Extracellular Histones are Major Mediators of Inflammation and Coagulation Contributing Directly to Organ Injury and Death  Charles Esmon  Member, OMRF AAST September 10, 2014 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

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:  Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA: Plasma DNA as a prognostic marker in trauma patients. Clin Chem 2000; 46:

BLOCKING HISTONE 4 PROTECTS BLOCKING HISTONE 4 PROTECTS AGAINST LPS SEPTIC SHOCK AGAINST LPS SEPTIC SHOCK

Cell death Extracellular Traps release Bactericidal activity Cytotoxicity Mediators of injury

Platelet activation, intravascular and intra- alveolar fibrin deposition in the lungs of histone-challenged mice Fibrin Anti mouse fibrinogen immunostaining Platelets Gp-III immunostaining Intra-alveolar fibrin Phosphotungstic acid staining Fibrin Fibrin Anti mouse fibrinogen Anti mouse fibrinogen Immunostaining Immunostaining Platelets Platelets Gp-III immunostaining 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 ControlHistone treated Immunostaining for neutrophil elastase Control Histone treated Immunostaining for neutrophil elastase

Flagellin HKLM Poly(I:C) LPS Loxoribine CL075 ODN2006 TNF α  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 Cells No Ligand Histones Control + 293/TLR cells TLR Ligand Screening SEAP Activity OD650nm  Histone infusion elevates cytokine levels in wild type but not TLR4 null mice

Histones Increase Thrombin Generation in Human PRP Thrombin (nmol L -1 ) Time (min) Thrombin (nmol L -1 ) D Time (min) Time (min) Thrombin nmol min Histones (  g mL -1 ) > Histones(  g mL -1 ) Time (min) Lagtime ttPeak Peak ETP H1 H3 Control H2A H2B H4 Histones  g mL 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 Thrombin (nmol L -1 ) Time (min) Thrombin (nmol L -1 ) Thrombin (nmol L -1 ) PolyP Drives Thrombin Generation in Histone-Treated PRP

Damage-associate molecular pattern molecules- DAMPS that Trigger Coagulation and Inflammation  RNA  DNA  Polyphosphate  Histones  HMGB1

Anti-Histone or Anti-polyP Antibodies Protect Against LPS Toxicity Days % Survival

Anti Polyphosphate Antibody Inhibits Polyphosphate Driven Coagulation PP2055 (50 ug/mL) No PolyP

Anti PolyP Antibody Protects Mice from LPS Control Anti PolyP n = 7 n = 6 Days % Survival

Suppression of Coagulation with Anti PolyP Antibody LPS+Anti PP LPS+IgG2a 1753 Control 72±29 TAT Levels

Suppression of Coagulation with Anti Histone Antibodies Control Anti H4 TAT LevelsAnti 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