Protein Protein Interactions:The Inflammasome

Mark D. Wewers, M.D.

Davis Heart and Lung Research Institute

Outline of Presentation

• Innate vs. Adaptive Immunity

• Focus on TLR and NLRs

• Pyrogens (endogenous and exogenous)

• Intracellular sensors/inflammasomes– Example of intracellular pathogen– Example of population differences in

pathogen responses (caspase-12)

Time Line for Innate vs. Adaptive Immunity

Cooper MD and Alder MN. Cell 124:815, 2006.

INFECTION

IL-1/TNFFever/Inflammation

• ARDS

• Multiple Organ Failure

Shock and Poor Tissue Perfusion May Lead to Distal Gangrene

25% of population died

Black Death

Hippocrates

5th century B.C.

Illness due to natural causes

Guns Germs and Steel Hypothesis

Jared Diamond

Lewis Thomas: The Lives of a Cell 1974

“Our arsenals for fighting off bacteria are so powerful andinvolve so many different defense mechanisms that we are more indanger from them than from the invaders.

We live in the midst of explosive devices, we are mined….When we sense lipopolysaccharide, we are likely to turn on everydefense at our disposal…”

Meningococcemia

Components of lipopolysaccharide

bacterial membrane

core polysaccharide

“O” antigen

lipid A

How can our defense repertoire cover the enormous diversity in pathogen

challenges?• Innate host response:

– Static defense system– Focus on immutable components of pathogens (e.g.,

cell wall component)

• Acquired host response:– Creative ability to change with pathogen– Depends upon the acquisition of recombinase gene

(RAG)

Innate Pathogen Sensors

NLR

TLR

Extracellular sensors

Intracellular sensors

TIRNB

LRRLRR

Family of TLR’s and Ligands

Beutler et al.

Akira et al. Nature Reviews (Immunology):4:499, 2004.

TLRs and their ligands

Medzhitov R. NEJM 343:338,2000.

The Signaling Pathway of Toll-like Receptors

Exogenous pyrogen induces endogenous pyrogen.

exogenous endogenous

Both Endogenous and Exogenous Pyrogens converge at NFkB

Figure 2-10Innate versus Adaptive Immunity Characteristics

Medzhitov R. NEJM 343:338,2000.

Bridge to Adaptive Immunity

News from the Genome Project that has expanded our understanding of the innate host response repertoire.

Plant Immunity: Pathogen sensing without Tolls

Dangl JL, Jones JDG. Nature 411:826, 2001.

NATURE | VOL 411 | 14 JUNE 2001

Guard Hypothesis

Dangl, J. et al.

NB-LRR protein dissociates due to pathogen

NB-LRR protein binds target complex due to pathogen binding to complex

Exogenous pyrogen induces endogenous pyrogen.

CN p20 p10

QACRG

p20p10

p20

p20

p10

p10

p45

CARD

Active ICE

Caspase-1 (ICE)

Caspase Recruitment Domains:

CARDs

CATERPILLER: A Large Family of Mammalian Genes Containing CARD, Pyrin, Nucleotide-binding, and Leucine-Rich Repeat Domains

Harton JA et al. J. Immunol. 169:4088, 2002.

CARD/PYD NBDLRR

10kD 20kD

caspase-1

caspase-5

ASC

NALP1NBD LRRpyrin CARD

CARD

20kD 10kD

Inflammasome Concept

Martinon, F et al. Mol.Cell 10:417-426, 2002.

Domain structure of CATERPILLERs

Toll like receptors and the Signalosome

signalosome

gene expression

proteasome

NFBNFBIB

IKK complex

TLR

*

NFBNFB

100’ s of genesincluding IL-1 and TNF

Cell Death and Differentiation (2006) 13, 816–825

Toll-like receptors

Rast,J.P. et al. Science 314:952, 2006.

Sea Urchin’s Pathogen Sensors

CARD NBDLRR

CARD NBDLRRCARD

NBDLRRpyrin

CARD NBDLRR

pyrin CARD

NBDLRRpyrin CARD

ASC

NALP1

IPAF

NOD1

NOD2

NALP3

IL-1

proIL-1

Caspase-5

NALP-1

NOD-2

TLR

ASC

CARD domain

pyrin domain

inflammasome

CASPASE-1

Martinon, F et al. Mol.Cell 10:417-426, 2002.

Multiprotein Complex Assembly requires ASC

• ASC (apoptosis associated speck forming complex) ASC

Sarkar A et al. J.Immunol. 176:4979, 2006.

ASC Critical to IL-1 Processing

ASC

Need for Intracellular Pathogen Sensors

• Phagosome escape

A B

DC

Francisella as Model of Inflammasome Activation

Gavrilin MA, PNAS 103:141, 2006.

F. novicida

E. coli

F. n. heat-killed

F. n. + cytochalasin D

-1-1

-1-1

-1-1

-1-1

-1-1

-1-1

-1-1

-1

-1-1

-1 -1-1-1-1

-1-1-1-1

-1-1

-1-1-1-1

-1-1

Phagosome Escape Induces Inflammasome Assembly

IL-1

proIL-1

phagosome

F.novicida

Mo LVS F.nov E.coli LVS F.nov E.coli LVS F.nov E.coli

live killed endotoxin

31 kDa

17 kDa

A. IL-1 mRNA B. IL-1 in media C. IL-1 in cell extract

0

1000

2000

3000

4000

Un

stim

ula

ted

LV

S

F.n

ov

E.c

oli

LV

S

F.n

ov

E.c

oli

LV

S

F.n

ov

E.c

oli

RC

N

live killed endotoxin

0

20

40

60

80

100

Uns

timul

ated

LVS

F.n

ov

E.c

oli

LVS

F.n

ov

E.c

oli

LVS

F.n

ov

E.c

oli

ng

/ml

live killed endotoxin

D. IL-8 mRNA E. IL-8 in media F. Protein/RNA Ratio

F. novicida induces both mRNA and processing and release of IL-1

Gavrilin MA, PNAS 103:141, 2006.

Toll like receptors and the Signalosome

gene expression

proteasome

NFBNFBIB

IKK complex

TLR

*

NFBNFB

100’s of genesincluding IL-1 and TNF

signalosome

proIL-1

IL-1

proIL-1

Caspase-5

NALP-1

TLR

ASC

CARD domain

pyrin domain

inflammasome

CASPASE-1

1

2

Molecular Cell 25, 713–724, March 9, 2007

Inflammasome structure

What about Septic Shock?

Figure 2-45 part 1 of 3

IL-1

proIL-1

CASPASE-1

Caspase-5

NALP-1

NOD-2

TLR

ASC

Caspase 1 and Sepsis

Caspase-1: Role in Sepsis Survival and Spleen Apoptosis

Sarkar A et al. AJRCCM 2006

IL-1

proIL-1

CASPASE-1

Caspase-5

NALP-1

NOD-2

TLR

ASC

Caspase-12

Caspase 12 and Sepsis

Saleh M. Nature 429:75, 2004.

Caspase-12 Comes in Two Forms

Caspase-12L (active) versus Caspase-12S (inactive) Allelic

DistributionThe American Journal of Human Genetics Volume 78 April 2006

Source Total (n)

Genotype Frequency (%) Allele Frequency (%)

T/T T/C C/C T C

Sepsis 38 60.5 29 10.5 75 25

Control 148 81.1 17.6 1.3 89.9 10.1

Caspase-12L Linked to Sepsis

Saleh M. Nature 429:75, 2004.

Caspase-1, 12

NLRP3, NLRP1 and Caspase-1 and 12

NLRP3

NLRP1

Summary

• Innate immune responses are critical to our host defense against pathogens.

• Innate mechanisms are probably more critical than previously recognized.

• Protein/protein interactions that are modified by pathogens (or their products) are central to pathogen sensing and signaling.