2013-06-25
1
Inflammasomes: Regulation and Function in Inflammatory
Lung Disease
Augustine M.K. Choi, MD
Parker B. Francis Professor of Medicine
Harvard Medical School
Chief, Pulmonary and Critical Care Medicine
Brigham and Women’s Hospital
Airway Vista 2013
Asan Medical Center
Seoul, Korea
March 30, 2013
TLR
Microbial molecules
(PAMPs)
Gene transcription
(NF-B)Pro IL-1, pro IL-18
Maturation of
IL-1, IL-18
Cleaved caspase-1
ASCNLP3
Pro caspase-1
P2X7 receptor
Inflammasome
ATP
secretion
Cytosol
Extracellular
Fig. A
TLR
Microbial molecules
(PAMPs)
Gene transcription
(NF-B)Pro IL-1, pro IL-18
Maturation of
IL-1, IL-18
Cleaved caspase-1
ASCNLP3
Pro caspase-1
P2X7 receptor
Inflammasome
ATP
secretion
Cytosol
Extracellular
Fig. A
2013-06-25
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Inflammasome
Inflammasomes are molecular platforms activated upon cellular infection or stress that
trigger the maturation of proinflammatory cytokines such as IL-1b and IL-18 to engage
innate immune defenses. (Cell 2010)
Members of the Nod-like receptor family, including NLRP1, NLRP3 and NLRC4, and the
adaptor ASC are critical components of the inflammasome that link microbial and
endogenous ‘danger’ signals to caspase-1 activation. (Nature Immunology 2009)
Associations between dysregulated inflammasome activity and inflammatory diseases.
Infection (bacteria, fungus, virus)
Crohn’s disease
Alzheimer’s disease
Metabolic syndrome (obesity, type2 diabetes)
Gout
Silicosis, asbestosis
Sunburn (UV)…….
Nature Immunology, 2012
2013-06-25
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Mechanical Ventilation Modulates Inflammasome-Related Gene Expression
Ex Vivo and In Vivo
Physiol Genomics, 2008
PLoS One, 2009
Gene expression of whole blood cells in sepsis/ARDS
2013-06-25
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AJRCCM, 2012
IL-18 in the Plasma Correlates with Patient Survival Outcomes
AJRCCM, 2012
2013-06-25
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IL-18 KO mice are more resistant to VILI
2013-06-25
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TLR
Microbial molecules
(PAMPs)
Gene transcription
(NF-B)Pro IL-1, pro IL-18
Maturation of
IL-1, IL-18
Cleaved caspase-1
ASCNLP3
Pro caspase-1
P2X7 receptor
Inflammasome
ATP
secretion
Cytosol
Extracellular
Fig. A
TLR
Microbial molecules
(PAMPs)
Gene transcription
(NF-B)Pro IL-1, pro IL-18
Maturation of
IL-1, IL-18
Cleaved caspase-1
ASCNLP3
Pro caspase-1
P2X7 receptor
Inflammasome
ATP
secretion
Cytosol
Extracellular
Fig. A
0 24 48 72 96 1200
50
100
150
IL-18 -/- Sham
IL-18 -/- CLP
C57 wt Sham
C57 wt CLP
CLP (Hrs)
Pe
rce
nt
su
rviv
al (%
)
P = 0.8890
0 24 48 72 96 1200
50
100
150
ASC -/- Sham
ASC -/- CLP
C57 wt Sham
C57 wt CLP
CLP (Hrs)
Pe
rce
nt
su
rviv
al (%
)
P < 0.0001
0 24 48 72 96 1200
50
100
150
Nlrp3 -/- Sham
Nlrp3 -/- CLP
C57 wt Sham
C57 wt CLP
CLP (Hrs)
Pe
rce
nt
su
rviv
al (%
)
P < 0.0001
Survival of NLRP3 inflammasome-deficient mice
80% Ligation
1 hole with 23G needle
Nature protocol (2009) 4: 31-36
Nature Reviews Immunology (2009) 9: 287-293
Nlrp3 -/- mice and ASC -/- mice showed higher survival rates compare with
that of C57 wt. However, IL-18 -/- mice had no phenotype.
after CLP
2013-06-25
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Pyroptosis a type of inflammatory programed cell death that is coordinated by inflammasome-mediated caspase-1 activation (JCB (2011) 195: 931-942)
a defining feature of pyroptosis
- activation of caspase-1 by inflammasome
- maturation and releasing of cytokines (e.g IL-1beta and IL-18)
- plasma membrane rupture
- DNA fragmentation
Nature Reviews Microbiology (2009) 9: 99-109
In vitro study of pyroptosis with BMDMs stimulated by LPS and ATP
(Comparison of WT to Nlrp3 -/- BMDM)
LPS (10ng/ml, 6hrs)
ATP (2mM, 15 and 30 mins)
Ctrl LPS
LPS+ATP
15 30
C57 wt BMDM
Ctrl
Nlrp3 -/- BMDM
LPS
LPS+ATP
15 30
B-Actin 40
Caspase-7
(cleaved) 20
Caspase-7
(total)
35
Caspase-1
(p10) 10
Caspase-1
(total) 45
NLRP3 110
LPS (10ng/ml, 6hrs)
ATP (4mM, 30 mins)
C57 wt BMDM LPS + ATP C57 wt BMDM Ctrl
Nlrp3 -/- BMBM LPS + ATP Nlrp3 -/- BMBM Ctrl
B A
2013-06-25
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TLR
Microbial molecules
(PAMPs)
Gene transcription
(NF-B)Pro IL-1, pro IL-18
Maturation of
IL-1, IL-18
Cleaved caspase-1
ASCNLP3
Pro caspase-1
P2X7 receptor
Inflammasome
ATP
secretion
Cytosol
Extracellular
Fig. A
TLR
Microbial molecules
(PAMPs)
Gene transcription
(NF-B)Pro IL-1, pro IL-18
Maturation of
IL-1, IL-18
Cleaved caspase-1
ASCNLP3
Pro caspase-1
P2X7 receptor
Inflammasome
ATP
secretion
Cytosol
Extracellular
Fig. A
??
Nat Immunol, 2011
2013-06-25
9
mtDNA Transfection Induces inflammasome cytokines in macrophages
mtDNA (mg/ml) 0 1.0 0 0.2 1.0 0 1.0 0 0.2 1.0 0 1.0 0 0.2 1.0
0
100
200
300
400
500
600
700
800
0
50
100
150
200
250
300
350
400
450
0
1000
2000
3000
4000
5000
6000
7000
IL-1
(pg/m
l)
TN
F-a
(pg/m
l)
IL-1
8 (
pg/m
l)
LPS - - + + + +
- DNase
+ DNase
- - + + + +
liposomes liposomes liposomes
- - + + + +
*
*
*
*
Nakahira, K. et al.
mtDNA Transfection Induces IL-1 and Caspase-1 Activation in Macrophages
2013-06-25
10
0
50
100
150
200
250
300
IL-1
(pg/m
l)
LPS - + + + +
mtDNA (mg/ml) 0 0 0.2 1.0 Poly(dA:dT)
Pro caspase-1
Cleaved caspase-1
LPS + + - + + + +
mtDNA (mg/ml) 0 0 0 0 0.2 1.0 Poly(dA:dT)
ATP + + - - - - -
J774A.1 r0 J774A.1
r0 J774A.1
*
*
Nakahira, K. et al.
mtDNA Transfection can restore caspase-1 in in r0 Macrophages
0
100
200
300
400
500
600
700
0
20
40
60
80
100
120
140
160
0
500
1000
1500
2000
2500
3000
3500
4000
4500
IL-1
8 (
ng/m
l)
IL-1
(pg/m
l)
TN
F-a
(pg/m
l)
LPS/ATP - + + +
Mito-TEMPO (mM) 0 0 100 500
- + + +
0 0 100 500
- + + +
0 0 100 500
*
*
*
*
Nakahira, K. et al.
Scavenging mitchondrial ROS attenuates inflammasome cytokines
2013-06-25
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0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Rela
tive m
tDN
A c
opy n
um
ber
in c
yto
sol
(Fold
incre
ase)
LPS/ATP - - + +
0
100
200
300
400
IL-1
(pg/m
l)
LPS/ATP - - + +
Mito-TEMPO - + - + Mito-TEMPO - + - +
Nakahira, K. et al.
*
*
Scavenging mitchondrial ROS attenuates mt DNA
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2013-06-25
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0
2000
4000
6000
8000
10000
mtNd1 DNA copy number in plasma
Co
py /m
l p
lasm
a
gp91
ALI/ARDS
COPD? Inflammasome
Translational research
ROS
LPS
Cleaved Caspase-1
IL-1, IL-18
P2X7 receptor
Inflammation
ALI/ARDS
Pro caspase-1
NLP3 ASC
Inflammasome
ATP
Mechanical
Ventilation
Mitochondrial
dysfunction
Lung injury & Inflammation
in vivo & in vitro
Mechanical
Ventilation Therapeutic potential
of P2X7 or IL-18
receptor inhibitor
Human ALI/ARDS
COPD
Human biomarker study
NADPH
oxidase p47
ROS Inflammasomes and
regulated cytokines
mtDNA
Human ALI/ARDS
2013-06-25
15
TEAM
Jeffrey A. Haspel, MD, PhD
Stefan W. Ryter, PhD
Kiichi Nakahira, MD, PhD
Seon Min Lee, PhD
Suzanne Cloonan, PhD
Kenji Mizumura, MD, PhD
Jong Seok Moon, PhD
Emeka Ifedidgo
BWH MICU RoCI