
8、tion of plasma membrane integrity duringnecroptosis(12)oruponchemical-orlaser-induceddamage(10,11).To investigate if cells repair membranes dam-aged by GSDMD pores,we imaged the disrup-tionoftheelectrochemicalgradient(byusingtheCa2+dye Fluo-8)and loss of membrane integrityby using propidium iodide(P
9、I)in mouse bonemarrowderived macrophages(BMDMs).Thesecells were either untreated or transfected withlipopolysaccharide(LPS)to activate caspase-11.Within 2 hours,up to 50%of LPS-transfectedwild-type(WT)BMDMs underwent pyroptosis,as demonstrated by a strong PI signal(PIhi)(fig.S1A).A marked spike of F
10、luo-8 signal precededthe PIhisignal,indicating a loss of membraneintegrityandCa2+influx(Fig.1,AandB;fig.S1,Bto D;and movies S1 and S2).In contrast,Fluo-8or PI signals did not change in Casp11/orGsdmd/BMDMs(Fig.1B;fig.S1,C and D;andmovies S1 and S2).Unexpectedly,we found thatCa2+influxnegative WT BMD
11、Ms that maintainedthe electrochemical gradient gradually acquiredlow levels of PI(PIlo)(Fig.1C).This PIlosignalreached only 10%of the PIhisignal observed inthe WT BMDMs that had lost membrane integ-rity upon LPStransfection.Furthermore,neitherunstimulated WT BMDMs(Fig.1A)nor LPS-transfected Casp11/o
12、r Gsdmd/cells showedRESEARCHRhl et al.,Science 362,956960(2018)23 November 20181 of 51Focal Area Infection Biology,Biozentrum,University ofBasel,Klingelbergstrasse 50/70,4056 Basel,Switzerland.2Department of Immunology,St.Jude Childrens ResearchHospital,Memphis,TN 38105,USA.3Department ofBiochemistr
13、y,University of Lausanne,Chemin desBoveresses 155,1066 Epalinges,Switzerland.*These authors contributed equally to this work.Corresponding author.Email:petr.brozunil.chALPS transfected(3.5 g/1 106 cells)LPS transfection(3.5 g/1 106 cells)Calcium influx negative cellsmockLPS transfection(16.6 g/1 106
14、 cells)DWTCasp11/BMDMsGAPDH0+1052.5LPS transfection(g/1 106 cells)Medium0.51.02.04.0 M BAPTA-AMEBAPTA-AM(3.3 M)Pro-GSDMD(anti-HA)GSDMDNT(anti-HA)HA-GSDMD-transgenic immortalized WT BMDMs FGHeLaMedium3.3 M BAPTA-AMmock1005075MOI sifA S.tm.MOI sifA S.typhimuriumGAPDHPro-GSDMD(anti-FLAG)GSDMDNT(anti-FL
15、AG)FLAG-GSDMD-transgenic HeLa BAPTA-AM(3.3 M)Time(minutes)Fluo8(Ft-F0)/F0PI(Ft-F0)/F0Time(minutes)+0+1007550+0204060Cell death(%LDH release)*0204060Cell death(%LDH release)ns*Wildtype BMDMs120Gsdmd/BMDMs120unstimulated90120Wildtype BMDMsGsdmd/BMDMsFluo8(Ft-F0)/F0PI(Ft-F0)/F0B-0.50.00.51.01.5-0.50.00
16、.51.01.5-0.50.00.51.01.5123456789123456789123456789PI(Ft-F0)/F0Wildtype BMDMsGsdmd/BMDMsCasp11/BMDMsCTime(minutes)Time(minutes)Time(minutes)306090-0.50.00.51.01.5-50510152025306090-0.50.00.51.01.5-50510152025-60-30 0 30 60-0.50.00.51.01.5-50510152025Time(minutes)306090-0.50.00.51.01.5-50510152025Tim
17、e(minutes)000Fig.1.GSDMD-induced calcium flux negatively regulates pyroptosis.(A and B)Fluo-8(Ca2+indicator)and PI signals in unstimulated or LPS-transfected WTand Gsdmd/BMDMs.Graphs show average data fromn=22,22,28,and 30 cells.Ft,Fluorescence at time t;F0,Fluorescence attime 0.(C)Acquisition of PI
18、losignal in Ca2+influxnegative WT,Casp11/,and Gsdmd/BMDMs treated as described for panel(A).Graphs showaverage data fromn=29,29,and26cells.(D)LDH release fromBAPTA-AMtreated BMDMs mock transfected or transfected with LPS for 2 hours.(E)GSDMD processing in hemagglutinin(HA)GSDMDtransgeniciBMDMs 2 hou
19、rs post-LPS transfection.GAPDH,glyceraldehyde-3-phosphate dehydrogenase.(F and G)LDH release and GSDMD process-ing in untreated or BAPTA-AMtreated HeLa cells infected with DsifAS.typhimurium for 5 hours.MOI,multiplicity of infection.The graphs inpanels(D)and(F)show means SD.*P 0.01;*P 0.001;*P 0.000
20、1;ns,not significant(Students t test).Results arerepresentative of at least three independent experiments.on December 28,2018 http:/science.sciencemag.org/Downloaded from this PIlosignal(Fig.1C).These findings con-firmed that the low-level PI influx was causedby GSDMD pores and suggested that the PI
21、locells had repaired their plasma membrane toprevent lysis,given the absence of a Fluo-8 sig-nal peak.We then used the Ca2+chelators BAPTA-AMand EDTA to block membrane repair,as pre-viouslyreported(10,11).Ca2+chelatorsincreasedthe levels of cell lysis and lactate dehydro-genase(LDH)release from WT B
22、MDMs afterLPS transfection in a dose-dependent manner(Fig.1D and figs.S1A and S2).Notably,Ca2+chelation was not cytotoxic(Fig.1D and fig.S2A)and did not enhance LPS transfection orcaspase-11 activation,as assessed by immuno-blotting for GSDMDNT(Fig.1E and fig.S3A).BAPTA-AM also increased caspase-4de
23、pendentpyroptosiswithoutsignificantly altering GSDMDprocessing in HeLa cells infected with a DsifAdeletion strain of Salmonella enterica serovarTyphimurium(hereafterS.typhimurium)(Fig.1,F and G,and fig.S3B)(13).Thus,Ca2+chelationenhanced pyroptosis in human andmouse cells,potentiallybypreventingthei
24、nitiationofplasmamembrane repair.ESCRTproteinstargetmembranesduringmem-brane repair to form a punctate pattern(10,12).Consistently,CHMP4 puncta were observed inHeLa cells expressing CHMP4 fused to greenfluorescent protein(CHMP4-GFP)from an en-dogenous promoter(14)after S.typhimuriuminfection,GSDMDNT
25、expression,orperforintreat-ment(control),in contrast with untreated cells(fig.S4 and movie S3).Moreover,Ca2+chelationsignificantly reduced the number of cells withCHMP4-GFPpositive puncta(Fig.2A),indicat-ing that ESCRT assembly requires Ca2+influxvia GSDMD pores.Next,we transfected CHMP4-mCherry or
26、CHMP4-GFP fusion proteins intohumanembryonickidney(HEK)293Tcellsstablyexpressing caspase-1 fused with a modified FKBPdomain(DmrBCasp-1).After dimerization andactivation of DmrBCasp-1 with B/B homodimer-izer,cells underwent pyroptosis as demonstratedby morphological changes and plasma membraneannexin
27、-Vstaining(Fig.2,BandC,andmoviesS4to S6).CHMP4 relocalized to a punctate patternin DmrBCasp-1-transgenic HEK293T cells ex-pressing either high Fig.2,B and C,and fig.S5,AtoC(arrows)orlow(fig.S5B)levelsofCHMP4.Rhl et al.,Science 362,956960(2018)23 November 20182 of 502550*DICCHMP4-mCherryGSDMD-GFPCHMP
28、4-mCherry+GSDMD-GFPCHMP4-mCherry+GSDMD-GFP+Annexin VMergedB00:0021:3023:0027:30CHMP4-mCherryCHMP4-mCherry+Annexin VCHMP4-mCherry+Annexin V+DIC-20:0032:30-20:0000:0040:00addition of 2 MB/B HomodimerizerDmrB-Caspase1-transgenic HEK293T D%of cells with CHMP4 specklesMedium6 M BAPTA-AM2 mM EDTACHMP4-GFP
29、-transgenic HeLaC00:00-20:00CHMP4-GFP+Annexin VCHMP4-GFP+Annexin V+DIC12:3010:0007:0005:00-20:0000:0012:30addition of 2 MB/B Homodimerizer+DoxDmrB-Caspase1-transgenic HEK293T AMedium+EDTA+BAPTA-AMMerged+DICCHMP4-GFPFig.2.The ESCRTmachinery translocates to the plasma membraneduring pyroptosis.(A)Fixe
30、d-cell microscopy images and CHMP4-GFPpuncta quantification of HeLa cells expressing CHMP4-GFP and Dox-inducibleGSDMDNTtreated with 1 mg/ml of Dox for 4.5 hours.DIC,differentialinterference contrast.(B and C)Time-lapse confocal images of eitherCHMP4-mCherryexpressing(B)or CHMP4-GFPexpressing(C)DmrBC
31、asp-1transgenic HEK293Tcells stained with annexin-V as membranemarker.Insets show CHMP4 localizing to the plasma membrane or necks ofbudding vesicles.(D)Close-up of annexin-V+vesicle released from CHMP4-mCherryexpressing and GSDMD-GFPinternalexpressing DmrBCasp-1transgenic HEK293Tcells after a 1-hou
32、r homodimerizer treatment.Graphsshow means SD.*P 0.05;*P 0.01(Students t test).Results arerepresentativeofatleastthreeindependentexperiments.ArrowheadsindicateCHMP4 puncta.Scale bars,5 mm(A),10 mm(B)and(C),or 1 mm(D).RESEARCH|REPORTon December 28,2018 http:/science.sciencemag.org/Downloaded from Sim
33、ilar CHMP4 puncta were detected after acti-vation of DmrBCasp-11(fig.S6A).Althoughmany CHMP4 puncta clearly localized to theplasma membrane(Fig.2,B and C;figs.S4 toS6;and movie S6),puncta also formed in thecytoplasm,presumably on intracellular organ-elles.During necroptosis,the ESCRT machin-erypromo
34、testheformationofannexin-V+plasmamembrane vesicles,which have been proposed toremove damaged areas of the plasma membrane(12).Similar annexin-V+vesicles were detectedbudding from HEK293T cells after activation ofDmrBCasp-1(Fig.2,C and D;fig.S5D;andmovie S5)or DmrBCasp-11(fig.S6B).Nota-bly,these vesi
35、clesshowed clear CHMP4 punctaat the neck(Fig.2C and figs.S5D and S6B).When GSDMD-GFPinternal(fig.S7A)was coex-pressed,it localized close to CHMP4-mCherrypuncta on the cell periphery(fig.S7B)and onthe membrane of annexin-V+vesicles(Fig.2Dand fig.S7C),suggesting that ESCRT-inducedectosomes remove GSDM
36、D pores from the plas-ma membrane.We then examined the impact of ESCRT-IIIinactivation on inflammasome effector functions.We transiently expressed either WT or dominant-negativeCHMP3orVPS4AinimmortalizedmouseBMDMs(iBMDMs)orHeLacells(fig.S8,AtoC),because prolonged ESCRT depletion is cytotoxic(10,12).
37、The expression of dominant-negativeVPS4AE228Qmutant protein(Glu228Gln)orthe CHMP31-179fragment inhibited ESCRT dis-assembly,as verified by a punctate pattern(fig.S8D),and reduced cell viability after 20 to24 hours of expression(fig.S8E).This promptedus to restrict our experiments to a maximum of15 h
38、ours postinduction with doxycycline(Dox).The expression of VPS4AE228Qin WT iBMDMsresulted in two-to fourfold-higher levels of celldeath after LPS transfection compared withVPS4AWT-expressing controls(Fig.3,A and B;fig.S9;andmoviesS7andS8)andalsodecreasedrecovery after LPS transfection(fig.S10).ESCRT
39、inactivation also strongly enhanced interleukin-1b(IL-1b)release after LPS transfection(Fig.3C),but notably,it enhanced neither LDH nor cy-tokine release from untreated,Casp11/,orGsdmd/cells(fig.S11).IL-1b release after non-canonical inflammasome activationdependsonGSDMD-induced K+efflux and subsequ
40、ent acti-vation of the NLRP3caspase-1 pathway(15,16).Because LPS-transfected WT iBMDMs expressingVPS4AE228Qhad higher levels of processed,acti-vated caspase-1 and released more mature IL-1bthan VPS4AWT-expressing control cells(Fig.3,Cand D),we hypothesized that this was due to en-hanced K+efflux via
41、 increased GSDMD poreRhl et al.,Science 362,956960(2018)23 November 20183 of 5050100150*01020304050*UnstimulatedLPS transfectionVPS4AWTVPS4AE228QVPS4AWTVPS4AE228QImmortalized WT BMDMs(5 g LPS/1 106 cells)3.6 2.7%3.7 3.5%19.5 5%50.6 0.6%ABDWTE228QWTE228QVPS4A-transgenicimmortalized WT macrophagesActi
42、nPro-Casp-1Casp-1 p20UTLPS transfection(5 g/1 106 cells)5WT5E228QWTE228QE228QWTE228QWTE228QWTE228QWT010010VPS4A-transgenicimmortalized WT macrophagesActinPro-GSDMDGSDMDNTFNT ControlALG2siRNA:ALG2+ALIXALIXCHMP3VPS4AVPS4BCell death(%LDH release)Gimmortalized WT BMDMsCimmortalized WT BMDMsEVPS4AWTVPS4A
43、E228QmediumMCC950VPS4AWTVPS4AE228QmediumMCC950immortalized Ripk3/BMDMsLPS transfection(g /1 106 cells)mockLPS transfection(g/1 106 cells)1052.50204060%of PIhi cells*UTLPSns0204060Cell death(%LDH release)*UTLPS transfection(g/1 106 cells)1052.5050100150IL-1 (pg/ml)*VPS4AWTVPS4AE228Qimmortalized WT BM
44、DMsVPS4AWTVPS4AE228Qnsns020406080nsnsnsnsnsnsCell death(%LDH release)nsns0100200300400*IL-1 (pg/ml)mockLPS transfection(g/1 106 cells)1052.5NT ControlALG2siRNA:ALG2+ALIXALIXCHMP3VPS4AVPS4BIL-1 (pg/ml)untreatedLPS transfection(10 g/1 106 cells)UTLPS transfection(g/1 106 cells)1052.5nsnsFig.3.ESCRT in
45、activation enhances noncanonical inflammasome-induced pyroptosis.(A to D)PI staining,LDH release,IL-1b release,andcaspase-1 processing from VPS4AWT-transgenic or VPS4AE228Q-transgeniciBMDMs 2 hours after LPS transfection.UT,untreated.(E and F)LDH release,IL-1b release,andGSDMDprocessingfromVPS4AWT-t
46、ransgenicorVPS4AE228Q-transgenic iBMDMs 2 hours post-LPS transfection in the presence or absenceof 2.5 mM MCC950.(G)LDH and IL-1b release 4 hours post-LPS transfectionfrom Ripk3/iBMDMstreated with thecorrespondingsmall interfering RNA.NT,nontargeting siRNA.Graphs show means SD.*P 0.01;*P 0.001,*P 0.
47、0001;ns,not significant(Students t test).Results are representativeof at least three independent experiments.VPS4AWT/E228Qexpression inpanels(A)to(F)was induced with 0.5 mg/ml of Dox for 6 hours.RESEARCH|REPORTon December 28,2018 http:/science.sciencemag.org/Downloaded from formation.Consistently,an
48、d in agreement withprevious reports(15),cytokine release but notcell death was attenuated by treatment with theNLRP3 inhibitor MCC950 or high extracellularK+(Fig.3E and fig.S12A).WenextassayedGSDMDprocessingandfoundthat GSDMDNTlevels were slightly enhanced inVPS4AE228Q-transgenic iBMDMs compared wit
49、hVPS4AWT-transgenic iBMDMs at high LPS con-centrations(Fig.3F).We postulated that theenhanced GSDMD processing was caused byenhanced NLRP3 inflammasome activation(Fig.3Eandfig.S12A)and was thus caspase-1 depen-dent.Indeed,GSDMD processing did not differbetween VPS4AE228Q-and VPS4AWT-expressingiBMDMs
50、 in the presence of MCC950(Fig.3F andfig.S12B),confirming that ESCRT negatively reg-ulates pyroptosis downstream of caspase-11 acti-vation and GSDMD processing.TostudytheroleofESCRT-associatedproteins,we knocked down the expression of ESCRT-I and-IIIproteinsandoftwoproteins,ALG2andALIX,that have bee
