extracellular histones are major mediators of inflammation and coagulation contributing directly to...
TRANSCRIPT
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
• 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
Cell death
Extracellular Traps release
Bactericidal activity
Cytotoxicity
Mediators of injury
Extracellular histones
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
Cell death
Extracellular Traps release
Bactericidal activity
Cytotoxicity
Mediators of injury
Extracellular histones
Platelet activation, intravascular and intra-alveolar fibrin deposition in the lungs of
histone-challenged mice
FibrinAnti mouse fibrinogen
immunostaining
Platelets Gp-III immunostaining
Intra-alveolar fibrinPhosphotungstic acid staining Fibrin
Anti mouse fibrinogen Immunostaining
Platelets Gp-III immunostaining
Intra-alveolar fibrin Phosphotungstic acid staining
Neutrophil infiltration in the lung of mice challenged with histones vs. controls
Control Histone treated
Immunostaining for neutrophil elastase
Control Histone treated
Immunostaining for neutrophil elastase
FlagellinHKLM
Poly(I:C)
LPS
Loxoribine
CL075
ODN2006
TNFα
Histones
Toll-like receptor screening identified TLR2 and TLR4 as functional receptors for histones
hTLR2
hTLR3
hTLR4(MD2-CD14)
hTLR5
hTLR7
hTLR8
hTLR9
NF-κB Control C
ells
1.000
0.000
0.500
1.500
2.000
2.500
3.500
3.000
No Ligand
HistonesControl +
293/TLR cells
TLR Ligand Screening
SE
AP
Act
ivit
y O
D65
0nm
Histone infusion elevates cytokine levels in wild type but not TLR4 null mice
Histones Increase Thrombin Generation in Human PRP
Thr
ombi
n (n
mol
L-1)
Thr
ombi
n (n
mol
L-1)
Tim
e (m
in)
Thr
ombi
n (n
mol
L-1)
Thr
ombi
n (n
mol
L-1) 140
0
D
80
60
40
20
100
120
140
0
0
20
40
60
80
100
120
140
160
0
100
120
20
806040
140160180
120100806040200
Time (min)60 80 1000 20 40
Time (min)
Throm
bin nmol m
in
2000
1500
1000
500
040 80 1200 160
Histones (g mL-1)
0
20
40
60
80
100
>180
40 80 1200 160
Histones(g mL-1)
0 20 40 60 80 100 120Time (min)
50
100
150
200
250
300
0
LagtimettPeak
PeakETP
H1
H3
Control
H2AH2B
H4
Histones g mL-1
20
0
40
510
80160
20
0
40
510
80160
Histones g mL-1
Histones increase thrombin generation in the presence of TM
Control
Histones
Histones + Psp
Control
PolyP
Histones
Histones + PolyP
Control
Histones
PolyP
Histones + PolyP
40
20
60
80
100
0
Thr
ombi
n (n
mol
L-1)
Time (min) Time (min) Time (min)
0
20
40
60
80
100
120
140
Thr
ombi
n (n
mol
L-1)
0
20
40
60
80
100
120
140
160
Thr
ombi
n (n
mol
L-1)
0 20 40 10060 80 120 140 160 180 0 20 40 10060 80 120 140 160 1800 20 40 10060 80 120 140 160 180
PolyP Drives Thrombin Generation in Histone-Treated PRP
Damage-associate molecular pattern molecules-DAMPS that Trigger Coagulation and Inflammation
RNA
DNA
Polyphosphate
Histones
HMGB1
0 1 2 3 4 5 6 70%
20%
40%
60%
80%
100%
120%
Lps+1754Lps+1947Lps+1753Lps+2055Lps+1989
Anti-Histone or Anti-polyP Antibodies Protect Against LPS Toxicity
Days
% S
urvi
val
Anti Polyphosphate Antibody Inhibits Polyphosphate Driven Coagulation
PP2055
(50
ug/mL)
No Poly
P
Anti PolyP Antibody Protects Mice from LPS
Control
Anti PolyP
n = 7
n = 6
Days
% S
urvi
val
Suppression of Coagulation with Anti PolyP Antibody
LPS+Anti PPLPS+IgG2a 1753 Control
72±29
TAT
Leve
ls
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
sera from trauma patients can kill endothelium
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
Jun Xu Florea Lupu Fabrizio Semeraro, Tiziana Ammollo Jim Morrissey Mitch Cohen