2013-06-25

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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

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AJRCCM, 2012

IL-18 in the Plasma Correlates with Patient Survival Outcomes

AJRCCM, 2012

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IL-18 KO mice are more resistant to VILI

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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

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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

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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

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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

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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

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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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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