HMGB1 is a ubiquitous nuclear protein present in almost all cell types. In addition to its intracellular functions, HMGB1 can be extracellularly released, where it mediates activation of innate ...immune responses, including chemotaxis and cytokine release. HMGB1 contains three conserved redox-sensitive cysteines (C23, C45, and C106); modification of these cysteines determines the bioactivity of extracellular HMGB1. Firstly, the cytokine-stimulating activity of HMGB1 requires C23 and C45 to be in a disulfide linkage, at the same time that C106 must remain in its reduced form as a thiol. This distinctive molecular conformation enables HMGB1 to bind and signal via the TLR4/MD-2 complex to induce cytokine release in macrophages. Secondly, for HMGB1 to act as a chemotactic mediator, all three cysteines must be in the reduced form. This all-thiol HMGB1 exerts its chemotactic activity to initiate inflammation by forming a heterocomplex with CXCL12; that complex binds exclusively to CXCR4 to initiate chemotaxis. Thirdly, binding of the HMGB1 to CXCR4 or to TLR4 is completely prevented by all-cysteine oxidation. Also, the initial post-translational redox modifications of HMGB1 are reversible processes, enabling HMGB1 to shift from acting as a chemotactic factor to acting as a cytokine and vice versa. Lastly, post-translational acetylation of key lysine residues within NLSs of HMGB1 affects HMGB1 to promote inflammation; hyperacetylation of HMGB1 shifts its equilibrium from a predominant nuclear location toward a cytosolic and subsequent extracellular presence. Hence, post-translational modifications of HMGB1 determine its role in inflammation and immunity.
High‐mobility group box‐1 (HMGB1) is a damage‐associated molecular pattern (DAMP) increased in response to liver injury. Because HMGB1 is a ligand for the receptor for advanced glycation endproducts ...(RAGE), we hypothesized that induction of HMGB1 could participate in the pathogenesis of liver fibrosis though RAGE cell‐specific signaling mechanisms. Liver HMGB1 protein expression correlated with fibrosis stage in patients with chronic hepatitis C virus (HCV) infection, primary biliary cirrhosis (PBC), or alcoholic steatohepatitis (ASH). Hepatic HMGB1 protein expression and secretion increased in five mouse models of liver fibrosis attributed to drug‐induced liver injury (DILI), cholestasis, ASH, or nonalcoholic steatohepatitis (NASH). HMGB1 was up‐regulated and secreted mostly by hepatocytes and Kupffer cells (KCs) following CCl4 treatment. Neutralization of HMGB1 protected, whereas injection of recombinant HMGB1 promoted liver fibrosis. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep) or in myeloid cells (Hmgb1ΔMye) partially protected, whereas ablation in both (Hmgb1ΔHepΔMye) prevented liver fibrosis in vivo. Coculture with hepatocytes or KCs from CCl4‐injected wild‐type (WT) mice up‐regulated Collagen type I production by hepatic stellate cells (HSCs); yet, coculture with hepatocytes from CCl4‐injected Hmgb1ΔHep or with KCs from CCl4‐injected Hmgb1ΔMye mice partially blunted this effect. Rage ablation in HSCs (RageΔHSC) and RAGE neutralization prevented liver fibrosis. Last, we identified that HMGB1 stimulated HSC migration and signaled through RAGE to up‐regulate Collagen type I expression by activating the phosphorylated mitogen‐activated protein kinase kinase (pMEK)1/2, phosphorylated extracellular signal‐regulated kinase (pERK)1/2 and pcJun signaling pathway. Conclusion: Hepatocyte and KC‐derived HMGB1 participates in the pathogenesis of liver fibrosis by signaling through RAGE in HSCs to activate the pMEK1/2, pERK1/2 and pcJun pathway and increase Collagen type I deposition.
Tissue damage causes inflammation, by recruiting leukocytes and activating them to release proinflammatory mediators. We show that high-mobility group box 1 protein (HMGB1) orchestrates both ...processes by switching among mutually exclusive redox states. Reduced cysteines make HMGB1 a chemoattractant, whereas a disulfide bond makes it a proinflammatory cytokine and further cysteine oxidation to sulfonates by reactive oxygen species abrogates both activities. We show that leukocyte recruitment and activation can be separated. A nonoxidizable HMGB1 mutant in which serines replace all cysteines (3S-HMGB1) does not promote cytokine production, but is more effective than wild-type HMGB1 in recruiting leukocytes in vivo. BoxA, a HMGB1 inhibitor, interferes with leukocyte recruitment but not with activation. We detected the different redox forms of HMGB1 ex vivo within injured muscle. HMGB1 is completely reduced at first and disulfide-bonded later. Thus, HMGB1 orchestrates both key events in sterile inflammation, leukocyte recruitment and their induction to secrete inflammatory cytokines, by adopting mutually exclusive redox states.
Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or ...passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2-deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2-HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4-MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness.
•HMGB1 is released in epileptic brain.•Disulfide HMGB1 contributes to seizure pathogenesis and comorbidites.•TLR4 mediate HMGB1’s effects in epilepsy.•HMGB1 isoforms may serve as disease ...biomarkers.•Anti-HMGB1 drugs affect epileptogenesis and reduce seizures and comorbidities.
Approximately 30% of epilepsy patients experience seizures that are not controlled by the available drugs. Moreover, these drugs provide mainly a symptomatic treatment since they do not interfere with the disease’s mechanisms. A mechanistic approach to the discovery of key pathogenic brain modifications causing seizure onset, recurrence and progression is instrumental for designing novel and rationale therapeutic interventions that could modify the disease course or prevent its development. In this regard, increasing evidence shows that neuroinflammation is a pathogenic factor in drug-resistant epilepsies. The High Mobility Group Box 1 (HMGB1)/Toll-like receptor 4 axis is a key initiator of neuroinflammation following brain injuries leading to epilepsy, and its activation contributes to seizure mechanisms in animal models. Recent findings have shown dynamic changes in HMGB1 and its isoforms in the brain and blood of animals exposed to acute brain injuries and undergoing epileptogenesis, and in surgically resected epileptic foci in humans. HMGB1 isoforms reflect different pathophysiological processes, and the disulfide isoform, which is generated in the brain during oxidative stress, is implicated in seizures, cell loss and cognitive dysfunctions. Interfering with disulfide HMGB1-activated cell signaling mediates significant therapeutic effects in epilepsy models. Moreover, both clinical and experimental data suggest that HMGB1 isoforms may serve as mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy.
These novel findings suggest that the HMGB1 system could be targeted to prevent seizure generation and may provide clinically useful prognostic biomarkers which may also predict the patient's response to therapy.
Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of ...HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.
Acetaminophen overdose is a common reason for hospital admission and the most frequent cause of hepatotoxicity in the Western world. Early identification would facilitate patient‐individualized ...treatment strategies. We investigated the potential of a panel of novel biomarkers (with enhanced liver expression or linked to the mechanisms of toxicity) to identify patients with acetaminophen‐induced acute liver injury (ALI) at first presentation to the hospital when currently used markers are within the normal range. In the first hospital presentation plasma sample from patients (n = 129), we measured microRNA‐122 (miR‐122; high liver specificity), high mobility group box‐1 (HMGB1; marker of necrosis), full‐length and caspase‐cleaved keratin‐18 (K18; markers of necrosis and apoptosis), and glutamate dehydrogenase (GLDH; marker of mitochondrial dysfunction). Receiver operator characteristic curve analysis and positive/negative predictive values were used to compare sensitivity to report liver injury versus alanine transaminase (ALT) and International Normalized Ratio (INR). In all patients, biomarkers at first presentation significantly correlated with peak ALT or INR. In patients presenting with normal ALT or INR, miR‐122, HMGB1, and necrosis K18 identified the development of liver injury (n = 15) or not (n = 84) with a high degree of accuracy and significantly outperformed ALT, INR, and plasma acetaminophen concentration for the prediction of subsequent ALI (n = 11) compared with no ALI (n = 52) in patients presenting within 8 hours of overdose. Conclusion: Elevations in plasma miR‐122, HMGB1, and necrosis K18 identified subsequent ALI development in patients on admission to the hospital, soon after acetaminophen overdose, and in patients with ALTs in the normal range. The application of such a biomarker panel could improve the speed of clinical decision‐making, both in the treatment of ALI and the design/execution of patient‐individualized treatment strategies. (Hepatology 2013;58:777–787)
In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of ...sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high-mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow-derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis.
Gas hydrates stored on continental shelves are susceptible to dissociation triggered by environmental changes. Knowledge of the timescales of gas hydrate dissociation and subsequent methane release ...are critical in understanding the impact of marine gas hydrates on the ocean-atmosphere system. Here we report a methane efflux chronology from five sites, at depths of 220-400 m, in the southwest Barents and Norwegian seas where grounded ice sheets led to thickening of the gas hydrate stability zone during the last glaciation. The onset of methane release was coincident with deglaciation-induced pressure release and thinning of the hydrate stability zone. Methane efflux continued for 7-10 kyr, tracking hydrate stability changes controlled by relative sea-level rise, bottom water warming and fluid pathway evolution in response to changing stress fields. The protracted nature of seafloor methane emissions probably attenuated the impact of hydrate dissociation on the climate system.
During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that ...generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor
is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4
CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.
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