Transcript Original goal of the research was to define why blood was

Extracellular Histones are Major
Mediators of Inflammation and
Coagulation Contributing Directly
to Organ Injury and Death
 Charles Esmon
 Member, OMRF
AAST September 10, 2014
UM1HL120877
COLLABORATORS
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Jim Morrissey
Mitch Cohen
Kaleb Freeman
Hunter Moore
Saulius Butenas
Ken Mann
Jeff Shupp (outside TACTIC)
AIMS
• To develop reagents to block the
toxic effects of histones and
polyphosphate
• To test these reagents in animal
models of trauma
• To assay histone and polyphosphate
levels in trauma patients and
determine the natural history of the
response
Bactericidal activity
Extracellular Traps release
Cytotoxicity
Mediators of injury
Cell death
In sepsis and trauma, circulating nucleosomal
DNA is positively correlated with disease
severity and adverse outcome

Zeerleder S, Zwart B, Wuillemin WA, Aarden LA,
Groeneveld AB, Caliezi C, van Nieuwenhuijze AE, van
Mierlo GJ, Eerenberg AJ, Lämmle B, Hack CE:
Elevated nucleosome levels in systemic inflammation
and sepsis. Crit Care Med 2003; 31:1947-1951.

Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA:
Plasma DNA as a prognostic marker in trauma
patients. Clin Chem 2000; 46:319-323.
BLOCKING HISTONE 4 PROTECTS
AGAINST LPS SEPTIC SHOCK
Bactericidal activity
Extracellular Traps release
Cytotoxicity
Mediators of injury
Cell death
Platelet activation, intravascular and intraalveolar fibrin deposition in the lungs of
histone-challenged mice
Fibrin
Fibrin
Anti
mouse
fibrinogen
Anti mouse fibrinogen
immunostaining
Immunostaining
Platelets
Platelets
Gp-III immunostaining
Intra-alveolar fibrin
Intra-alveolar
fibrin
Phosphotungstic
acid
staining
Phosphotungstic acid staining
Neutrophil infiltration in the lung of mice
challenged with histones vs. controls
Control
Control
Histone treated
Histone treated
Immunostaining for neutrophil elastase
Immunostaining
for neutrophil elastase

Histone infusion elevates cytokine levels in wild
type but not TLR4 null mice
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Toll-like receptor screening identified TLR2 and
TLR4 as functional receptors for histones
SEAP Activity OD650nm
TLR Ligand Screening
Poly(I:C)
Flagellin
HKLM
LPS
CL075
TNFα
Loxoribine
ODN2006
Control +
Histones
Histones
No Ligand
293/TLR cells
Histones g mL-1
0
5
10
20
40
80
160
180
160
140
120
100
80
60
40
20
0
0
20
40
60
80
Thrombin (nmol L-1)
Thrombin (nmol L-1)
Histones Increase Thrombin Generation in Human PRP
Histones g mL-1
0
5
10
20
40
80
160
160
140
120
100
80
60
40
20
0
100
0
20
Time (min)
Thrombin (nmol L-1)
80
60
40
20
0
80
120
80
100 120
Peak 2000
ETP
300
250
1500
200
1000
150
100
500
50
0
160
0
Histones (g mL-1)
40
Control
H1
H2A
H2B
H3
H4
120
100
80
60
40
20
00
0
80
120
Histones(g mL-1)
D
140
140
Thrombin (nmol L-1)
Time (min)
100
40
60
20
40
60
80
Time (min)
100 120
160
0
Thrombin nmol min
Lagtime
ttPeak
>180
0
40
Time (min)
Histones increase thrombin generation
in the presence of TM
PolyP Drives Thrombin Generation in
Histone-Treated PRP
80
60
40
20
0
Control
140
120
PolyP
Histones
100
Histones + PolyP
80
60
40
20
20
40 60
80 100 120 140 160 180
Time (min)
140
120
100
80
60
40
20
0
0
0
Control
PolyP
Histones
Histones + PolyP
160
Thrombin (nmol L-1)
Control
Histones
Histones + Psp
Thrombin (nmol L-1)
Thrombin (nmol L-1)
100
0
20
40
60
80 100 120 140 160 180
Time (min)
0
20 40 60
80 100 120 140 160 180
Time (min)
Damage-associate molecular pattern moleculesDAMPS that Trigger Coagulation and Inflammation
 RNA
 DNA
 Polyphosphate
 Histones
 HMGB1
Anti-Histone or Anti-polyP Antibodies
Protect Against LPS Toxicity
120%
100%
% Survival
80%
Lps+1754
Lps+1947
60%
Lps+1753
Lps+2055
Lps+1989
40%
20%
0%
0
1
2
3
4
Days
5
6
7
Anti Polyphosphate Antibody Inhibits
Polyphosphate Driven Coagulation
Anti PolyP Antibody Protects Mice from LPS
% Survival
n=7
Control
Anti PolyP
n=6
Days
TAT Levels
Suppression of Coagulation with Anti PolyP Antibody
72±29
LPS+IgG2a 1753 Control
LPS+Anti PP
Suppression of Coagulation with Anti Histone Antibodies
Control
Anti H4 TAT Levels
Anti H3 TAT Levels
We showed that

there are exceedingly high levels of nucleosomes
in trauma patients

histones kill endothelium
Chen-Hoc Toh’s laboratory demonstrated that
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sera from trauma patients can kill endothelium
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histones contribute to trauma induced lung injury
in mice. (Abrams et al: Circulating histones are
mediators of trauma-associated lung injury.
Am J Respir Crit Care Med 187(2):160-9, 2013.)
Median 0- and 6-hour histone levels by injury severity. *p <0.05 between
categories by Mann-Whitney U-test; †p <0.05 between time points by paired
signed rank test.
Admission international normalized ratio ([INR] A) and partial thromboplastin
times ([PTT] B) by admission histone level. *p <0.05 by Wilcoxon rank-sum
testing.
Aknowledgements


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
Jun Xu
Florea Lupu
Fabrizio Semeraro, Tiziana Ammollo
Jim Morrissey
Mitch Cohen