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