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1.  High Mobility Group Box Protein 1 (HMGB1)-Partner Molecule Complexes Enhance Cytokine Production by Signaling Through the Partner Molecule Receptor 
Molecular Medicine  2011;18(1):224-230.
The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain–containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain–containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.
doi:10.2119/molmed.2011.00327
PMCID: PMC3320135  PMID: 22076468
2.  Release of Neuronal HMGB1 by Ethanol through Decreased HDAC Activity Activates Brain Neuroimmune Signaling 
PLoS ONE  2014;9(2):e87915.
Neuroimmune gene induction is involved in many brain pathologies including addiction. Although increased expression of proinflammatory cytokines has been found in ethanol-treated mouse brain and rat brain slice cultures as well as in post-mortem human alcoholic brain, the mechanisms remain elusive. High-mobility group box 1 (HMGB1) protein is a nuclear protein that has endogenous cytokine-like activity. We previously found increased HMGB1 in post-mortem alcoholic human brain as well as in ethanol treated mice and rat brain slice cultures. The present study investigated the mechanisms for ethanol-induced release of HMGB1 and neuroimmune activation in a model of rat hippocampal-entorhinal cortex (HEC) brain slice cultures. Ethanol exposure triggered dose-dependent HMGB1 release, predominantly from neuronal cells. Inhibitors of histone deacetylases (HDACs) promoted nucleocytoplasmic mobilization of HDAC1/4 and HMGB1 resulting in increased total HMGB1 and acetylated HMGB1 release. Similarly, ethanol treatment was found to induce the translocation of HDAC1/4 and HMGB1 proteins from nuclear to cytosolic fractions. Furthermore, ethanol treatment reduced HDAC1/4 mRNA and increased acetylated HMGB1 release into the media. These results suggest decreased HDAC activity may be critical in regulating acetylated HMGB1 release from neurons in response to ethanol. Ethanol and HMGB1 treatment increased mRNA expression of proinflammatory cytokines TNFα and IL-1β as well as toll-like receptor 4 (TLR4). Targeting HMGB1 or microglial TLR4 by using siRNAs to HMGB1 and TLR4, HMGB1 neutralizing antibody, HMGB1 inhibitor glycyrrhizin and TLR4 antagonist as well as inhibitor of microglial activation all blocked ethanol-induced expression of proinflammatory cytokines TNFα and IL-1β. These results support the hypothesis that ethanol alters HDACs that regulate HMGB1 release and that danger signal HMGB1 as endogenous ligand for TLR4 mediates ethanol-induced brain neuroimmune signaling through activation of microglial TLR4. These findings provide new therapeutic targets for brain neuroimmune activation and alcoholism.
doi:10.1371/journal.pone.0087915
PMCID: PMC3925099  PMID: 24551070
3.  Intravenous immunoglobulin (IVIg) dampens neuronal toll-like receptor-mediated responses in ischemia 
Background
Ischemic stroke causes a high rate of deaths and permanent neurological damage in survivors. Ischemic stroke triggers the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1), which activate toll-like receptors (TLRs) and receptor for advanced glycation endproducts (RAGE) in the affected area, leading to an exaggerated inflammatory response and cell death. Both TLRs and RAGE are transmembrane pattern recognition receptors (PRRs) that have been shown to contribute to ischemic stroke-induced brain injury. Intravenous immunoglobulin (IVIg) preparations obtained by fractionating human blood plasma are increasingly being used as an effective therapeutic agent in the treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke has been proposed, but little is known about the direct neuroprotective mechanisms of IVIg. We therefore investigate whether IVIg exerts its beneficial effects on the outcome of neuronal injury by modulating HMGB1-induced TLR and RAGE expressions and activations.
Methods
Primary cortical neurons were subjected to glucose deprivation or oxygen and glucose deprivation conditions and treated with IVIg and recombinant HMGB1. C57/BL6J mice were subjected to middle cerebral artery occlusion, followed by reperfusion, and IVIg was administered intravenously 3 h after the start of reperfusion. Expression of TLRs, RAGE and downstream signalling proteins in neurons and brain tissues were evaluated by immunoblot.
Results
Treatment of cultured neurons with IVIg reduced simulated ischemia-induced TLR2, TLR4, TLR8 and RAGE expressions, pro-apoptotic caspase-3 cleavage and phosphorylation of the cell death-associated kinases such as c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) as well as the p65 subunit of nuclear factor kappa B (NF-κB). These results were recapitulated in an in vivo model of stroke. IVIg treatment also upregulated the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) in cortical neurons under ischemic conditions. Finally, IVIg protected neurons against HMGB1-induced neuronal cell death by modulating TLR and RAGE expressions and signalling pathways.
Conclusions
Taken together, these results provide a rationale for the potential use of IVIg to target inappropriately activated components of the innate immune system following ischemic stroke.
doi:10.1186/s12974-015-0294-8
PMCID: PMC4409750  PMID: 25886362
TLRs; IVIg; Neuronal death; HMGB1; Ischemic stroke
4.  HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression 
PLoS Medicine  2009;6(1):e1000010.
Background
Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.
Methods and Findings
Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4+ and CD8+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.
Conclusions
Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.
Maria Castro and colleagues use cell line and transgenic mouse approaches to study the mechanisms underlying the immune response to glioblastoma multiforme.
Editors' Summary
Background.
Every year, more than 175,000 people develop a primary brain tumor (a cancer that starts in the brain rather than spreading in from elsewhere). Like all cancers, brain tumors develop when a cell acquires genetic changes that allow it to grow uncontrollably and that change other aspects of its behavior, including the proteins it makes. There are many different types of cells in the brain and, as a result, there are many different types of brain tumors. However, one in five primary brain tumors is glioblastoma multiforme (GBM; also known as grade 4 astrocytoma), a particularly aggressive cancer. With GBM, the average time from diagnosis to death is one year and only one person in 20 survives for five years after a diagnosis of GBM. Symptoms of GBM include headaches, seizures, and changes in memory, mood, or mental capacity. Treatments for GBM, which include surgery, radiotherapy, and chemotherapy, do not “cure” the tumor but they can ease these symptoms.
Why Was This Study Done?
Better treatments for GBM are badly needed, and one avenue that is being explored is immunotherapy—a treatment in which the immune system is used to fight the cancer. Because many tumors make unusual proteins, the immune system can sometimes be encouraged to recognize tumor cells as foreign invaders and kill them. Unfortunately, attempts to induce a clinically useful anti-GBM immune response have been unsuccessful, partly because the brain contains very few dendritic cells, a type of immune system cell that kick-starts effective immune responses by presenting foreign proteins to other immune system cells. Another barrier to immunotherapy for GBM is immune evasion by the tumor. Many tumors develop ways to avoid the immune response as they grow. For example, they sometimes reduce the expression of proteins that the immune system might recognize as foreign. In this study, the researchers test a new combined treatment strategy for GBM in which dendritic cells are encouraged to enter the brain and tumor cells are killed to release proteins capable of stimulating an effective antitumor immune response.
What Did the Researchers Do and Find?
The researchers first established brain tumors in mice. Then, they injected harmless viruses carrying the genes for Fms-like tyrosine kinase 3 ligand (Ftl3L; a protein that attracts dendritic cells) and for thymidine kinase (TK; cells expressing TK are killed by a drug called gancyclovir) into the tumor. Expression of both Flt3L and TK (but not of either protein alone) plus gancyclovir treatment shrank the tumors and greatly improved the survival of the mice. The researchers show that their strategy increased the migration of dendritic cells into the tumor provided they expressed an immune system protein called Toll-like receptor 2 (TLR2). TLR2 expression on the dendritic cells was also needed for an effective anti-tumor immune response and for tumor regression. TLR2 normally activates dendritic cells by binding to specific proteins on invading pathogens, so what was TLR2 binding to in the mouse tumors? The researchers reveal that TLR2 was responding to high-mobility-group box 1 (HMGB1), a protein released by the dying tumor cells by showing that treatment of the tumor-bearing mice with the HMGB1 inhibitor glycyrrhizin blocked the therapeutic effect of Flt3L/TK expression. Finally, the researchers report that other tumor cell types release HMGB1 when they are killed and that the Flt3L/TK expression strategy can also kill other tumors growing in mouse brains.
What Do These Findings Mean?
Results obtained in mouse models of human diseases do not always lead to effective treatments for human patients. Nevertheless, the findings of this study provide new insights into how an effective immune response against brain tumors might be brought about. Most importantly, they show that an effective strategy might need to both attract dendritic cells into the brain tumor and to kill tumor cells, so they release proteins that can activate the dendritic cells. That is, the authors suggest it's important to combine immunotherapies with tumor-killing strategies to provide effective treatments for primary and metastatic brain tumors
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000010.
The US National Cancer Institute provides information about brain tumors for patients and health professionals and about the the immune system and how it can be harnessed to fight cancer (in English and Spanish)
Cancer Research UK provides information on all aspects of brain tumors for patients and their caregivers
MedlinePlus provides links to further information about brain cancer, (including some links to information in Spanish)
The American Brain Tumor Association provides brain tumor resources and information
The National Brain Tumor Society provides educational and support services regarding brain tumors
doi:10.1371/journal.pmed.1000010
PMCID: PMC2621261  PMID: 19143470
5.  Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis 
Xu, J | Jiang, Y | Wang, J | Shi, X | Liu, Q | Liu, Z | Li, Y | Scott, M J | Xiao, G | Li, S | Fan, L | Billiar, T R | Wilson, M A | Fan, J
Cell Death and Differentiation  2014;21(8):1229-1239.
Macrophages can be activated and regulated by high-mobility group box 1 (HMGB1), a highly conserved nuclear protein. Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors, including the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, TLR4, and TLR9. Pyroptosis is a caspase-1-dependent programmed cell death, which features rapid plasma membrane rupture, DNA fragmentation, and release of proinflammatory intracellular contents. Pyroptosis can be triggered by various stimuli, however, the mechanism underlying pyroptosis remains unclear. In this study, we identify a novel pathway of HMGB1-induced macrophage pyroptosis. We demonstrate that HMGB1, acting through RAGE and dynamin-dependent signaling, initiates HMGB1endocytosis, which in turn induces cell pyroptosis. The endocytosis of HMGB1 triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. We further confirm that HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for this form of pyroptosis in the development of inflammation. These findings shed light on the regulatory role of ligand-receptor internalization in directing cell fate, which may have an important role in the progress of inflammation following infection and injury.
doi:10.1038/cdd.2014.40
PMCID: PMC4085529  PMID: 24769733
6.  HMGB1/TLR Receptor Danger Signaling Increases Brain Neuroimmune Activation in Alcohol Dependence 
Biological psychiatry  2012;73(7):602-612.
Background
Innate immune gene expression is regulated in part through high mobility group box 1(HMGB1), an endogenous proinflammatory cytokine, that activates multiple members of the interleukin-1/Toll-like receptor (IL-1/TLR) family associated with danger signaling. We investigated expression of HMGB1, TLR2, TLR3 and TLR4 in chronic ethanol treated mouse brain, post-mortem human alcoholic brain, and rat brain slice culture to test the hypothesis that neuroimmune activation in alcoholic brain involves ethanol activation of HMGB1/TLR danger signaling.
Methods
Protein levels were assessed using Western blot, ELISA, immunohistochemical immunoreactivity (+IR), and mRNA levels were measured by real time PCR in ethanol-treated mice (5 g/kg/day, i.g., 10 days + 24 hr), rat brain slice culture, and post-mortem human alcoholic brain.
Results
Ethanol treatment of mice increased brain mRNA and +IR protein expression of HMGB1, TLR2, TLR3, and TLR4. Post-mortem human alcoholic brain also showed increased HMGB1, TLR2, TLR3, and TLR4+IR cells that correlated with lifetime alcohol consumption as well as each other. Ethanol treatment of brain slice culture released HMGB1 into the media and induced the proinflammatory cytokine, IL-1β. Neutralizing antibodies to HMGB1 and small inhibitory mRNA to HMGB1 or TLR4 blunted ethanol induction of IL-1β.
Conclusions
Ethanol-induced HMGB1/TLR signaling contributes to induction of the proinflammatory cytokine, IL-1β. Increased expression of HMGB1, TLR2, TLR3, and TLR4 in alcoholic brain and in mice treated with ethanol suggests that chronic alcohol-induced brain neuroimmune activation occurs through HMGB1/TLR signaling.
doi:10.1016/j.biopsych.2012.09.030
PMCID: PMC3602398  PMID: 23206318
alcoholism; Toll-like receptors; ethanol; cytokines; interleukin-1; cerebral cortex
7.  HMGB1 and TLR4 mediate skeletal muscle recovery in a murine model of hindlimb ischemia 
Journal of vascular surgery  2013;58(2):460-469.
Introduction
We have previously shown that the danger signal High Mobility Group Box 1 (HMGB1) promotes angiogenesis when administered to ischemic muscle. HMGB1 signals through Toll-like receptor 4 (TLR4) as well as the receptor for advanced glycation end-products (RAGE). However, the actions of these receptors in ischemic injury and muscle recovery are not known. We hypothesize that TLR4 mediates tissue recovery and angiogenesis in response to ischemia.
Methods
Femoral artery ligation was performed in control, TLR4 competent (C3H/HeOuJ), and incompetent (C3H/HeJ) mice, as well as RAGE knockout mice and their C57B6 control counterparts. In other experiments, control mice were pretreated with anti-HMGBI neutralizing antibody before femoral artery ligation. After two weeks, limb perfusion was evaluated using laser Doppler perfusion imaging (LDPI) and reported as the ratio of blood flow in the ischemic to nonischemic limb. Muscle necrosis, fat replacement, and vascular density in the anterior tibialis muscle were quantified histologically. In vitro, TLR4 and RAGE expression was evaluated in human dermal microvascular endothelial cells (HDMVECs) in response to hypoxia. HDMVECs treated with HMGB1 alone and in the presence of anti-TLR4 antibody were probed for phosphorylated ERK (p-ERK), a signaling molecule critical to EC angiogenic behavior.
Results
Both anti-HMGB1 antibody as well as defective TLR4 signaling in HeJ mice resulted in prominent muscle necrosis two weeks after femoral artery ligation. Control HeOuJ mice had less necrosis than TLR4 incompetent HeJ mice, but a greater amount of fat replacement. In contrast to control C3H mice, control C57B6 mice demonstrated prominent muscle regeneration with very little necrosis. Muscle regeneration was not dependent on RAGE. While vascular density did not differ between strains, mice with intact RAGE and TLR4 signaling had less blood flow in ischemic limbs compared to mutant strains. In vitro, EC TLR4 expression increased in response to hypoxia while TLR4 antagonism decreased HMGB1-induced activation of ERK.
Conclusion
Both HMGB1 and TLR4 protect against muscle necrosis after hindlimb ischemia. However, muscle regeneration does not appear to be tied to vascular density. HMGB1 likely activates angiogenic behavior in EC in vitro, and this activation may be modulated by TLR4. The improvement in blood flow seen in mice with absent TLR4 and RAGE signaling may suggest anti-angiogenic roles for both receptors, or vasoconstriction induced by TLR4 and RAGE mediated inflammatory pathways.
doi:10.1016/j.jvs.2012.11.071
PMCID: PMC3657591  PMID: 23414695
8.  Stepwise Release of Biologically Active HMGB1 during HSV-2 Infection 
PLoS ONE  2011;6(1):e16145.
Background
High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells.
Methodology/Principal Findings
Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data.
Conclusions
These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection.
doi:10.1371/journal.pone.0016145
PMCID: PMC3023802  PMID: 21283827
9.  High-Mobility Group Box 1 Inhibits Gastric Ulcer Healing through Toll-Like Receptor 4 and Receptor for Advanced Glycation End Products 
PLoS ONE  2013;8(11):e80130.
High-mobility group box 1 (HMGB1) was initially discovered as a nuclear protein that interacts with DNA as a chromatin-associated non-histone protein to stabilize nucleosomes and to regulate the transcription of many genes in the nucleus. Once leaked or actively secreted into the extracellular environment, HMGB1 activates inflammatory pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 2, TLR4, and receptor for advanced glycation end products (RAGE), leading to tissue injury. Although HMGB1’s ability to induce inflammation has been well documented, no studies have examined the role of HMGB1 in wound healing in the gastrointestinal field. The aim of this study was to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1’s effects on ulcer healing. Gastric ulcers were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation. The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and the expression of tumor necrosis factor α (TNFα) mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity and TNFα expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO activity and TNFα expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNFα, whereas ulcer healing in TLR2 knockout (KO) mice was similar to that in wild-type mice. In TLR4 KO and RAGE KO mice, exogenous HMGB1 did not affect ulcer healing and TNFα expression. Thus, we showed that HMGB1 is a complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive inflammatory responses.
doi:10.1371/journal.pone.0080130
PMCID: PMC3823709  PMID: 24244627
10.  High-mobility group box-1 impairs memory in mice through both toll-like receptor 4 and Receptor for Advanced Glycation End Products 
Experimental neurology  2011;232(2):143-148.
High-mobility group box-1 (HMGB1) is a nuclear protein with cytokine-type functions upon its extracellular release. HMGB1 activates inflammatory pathways by stimulating multiple receptors, chiefly toll-like receptor 4 (TLR4) and Receptor for Advanced Glycation End Products (RAGE). TLR4 and RAGE activation has been implicated in memory impairments, although the endogenous ligand subserving these effects is unknown. We examined whether HMGB1 induced memory deficits using novel object recognition test, and which of the two receptor pathways was involved in these effects. Non-spatial long-term memory was examined in wild type, TLR4 knockout, and RAGE knockout mice. Recombinant HMGB1 (10 μg, intracerebroventricularly, i.c.v.) disrupted memory encoding equipotently in wild type, TLR4 knockout and RAGE knockout animals, but affected neither memory consolidation, nor retrieval. Neither TLR4 knockout nor RAGE knockout mice per se, exhibited memory deficits. Blockade of TLR4 in RAGE knockout mice using Rhodobacter sphaeroides lipopolysaccharide (LPS-Rs; 20 μg, i.c.v.) prevented the detrimental effect of HMGB1 on memory. These data show that elevated brain levels of HMGB1 induce memory abnormalities which may be mediated by either TLR4, or RAGE. This mechanism may contribute to memory deficits under various neurological and psychiatric conditions associated with the increased HMGB1 levels, such as epilepsy, Alzeheimer’s disease and stroke.
doi:10.1016/j.expneurol.2011.08.012
PMCID: PMC3202022  PMID: 21884699
Inflammation; High-mobility group box-1; toll-like receptor 4; Receptor for Advanced Glycation End Products; memory; novel object recognition test
11.  Toll-Like Receptor 4 Mediates Acute Lung Injury Induced by High Mobility Group Box-1 
PLoS ONE  2013;8(5):e64375.
Background
Acute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process.
Methods
Recombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo.
Results
Features of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro.
Conclusion
HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.
doi:10.1371/journal.pone.0064375
PMCID: PMC3656835  PMID: 23691208
12.  Overexpression of HMGB1 A-box reduced lipopolysaccharide-induced intestinal inflammation via HMGB1/TLR4 signaling in vitro 
AIM: To investigate the inhibitory effects and mechanism of high mobility group box (HMGB)1 A-box in lipopolysaccharide (LPS)-induced intestinal inflammation.
METHODS: Overexpression of HMGB1 A-box in human intestinal epithelial cell lines (SW480 cells) was achieved using the plasmid pEGFP-N1. HMGB1 A-box-overexpressing SW480 cells were stimulated with LPS and co-culturing with human monocyte-like cell lines (THP-1 cells) using a Transwell system, compared with another HMGB1 inhibitor ethyl pyruvate (EP). The mRNA and protein levels of HMGB1/toll-like receptor (TLR) 4 signaling pathways [including HMGB1, TLR4, myeloid differentiation factor88 (MYD88), Phosphorylated Nuclear Factor κB (pNF-κB) p65] in the stimulated cells were determined by real-time polymerase chain reaction and Western blotting. The levels of the proinflammatory mediators [including HMGB1, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α] in the supernatants of the stimulated cells were determined by ELISA.
RESULTS: EP downregulated the mRNA and protein levels of HMGB1, inhibited the TLR4 signaling pathways (TLR4, MYD88 and pNF-κB p65) and reduced the secretion of proinflammatory mediators (HMGB1, IL-1β, IL-6 and TNF-α) in the SW480 and THP-1 cells activated by LPS but not in the unstimulated cells. Activated by LPS, the overexpression of HMGB1 A-box in the SW480 cells also inhibited the HMGB1/TLR4 signaling pathways and reduced the secretion of these proinflammatory mediators in the THP-1 cells but not in the transfected and unstimulated cells.
CONCLUSION: HMGB1 A-box, not only EP, can reduce LPS-induced intestinal inflammation through inhibition of the HMGB1/TLR4 signaling pathways.
doi:10.3748/wjg.v21.i25.7764
PMCID: PMC4491963  PMID: 26167076
High mobility group box 1; Toll-like receptor 4; HMGB1 A-box; Ethyl pyruvate; Inflammatory bowel disease
13.  NIASPAN REDUCES HIGH-MOBILITY GROUP BOX 1/RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS AFTER STROKE IN TYPE-1 DIABETIC RATS 
Neuroscience  2011;190:339-345.
Objective
High-mobility group box 1 (HMGB1), an active receptor for advanced glycation endproducts (RAGE), functions as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of vasculature. Diabetes mellitus (DM) is associated with accelerated development of both microvascular and macrovascular disease and increases the risk of ischemic stroke. Using a model of streptozotocin-induced type-1 diabetes (T1DM) in rats, we investigated the changes in HMGB and RAGE and tested the effects of Niaspan, a slow release form of niacin, on the expression of pro-inflammatory proteins in rats after stroke.
Research design and methods
T1DM rats were subjected to transient middle cerebral artery occlusion (MCAo) and treated without or with Niaspan (40 mg/kg) daily for 14 days after MCAo. Non-streptozotocin rats (WT) were also subjected to MCAo. Immunostaining for inflammatory mediators including HMGB1, RAGE, matrix metalloproteinase-9 (MMP-9) and toll-like receptor 4 (TLR4) immunostaining (n=8/group) and Western blotting (n=4/group) were performed.
Results
Compared to WT-MCAo rats, T1DM-MCAo rats showed an increased expression of HMGB1 (0.82±0.07 vs. 1.81±0.98, P<0.05), RAGE (1.31±0.22 vs. 3.77±0.72, P<0.05), MMP-9 (0.74±0.08 vs. 1.61±0.09, P<0.05) and TLR4 (2.85±0.22 vs. 6.72±0.44, P<0.05) after stroke. Niaspan treatment significantly attenuated the expression of HMGB1 (1.80±0.98 vs. 1.31±0.01, P<0.05), RAGE (3.77±0.71 vs. 1.78±0.45, P<0.05), MMP-9 (1.61±0.09 vs. 0.97±0.07, P<0.05) and TLR4 (6.72±0.44 vs. 2.28±0.43, P<0.05) in the ischemic brain in T1DM-MCAo rats.
Conclusions
T1DM increases HMGB1/RAGE, TLR4 and MMP-9 expression after stroke. Niaspan treatment of stroke in T1DM rats inhibits HMGB1/RAGE, TLR4 and MMP-9 expression which may contribute to the reduced inflammatory response after stroke in T1DM rats.
doi:10.1016/j.neuroscience.2011.06.004
PMCID: PMC3260534  PMID: 21683770
type-1 diabetes rats; stroke; RAGE; Niaspan; HMGB1; MMP-9
14.  Tanshinone IIA sodium sulfonate facilitates endocytic HMGB1 uptake 
Biochemical pharmacology  2012;84(11):1492-1500.
Our seminal discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation has prompted a new field of investigation for the development of experimental therapeutics. We previously reported that a major Danshen ingredient, tanshinone IIA sodium sulfonate (TSN-SS), selectively inhibited endotoxin-induced HMGB1 release and conferred protection against lethal endotoxemia and sepsis. To investigate the underlying mechanisms by which TSN-SS effectively inhibits HMGB1 release, we examined whether TSN-SS stimulates HMGB1 uptake by macrophages and whether genetic depletion of HMGB1 receptors [e.g., toll-like receptors (TLR)2, TLR4, or the receptor for advanced glycation end product (RAGE)] or pharmacological inhibition of endocytosis impairs TSN-SS-facilitated HMGB1 cellular uptake. TSN-SS stimulated internalization of exogenous HMGB1 protein into macrophage cytoplasmic vesicles that subsequently co-localized with microtubule-associated protein light chain 3 (LC3)-positive punctate structures (likely amphisomes). Meanwhile, it time-dependently elevated cellular levels of internalized HMGB1, leading to elevated LC3-II production and aggregation. Although genetic depletion of TLR2, TLR4, and/or RAGE did not impair TSN-SS-mediated HMGB1 uptake, specific inhibitors of the clathrin- and caveolin-dependent endocytosis significantly impaired TSN-SS-mediated HMGB1 uptake. Co-treatment with a lysosomal inhibitor, bafilomycin A1, led to enhanced accumulation of endogenous LC3-II and internalized exogenous HMGB1 in TSN-SS/rHMGB1-treated macrophages. Taken together, these findings suggest that TSN-SS may facilitate HMGB1 endocytic uptake, and subsequently delivered it to LC3-positive vacuoles (possibly amphisomes) for degradation via a lysosome-dependent pathway.
doi:10.1016/j.bcp.2012.09.015
PMCID: PMC3491099  PMID: 23022229
15.  Poly(ADP-Ribosyl)ation of High Mobility Group Box 1 (HMGB1) Protein Enhances Inhibition of Efferocytosis 
Molecular Medicine  2011;18(1):359-369.
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.
doi:10.2119/molmed.2011.00203
PMCID: PMC3356430  PMID: 22204001
16.  High-mobility group protein box-1 and its relevance to cerebral ischemia 
High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant, nonhistone DNA-binding protein. It has well-established functions in the maintenance of nuclear homeostasis. The HMGB1 can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatory signals. Extracellular HMGB1 interacts with receptors, including those for advanced glycation endproducts (RAGEs) as well as Toll-like receptor 2 (TLR2) and TLR4. The HMGB1 functions in a synergistic manner with other proinflammatory mediators and acts as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Numerous reports point to HMGB1 as a novel player in the ischemic brain. This review provides an appraisal of the emerging roles of HMGB1 in cerebral ischemia injury, highlighting the relevance of HMGB1-blocking agents as potent therapeutic tools for neuroprotection.
doi:10.1038/jcbfm.2009.202
PMCID: PMC2949127  PMID: 19794402
cerebral ischemia; HMGB1; inflammation; RAGE; TLR
17.  High-mobility group box protein 1 (HMGB1): an alarmin mediating the pathogenesis of rheumatic disease 
High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. To function as an alarmin, HMGB1 translocates from the nucleus of the cell to the extra-cellular milieu, a process that can take place with cell activation as well as cell death. HMGB1 can interact with receptors that include RAGE (receptor for advanced glycation endproducts) as well as Toll-like receptor-2 (TLR-2) and TLR-4 and function in a synergistic fashion with other proinflammatory mediators to induce responses. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. New approaches to therapy for these diseases may involve strategies to inhibit HMGB1 release from cells, its interaction with receptors, and downstream signaling.
doi:10.1186/ar2440
PMCID: PMC2483460  PMID: 18598385
18.  Toll-like receptor 4 on islet β cells senses expression changes in high-mobility group box 1 and contributes to the initiation of type 1 diabetes 
Experimental & Molecular Medicine  2012;44(4):260-267.
Type 1 diabetes mellitus is caused by the autoimmune destruction of β cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were β cells; few α cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on β cells and that HMGB1 may signal via TLR4 to selectively damage β cells rather than α cells during the development of type 1 diabetes mellitus.
doi:10.3858/emm.2012.44.4.021
PMCID: PMC3349908  PMID: 22217446
diabetes mellitus, type 1; HMGB1 protein; islets of Langerhans; mice, inbred NOD; toll-like receptor 4
19.  Increased Receptor for Advanced Glycation End Product Expression in the Human Alcoholic Prefrontal Cortex is Linked to Adolescent Drinking 
Neurobiology of disease  2013;59:52-62.
Adolescence is characterized behaviorally by increased impulsivity and risk-taking that declines in parallel with maturation of the prefrontal cortex and executive function. In the brain, the receptor for advanced glycation end products (RAGE) is critically involved in neurodevelopment and neuropathology. In humans, the risk of alcoholism is greatly increased in those who begin drinking between 13 and 15 years of age, and adolescents binge drink more than any other age group. We have previously found that alcoholism is associated with increased expression of neuroimmune genes. This manuscript tested the hypothesis that adolescent binge drinking upregulates RAGE and Toll-like receptor (TLR) 4 as well as their endogenous agonist, high-mobility group box 1 (HMGB1). Immunohistochemistry, Western blot, and mRNA analyses found that RAGE expression was increased in the human post-mortem alcoholic orbitofrontal cortex (OFC). Further, an earlier age of drinking onset correlated with increased expression of RAGE, TLR4, and HMGB1. To determine if alcohol contributed to these changes, we used an adolescent binge ethanol model in rats (5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) and assessed neuroimmune gene expression. We found an age-associated decline of RAGE expression from late adolescence (P56) to young adulthood (P80). Adolescent intermittent ethanol exposure did not alter RAGE expression at P56, but increased RAGE in the young adult PFC (P80). Adolescent intermittent ethanol exposure also increased TLR4 and HMGB1 expression at P56 that persisted into young adulthood (P80). Assessment of young adult frontal cortex mRNA (RT-PCR) found increased expression of proinflammatory cytokines, oxidases, and neuroimmune agonists at P80, 25 days after ethanol treatment. Together, these human and animal data support the hypothesis that an early age of drinking onset upregulates RAGE/TLR4-HMGB1 and other neuroimmune genes that persist into young adulthood and could contribute to risk of alcoholism or other brain diseases associated with neuroinflammation.
doi:10.1016/j.nbd.2013.07.002
PMCID: PMC3775891  PMID: 23867237
Ethanol; neuroimmune; innate immunity; cytokine; adolescence; alcoholism; Toll-like receptor; HMGB1; binge drinking
20.  Extracellular High-Mobility Group Box 1 Protein (HMGB1) as a Mediator of Persistent Pain 
Molecular Medicine  2015;20(1):569-578.
Although originally described as a highly conserved nuclear protein, high-mobility group box 1 protein (HMGB1) has emerged as a danger-associated molecular pattern molecule protein (DAMP) and is a mediator of innate and specific immune responses. HMGB1 is passively or actively released in response to infection, injury and cellular stress, providing chemotactic and cytokine-like functions in the extracellular environment, where it interacts with receptors such as receptor for advanced glycation end products (RAGE) and several Toll-like receptors (TLRs). Although HMGB1 was first revealed as a key mediator of sepsis, it also contributes to a number of other conditions and disease processes. Chronic pain arises as a direct consequence of injury, inflammation or diseases affecting the somatosensory system and can be devastating for the affected patients. Emerging data indicate that HMGB1 is also involved in the pathology of persistent pain. Here, we give an overview of HMGB1 as a proinflammatory mediator, focusing particularly on the role of HMGB1 in the induction and maintenance of hypersensitivity in experimental models of pain and discuss the therapeutic potential of targeting HMGB1 in conditions of chronic pain.
doi:10.2119/molmed.2014.00176
PMCID: PMC4365060  PMID: 25222915
21.  High mobility group box 1 contributes to anti-neutrophil cytoplasmic antibody-induced neutrophils activation through receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 
Introduction
High mobility group box-1 (HMGB1), a typical damage-associated molecular pattern (DAMP) protein, is associated with inflammatory conditions and tissue damage. Our recent study found that circulating HMGB1 levels could reflect the disease activity of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate whether HMGB1 participated in ANCA-induced neutrophil activation, which is one of the most important pathogenic aspects in the development of AAV.
Methods
The various effects of HMGB1 in ANCA-induced neutrophil activation were measured. Antagonists for relevant receptors and signaling molecules were employed.
Results
ANCA antigens translocation on neutrophils primed with HMGB1 was significantly higher than non-primed neutrophils. The levels of respiratory burst and degranulation increased significantly in HMGB1-primed neutrophils activated with ANCA-positive IgG, as compared with non-primed neutrophils. Furthermore, blocking Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE), rather than TLR2, resulted in a significant decrease in HMGB1-induced ANCA antigens translocation, respiratory burst and degranulation. Similar effects were also found when blocking MyD88 and NF-κB.
Conclusions
HMGB1 could prime neutrophils by increasing ANCA antigens translocation, and the primed neutrophils could be further induced by ANCA, resulting in the respiratory burst and degranulation. This process is TLR4- and RAGE-dependent through the MyD88/NF-κB pathway.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0587-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-015-0587-4
PMCID: PMC4382936  PMID: 25889374
22.  Preconditioning with high mobility group box 1 (HMGB1) induces lipoteichoic acid (LTA) tolerance 
High mobility group box chromosomal protein 1 (HMGB1) is a DNA binding protein that exhibits pro-inflammatory properties when present in the extracellular compartment. Putative receptors for HMGB1 include Toll-like receptor (TLR) 4, TLR2 and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce tolerance to the bacterial product, lipoteichoic acid (LTA). Pretreatment of human monocyte-like THP-1 cells with 1 μg/ml HMGB1 18 h before exposure to LTA (10 μg/ml) decreased secretion of TNF, NF-κB DNA-binding, and degradation of IκBα. Denaturation of HMGB1 with boiling water or co-incubation with anti-HMGB1 antibody abrogated the induction of tolerance to LTA. In contrast, co-incubation with polymyxin B failed to diminish HMGB1-induced tolerance to LTA. These findings support the view that the induction of LTA tolerance by HMGB1 was not due to LPS contamination. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type and RAGE-deficient mice became LTA-tolerant following HMGB1 exposure ex vivo. We were unable to demonstrate LTA tolerance in TLR2 and TLR4-deficient macrophages, as they are hyporesponsive to LTA. These findings suggest that extracellular HMGB1 induces LTA tolerance, and RAGE receptor is not required for this induction.
doi:10.1097/CJI.0b013e3181dcd111
PMCID: PMC2954270  PMID: 20664360
High mobility group box chromosomal protein 1 (HMGB1); Lipoteichoic acid; tolerance; signal transduction; Toll-like receptor -2
23.  Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling 
Molecular Medicine  2015;20(1):639-648.
Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1–TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.
doi:10.2119/molmed.2014.00076
PMCID: PMC4365061  PMID: 25375408
24.  Inhibitor of NF-κB Kinases α and β Are Both Essential for High Mobility Group Box 1-Mediated Chemotaxis 
Inhibitor of NF-κB kinases β (IKKβ) and α (IKKα) activate distinct NF-κB signaling modules. The IKKβ/canonical NF-κB pathway rapidly responds to stress-like conditions, whereas the IKKα/noncanonical pathway controls adaptive immunity. Moreover, IKKα can attenuate IKKβ-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKα and IKKβ are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKα and IKKβ are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKα-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKα and IKKβ, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKβ, but not IKKα, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKβ target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKβ, but not IKKα, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-κB signaling pathways.
doi:10.4049/jimmunol.0903131
PMCID: PMC2915896  PMID: 20231695
25.  Novel insights for high mobility group box 1 protein-mediated cellular immune response in sepsis: A systemic review 
BACKGROUND:
High mobility group box 1 protein (HMGB1) is a highly conserved, ubiquitous protein in the nuclei and cytoplasm of nearly all cell types. HMGB1 is secreted into the extracellular milieu and acts as a proinfl ammatory cytokine. In this article we reviewed briefl y the cellular immune response mediated by HMGB1 in infl ammation and sepsis.
METHODS:
This systemic review is mainly based on our own work and other related reports.
RESULTS:
HMGB1 can actively affect the immune functions of many types of cells including T lymphocytes, regulatory T cells (Tregs), dendritic cells (DCs), macrophages, and natural killer cells (NK cells). Various cellular responses can be mediated by HMGB1 which binds to cell-surface receptors [e.g., the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, and TLR4]. Anti-HMGB1 treatment, such as anti-HMGB1 polyclonal or monoclonal antibodies, inhibitors (e.g., ethyl pyruvate) and antagonists (e.g., A box), can protect against sepsis lethality and give a wider window for the treatment opportunity.
CONCLUSION:
HMGB1 is an attractive target for the development of new therapeutic strategies in the treatment of patients with septic complications.
doi:10.5847/wjem.j.issn.1920-8642.2012.03.001
PMCID: PMC4129786  PMID: 25215057
High mobility group box 1 protein; Sepsis; Immunological effect; Cytokine; Signal transduction

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