DAMPS in Ischemic Liver Injury

Allan Tsung, MDDepartment of Surgery

University of Pittsburgh

Our Immune System at Work

Organ Dysfunction

Infection

Injury

Immune Activation

Organ Dysfunction

Systemic Inflammatory Response

Infection

Injury

PAMPs+ Receptors (e.g. TLRs)

Classes of Molecules That Initiate The Innate Immune Response

Damage-associated Molecular Patterns (DAMPs):

Endogenous molecules that are normally unavailable to the immune system that are released and recognized by immune cells following tissue injury.

Pathogen-associated Molecular Patterns (PAMPs):

Exogenous molecules expressed or released by invading microorgansims that are structurally unique to the pathogen.

Sources of Endogenous Danger Signals(Damage Associate Molecular Pattern (DAMP) Molecules)

Damaged or Dying Cells

Secreted From Stressed Cells

Degradation of Tissue Matrix

DAMPs

Pattern Recognition

Receptor

PMN

Protease

DAMP-TLR Interactions: DAMP Molecular Classification

Piccinini & Midwood, 2010

Extracellular Functions of HMGB1

Hepatocyte

Ischemia-Reperfusion

TLR4

HDACsHATs

HMGB1 Acetyl-HMGB1

Kupffer Cellor

Dendritic Cell

PMNs

Inflammatory cell activation, differentiation,and infiltration

ROSCa+2

CaMKs

Nuclear DAMPs

http://www.fastbleep.com/biology-notes/40/116/1191

QuestionWhat is the role of histones in liver I/R injury?

Clamp

Ischemia time

Reperfusion time

Clamp removed

1hr

6hr

* P<0.05

Sham 1h 3h 6h0

50

100

150

200

250

300

350

400

450

500

pg

/L

**

*

Histones are released from the liver after hepatic I/R injury

Histones are released from the liver after hepatic I/R injury

Sham 40×

Liver I/R 40×

Sham 40×

Liver I/R 40×

Histone-redNuclei-blueActin-green

Histones are released from hepatocytes after hypoxia in vitro

0 1 3 6 12 24 48

Hypoxia (1%)

H3

H4

17kDa

11kDa

Histone H3Hypoxia

Histone H3Normoxia

Histone H4Hypoxia

Histone H4Normoxia

Histone-greenNuclei-blueActin-red

Sham I/R0

500

1000

1500

2000

2500

3000

IgG

Anti-H3

Anti-H4

sA

LT

(IU

/L)

Neutralizing histones is protective in hepatic I/R injury

**

* P<0.05

Sham IgG

Anti-H4Anti-H3

18.7±4.5%

5.8±2.6%6.7±2.1%

Neutralizing histones is protective in hepatic I/R injury

sham IgG Anti-H3 Anti-H40

2

4

6

8

10

12

IL-6

mR

NA

fo

ld in

cre

as

e

sham IgG Anti-H3 Anti-H40

2

4

6

8

TNF-α

mR

NA

fo

ld in

cre

as

e

** *

*

* P<0.05

Neutralizing histones is protective in hepatic I/R injury

* P<0.05

Exogenous histones exacerbate hepatic I/R injury

sham 1h 3h 6h0

1000

2000

3000

4000

5000

6000

Histone

PBS

sA

LT

(IU

/L)

*

*

*

PBS

Histone

13±7%

50±10%

Histones modulate inflammatory signaling pathways.

P-p38

Total-p38

ShamI/R

PBSI/R

Histone

P-JNK

Total-JNK

P-ERK

Total-ERK

sham liver I/R (6h)0

3

6

9

12

15

18

TNF-α

Histone

PBS

mR

NA

fo

ld in

cre

as

e

sham Liver I/R (6h)0

5

10

15

20

25

30

IL-6

HistonePBS

mR

NA

fo

ld in

cre

as

e

Question

Which pattern recognition receptor is involved in histone signaling pathway in liver I/R?

Toll-like receptor 9 (TLR9)

recognizes both bacterial DNA rich in unmethylated CpG motifs and endogenous DNA from mammalian

cellsTian J et al. Nat Immunol.2007

Klinman DM. Nat Rev Immunol.2004

Histones mediate hepatic I/R injury through TLR9

WT

TLR9 -/-

WT

TLR9 -/-

sham I/R

0

1000

2000

3000

4000

5000

6000PBS

Histone

sA

LT

(IU

/L)

WT

TLR9 -/-

WT

TLR9 -/-

Sham I/R

0

1000

2000

3000

4000

5000

6000

7000

IgG

Anti-H3

Anti-H4

sA

LT

(IU

/L)

* P<0.05

*

**

Histone-mediated Inflammatory Signaling: Proposed Mechanism

Extracellular

Intracellular

NPC

Ischemichepatocyte

Endosome

TLR9

MyD88IRAK

TRAF6

P

JNK/P38/ERK

AP1

P

IRF7 IKK complex

P

IκBNFκB

Pro-inflammatory gene expression

Histones

Huang H et al.Hepatology 2011

Question

Besides MAPK and NF-κB mediated cytokine production, do histones activate other inflammatory signaling pathway during liver I/R injury?

Inflammasome

Davis BK. et al. Annu Rev Immunol 2011

• Inflammasome is a cytosolic multi-protein complex

• The sensor protein (NLRP3)• The adaptor protein ASC• The inflammatory protease caspase-1

• Activated form senses a diverse range of microbial and non-microbial cellular stress and damages

• Platform of activating caspase-1, cleaves the biologically inactive precursors of IL-1β and IL-18

Genetic Deletion of nlrp3 (NLRP3 KO) or caspase-1 (Caspase-1 KO) Is Protective in Liver I/R Injury

NALP3 Caspase-10

500

1000

1500

2000

2500

3000

3500

Serum ALT (IU/L)

WT

KO

IU/L

*

*

* P<0.05

• Do histones activate the NLRP3 inflammasome in liver I/R?

Question

Extracellular Histones Activate the Inflammasome during liver I/R.

Histones

Sham

Histo

nes

PBS

I/R

Casp-1p20

Cleaved IL-1β

Cleaved IL-18

β-actin

Inhibition of Histones Decreases the Activation of the Inflammasome

Anti-

histo

ne H3

Anti-

histo

ne H4

Casp-1p20

Cleaved IL-1β

Cleaved IL-18

IgG

Sham

I/R

β-actin

Liver I/R

Anti-histones Ab

Histone Mediated Liver I/R Injury is Dependent on the NLRP3 Inflammasome.

WT NLRP3 KO0

2000

4000

6000

Serum ALT (IU/L)

PBSHistones

IU/L

WT NLRP3 KO0

20

40

60

80

100

120

Serum IL-1β

PBS

Histones

pg

/mL

N.S. N.S.

Histones Activate the Inflammasome through TLR9 signaling

sham Liver I/RWT TLR9 KO

WT TLR9 KOPBS Histones PBS Histones

Casp-1 p20

Cleaved IL-1β

Cleaved IL-18

β-actin

Histones

TLR9

• What liver cell types mediate histone activation of the Inflammasome in liver I/R?

Question

Parenchymal cells (e.g. Hepatocytes)

Bone-marrow derived cells(e.g. Kupffer cell, Neutrophils, Dendritic cell)

Chimeric mice

Steiner AA, et al. Blood 2005

Caspase-1 KOCaspase-1 WT

WT/WT WT/KO KO/WT KO/KO

Bone marrow derived cells from caspase-1 KO mice confer protection after liver I/R

WT/WT WT/KO KO/KO KO/WT0

2000

4000

6000

8000

10000

sALT (IU/L)

IU/L

WT/W

T

KO/WT

WT/K

O

KO/KO

Cleaved IL-1β

Cleaved IL-18

β-actin

**

The Inflammasome in Kupffer Cells is Activated by Histones

Histones (μg/mL)

0 5 25 50

Casp-1 p20

β-actin

Cleaved IL-1β

Cleaved IL-18

PB

SH

isto

ne

s

MergeActivated caspase-1

+Actin

Kupffer cells

NLRP3

ASCTXNIP

TXNIPTRX

TLR9

Histones

MyD88

Endosome

JNKP

ROS

NF-κBPro-IL-1βTranscription

Pro-caspase-1

Activated-caspase-1

IL-1β

Ischemic Hepatocytes

Pro-IL-18

IL-18Innate immune cells recruitment

Dendritic cells

Inflammatory monocytes

Neutrophils

IL-6TNF-α

Huang H et al.Journal of Immunology 2013

Histone-mediated Inflammatory Signaling: Proposed Mechanism

Acknowledgement

Hai Huang

Doris Chen

John Evankovich

Gary Nace

Timothy Billiar

Charles Esmon

Donna Stolz

Xinhua Liao

Nicole Hays

Funding NIH

HHMISociety of University Surgeons