Allan Tsung, MD Department 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 HDACs HATs HMGB1 Acetyl-HMGB1 Kupffer Cell or Dendritic Cell PMNs Inflammatory cell activation, differentiation, and infiltration ROS Ca + 2 CaMKs

What is the role of histones in liver I/R injury? ClampClamp Ischemia time Reperfusion time Clamp removed removedClamp 1hr1hr 6hr6hr

* P<0.05 * * *

Sham 40× Liver I/R 40× Sham 40× Liver I/R 40× Histone-red Nuclei-blue Actin-green

Hypoxia (1%) H3 H4 17kDa 11kDa Histone H3 Hypoxia Histone H3 Normoxia Histone H4 Hypoxia Histone H4 Normoxia Histone-green Nuclei-blue Actin-red

* * * P<0.05

Sham IgG Anti-H4 Anti-H3 18.7±4.5% 5.8±2.6% 6.7±2.1%

* * * * * P<0.05

* * * PBS Histone 13±7% 50±10%

P-p38 Total-p38 Sham I/R PBS I/R Histone P-JNK Total-JNK P-ERK Total-ERK

Which pattern recognition receptor is involved in histone signaling pathway in liver I/R?

recognizes both bacterial DNA rich in unmethylated CpG motifs and endogenous DNA from mammalian cells Tian J et al. Nat Immunol.2007 Klinman DM. Nat Rev Immunol.2004

* P<0.05 * * *

Extracellular Intracellular NPC Ischemic hepatocyte Endosome TLR9 MyD88 IRAK TRAF6 P JNK/P38/ERK AP1 P IRF7 IKK complex P IκBIκBNFκB Pro-inflammatory gene expression Histones Huang H et al. Hepatology 2011

Besides MAPK and NF-κB mediated cytokine production, do histones activate other inflammatory signaling pathway during liver I/R injury?

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

* * * P<0.05

Do histones activate the NLRP3 inflammasome in liver I/R? Do histones activate the NLRP3 inflammasome in liver I/R?

Histones Sham Histones PBS I/R Casp-1p20 Cleaved IL-1β Cleaved IL-18 β-actin

Anti- histone H3 Anti- histone H4 Casp-1p20 Cleaved IL-1β Cleaved IL-18 IgG Sham I/R β-actin Liver I/R Anti-histones Ab

N.S.

shamLiver I/R WT TLR9 KO WTTLR9 KO PBSHistones 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? What liver cell types mediate histone activation of the Inflammasome in liver I/R? Parenchymal cells (e.g. Hepatocytes) Bone-marrow derived cells (e.g. Kupffer cell, Neutrophils, Dendritic cell)

Steiner AA, et al. Blood 2005 Caspase-1 KO Caspase-1 WT WT/WT WT/KOKO/WT KO/KO

WT/WT KO/WT WT/KO KO/KO Cleaved IL-1β Cleaved IL-18 β-actin * *

Histones (μg/mL) Casp-1 p20 β-actin Cleaved IL-1β Cleaved IL-18 PBS Histones Merge Activated caspase-1 +Actin

Kupffer cells NLRP3 ASC TXNIP TRX TLR9 Histones MyD88 Endosome JNK P ROS NF-κB Pro-IL-1β Transcription Pro- caspase-1 Activated- caspase-1 IL-1β Ischemic Hepatocytes Pro-IL-18 IL-18 Innate immune cells recruitment Dendritic cells Inflammatory monocytes Neutrophils IL-6 TNF-α Huang H et al. Journal of Immunology 2013

Hai Huang Doris Chen John Evankovich Gary Nace Timothy Billiar Charles Esmon Donna Stolz Xinhua Liao Nicole Hays NIHHHMI Society of University Surgeons