allan tsung, md department of surgery university of pittsburgh
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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
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