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
Fever/Inflammation
IL-1/TNF
• ARDS
• Multiple Organ Failure
Shock and Poor Tissue Perfusion
May Lead to Distal Gangrene
Black Death
25% of population died
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 and
involve so many different defense mechanisms that we are more in
danger 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 every
defense at our disposal…”
Meningococcemia
core
polysaccharide
bacterial
membrane
lipid A
“O”
antigen
Components of
lipopolysaccharide
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
LRR
LRR
TLR
TIR
NLR
NB
Extracellular
sensors
Intracellular
sensors
Family of TLR’s and Ligands
Beutler et al.
TLRs and their ligands
Akira et al. Nature
Reviews
(Immunology):4:499, 2004.
The Signaling
Pathway of Toll-like
Receptors
Medzhitov R. NEJM 343:338,2000.
Exogenous pyrogen induces endogenous
pyrogen.
exogenous
endogenous
Both Endogenous
and Exogenous
Pyrogens converge
at NFkB
Innate versus Adaptive Immunity Characteristics
Figure 2-10
Bridge to Adaptive Immunity
Medzhitov R. NEJM 343:338,2000.
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.
Guard Hypothesis
NB-LRR protein
dissociates due to
pathogen
NB-LRR protein binds
target complex due to
pathogen binding to
complex
Dangl, J. et al.
NATURE | VOL 411 | 14 JUNE 2001
Exogenous pyrogen induces endogenous
pyrogen.
Caspase-1 (ICE)
CARD
p45 N
QACRG
p20
p10
C
p20
p10
p20
p10
p20
p10
Active
ICE
Caspase Recruitment Domains:
CARDs
CATERPILLER: A Large Family of Mammalian
Genes Containing CARD, Pyrin, Nucleotide-binding,
and Leucine-Rich Repeat Domains
CARD/PYD
NBD
Harton JA et al. J. Immunol. 169:4088, 2002.
LRR
Inflammasome Concept
NALP1
pyrin
NBD
LRR
CARD
CARD
10kD
ASC
20kD
caspase-5
20kD
10kD
caspase-1
Domain structure of CATERPILLERs
Martinon, F et al. Mol.Cell 10:417-426, 2002.
Toll like receptors and the
Signalosome
TLR
signalosome
IKK complex
b
a
*
IkBa
g
NFkB
100’s of genes
including IL-1b and TNF
proteasome
gene
expression
NFkB
Toll-like receptors
Cell Death and Differentiation (2006) 13, 816–825
Sea Urchin’s Pathogen Sensors
Rast,J.P. et al. Science 314:952, 2006.
pyrin CARD
ASC
pyrin
NBD
CARD
LRR
NALP1
CARD
NBD
LRR
IPAF
CARD
NBD
CARD CARD
NBD
LRR
NOD1
LRR
NOD2
pyrin
NALP3
NBD
LRR
TLR
inflammasome
NOD-2
ASC
CARD domain
pyrin domain
CASPASE-1
Caspase-5
proIL-1b
IL-1b
Martinon, F et al. Mol.Cell 10:417-426, 2002.
Multiprotein Complex Assembly
requires ASC
• ASC (apoptosis associated
speck forming complex)
ASC
ASC Critical to IL-1b Processing
ASC
Sarkar A et al. J.Immunol. 176:4979, 2006.
Need for Intracellular Pathogen
Sensors
• Phagosome escape
Francisella as Model of
Inflammasome Activation
A
B
F. novicida
F. n. heatkilled
C
E. coli
D
F. n. +
cytochalasin D
Gavrilin MA, PNAS 103:141, 2006.
Phagosome Escape Induces
Inflammasome Assembly
-1
-1
-1
F.novicida
proIL-1b
-1
-1
-1
-1
-1
phagosome
-1
-1
-1
-1
-1
IL-1b
F. novicida induces both mRNA and
processing and release of IL-1b
B. IL-1b in media
4000
100
3000
80
ng/ml
2000
1000
60
31 kDa
40
20
live
killed
endotoxin
live
D. IL-8 mRNA
E. IL-8 in media
6000
500
killed
E.coli
F.nov
LVS
E.coli
F.nov
LVS
E.coli
F.nov
17 kDa
LVS
E.coli
F.nov
LVS
E.coli
F.nov
LVS
E.coli
F.nov
LVS
0
Unstimulated
0
C. IL-1
Mo L
Unstimulated
RCN
A. IL-1b mRNA
endotoxin
F. Pro
300 MA, PNAS 103:141, 2006.
Gavrilin
ng/ml
RCN
4000
200
1b Ratio
400
70
60
50
40
30
Toll like receptors and the
Signalosome
TLR
signalosome
IKK complex
b
a
*
g
IkBa
NFkB
100’s of genes
proIL-1b
proteasome
including IL-1b and TNF
gene
expression
NFkB
1
TLR
inflammasome
ASC
2
CARD domain
pyrin domain
CASPASE-1
Caspase-5
proIL-1b
IL-1b
Molecular Cell 25, 713–724, March 9, 2007
Inflammasome structure
What about Septic Shock?
Figure 2-45 part 1 of 3
Caspase 1 and Sepsis
TLR
NOD-2
ASC
CASPASE-1
Caspase-5
proIL-1b
IL-1b
Caspase-1: Role in Sepsis Survival and Spleen Apoptosis
Sarkar A et al. AJRCCM 2006
Caspase 12 and Sepsis
Caspase-12
TLR
NOD-2
ASC
CASPASE-1
Caspase-5
proIL-1b
IL-1b
Caspase-12 Comes in Two
Forms
Saleh M. Nature 429:75, 2004.
Caspase-12L (active) versus Caspase-12S
(inactive) Allelic Distribution
The American Journal of Human Genetics Volume 78 April 2006
Caspase-12L Linked to Sepsis
Source
Total
(n)
Genotype Frequency (%) Allele
Frequency
(%)
Sepsis
38
T/T
60.5
Control
148
81.1
T/C
29
C/C
10.5
T
75
C
25
17.6
1.3
89.9
10.1
Saleh M. Nature 429:75, 2004.
NLRP3, NLRP1 and Caspase-1 and 12
NLRP1
Caspase-1, 12
NLRP3
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.