allan tsung, md department of surgery university of pittsburgh
TRANSCRIPT
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DAMPS in Ischemic Liver Injury
Allan Tsung, MDDepartment of Surgery
University of Pittsburgh
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Our Immune System at Work
Organ Dysfunction
Infection
Injury
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Immune Activation
Organ Dysfunction
Systemic Inflammatory Response
Infection
Injury
PAMPs+ Receptors (e.g. TLRs)
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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.
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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
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DAMP-TLR Interactions: DAMP Molecular Classification
Piccinini & Midwood, 2010
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Extracellular Functions of HMGB1
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Hepatocyte
Ischemia-Reperfusion
TLR4
HDACsHATs
HMGB1 Acetyl-HMGB1
Kupffer Cellor
Dendritic Cell
PMNs
Inflammatory cell activation, differentiation,and infiltration
ROSCa+2
CaMKs
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Nuclear DAMPs
http://www.fastbleep.com/biology-notes/40/116/1191
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QuestionWhat is the role of histones in liver I/R injury?
Clamp
Ischemia time
Reperfusion time
Clamp removed
1hr
6hr
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* 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
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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
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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
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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
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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
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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
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* 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%
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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
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Question
Which pattern recognition receptor is involved in histone signaling pathway in liver I/R?
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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
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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
*
**
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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
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Question
Besides MAPK and NF-κB mediated cytokine production, do histones activate other inflammatory signaling pathway during liver I/R injury?
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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
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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
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• Do histones activate the NLRP3 inflammasome in liver I/R?
Question
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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
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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
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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.
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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
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• 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)
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Chimeric mice
Steiner AA, et al. Blood 2005
Caspase-1 KOCaspase-1 WT
WT/WT WT/KO KO/WT KO/KO
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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
**
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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
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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
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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