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The NALP3/cias1/Cryopyrin
Inflammasome
Martinon Fabio
FMFSAID April 2008
London/LeedsMicheal McDermottPhilip Hawkins
UtrechtAlain Kummer
Dept. BiochemistryJürg Tschopp
Virginie PétrilliAnnick MayorMarie MichalletManuele RebsamenStéphanie PapinCatherine DostertFrancesco StaehliDaniel RossiEmmanuelle LogetteLeo HeinzPhilippe MenuRongbin ZhouAmir YazdiAndrea D’OsualdoGreta GuardaMarlène BrasMirjam EckertAkihito Nakajima
Apoxis (Lausanne)Thibaud De Smedt
CHUV-LausanneAlexander So
CalgaryDan Muruve
Interleukin-1 (Interleukin-1 (IL-1IL-1ββ))
>Key player in Inflammation
>Important player in various aspect of cell regulation
Known as
•Lymphocyte activating factor (LAF)•Mitogenic protein (MP)•T cell replacing factor III (TRFIII)•B-cell activating factor (BAF)•B-cell differentiation factor (BDF) •“Heidikine”
•Endogenous Pyrogen (EP)
IL-1
NFκB; JNK; p38; INFβ …
IL-1β
IL-1R complex
TIR
IL-1IL-1ββ ProductionProduction
NFκB JNK P38..
Pro-inflammatory stimuli
Caspase-1NALPs/Ipaf
Inflammasome
Pro-IL-1β1)
1) Transcription
IL-1β
2)
2) MaturationIL-1β
3) 3) Export
IL-1β
IL-1R complex
TIR
Convergences between Convergences between InflammasomesInflammasomes and and TLRsTLRs
Martinon and Tschopp Trend in Immunology 2005 vol. 26 (8) pp. 447-54
Inflammasome
NALP1-INFLAMMASOME
IPAF-INFLAMMASOME
Caspase-1/5
NALP1
ASC
CARDCasp1
CARD
PYD CARD
Casp5
NODNODPYD
NAD
Caspase-1/1
NALP3
ASC
CARDCasp1
CARD
PYD CARD
Casp1
CARDFIINDNODNODPYD
NAD CARDINAL
IL-1βIL-18IL-33
CARDFIIND
NALP3-INFLAMMASOME
IL-1βIL-18IL-33
Cell deathIL-1β
IPAF
NODNOD
NAD
CARDCasp1
CARDCaspase-1
The The InflammasomesInflammasomes
NALP3 (NALP3 (CIAS1; CIAS1; cryopyrincryopyrin) and ) and autoinflammatoryautoinflammatory diseases diseases
Localized on Chr: 1q44 a periodic fever locus
Suscseptibility locus• 1q44
Pathology• Rashes• Fever• Amyloidosis• Sensorineural Deafness (MWS)• Symptoms provoked in the cold (FCU)• Neurological involvement (CINCA)
• Autosomal dominant
• Familial cold urticaria (FCU)• Muckle-wells syndrome (MWS)• Chronic infantile neurological cutaneous arthropathy (CINCA)
Treatment of patients with IL1ra (Treatment of patients with IL1ra (AnakinraAnakinra))
NACHT
PYD
NAD
CARD
PYD
CARD Casp1
IL-1β
CARD
FIIND
CARD
Casp1
TNFproIL-1
SAA
IL-6 …
…
IL1ra
IL-1 a target for therapy Hawkins et al. N Engl J Med. 2003 Jun 19;348(25):2583-4 Hawkins et al. Arthritis Rheum. 2004 Feb;50(2):607-12.
IL1ra
IL-1βPro- IL-1β
NO
DN
OD
PYD
NAD
Caspase-1
PAMPS DAMPS
DAMPSDAMPS
Hypotonic stressHypotonic stressATP /P2X7ATP /P2X7Uric Uric AcidAcidUVBUVB
PAMPSPAMPS
Bacterial cell wallBacterial cell wallcomponent (component (PeptidoglycansPeptidoglycans))RNARNAAdenovirusesAdenovirusesPoxvriusesPoxvriuses
ShigellaShigella/ / IpaBIpaBFrancisellaFrancisellaListeriaListeria
Activators of the InflammasomeActivators of the Inflammasome Other DAMP: Uric acid crystals (MSU)....Other DAMP: Uric acid crystals (MSU)....
Molecular identification of a danger signal that alerts the immune system to dying cells.Nature. 2003 Oct 2;425(6957):516-21.Shi Y, Evans JE, Rock KL.
...the etiologic agent of gout ...the etiologic agent of gout
-Autoinflammatory disorder
-pain
-inflammation of the articular and periarticular tissues
Does MSU activates the NALP3 inflammasome?
PBS
1µg
10µ
g 10
0µg
MSU
PBS
1µg
10µ
g
100µ
g
MSU
z-YVAD-fmk
SN
WB:D116IL-1β p17
Cell
WB:IL-1βproIL-1β
MSU
NALP
Caspase-1
IL-1Nature Vol. 440, 237–241, march 2006,
MSU
NALP
ASC
Caspase-1
IL-1b
MSU activation of IL-1β is ASC dependent
WB:ASC
IL-1β p17SN
WB: IL-1β
ASC +/+
MSU
1h
PBS
MSU
3h
MSU
6h
MSU
16h
MSU
1h
PBS
MSU
3h
MSU
6h
MSU
16h
ASC -/-
Cell
CellWB:Casp1
Casp1
ASC
MSU
NALP3
ASC
Caspase-1
IL-1b
MSU activation of IL-1β is NALP3 dependent
NALP3 +/+
MSU
1h
PBS
MSU
3h
MSU
6h
MSU
16h
MSU
1h
PBS
MSU
3h
MSU
6h
MSU
16h
NALP3 -/-
SN
WB:IL-1β
CellWB:IL-1β
IL-1β p17
proIL-1β p35
MSU
i.p
study neutrophils
influx
0
1
2
3
4
5
6
ASC +/+ASC -/-
ZYM
neut
roph
ils (x
106 )
0.0
0.5
1.0
1.5
2.0
2.5
3.0
ZYM
IL-1R +/+IL-1R -/-
neut
roph
ils (x
106 )
0.00.5
1.01.52.0
2.5ASC +/+ASC -/-
neut
roph
ils (x
106 )
p=0.01
0.00.51.01.52.02.53.03.54.0
neut
roph
ils (x
106 ) p<0.01
p<0.01
IL-1R +/+IL-1R -/-
MSU Zymosan (TLR2)
MSU CPPD
Inflammasome dependent inflammation in vivo
Nature Vol. 440, 237–241, march 2006,
Gout
Martinon and Glimcher J. Clin Invest. 116 (8) 2073-5
CPPD crystals and CPPD crystals and pseudogoutpseudogout
SN
WB:IL-1β
CellWB:IL-1β
IL-1β p17
proIL-1β p35
CPPD (Calcium Pyrophosphade Dehydrate) crystal depositiondisease causes pain, swelling, and sometimes redness and/orheat in one or many joints
OtherOther crystals: Asbestos and Silica crystals: Asbestos and Silica
C. Dostert et al. in press.
...the etiologic ...the etiologic agents agents ofof asbestosis and silicosis asbestosis and silicosis
-Inflammatory diseases of the lung
-acute or chronic
OtherOther crystals: Asbestos and Silica crystals: Asbestos and Silica
C. Dostert et al. in press.
...the etiologic ...the etiologic agents agents ofof asbestosis and silicosis asbestosis and silicosis
SN
Cell
-IL-1β p17
-proIL-1β
shMyD88
Ø Asbestos
MSUNigerici
n
Ø Asbestos
MSUNigerici
nØ Asbesto
s
MSUNigerici
n
Ø Asbestos
MSUNigerici
n
Ø Asbestos
MSUNigerici
n
shNalp3mock shASC shcasp1
SN -IL-1βp17
-Casp1
-Casp1p20
.2 .1 .05 .2
Asbestos
Ø
Silica
.15 .15
MSU
.5 .2 .1 .5
zVAD + + + .2 .1 .05 .2
Asbestos
Ø
Silica
.15 .15
MSU
.5 .2 .1 .5
+ + + .2 .1 .05 .2
Asbestos
Ø
Silica
.15 .15 (mg/ml)
MSU
.5 .2 .1 .5
+ + +
Human
Mouse
Asc-/-Nalp3-/-
AsbestosSilica
NALP3
ASC
Caspase-1
IL-1b
Autoinflammatory and chronic inflammatory diseases
NACHT
PYD
NAD
CARD
PYD
CARD Casp1
IL-1β
CARD
FIIND
CARD
Casp1
IL-1RMyD88
NACHT
PYD
NAD
CARD
PYD
CARD Casp1
IL-1β
CARD
FIIND
CARD
Casp1
IL-1RMyD88
Periodic fevers
Gout / Pseudogout/Asbestosis/ Silicosis
IL1ra
C1ASCNALP3ATP Pannexin1P2X7
Activator Intermediate Inflammasome
C1ASCNALP3
MDP ?NOD2 Rip2
C1ASCNALP3NALP3mutations
Result (if deregulated)
Hereditary periodic fevers suchas MWS, FCU and CINCA
C1ASCNALP3
NigericinMaitotoxin Pannexin1
C1ASCNALP3
ListeriaStaphylococcus ?
C1NALP1MDP
C1NALP1bAnthraxLeTx ? Macrophage death
C1NALP1NALP1mutations
Vitiligo
C1NALP7NALP7mutations
Hydatidiform moleASC
C1ASC?Francisella
IFNβ
Bacteria or bacterial product
Genetic mutation
Known adjuvant
Chemical
Danger signal
C1ASCNALP3MSUCPPD ?
Gout, Pseudogout,
C1ASCNALP3
TNP-CLTNCBDNFB ?
Contact Hypersensitivity
NALP NALP activators activators
Bacteria/Bacteria/InflammasomeInflammasome interaction interactionNod-like receptors (Nod-like receptors (NLRsNLRs); ); NBS-LRR; NBS-LRR; CatterpilersCatterpilers
R genes Plants
NO
DN
BAR
CTIR
NALP2-14NALP1
CAR
DFI
IND
NO
DN
AC
HT
PYD
NAD
NO
DN
AC
HT
PYD
NAD
Caspase Caspase
NAIPIPAF
NAC
HT
CAR
D
NO
DN
AC
HT
BIR
CIITA
NO
DN
AC
HT
CAR
D
MHC ClassII MAPK
NOD1,2
NO
DN
AC
HT
NAD
RIP-Kinase
CAR
D
Crohn’s dis.,Blau s.
MWS,FCU,CINCA
SMA,Legionnellasuceptibity
BLSII <--DISEASE
Resistance (R) genes in PlantsResistance (R) genes in Plants
Hypersensitive Response
PLANT
AvirulanceFactors (Avr)
LRR
MAPK
Kin
Virus
AvrRpsV4 AvrRpt2
RPS2LRR
Resistance genes (>100)
TIR
NB-A
RC
CC
NB-A
RC
Virus
Transcription
RPSV4
Direct detection orIndirect Detection (Danger Signal)
Identification of various regulators of activation:
RAR1SGT1HSP90
1034PYD NACHT NAD
LRR
NALP3
TPR CS/p23 SGSSGT1 395
SGT1 interacts with the LRR of SGT1 interacts with the LRR of NLRsNLRs
NALP
3NA
LP3Δ
LRR
NACH
TPY
DLR
R
IP: α-Flag
WB: IP SGT1bSGT1a
HSP90
moc
kN3
LRR
NALP
3NA
LP2
NALP
4NA
LP12
NOD1
NOD2
RIP1
SDS2
2pp
32IP: α-Flag
WB: IP
HSP90
SGT1bSGT1a
IPAF
Immunoprecipitation
SGT1 and HSP90 SGT1 and HSP90 cooperatecooperate for binding to the NALP3-LRR for binding to the NALP3-LRR
NO
DN
OD
PYD
NAD
HSP90SGT1HSP90
HSP90 WB: XT
NALP
3-LR
R
IP: α-Flag
WB: IP
SGT1bSGT1a
SGT1bSGT1a
NALP3-LRR(Flag)
Moc
kNA
LP3-
LRR
Moc
k
RNAi: scr SGT1
RNAi IP: α-Flag NA
LP3-
LRR
NALP
3-LR
R
SGT1bSGT1a
SGT1bSGT1aHSP90
Flag-Proteins
WB: XT
GA 200nM - +
WB: IP
geldanamycin
SGT1scrambled
Stimulation:RNAi:
IL-1β
(pg/
ml)
---
LPS
LPS
--- LPS
LPS
6h 2h 6h 2h
PGN
PGN
PGN
PGN
0
100200
300400
500600
700800900
MSU PG
N
MSU PG
N
moc
k
moc
k
WB: XT
WB: SN IL-1β
SGT1aSGT1b
RNAi SGT1RNAi scr + + +
+ + +
proIL-1β
020406080
100120140
mock MSU PGN
TNF
(pg/
ml)
siScrsiSGT1
SGT1 SGT1 RNAiRNAi blocks blocks Inflammasome/IL-1Inflammasome/IL-1 activation activation
NO
DN
OD
PYD
NAD
HSP90SGT1
RNAi
THP1
ATP
MSU
PGN
GA 200nM + ++
Moc
k
---- +
IL-1βWB: SN
proIL-1β
MSU
100
µg
MSU
50µ
g
IL-1β
ASC
Nalp3
GA 200nM
WB: SN
WB: XT
+ + + + + +
moc
k
ATP
30ʼ
ATP
4h
PGN
- - - - - -
NO
DN
OD
PYD
NAD
HSP90SGT1
geldanamycin
Human monocytes
THP1
GeldanamycinGeldanamycin blocks blocks Inflammasome/IL-1Inflammasome/IL-1 activation activation
n=12
17-DMAG blocks inflammation in a 17-DMAG blocks inflammation in a Gout-NALP3Gout-NALP3model model in vivoin vivo
NO
DN
OD
PYD
NAD
HSP90SGT1
17-DMAGMSU
i.p
study neutrophils
influxMSU MSU PBSMSU MSU
WT NALP3-/-
+17-
DMAG
+17-
DMAG
?K+ efflux<100 mM
Caspase-1
NALP3 inflammasome
Asbetos Silica
MSU
ATP
PGNDNA
CPPD
IL-1bPro- IL-1b
DAMPs
PAMPs
NO
DN
OD
PYD
NAD
HSP90 SGT117-DMAG
IL-1ra (Anakinra)IL-1b
SummarySummary
Inflammation, periodic fevers, Gout…
VX-765
AdenovirusesListeria
….
Pathogens
London/LeedsMicheal McDermottPhilip Hawkins
UtrechtAlain Kummer
Dept. BiochemistryJürg Tschopp
Virginie PétrilliAnnick MayorMarie MichalletManuele RebsamenStéphanie PapinCatherine DostertFrancesco StaehliDaniel RossiEmmanuelle LogetteLeo HeinzPhilippe MenuRongbin ZhouAmir YazdiAndrea D’OsualdoGreta GuardaMarlène BrasMirjam EckertAkihito Nakajima
Apoxis (Lausanne)Thibaud De Smedt
CHUV-LausanneAlexander So
CalgaryDan Muruve
Use of IL-1 inhibitors inUse of IL-1 inhibitors in Gout Gout patientspatients
So, A., De Smedt, T., Revaz, S., Tschopp, J.A pilot study of IL-1 inhibition by anakinra in acute gout. Arthritis Res Ther. 2007;9(2):R28.
McGonagle, D. Tan, A. Shankaranarayana, S. Madden, J. Emery, P. Mcdermott, M.Management of treatment resistant inflammation of acute on chronic tophaceous gout withanakinraAnn Rheum Dis 2007;66:1683-1684
Use of IL-1 inhibitors inUse of IL-1 inhibitors in pseudogoutpseudogout patientspatients
McGonagle, D, Tan, A, Madden, J, Emery, P, McDermott, MSuccessful treatment of resistant pseudogout with anakinraArthritis Rheum 2008 vol. 58 (2) pp. 631-633
SN
WB:IL-1β
CellWB:IL-1β
IL-1β p17
proIL-1β p35
CPPD (Calcium Pyrophosphade Dehydrate) crystal depositiondisease causes pain, swelling, and sometimes redness and/orheat in one or many joints
Systems for the sensing of MicrobesSystems for the sensing of Microbes
Microbial world
Detection System
Immune Response(pathogen)
Symbiotic Response(commensal)
TLRs
TIR
TIR
TIR
TIR
LPS
NFκB; JNK; p38; INFβ …
ZymosanCpG dsRNA
Patterns Recognized
Receptors
Response
Toll-like receptors (TLRs) and Nod-like receptors (NLRs)Toll-like receptors (TLRs) and Nod-like receptors (NLRs)
Innate Immunity; Inflammation
dsRNA,LPS, ssRNA, Flagellin, etc.
PRR (Pathogen/microbe recognition receptors)
MYD88
MAPKNFκB
PAMP (Pathogen/microbe -associate molecular patterns)
TIR
Toll-like receptors
(TLR)
RIP
PAMP DAMP
Nod-likereceptors
(NLR)
NO
DN
AC
HT
DF
Caspases
NALP3 InflammasomeNALP3 Inflammasome
NACHT
PYD
NAD
CARD
PYD
CARD
CARD
Casp1
FIIND
CARD
Casp5Caspase-5
Caspase-1
ASC
NALP1
IL-1β
NALP1 inflammasome NALP3 inflammasome
NACHT
PYD
NAD
CARD
PYD
CARD Casp1Caspase-1
ASC
NALP3
IL-1β
CARD
FIIND
CARD
Casp1Caspase-1
Cardinal
SalmonellaShigella
ListeriaStreptomyces
StaphylococcusBacillus brevis
AeromonasE.coliAnthrax
Inflammasome
Legionella
DAMPs
?
AdenovirusSendai
DANGERVIRUSBACTERIA
IL-1βPro- IL-1βCell death
IL-18/33
Francisella
PAMPs
NALP3
ASC
Casp1
IPAF
NAIP
Casp1
NALP1
Casp1/5
NALPx
ASC
Casp1
? Flagellin LT Pore forming toxins RNA?PGN
ACTIVATORS OFINFLAMMASOMES
ATPNADMSUCPPDR837TNCB
UrushiolUVBEtc.
IL-1βPro- IL-1β
NO
DN
OD
CARD
Role of K+ in inflamasomeactivation?
Caspase-1
NALP3 inflammasome
K+ (143 mM)
K+ efflux
P2x7
ATPBacterial toxins
Nigericin 130 mM KClextracellularK+ (4 mM)
Role of K+ in inflammasomeactivation?
Nige
ricin
Poly(
I:C)
ATP
--- MSU
SN
XT ProIL-1β
IL-1βp17
KCl (130mM)
Nige
ricin
ATP
--- MSU
Poly(
I:C)
R84
8Ba
ct. R
NA
PGN
MSU
Nig
eric
in
R84
8
PGN
MSU
Nig
eric
in
Bact
. RN
A
--- ---
IL-1βp17
+ KCl
NALP3+/+ NALP3-/-
SN
SN
IL-1β
Casp1
Pro- IL-1β
Casp1/5
Activator130 mM KCl
Role of K+ in IPAFinflammasome activation?
IL-1β
Casp1
Pro- IL-1β
Casp1/5
Activator130 mM KCl
Salmonella activates the IPAF inflammasome
•K+ efflux is specific for NALP3 inflammasome(not IPAF)
--- Salm
oSa
lmo
Salm
o
IPAF+/+ IPAF-/-
--- Salm
oSa
lmo
Salm
o
KCl + +Time (min) 90 180 180 90 180 180
ProIL-1βIL-1βp17
An in vitro system for Inflammasome activationAn in vitro system for Inflammasome activation
30°C 60 (min)
0°C
Stress(K+low,
ROS,etc.)
Hypotonic lysis
THP1
THP1
0°C
30°C 10 30 60 (min)
Molecular Cell, Vol. 10, 417–426, August, 2002,
NALPsNALPs
NALP3
NO
DN
AC
HT
PYD
NAD
-14 members in human
-Characterized by a PYD, NACHT and LRR domain
-Present in all vertebrates
-Display a pattern of rapid evolution.
The adaptor ASC is essential in vitroThe adaptor ASC is essential in vitro
Caspase-1
NODNACHTPYD
NAD
CARD
PYD
CARD
CARD
Casp1
FIIND
CARD
Casp1
Caspase-5
ASC
NALP1
Inflammasome
Anti-ASC
Caspase-1
NODNACHTPYD
NAD
CARD
PYD
CARD
CARD
Casp1
FIIND
CARD
Casp1
Caspase-5
ASC
NALP1
Immunodepletion of ASC
+ ASC - ASC
activation - + - +
WB: ASC
WB:Caspase-1
Casp1
p35
p10
ASC
30°C60 (min)
0°C
Molecular Cell, Vol. 10, 417–426, August, 2002,
NALP3 (CIAS1) is mutated in Periodic FeversNALP3 (CIAS1) is mutated in Periodic Fevers
L305P
A352V A439V E627G
T348MD303N
A439T G569R
D303N
H358R M573S T436R M662TQ306L
R260WR260W
V198M
F309S
V198M
FCAS/FCU CINCA
MWS
Hofmann et al.McDermott et al.Feldman et al.Dode et al.
NACHTPYD NAD
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