Alarmin High Mobility Group Box Research Articles

Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

Ultraviolet light exposure stimulates HMGB1 release by keratinocytes

The primary cause of non-melanoma skin cancer is ultraviolet (UV) light from the sun. Many studies have demonstrated that cutaneous inflammation resulting from UV exposure is important for the development of skin cancer. In fact, anti-inflammatory drugs have been shown to be effective in preventing skin cancer in animal models and in clinical trials. One new class of inflammatory mediators that could regulate UV-induced inflammation and skin carcinogenesis is alarmins. Alarmins are endogenous molecules that act as potent pro-inflammatory mediators when they are released by cells or accumulate extracellularly. The purpose of the current studies was to examine the expression and release of the alarmin high mobility group box 1 (HMGB1) after acute and chronic UV irradiation. Acute UV exposure stimulated the release of HMGB1 in cultured human keratinocytes and epidermal keratinocytes in murine skin. HMGB1 release correlated with pro-inflammatory cytokine production in vitro and inflammatory cell infiltration in vivo. HMGB1 was also examined in tumors arising in chronically irradiated murine skin. HMGB1 protein expression in low grade, benign papillomas was similar to adjacent skin. However, HMGB1 staining was more widespread with a higher number of HMGB1-positive cells observed in high grade papillomas and malignant tumors. Overall, the data suggest that HMGB1 may be an important regulator of UV-induced cutaneous inflammation and tumor formation. Additional studies are needed to assess whether targeting HMGB1 would be a useful strategy to prevent tumors from developing in response to chronic UV exposure.

P53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

Cellular senescence irreversibly arrests proliferation in response to potentially oncogenic stress. Senescent cells also secrete inflammatory cytokines such as IL-6, which promote age-associated inflammation and pathology. HMGB1 (high mobility group box 1) modulates gene expression in the nucleus, but certain immune cells secrete HMGB1 as an extracellular Alarmin to signal tissue damage. We show that nuclear HMGB1 relocalized to the extracellular milieu in senescent human and mouse cells in culture and in vivo. In contrast to cytokine secretion, HMGB1 redistribution required the p53 tumor suppressor, but not its activator ATM. Moreover, altered HMGB1 expression induced a p53-dependent senescent growth arrest. Senescent fibroblasts secreted oxidized HMGB1, which stimulated cytokine secretion through TLR-4 signaling. HMGB1 depletion, HMGB1 blocking antibody, or TLR-4 inhibition attenuated senescence-associated IL-6 secretion, and exogenous HMGB1 stimulated NF-κB activity and restored IL-6 secretion to HMGB1-depleted cells. Our findings identify senescence as a novel biological setting in which HMGB1 functions and link HMGB1 redistribution to p53 activity and senescence-associated inflammation.

The alarmin HMGB‐1 influences healing outcomes in fetal skin wounds

In mice, cutaneous wounds generated early in development (embryonic day 15, E15) heal scarlessly, while wounds generated late in gestation (embryonic day 18, E18) heal with scar formation. Even though both types of wounds are generated in the same sterile uterine environment, scarless fetal wounds heal without inflammation, but a strong inflammatory response is observed in scar-forming fetal wounds. We hypothesized that altered release of alarmins, endogenous molecules that trigger inflammation in response to damage, may be responsible for the age-related changes in inflammation and healing outcomes in fetal skin. The purpose of this study was to determine whether the alarmin high-mobility group box-1 (HMGB-1) is involved in fetal wound repair. Immunohistochemical analysis showed that in unwounded skin, E18 keratinocytes expressed higher levels of HMGB-1 compared with E15 keratinocytes. After injury, HMGB-1 was released to a greater extent from keratinocytes at the margin of scar-forming E18 wounds, compared with scarless E15 wounds. Furthermore, instead of healing scarlessly, E15 wounds healed with scars when treated with HMGB-1. HMGB-1-injected wounds also had more fibroblasts, blood vessels, and macrophages compared with control wounds. Together, these data suggest that extracellular HMGB-1 levels influence the quality of healing in cutaneous wounds.

Clinical chorioamnionitis is characterized by changes in the expression of the alarmin HMGB1 and one of its receptors, sRAGE

Objective: High mobility group box-1 (HMGB1) protein is an alarmin, a normal cell constituent, which is released into the extracellular environment upon cellular stress/damage and capable of activating inflammation and tissue repair. The receptor for advanced glycation end products (RAGE) can bind HMGB1. RAGE, in turn, can induce the production of pro-inflammatory cytokines; this may be modulated by the soluble truncated forms of RAGE, including soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE). The objectives of this study were to determine whether: 1) clinical chorioamnionitis at term is associated with changes in amniotic fluid concentrations of HMGB1, sRAGE and esRAGE; and 2) the amniotic fluid concentration of HMGB1 changes with labor or as a function of gestational age. Methods: Amniotic fluid samples were collected from the following groups: 1) mid-trimester (n = 45); 2) term with (n = 48) and without labor (n = 22) without intra-amniotic infection; and 3) term with clinical chorioamnionitis (n = 46). Amniotic fluid concentrations of HMGB1, sRAGE and esRAGE concentrations were determined by ELISA. Results: 1) the median amniotic fluid HMGB1 concentration was higher in patients at term with clinical chorioamnionitis than in those without this condition (clinical chorioamnionitis: median 3.8 ng/mL vs. term in labor: median 1.8 ng/mL, p = 0.007; and vs. term not in labor: median 1.1 ng/mL, p = 0.003); 2) in contrast, patients with clinical chorioamnionitis had a lower median sRAGE concentration than those without this condition (clinical chorioamnionitis: median 9.3 ng/mL vs. term in labor: median 18.6 ng/mL, p = 0.001; and vs. term not in labor median: 28.4 ng/mL, p < 0.001); 3) amniotic fluid concentrations of esRAGE did not significantly change in patients with clinical chorioamnionitis at term (clinical chorioamnionitis: median 5.4 ng/mL vs. term in labor: median 6.1 ng/mL, p = 0.9; and vs. term not in labor: median 9.5 ng/mL, p = 0.06); and 4) there was no significant difference in the median AF HMGB1 concentration between women at term in labor and those not in labor (p = 0.4) and between women in the mid-trimester and those at term not in labor (mid-trimester: median 1.5 ng/mL; p = 0.2). Conclusion: An increase in the amniotic fluid HMGB1 concentration and a decrease in sRAGE were observed in clinical chorioamnionitis at term. This finding provides evidence that an alarmin, HMGB1, and one of its receptors, sRAGE, are engaged in the process of clinical chorioamnionitis at term. These changes are quite different from those observed in cases of intra-amniotic infection/inflammation in preterm gestations.

Abstract LB-483: p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

Abstract Cellular senescence irreversibly arrests the proliferation of cells at risk for malignant transformation in part through activities of the transcriptional regulator and tumor suppressor p53. Cells that senesce owing to DNA damage also secrete many biologically active factors, including inflammatory cytokines such as IL-6. The High Mobility Group Box 1 (HMGB1) protein is unusual in having two distinct functions. Intracellularly, it binds chromatin and modulates transcription, including stimulating p53 activity. In addition, necrosis or microbial infection causes HMGB1 leakage or active secretion, respectively, whereupon it functions as an extracellular Alarmin to signal tissue damage and promote tissue regeneration, stem cell recruitment and immune activation. We show that HMGB1 is largely nuclear in non-senescent human and mouse fibroblasts and epithelial cells, but is actively exported from the nucleus and secreted by senescent cells. In culture and in vivo, HMGB1 re-localization occurred prior to the appearance of other hallmarks of senescence, and depended on the function of p53, but not the upstream p53 activator ATM. Old mice contained significantly higher levels of HMGB1 in serum compared to young animals. Disruption of HMGB1 stoichiometry, either by overexpression or depletion, induced a p53-dependent senescence growth arrest, but only HMGB1 overexpression, not HMGB1 depletion, promoted IL-6 secretion. Senescence-associated secretion required endogenous and secreted HMGB1 because deletion of endogenous HMGB1 – or addition of an HMGB1 blocking antibody - attenuated IL-6 secretion. Recombinant HMGB1 protein induced IL-6 secretion in cells depleted, but not harboring, endogenous HMGB1. Depletion of endogenous HMGB1 promoted NF-κ B transcriptional activity in cells cultured with recombinant HMGB1. Our findings identify a novel biological setting (senescence), independent of necrosis or microbial infection, in which HMGB1 secretion occurs, and link senescence-dependent HMGB1 redistribution to p53 activity and inflammatory cytokine secretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-483. doi:1538-7445.AM2012-LB-483

Neutrophil Extracellular Trap Formation Is Associated with IL-1β and Autophagy-Related Signaling in Gout

BackgroundGout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.Methodology/Principal FindingsSynovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.Conclusions/SignificanceThese findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.

Damage-associated molecular patterns (DAMPs) in preterm labor with intact membranes and preterm PROM: a study of the alarmin HMGB1

Objective. Preterm parturition is a syndrome caused by multiple etiologies. Although intra-amniotic infection is causally linked with intrauterine inflammation and the onset of preterm labor, other patients have preterm labor in the absence of demonstrable infection. It is now clear that inflammation may be elicited by activation of the Damage-Associated Molecular Patterns (DAMPs), which include pathogen-associated molecular patterns (PAMPs) as well as “alarmins” (endogenous molecules that signal tissue and cellular damage). A prototypic alarmin is high-mobility group box 1 (HMGB1) protein, capable of inducing inflammation and tissue repair when it reaches the extracellular environment. HMGB1 is a late mediator of sepsis, and blockade of HMGB1 activity reduces mortality in an animal model of endotoxemia, even if administered late during the course of the disorder. The objectives of this study were to: (1) determine whether intra-amniotic infection/inflammation (IAI) is associated with changes in amniotic fluid concentrations of HMGB1; and (2) localize immunoreactivity of HMGB1 in the fetal membranes and umbilical cord of patients with chorioamnionitis. Methods. Amniotic fluid samples were collected from the following groups: (1) preterm labor with intact membranes (PTL) with (n = 42) and without IAI (n = 84); and (2) preterm prelabor rupture of membranes (PROM) with (n = 38) and without IAI (n = 35). IAI was defined as either a positive amniotic fluid culture or amniotic fluid concentration of interleukin-6 (IL-6) ≥2.6 ng/mL. HMGB1 concentrations in amniotic fluid were determined by ELISA. Immunofluorescence staining for HMGB1 was performed in the fetal membranes and umbilical cord of pregnancies with acute chorioamnionitis. Results. (1) Amniotic fluid HMGB1 concentrations were higher in patients with IAI than in those without IAI in both the PTL and preterm PROM groups (PTL IAI: median 3.1 ng/mL vs. without IAI; median 0.98 ng/mL; p < 0.001; and preterm PROM with IAI median 7.3 ng/mL vs. without IAI median 2.6 ng/mL; p = 0.002); (2) patients with preterm PROM without IAI had a higher median amniotic fluid HMGB1 concentration than those with PTL and intact membranes without IAI (p < 0.001); and (3) HMGB1 was immunolocalized to amnion epithelial cells and stromal cells in the Wharton’s jelly (prominent in the nuclei and cytoplasm). Myofibroblasts and macrophages of the chorioamniotic connective tissue layer and infiltrating neutrophils showed diffuse cytoplasmic HMGB1 immunoreactivity. Conclusions. (1) intra-amniotic infection/inflammation is associated with elevated amniotic fluid HMGB1 concentrations regardless of membrane status; (2) preterm PROM was associated with a higher amniotic fluid HMGB1 concentration than PTL with intact membranes, suggesting that rupture of membranes is associated with an elevation of alarmins; (3) immunoreactive HMGB1 was localized to amnion epithelial cells, Wharton’s jelly and cells involved in the innate immune response; and (4) we propose that HMGB1 released from stress or injured cells into amniotic fluid may be responsible, in part, for intra-amniotic inflammation due to non-microbial insults.

Abstract A3: p53-dependent release of alarmin HMGB1 is a central mediator of senescent phenotypes

Abstract Cellular senescence irreversibly arrests the proliferation of cells at risk for malignant transformation in part through activities of the transcriptional regulator and tumor suppressor p53. Cells that senesce owing to DNA damage also secrete many biologically active factors, including inflammatory cytokines such as IL-6. Some data suggest that the senescence associated secretory phenotype (SASP) creates a tumor permissive environment. However, we find that senescent cells secrete a potent bioactive molecule—High Mobility Group Box 1 (HMGB1) protein, which is unusual in having two distinct functions. Intracellularly, it binds chromatin and modulates transcription, including stimulating p53 activity. In addition, necrosis or microbial infection causes HMGB1 leakage or active secretion, respectively, whereupon it functions as an extracellular alarmin to signal tissue damage and promote tissue regeneration, stem cell recruitment and immune activation. We show that HMGB1 is largely nuclear in non-senescent human and mouse fibroblasts and epithelial cells, but is actively exported from the nucleus and secreted by senescent cells. In culture and in vivo, HMGB1 re-localization occurred prior to the appearance of other hallmarks of senescence, and depended on the function of p53, but not the upstream p53 activator ATM, which distinguished HMGB1 secretion from the SASP. Aged mice or human sera contained significantly higher levels of circulating HMGB1 compared to sera from young mice or human subjects. Disruption of HMGB1 stoichiometry, either by overexpression or depletion, induced a p53-dependent senescence growth arrest, but only HMGB1 overexpression, not HMGB1 depletion, promoted IL-6 secretion. Senescence-associated secretion required endogenous and secreted HMGB1 because deletion of endogenous HMGB1— or addition of an HMGB1 blocking antibody – attenuated IL-6 secretion. Recombinant HMGB1 protein induced IL-6 secretion in cells depleted, but not harboring, endogenous HMGB1. Depletion of endogenous HMGB1 promoted NF-κ B transcriptional activity in cells cultured with recombinant HMGB1. Our findings identify a novel biological setting (senescence), independent of necrosis or microbial infection, in which HMGB1 secretion occurs in vitro and in vivo, and link senescence-dependent HMGB1 redistribution to p53 activity and inflammatory cytokine secretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A3.

Natural killer cells, dendritic cells, and the alarmin high-mobility group box 1 protein

Natural killer (NK) cells promote antiviral immunity by producing proinflammatory cytokines and by lysing infected cells. In addition, NK cells can modulate dendritic cell functions. NK-dendritic cell crosstalk results in activation of both cell types, with dendritic cells promoting NK-cell activity and NK cells inducing further maturation of dendritic cells. Here we review the recent evidence suggesting that NK-dendritic cell crosstalk is disrupted during HIV-1 infection and we discuss the consequences on HIV persistence in dendritic cells. NK cell-mediated dendritic cell editing is compromised during HIV-1 infection, and NK cells from viremic individuals show a decreased ability to kill immature dendritic cells. The defect is associated with impaired NKp30 function. Moreover, the resistance of HIV-1-infected dendritic cells to NK-mediated lysis is associated with the upregulation of apoptosis inhibitors, thus protecting infected dendritic cells from TRAIL-dependent apoptosis. These inhibitors are upregulated by the high-mobility group box 1 protein (HMGB1), an alarmin produced at NK-dendritic cell synapse that is essential for NK-dependent dendritic cell maturation, but also promotes viral replication in infected dendritic cells. HIV-1-induced impairment of NK-dendritic cell crosstalk may significantly alter both innate and adaptive immunity. It may also contribute to HIV persistence in dendritic cells through an HMGB1-dependent mechanism.

The Effect of HMGB1, a Damage-Associated Molecular Pattern Molecule, on Polymorphonuclear Neutrophil Migration Depends on Its Concentration

Polymorphonuclear neutrophils (PMN) play a key role in host defenses against invading microorganisms but also potentiate inflammatory reactions in case of excessive or misdirected responses. Release of the alarmin high-mobility group box 1 (HMGB1) by cells that die at an inflammatory site may act as an alert signal for the immune system. We studied the effect of HMGB1 on human PMN migration, using whole-blood samples to avoid cell activation associated with isolation procedures. HMGB1 50–100 ng/ml reduced baseline PMN migration as well as formyl-methionyl-leucyl-phenylalanine- and IL-8-induced PMN chemotaxis. This inhibitory effect was mediated by the RAGE receptor. In contrast, a higher HMGB1 concentration (5,000 ng/ml) had a chemoattractant effect on PMN through IL-8 production. This effect required the engagement of Toll-like receptors 2 and 4 in addition to the RAGE receptor. The A box component of HMGB1, which antagonizes the endogenous protein, reduced chemotaxis and also strongly inhibited the enhancement of PMN migration observed with the highest HMGB1 concentration. In contrast, the B box, reported to be the active form of HMGB1, exerted a chemoattractant effect. These results strongly point to a key regulatory role of HMGB1 in PMN recruitment to inflammatory tissues. The A box component could potentially serve to inhibit inappropriate PMN recruitment during chronic inflammatory disorders associated with excessive HMGB1 release.

Alarmin HMGB1 Is Released in the Small Intestine of Gnotobiotic Piglets Infected with Enteric Pathogens and Its Level in Plasma Reflects Severity of Sepsis

Alarmin high mobility group box 1 (HMGB1) is essential for correct DNA folding and transcription. It can be released from damaged cells or secreted by stimulated cells. HMGB1 has been detected in serum or plasma as a late marker of sepsis, but its suitability as a marker of sepsis has been disputed. One-week-old germ-free piglets were orally infected/colonized with enteric bacterial pathogens (Salmonella Typhimurium or Escherichia coli O55) or with probiotic bacteria (E. coli Nissle 1917) for 24 h. The transcriptions of HMGB1, interleukin (IL)-8, tumor necrosis factor (TNF)-α, and IL-10 (quantitative reverse transcription and polymerase chain reaction), their protein levels (ELISA), and clinical state of the piglets (somnolence, anorexia, diarrhea, tachycardia, tachypnea, and tremor) were estimated. The piglets infected with enteric pathogens suffered from infections. HMGB1 was transcribed in the terminal ileum constitutively, regardless of any bacterial presence. In contrast, the transcription of cytokines was upregulated by virulent bacteria. HMGB1, IL-8, and TNF-α levels in the ileum were increased by both enteric pathogens, while IL-10 levels increased in E. coli O55-infected piglets only. HMGB1 significantly increased in the plasma of piglets infected with virulent E. coli only, but cytokine levels were in most cases increased by both virulent bacteria. HMGB1 and cytokine levels in ileum lavages and plasma of piglets colonized with probiotic E. coli remained comparable to those of the non-stimulated germ-free piglets. The local and systemic expression of HMGB1, its relationship to the inflammatory cytokines, and clinical findings showed HMGB1 as a suitable marker of severity of sepsis in the gnotobiotic piglet infection model.

Inflammasomes: Caspase-1-Activating Platforms with Critical Roles in Host Defense

Activation of the inflammatory cysteine protease caspase-1 in inflammasome complexes plays a critical role in the host response to microbial infections. Inflammasome activation induces inflammation through secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and through extracellular release of the alarmin high mobility group box 1. Moreover, caspase-1 activation by inflammasomes counters bacterial replication and induces pyroptosis, a specialized cell death program that removes infected immune cells as part of the host defense system. It is thus not surprising that bacterial and viral pathogens evolved virulence factors targeting inflammasome activation and activity. Here, we provide an overview of the distinct inflammasome complexes that are activated in a pathogen-specific manner and discuss the diverse strategies employed by viruses and bacteria to modulate inflammasome function.

Islet‐expressed TLR2 and TLR4 sense injury and mediate early graft failure after transplantation

Although islet transplantation is an effective treatment for Type 1 diabetes, primary engraftment failure contributes to suboptimal outcomes. We tested the hypothesis that islet isolation and transplantation activate innate immunity through TLR expressed on islets. Murine islets constitutively express TLR2 and TLR4, and TLR activation with peptidoglycan or LPS upregulates islet production of cytokines and chemokines. Following transplantation into streptozotocin-induced diabetic, syngeneic mice, islets exposed to LPS or peptidoglycan had primary graft failure with intra- and peri-islet mononuclear cell inflammation. The use of knockout mice showed that recipient CD8(+) T cells caused engraftment failure and did so in the absence of islet-derived DC. To mimic physiological islet injury, islets were transplanted with exocrine debris. Transplantation of TLR2/4(-/-) islets reduced proinflammatory cytokine production and improved islet survival. Stressed islets released the alarmin high-mobility group box protein 1 (HMGB1) and recombinant HMGB1 (rHMGB1) induced NFkB activation. NFkB activation was prevented in the absence of both TLR2 and TLR4. rHMGB1 pretreatment also prevented primary engraftment through a TLR2/4-dependent pathway. Our results show that islet graft failure can be initiated by TLR2 and TLR4 signaling and suggest that HMGB1 is one likely early mediator. Subsequent downstream signaling results in intra-islet inflammation followed by T-cell-mediated graft destruction.

HMGB1-Directed Drug Discovery Targeting Cutaneous Inflammatory Dysregulation

Extracellular cytokine function of the non-histone nuclear protein high-mobility group box 1 (HMGB1) has recently been recognized as an important drug target for novel anti-inflammatory therapeutics. Accumulating evidence supports the mechanistic involvement of the alarmin HMGB1 in skin response to microbial infection and ultraviolet-induced solar damage. Moreover, HMGB1 modulation of inflammatory signaling and tissue remodeling is now emerging as a causative factor in wound repair, autoimmune dysregulation, and skin carcinogenesis, representing cutaneous pathologies that affect large patient populations with unmet therapeutic needs. Recent structure-based drug discovery efforts have aimed at increasing the number of small molecule- and biologics-based prototype therapeutics targeting HMGB1. Small molecule drugs that may provide therapeutic benefit through HMGB1-directed mechanisms involve HMGB1 inhibitory ligands, Toll-like receptor antagonists, RAGE antagonists, alpha7 nicotinic acetylcholine receptor agonists, G2A antagonists, serine protease inhibitors, and alpha-dicarbonyl-based soft electrophiles. Using some of these agents, pharmacological modulation of HMGB1-associated cutaneous pathology has been achieved with an acceptable toxicity profile, and preclinical proof-of-concept experimentation has demonstrated feasibility of developing HMGB1-modulators into novel systemic and topical therapeutics that target cutaneous inflammatory dysregulation.

Effects of HMGB1 on in vitro responses of isolated muscle fibers and functional aspects in skeletal muscles of idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are heterogeneous rheumatic disorders of unknown cause characterized by muscle weakness, inflammatory cell infiltrates, and major histocompatibility complex (MHC) class I expression on muscle fibers. The nonhistone nuclear protein alarmin high-mobility group box 1 protein (HMGB1) has been detected extranuclearly in muscle biopsies from patients with IIMs. We hypothesize that HMGB1 has a central role in the cause of muscle weakness, particularly in the early phases of IIMs. Experiments were performed on skeletal muscle fibers isolated from adult mice, which were exposed to recombinant interferon (IFN)-gamma or HMGB1. The myoplasmic free [Ca(2+)] was measured. Stimulation with IFN-gamma resulted in increased HMGB1 expression in muscle nuclei and the myoplasm. Exposure to HMGB1 induced a reversible up-regulation of MHC class I in the muscle fibers. However, HMGB1 exposure caused an irreversible decrease in Ca(2+) release from the sarcoplasmic reticulum during fatigue, induced by repeated tetanic contractions. HMGB1 and MHC class I were frequently colocalized in the myoplasm of muscle fibers in muscle biopsies from patients with early IIMs. However, HMGB1-expressing fibers outnumbered fibers expressing MHC class I. Our data indicate that HMGB1 could be an early inducer of skeletal muscle dysfunction in IIMs.

Oxidation of the alarmin high-mobility group box 1 protein (HMGB1) during apoptosis

The architectural chromosomal protein high-mobility group box 1 protein (HMGB1) acts as an alarmin when released from cells. It is involved in the pathogenesis of inflammatory and autoimmune diseases. HMGB1 can undergo post-translational modifications including oxidation. However, the mechanisms and functional relevance of HMGB1 oxidation are not yet understood. Increased concentrations of reactive oxygen species (ROS) have been reported during apoptosis and necrosis. Hence, we investigated the oxidative status of HMGB1 in dead cells. Immunoblot analyses under reducing and non-reducing conditions revealed that HMGB1 is oxidized in dead cells. Moreover, tagging of oxidized cysteine residues by a maleimide moiety linked to polyethylene glycol showed that HMGB1 passively released from primary and secondary necrotic cells was predominantly oxidized. Also HMGB1 in plasma of patients with systemic lupus was reversibly oxidized. In conclusion, HMGB1 undergoes reversible oxidative modifications at cysteine residues during cell death, which may modulate its biological properties.