John R. Klune

HMGB1 release induced by liver ischemia involves Toll-like receptor 4–dependent reactive oxygen species production and calcium-mediated signaling

Ischemic tissues require mechanisms to alert the immune system of impending cell damage. The nuclear protein high-mobility group box 1 (HMGB1) can activate inflammatory pathways when released from ischemic cells. We elucidate the mechanism by which HMGB1, one of the key alarm molecules released during liver ischemia/reperfusion (I/R), is mobilized in response to hypoxia. HMGB1 release from cultured hepatocytes was found to be an active process regulated by reactive oxygen species (ROS). Optimal production of ROS and subsequent HMGB1 release by hypoxic hepatocytes required intact Toll-like receptor (TLR) 4 signaling. To elucidate the downstream signaling pathways involved in hypoxia-induced HMGB1 release from hepatocytes, we examined the role of calcium signaling in this process. HMGB1 release induced by oxidative stress was markedly reduced by inhibition of calcium/calmodulin-dependent kinases (CaMKs), a family of proteins involved in a wide range of calcium-linked signaling events. In addition, CaMK inhibition substantially decreased liver damage after I/R and resulted in accumulation of HMGB1 in the cytoplasm of hepatocytes. Collectively, these results demonstrate that hypoxia-induced HMGB1 release by hepatocytes is an active, regulated process that occurs through a mechanism promoted by TLR4-dependent ROS production and downstream CaMK-mediated signaling.

High Mobility Group Box 1 Release from Hepatocytes during Ischemia and Reperfusion Injury Is Mediated by Decreased Histone Deacetylase Activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.

Noninvasive radiofrequency ablation of cancer targeted by gold nanoparticles

INTRODUCTION Current radiofrequency ablation (RFA) techniques require invasive needle placement and are limited by accuracy of targeting. The purpose of this study was to test a novel non invasive radiowave machine that uses RF energy to thermally destroy tissue. Gold nanoparticles were designed and produced to facilitate tissue heating by the radiowaves. METHODS A solid state radiowave machine consisting of a power generator and transmitting/receiving couplers which transmit radiowaves at 13.56 MHz was used. Gold nanoparticles were produced by citrate reduction and exposed to the RF field either in solutions testing or after incubation with HepG2 cells. A rat hepatoma model using JM-1 cells and Fisher rats was employed using direct injection of nanoparticles into the tumor to focus the radiowaves for select heating. Temperatures were measured using a fiber-optic thermometer for real-time data. RESULTS Solutions containing gold nanoparticles heated in a time- and power-dependent manner. HepG2 liver cancer cells cultured in the presence of gold nanoparticles achieved adequate heating to cause cell death upon exposure to the RF field with no cytotoxicity attributable to the gold nanoparticles themselves. In vivo rat exposures at 35 W using direct gold nanoparticle injections resulted in significant temperature increases and thermal injury at subcutaneous injection sites as compared to vehicle (water) injected controls. DISCUSSION These data show that non invasive radiowave thermal ablation of cancer cells is feasible when facilitated by gold nanoparticles. Future studies will focus on tumor selective targeting of nanoparticles for in vivo tumor destruction.

Increasing numbers of hepatic dendritic cells promote HMGB1-mediated ischemia-reperfusion injury

Endogenous ligands released from damaged cells, so‐called damage‐associated molecular pattern molecules (DAMPs), activate innate signaling pathways including the TLRs. We have shown that hepatic, warm ischemia and reperfusion (I/R) injury, generating local, noninfectious DAMPs, promotes inflammation, which is largely TLR4‐dependent. Here, we demonstrate that increasing dendritic cell (DC) numbers enhance inflammation and organ injury after hepatic I/R. High‐mobility group box 1 (HMGB1), a NF released by necrotic cells or secreted by stimulated cells, is one of a number of ligands promoting TLR4 reactivity. Augmentation of DC numbers in the liver with GM‐CSF hydrodynamic transfection significantly increased liver damage after I/R when compared with controls. TLR4 engagement on hepatic DC was required for the I/R‐induced injury, as augmentation of DC numbers in TLR4 mutant (C3H/HeJ) mice did not worsen hepatic damage. It is interesting that TLR4 expression was increased in hepatic DC following HMGB1 stimulation in vitro, suggesting a mechanism for the increased liver injury following I/R. It thus appears that functional TLR4 on DC is required for I/R‐induced injury. Furthermore, HMGB1 may direct the inflammatory responses mediated by DC, at least in part, by enhancing TLR4 expression and reactivity to it and other DAMPs.

Hepatocyte-specific high-mobility group box 1 deletion worsens the injury in liver ischemia/reperfusion: a role for intracellular high-mobility group box 1 in cellular protection.

High‐mobility group box 1 (HMGB1) is an abundant chromatin‐associated nuclear protein and released into the extracellular milieu during liver ischemia‐reperfusion (I/R), signaling activation of proinflammatory cascades. Because the intracellular function of HMGB1 during sterile inflammation of I/R is currently unknown, we sought to determine the role of intracellular HMGB1 in hepatocytes after liver I/R. When hepatocyte‐specific HMGB1 knockout (HMGB1‐HC‐KO) and control mice were subjected to a nonlethal warm liver I/R, it was found that HMGB1‐HC‐KO mice had significantly greater hepatocellular injury after I/R, compared to control mice. Additionally, there was significantly greater DNA damage and decreased chromatin accessibility to repair with lack of HMGB1. Furthermore, lack of hepatocyte HMGB1 led to excessive poly(ADP‐ribose)polymerase 1 activation, exhausting nicotinamide adenine dinucleotide and adenosine triphosphate stores, exacerbating mitochondrial instability and damage, and, consequently, leading to increased cell death. We found that this was also associated with significantly more oxidative stress (OS) in HMGB1‐HC‐KO mice, compared to control. Increased nuclear instability led to a resultant increase in the release of histones with subsequently more inflammatory cytokine production and organ damage through activation of Toll‐like receptor 9. Conclusion: The lack of HMGB1 within hepatocytes leads to increased susceptibility to cellular death after OS conditions. (Hepatology 2014;59:1984–1997)

Cellular-specific role of toll-like receptor 4 in hepatic ischemia-reperfusion injury in mice.

Ischemia‐reperfusion (I/R) injury is a process whereby an initial hypoxic insult and subsequent return of blood flow leads to the propagation of innate immune responses and organ injury. The necessity of the pattern recognition receptor, Toll‐like receptor (TLR)4, for this innate immune response has been previously shown. However, TLR4 is present on various cell types of the liver, both immune and nonimmune cells. Therefore, we sought to determine the role of TLR4 in individual cell populations, specifically, parenchymal hepatocytes (HCs), myeloid cells, including Kupffer cells, and dendritic cells (DCs) subsequent to hepatic I/R. When HC‐specific (Alb‐TLR4−/−) and myeloid‐cell–specific (Lyz‐TLR4−/−) TLR4 knockout (KO) mice were subjected to warm hepatic ischemia, there was significant protection in these mice, compared to wild type (WT). However, the protection afforded in these two strains was significantly less than global TLR4 KO (TLR4−/−) mice. DC‐specific TLR4−/− (CD11c‐TLR4−/−) mice had significantly increased hepatocellular damage, compared to WT mice. Circulating levels of high‐mobility group box 1 (HMGB1) were significantly reduced in Alb‐TLR4−/− mice, compared to WT, Lyz‐TLR4−/−, CD11c‐TLR4−/− mice and equivalent to global TLR4−/− mice, suggesting that TLR4‐mediated HMGB1 release from HCs may be a source of HMGB1 after I/R. HCs exposed to hypoxia responded by rapidly phosphorylating the mitogen‐activated protein kinases, c‐Jun‐N‐terminal kinase (JNK) and p38, in a TLR4‐dependent manner; inhibition of JNK decreased release of HMGB1 after both hypoxia in vitro and I/R in vivo. Conclusion: These results provide insight into the individual cellular response of TLR4. The parenchymal HC is an active participant in sterile inflammatory response after I/R through TLR4‐mediated activation of proinflammatory signaling and release of danger signals, such as HMGB1. (HEPATOLOGY 2013)

Interferon regulatory factor 1 mediates acetylation and release of high mobility group box 1 from hepatocytes during murine liver ischemia-reperfusion injury.

Damage-associated molecular patterns (DAMPs) initiate inflammatory pathways that are common to both sterile and infectious processes. The DAMP, high-mobility group box 1 (HMGB1), and the transcription factor, interferon regulatory factor 1 (IRF-1), have been independently associated as key players in ischemia-reperfusion (I/R) injury. Our study demonstrates that IRF-1 contributes to hepatocellular release of HMGB1 and further that IRF-1 is a necessary component of HMGB1 release in response to hypoxia or after liver I/R. We also link the nuclear upregulation of IRF-1 to the presence of functional Toll-like receptor 4 (TLR4), a pattern recognition receptor also important in sterile and infectious processes. Using IRF-1 chimeric mice, we show that IRF-1 upregulation in hepatic parenchymal cells, and not in the bone marrow-derived immune cells, is responsible for HMGB1 release during ischemic liver injury. Finally, our study also demonstrates a role for IRF-1 in modulating the acetylation status and subsequent release of HMGB1 through histone acetyltransferases. We found that serum HMGB1 is acetylated after liver I/R and that this process was dependent on IRF-1. Additionally, liver I/R induced a direct association of IRF-1 and the nuclear histone acetyltransferase enzyme p300. Together, these findings suggest that I/R-induced release of acetylated HMGB1 is a process that is dependent on TLR4-mediated upregulation of IRF-1.ABBREVIATIONS-IRF-1-interferon regulatory factor 1; HMGB1-high-mobility group box 1; I/R-ischemia-reperfusion; PRR-pattern recognition receptor; DAMP-damage-associated molecular pattern; TLR4-Toll-like receptor-4; HAT-histone acetyltransferase; HDAC-histone deacetylase; WT-wild-type; MOI-multiplicity of infection

Critical role of interferon regulatory factor‐1 in murine liver transplant ischemia reperfusion injury

Interferon regulatory factor‐1 (IRF‐1) is a transcription factor that regulates gene expression during immunity. We hypothesized that IRF‐1 plays a pivotal role in liver transplant (LTx) ischemia/reperfusion (I/R) injury. Mouse orthotopic LTx was conducted after 24 hours cold storage in University of Wisconsin (UW) solution in wildtype (WT) C57BL/6 and IRF‐1 knockout (KO) mice. IRF‐1 deficiency in liver grafts, but not in recipients, resulted in significant reduction of hepatocyte apoptosis and liver injury, as well as improved survival. IRF‐1 mRNA up‐regulation was typically seen in graft hepatocytes in WT→WT LTx. Deficiency of IRF‐1 signaling in graft resulted in significantly reduced messenger RNA (mRNA) levels for death ligands and death receptors in hepatocytes, as well as decreased caspase‐8 activities, indicating that IRF‐1 mediates death ligand‐induced hepatocyte death. Further, a smaller but significant IRF‐1 mRNA up‐regulation was seen in WT graft nonparenchymal cells (NPC) and associated with interferon gamma (IFN‐γ) mRNA up‐regulation exclusively in NPC. IFN‐γ mRNA was significantly reduced in IRF‐1 KO graft. Thus, IRF‐1 in graft hepatocytes and NPC has distinct effects in hepatic I/R injury. However, LTx with chimeric liver grafts showed that grafts lacking hepatocellular IRF‐1 had better protection compared with those lacking IRF‐1 in NPC. The study identifies a critical role for IRF‐1 in liver transplant I/R injury. (HEPATOLOGY 2010.)

Arginase blockade protects against hepatic damage in warm ischemia-reperfusion.

BACKGROUND Liver ischemia reperfusion (I/R) injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. Nitric oxide (NO), shown to have protective effects in I/R, is produced by nitric oxide synthase (NOS) from the substrate arginine. The purpose of this study was to determine if nor-NOHA, a novel arginase inhibitor, would be able to increase circulating arginine levels and decrease hepatic damage following warm I/R. METHODS C57BL/6 mice underwent partial liver warm I/R and were treated intraperitoneally with either nor-NOHA (100mg/kg) or saline. Serum and tissue samples were collected to measure liver enzyme levels, amino acids, and inflammatory mediators. The agent nor-NOHA (100mg/kg) was administered 15 min before ischemia and immediately after reperfusion. Serum amino acid analysis was performed using HPLC. RESULTS Arginase activity after hepatic I/R peaked at 3-6h after reperfusion and resulted in a 10-fold drop in circulating arginine levels. Treatment with nor-NOHA inhibited arginase activity and reversed the arginine depletion after I/R while simultaneously increasing serum nitric oxide. In addition, circulating citrulline, a product of NOS activity, was increased in nor-NOHA-treated animals compared to controls. Inhibition of arginase also resulted in protection from hepatic I/R-induced damage in association with markedly lower hepatic TNF, IL-6, and inducible NOS mRNA levels compared to controls. CONCLUSION Arginase blockade represents a potentially novel strategy to combat liver injury under conditions of arginine deficiency. This protection may be mediated through the arginine-NO pathway.

A novel inhibitory peptide of Toll-like receptor signaling limits lipopolysaccharide-induced production of inflammatory mediators and enhances survival in mice.

Sepsis resulting from gram-negative bacterial infections is characterized by an excessive inflammatory immune response initiated by exposure of the host innate immune system to either bacteria or bacterial products, primarily lipopolysaccharide (LPS). Engagement of the Toll-like receptor (TLR) 4 on immune cells by LPS induces production of inflammatory mediators, leading to tissue damage. We recently identified a peptide, termed P13, which was previously shown to be a potent inhibitor of in vitro TLR signaling. In this study, we demonstrate that the use of this novel peptide significantly reduces the in vitro production of inflammatory mediators seen after exposure of hepatocytes/nonparenchymal cell cocultures and endothelial cells to LPS. In addition, in vivo treatment of mice with this peptide was effective at inhibiting LPS-induced production of inflammatory mediators and significantly limited liver damage. Peptide treatment significantly increased survival of LPS-/D-galactosamine-treated mice and mice treated with high-dose LPS. These results demonstrated the therapeutic potential of peptide P13 to limit an LPS-induced inflammatory response and enhance survival in murine models of inflammation.