Poly(ADP-ribosyl)ation of high mobility group box 1 (HMGB1) protein enhances inhibition of efferocytosis

Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.     

Mycobacterial infection induces the secretion of high-mobility group box 1 protein

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also release HMGB1 at the site of tissue damage which induces a variety of cellular responses, including the expression of pro-inflammatory mediators. This study investigated the secretion of HMGB1 in mycobacterial infection by macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection by mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis induced maximum release of HMGB1 compared with different subcellular fractions of mycobacterium. We demonstrated that HMGB1 is released in lungs during infection of M. tuberculosis in guinea pigs and increased HMGB1 secretion in lungs of guinea pigs was delayed by prior vaccination with Mycobacterium bovis BCG. The secretion of cytokines like tumour necrosis factor alpha (TNF-alpha) and Interleukin-1beta was significantly increased when M. bovis BCG-infected cultures of J774A.1 cells were incubated with HMGB1. Among different mycobacterial toll-like receptor ligands, heat-shock protein 65 (HSP65) was found to be more potent in inducing HMGB1 secretion in RAW 264.7 cells. Pharmacological suppression of p38 or extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases with specific inhibitors failed to inhibit HSP65-induced HMGB1 release, but inhibition of c-Jun NH(2)-terminal kinase activation attenuated HMGB1 release. Inhibition of the inducible NO synthase and neutralizing antibodies against TNF-alpha also reduced HMGB1 release stimulated by HSP65. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and enhances immune response.     

High Systemic Levels of the Cytokine-Inducing HMGB1 Isoform Secreted in Severe Macrophage Activation Syndrome

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced “all-thiol HMGB1” exerts chemotactic activity; “disulfide HMGB1” has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized “sulfonyl HMGB1” lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.     

HMGB1, an architectural chromatin protein and extracellular signalling factor,has a spatially and temporally restricted expression pattern in mouse brain

HMGB1 is an abundant chromatin component, so far considered ubiquitous. HMGB1 also has an extracellular signalling role: when passively released by necrotic cells, it triggers inflammation; moreover, it can be actively secreted by myeloid cells, neurons and neuronal cancer cells. We show here that HMGB1 protein is undetectable in most cells in adult mouse brain, and is present in a subset of brain cells during development, with a very complex temporal, spatial and subcellular expression pattern. HMGB1 is expressed in the cortical plate of E14.5 embryos, predominantly in the nucleus, although roughly 1% of cells show a cytoplasmic localization as well. In E16 embryos, HMGB1 is nuclearly expressed in scattered cells apparently moving from the ventricular zone to the cortical plate. HMGB1 expression is strongly down-regulated at later developmental stages; in adult mice significant expression is maintained only in areas of continuing neurogenesis. Finally, HMGB1 subcellular localization changes during retinoic acid induced differentiation of P19 neuroblastoma cells.     

High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine

Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with an unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. High mobility group protein-1 (HMGB1), a nuclear nonhistone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and fluorescence resonance energy transfer demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage fluid from Scnn(+) mice, a murine model of cystic fibrosis lung disease which contains elevated concentrations of HMGB1, inhibits neutrophil efferocytosis. Anti-HMGB1 Abs reverse the inhibitory effect of Scnn(+) bronchoalveolar lavage on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses.     

High mobility group B1 protein suppresses the human plasmacytoid dendritic cell response to TLR9 agonists

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-alpha, IL-6, TNF-alpha, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-gamma-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.     

Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1)

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.     

Identification of Hemopexin as an Anti-Inflammatory Factor That Inhibits Synergy of Hemoglobin with HMGB1 in Sterile and Infectious Inflammation

Hemoglobin is released from lysed RBCs in numerous clinical settings. High mobility group box 1 (HMGB1) is a nuclear and cytosolic DNA-binding protein released from injured cells that has been shown to play an important role in inducing inflammation. Because both of these endogenous molecules are frequently present in sites of necrosis and inflammation, we studied their interaction on the activation of macrophages. We report in this article that hemoglobin and HMGB1 synergize to activate mouse macrophages to release significantly increased proinflammatory cytokines. Addition of microbial ligands that activate through TLR2 or TLR4 resulted in further significant increases, in a "three-way" synergy between endogenous and microbial ligands. The synergy was strongly suppressed by hemopexin (Hx), an endogenous heme-binding plasma protein. The findings suggest that hemoglobin may play an important role in sterile and infectious inflammation, and that endogenous Hx can modulate this response. Administration of Hx may be beneficial in clinical settings characterized by elevated extracellular hemoglobin and HMGB1.     

HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers

High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with "cytomix" (a mixture of TNF, IL-1, and IFN-) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop. exosome; toll-like receptor; flagellin     

Essential roles of high-mobility group box 1 in the development of murine colitis and colitis-associated cancer

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as a proinflammatory cytokine. We measured the HMGB1 concentration in the sera of mice with chemically induced colitis (DSS; dextran sulfate sodium salt) and found a marked increase. Inhibition of HMGB1 by neutralizing anti-HMGB1 antibody resulted in reduced inflammation in DSS-treated colons. In macrophages, HMGB1 induces several proinflammatory cytokines, such as IL-6, which are regulated by NF-kappaB activation. Two putative sources of HMGB1 were explored: in one, bacterial factors induce HMGB1 secretion from macrophages and in the other, necrotic epithelial cells directly release HMGB1. LPS induced a small amount of HMGB1 in macrophages, but macrophages incubated with supernatant prepared from necrotic cells and containing large amounts of HMGB1 activated NF-kappaB and induced IL-6. Using the colitis-associated cancer model, we demonstrated that neutralizing anti-HMGB1 antibody decreases tumor incidence and size. These observations suggest that HMGB1 is a potentially useful target for IBD treatment and the prevention of colitis-associated cancer.     

A highly purified vegetal fraction able to modulate HMGB1 and to attenuate septic shock in mice

High-mobility group box protein 1 (HMGB1) is an intracellular protein that may be released actively from monocytes and macrophages or passively from necrotic or damaged cells. Its inhibition in animal experiments, even in the late phase of septic shock, significantly enhanced the survival rate of rodents. The aim of our study was to investigate the effect of a vegetal fraction isolated and highly purified from Helleborus purpurascens regarding the modulation of HMGB1 release either from tumor cells or human blood mononuclear cells. Our results showed that the vegetal fraction was able to down-regulate the release of HMGB1 from activated human blood mononuclear cells (PBMCs) and tumor cells. By combining the purified fraction with Cyclophosphamide the release of HMGB1 from tumor cells was strongly decreased. This synergism was not noticed when the ve getal product was associated with Doxorubicin. We also studied the effect of the purified fraction in mice with septic shock induced by cecal ligation and puncture (CLP) method. The tested vegetal product increased significantly the survival rate of animals compared to the mice not treated with it. Our data suggest that the purified vegetal fraction may modulate inflammation by down-regulating the HMGB1, which can also explain its efficacy in septic shock in mice.     

低血压灌注对无心跳大鼠肺支气管肺泡灌洗液中不同白细胞及HMGB1 的影响

目的:研究低血压灌注对无心跳大鼠肺支气管肺泡灌洗液中不同白细胞及HMGB1的影响。方法:将32只180-200g SD雄性大鼠随机分成对照组[sham]、缺血30min组[30I]、缺血60 min组[60I]和缺血90 min组[90I](n=8),4组大鼠分别接受麻醉后持续机械通气,测定并统计各组支气管肺泡灌洗液中总白细胞计数及不同白细胞计数,免疫印迹法检测支气管肺泡灌洗液中HMGB1蛋白的表达。结果:总白细胞、巨噬细胞、淋巴细胞计数及HMGB1蛋白表达均随着低血压灌注时间的延长而逐渐增加,尤其在[60I]和[90I]两组,灌洗液中巨噬细胞和淋巴细胞计数有显著增高,并且巨噬细胞和淋巴细胞与HMGB1蛋白表达呈正相关。结论:本研究证实HMGB1蛋白表达和巨噬细胞,淋巴细胞计数在低血压灌注缺血期内升高,提示HMGB1蛋白可能具有驱动炎症反应的作用。     

Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion

The high mobility group box 1 (HMGB1) protein can be secreted by activated monocytes and macrophages and functions as a late mediator of sepsis. HMGB1 contains two nuclear localization signals (NLSs) for controlled nuclear transport, and acetylation of both NLSs of HMGB1 is involved in nuclear transport toward secretion. However, phosphorylation of HMGB1 and its relation to nuclear transport have not been shown. We show here that HMGB1 is phosphorylated and dynamically shuttled between cytoplasmic and nuclear compartments according to its phosphorylation state. Phosphorylation of HMGB1 was detected by metabolic labeling and Western blot analysis after treatments with TNF-alpha and okadaic acid, a phosphatase inhibitor. Hyperphosphorylated HMGB1 in RAW 264.7 and human monocytes was relocated to the cytoplasm. In a nuclear import assay, phosphorylated HMGB1 in the cytoplasm did not enter the nucleus. We mutated serine residues of either or both NLSs of HMGB1 to glutamic acid to simulate a phosphorylated state and examined the binding of HMGB1 to karyopherin-alpha1, which was identified as the nuclear import protein for HMGB1 in this study. Substitution to glutamic acid in either NLSs decreased the binding with karyopherin-alpha1 by approximately 50%; however, substitution of both NLSs showed no binding, and HMGB1 was relocated to the cytoplasm and subsequently secreted. These data support the hypothesis that HMGB1 could be phosphorylated and that the direction of transport is regulated by phosphorylation of both NLS regions.     

High mobility group box 1 potentiates the pro-inflammatory effects of interleukin-1β in osteoarthritic synoviocytes

Introduction  

High mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1β) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).     

The nuclear protein HMGB1 is secreted by monocytes via a non-classical,vesicle-mediated secretory pathway

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.