John Evankovich

Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice.

Sterile inflammatory insults are known to activate innate immunity and propagate organ damage through the recognition of extracellular damage‐associated molecular pattern (DAMP) molecules. Although DAMPs such as endogenous DNA and nuclear high‐mobility group box 1 have been shown to be critical in sterile inflammation, the role of nuclear histone proteins has not yet been investigated. We report that endogenous histones function as DAMPs after ischemic injury through the pattern recognition receptor Toll‐like receptor (TLR) 9 to initiate inflammation. Using an in vivo model of hepatic ischemia/reperfusion (I/R) injury, we show that levels of circulating histones are significantly higher after I/R, and that histone neutralization significantly protects against injury. Injection of exogenous histones exacerbates I/R injury through cytotoxic effects mediated by TLR9 and MyD88. In addition, histone administration increases TLR9 activation, whereas neither TLR9 nor MyD88 mutant mice respond to exogenous histones. Furthermore, we demonstrate in vitro that extracellular histones enhance DNA‐mediated TLR9 activation in immune cells through a direct interaction. Conclusion: These novel findings reveal that histones represent a new class of DAMP molecules and serve as a crucial link between initial damage and activation of innate immunity during sterile inflammation. (HEPATOLOGY 2011; 54:999–1008)

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.

Histones Activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests that it also plays a role in inflammation driven by endogenous danger-associate molecular pattern molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. In this article, we report that both NLRP3 and its downstream target caspase-1 are activated during I/R and are essential for hepatic I/R injury, because both NLRP3 and caspase-1 knockout mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on caspase-1 expression in liver nonparenchymal cells. Although upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through TLR9. This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and the activation of innate immunity during sterile inflammation.

Dendritic Cells and Damage-Associated Molecular Patterns: Endogenous Danger Signals Linking Innate and Adaptive Immunity

Dendritic cells (DCs) are potent antigen-presenting cells critical in regulating the adaptive immune response. The role of DCs is dichotomous; they may both present antigens and the appropriate stimulatory molecules to initiate an adaptive immune response, or they may induce tolerance and release anti-inflammatory signals. The activation of immature DCs, required for the expression of the necessary costimulatory T cell molecules, is dependent on pattern recognition receptors. In addition to the pathogen-derived ligands of pattern recognition receptors, several damage-associated molecular patterns (DAMPs) have recently been shown to interact with DCs and dramatically affect their ultimate function. The complex interplay of DAMPs on DCs is clinically important, with implications for transplantation, tumor immunity, autoimmunity, chronic inflammation and other conditions of sterile inflammation such as ischemia reperfusion injury. In this review, we will focus on the role of DAMPs in DC function.

Interferon-gamma induces autophagy with growth inhibition and cell death in human hepatocellular carcinoma (HCC) cells through interferon-regulatory factor-1 (IRF-1)

Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, anti-viral, and anti-proliferative effects. In this study, we examined the effects of IFN-γ on autophagy and cell growth in human hepatocellular carcinoma (HCC) cells. IFN-γ inhibited cell growth of Huh7 cells with non-apoptotic cell death. IFN-γ induced autophagosome formation and conversion/turnover of microtubule associated protein 1 light chain 3 (LC3) protein. Furthermore, overexpression of IRF-1 also induced autophagy in Huh7 cells. Silencing IRF-1 expression with target small hairpin RNA blocked autophagy induced by IFN-γ. Silencing of the autophagy signals Beclin-1 or Atg5 attenuated the inhibitory effect of IFN-γ on Huh7 cells with decreased cell death. Additionally, IFN-γ activated autophagy in freshly cultured human HCC cells. Together, these findings show that IFN-γ induces autophagy through IRF-1 signaling pathway and the induction of autophagy contributes to the growth-inhibitory effect of IFN-γ with cell death in human liver cancer cells.

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

Calcium/calmodulin-dependent protein kinase IV limits organ damage in hepatic ischemia-reperfusion injury through induction of autophagy

Sterile inflammatory insults, such as ischemia-reperfusion (I/R) injury, result from pathogenic factors, including damage-associated molecular pattern signaling, activation of innate immunity, and upregulation of proinflammatory cytokines. At the same time, a number of protective, or prosurvival, pathways are also activated, and the extent of end-organ damage is ultimately determined by the balance between these two systems. In liver I/R, members of the calcium/calmodulin-dependent protein kinase (CaMK) family are known to be activated, but their individual roles are largely unknown. In this study, we show that one CaMK member, CaMKIV, is protective in hepatic I/R by activating the prosurvival pathway of autophagy in hepatocytes. CaMKIV knockout mice experience significantly worse organ damage after I/R and are deficient in hepatocyte autophagic signaling. Restoration of autophagic signaling with rapamycin reduces organ damage in CaMKIV knockout mice to wild-type levels. In vitro, we show that CaMKIV activation induces autophagy in mouse hepatocytes, and that CaMKIV activation protects hepatocytes from oxidative stress-induced cell death. In conclusion, the protective autophagic signaling pathway serves to reduce organ damage following I/R and is regulated by activation of CaMKIV signaling in hepatocytes.

Interferon Regulatory Factor-1 Regulates the Autophagic Response in LPS-Stimulated Macrophages through Nitric Oxide

The pathogenesis of sepsis is complex and, unfortunately, poorly understood. The cellular process of autophagy is believed to play a protective role in sepsis; however, the mechanisms responsible for its regulation in this setting are ill defined. In the present study, interferon regulatory factor 1 (IRF-1) was found to regulate the autophagic response in lipopolysaccharide (LPS)-stimulated macrophages. In vivo, tissue macrophages obtained from LPS-stimulated IRF-1 knockout (KO) mice demonstrated increased autophagy and decreased apoptosis compared to those isolated from IRF-1 wild-type (WT) mice. In vitro, LPS-stimulated peritoneal macrophages obtained from IRF-1 KO mice experienced increased autophagy and decreased apoptosis. IRF-1 mediates the inhibition of autophagy by modulating the activation of the mammalian target of rapamycin (mTOR). LPS induced the activation of mTOR in WT peritoneal macrophages, but not in IRF-1 KO macrophages. In contrast, overexpression of IRF-1 alone increased the activation of mTOR and consequently decreased autophagic flux. Furthermore, the inhibitory effects of IRF-1 mTOR activity were mediated by nitric oxide (NO). Therefore, we propose a novel role for IRF-1 and NO in the regulation of macrophage autophagy during LPS stimulation in which IRF-1/NO inhibits autophagy through mTOR activation.

Interferon regulatory factor-2 is protective against hepatic ischemia-reperfusion injury

Interferon regulatory factor (IRF)-1 is a nuclear transcription factor that induces inflammatory cytokine mediators and contributes to hepatic ischemia-reperfusion (I/R) injury. No strategies to mitigate IRF1-mediated liver damage exist. IRF2 is a structurally similar endogenous protein that competes with IRF1 for DNA binding sites in IRF-responsive target genes and acts as a competitive inhibitor. However, the role of IRF2 in hepatic injury during hypoxic or inflammatory conditions is unknown. We hypothesize that IRF2 overexpression may mitigate IRF1-mediated I/R damage. Endogenous IRF2 is basally expressed in normal livers and is mildly increased by ischemia alone. Overexpression of IRF2 protects against hepatic warm I/R injury. Furthermore, we demonstrate that IRF2 overexpression limits production of IRF1-dependent proinflammatory genes, such as IL-12, IFNβ, and inducible nitric oxide synthase, even in the presence of IRF1 induction. Additionally, isograft liver transplantation with IRF2 heterozygote knockout (IRF2(+/-)) donor grafts that have reduced endogenous IRF2 levels results in worse injury following cold I/R during murine orthotopic liver transplantation. These findings indicate that endogenous intrahepatic IRF2 protein is protective, because the IRF2-deficient liver donor grafts exhibited increased liver damage compared with the wild-type donor grafts. In summary, IRF2 overexpression protects against I/R injury by decreasing IRF1-dependent injury and may represent a novel therapeutic strategy.

Delayed operating room availability significantly impacts the total hospital costs of an urgent surgical procedure

BACKGROUND Financial pressures drive efforts to optimize hospital resource use, but inefficiencies occur in systems as volume nears total capacity. We examined how operating room use impacts efficiency and costs of treating an urgent surgical condition. METHODS A retrospective review of patients who underwent appendectomy for appendicitis at a single hospital from 2004 to 2009 was performed. Patient demographics, operative characteristics, pathologic diagnoses, hospital time intervals, and costs were analyzed. Gap time (time from case booking to surgery start) was used to measure operating room availability. RESULTS In all, 453 patients met inclusion criteria. Longer gap times were associated with increased hospital-based costs. A gap time of greater than 2 h was associated with 39% higher costs to the hospital, which could not be accounted for by any single cost center. The patients in the 2 groups had similar medical and surgical complexity, as well as similar clinical outcomes and hospital duration of stay. Gap times were greatest during peak elective operating room activity (7 am to 11 pm); however, the total hospital costs were not related to the time of day of the case. CONCLUSION A short delay in operating room availability for urgent cases is associated with significantly increased total hospital costs. Our data suggest this finding is attributable to inefficient care when the operating room volume nears total capacity.