Blockade Of High-mobility Group Box 1 Research Articles

IL-33 and HMGB1 modulate the progression of EAE via oppositely regulating each other

Interleukin-33 (IL-33) and high mobility group box 1 (HMGB1) have been reported to play crucial and distinct roles in experimental autoimmune encephalomyelitis (EAE). However, little is known about their interaction in the progression of EAE. In this study, the dynamic expression and release of IL-33 and HMGB1 in different stages of EAE in vivo, and their interaction in vitro were explored. We found that HMGB1 was dominant in pre-onset stage of EAE, while IL-33 was dominant in peak stage. Moreover, both blockade of extracellular HMGB1 in the central nervous system (CNS) and conditional knockout of HMGB1 in astrocytes decreased IL-33 release. HMGB1 promoted the release of IL-33, while IL-33 reduced the release of HMGB1 from primary astrocytes in vitro. Taken together, IL-33 and HMGB1 in the CNS jointly participate in the EAE progression and the inhibitory effect of IL-33 on HMGB1 may be involved in the self-limiting of EAE.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

CXCL12/CXCR4 Axis is Involved in the Recruitment of NK Cells by HMGB1 Contributing to Persistent Airway Inflammation and AHR During the Late Stage of RSV Infection.

We previously showed that both high-mobility group box-1 (HMGB1) and natural killer (NK) cells contribute to respiratory syncytial virus (RSV)-induced persistent airway inflammation and airway hyperresponsiveness (AHR). Meanwhile, Chemokine (C-X-C motif) ligand 12 (CXCL12) and its specific receptor (chemokine receptor 4, CXCR4) play important roles in recruitment of immune cells. CXCL12 has been reported to form a complex with HMGB1 that binds to CXCR4 and increases inflammatory cell migration. The relationship between HMGB1, NK cells and chemokines in RSV-infected model remains unclear. An anti-HMGB1 neutralizing antibody and inhibitor of CXCR4 (AMD3100) was administered to observe changes of NK cells and airway disorders in nude mice and BALB/c mice. Results showed that the mRNA expression and protein levels of HMGB1 were elevated in late stage of RSV infection and persistent airway inflammation and AHR were diminished after administration of anti-HMGB1 antibodies, with an associated significant decrease in CXCR4+ NK cells. In addition, CXCL12 and CXCR4 were reduced after HMGB1 blockade. Treatment with AMD3100 significantly suppressed the recruitment of NK cells and alleviated the airway disorders. Thus, CXCL12/CXCR4 axis is involved in the recruitment of NK cells by HMGB1, contributing to persistent airway inflammation and AHR during the late stage of RSV infection.

Tubular Epithelial Cell HMGB1 Promotes AKI-CKD Transition by Sensitizing Cycling Tubular Cells to Oxidative Stress: A Rationale for Targeting HMGB1 during AKI Recovery.

Cells undergoing necrosis release extracellular high mobility group box (HMGB)-1, which triggers sterile inflammation upon AKI in mice. Neither deletion of HMGB1 from tubular epithelial cells, nor HMGB1 antagonism with small molecules, affects initial ischemic tubular necrosis and immediate GFR loss upon unilateral ischemia/reperfusion injury (IRI). On the contrary, tubular cell-specific HMGB1 deficiency, and even late-onset pharmacological HMGB1 inhibition, increased functional and structural recovery from AKI, indicating that intracellular HMGB1 partially counters the effects of extracellular HMGB1. In vitro studies indicate that intracellular HMGB1 decreases resilience of tubular cells from prolonged ischemic stress, as in unilateral IRI. Intracellular HMGB1 is a potential target to enhance kidney regeneration and to improve long-term prognosis in AKI. Late diagnosis is a hurdle for treatment of AKI, but targeting AKI-CKD transition may improve outcomes. High mobility group box-1 (HMGB1) is a nuclear regulator of transcription and a driver of necroinflammation in AKI. We hypothesized that HMGB1 would also modulate AKI-CKD transition in other ways. We conducted single-cell transcriptome analysis of human and mouse AKI and mouse in vivo and in vitro studies with tubular cell-specific depletion of Hmgb1 and HMGB1 antagonists. HMGB1 was ubiquitously expressed in kidney cells. Preemptive HMGB1 antagonism with glycyrrhizic acid (Gly) and ethyl pyruvate (EP) did not affect postischemic AKI but attenuated AKI-CKD transition in a model of persistent kidney hypoxia. Consistently, tubular Hmgb1 depletion in Pax8 rtTA, TetO Cre, Hmgb1fl/fl mice did not protect from AKI, but from AKI-CKD transition. In vitro studies confirmed that absence of HMGB1 or HMGB1 inhibition with Gly and EP does not affect ischemic necrosis of growth-arrested differentiated tubular cells but increased the resilience of cycling tubular cells that survived the acute injury to oxidative stress. This effect persisted when neutralizing extracellular HMGB1 with 2G7. Consistently, late-onset HMGB1 blockade with EP started after the peak of ischemic AKI in mice prevented AKI-CKD transition, even when 2G7 blocked extracellular HMGB1. Treatment of AKI could become feasible when ( 1 ) focusing on long-term outcomes of AKI; ( 2 ) targeting AKI-CKD transition with drugs initiated after the AKI peak; and ( 3 ) targeting with drugs that block HMGB1 in intracellular and extracellular compartments.

High-mobility group box 1-mediated hippocampal microglial activation induces cognitive impairment in mice with neuropathic pain

Clinical evidence indicates that cognitive impairment is a common comorbidity of chronic pain, including neuropathic pain, but the mechanism underlying cognitive impairment remains unclear. Neuroinflammation plays a critical role in the development of both neuropathic pain and cognitive impairment. High-mobility group box 1 (HMGB1) is a proinflammatory molecule and could be involved in neuroinflammation-mediated cognitive impairment in the neuropathic pain state. Hippocampal microglial activation in mice has been associated with cognitive impairment. Thus, the current study examined a potential role of HMGB1 and microglial activation in cognitive impairment in mice with neuropathic pain due to a partial sciatic nerve ligation (PSNL). Mice developed cognitive impairment over two weeks, but not one week, after nerve injury. Nerve-injured mice demonstrated decreased nuclear fraction HMGB1, suggesting increased extracellular release of HMGB1. Furthermore, two weeks after PSNL, significant microglia activation was observed in hippocampus. Inhibition of microglial activation with minocycline, local hippocampal microglia depletion with clodronate liposome, or blockade of HMGB1 with either glycyrrhizic acid (GZA) or anti-HMGB1 antibody in PSNL mice reduced hippocampal microglia activation and ameliorated cognitive impairment. Other changes in the hippocampus of PSNL mice potentially related to cognitive impairment, including decreased hippocampal neuron dendrite length and spine densities and decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor (AMPAR) subunits, were prevented with anti-HMGB1 antibody treatment. The current findings suggest that neuro-inflammation involves a number of cellular-level changes and microglial activation. Blocking neuro-inflammation, particularly through blocking HMGB1 could be a novel approach to reducing co-morbidities such as cognitive impairment associated with neuropathic pain.

High-Mobility Group Box-1 Is Critical in the Pathogenesis of Mouse Experimental Necrotizing Enterocolitis.

Although high-mobility group box-1 (HMGB1) is related to the persistent intestinal inflammation in the development of necrotizing enterocolitis (NEC), the role of HMGB1 in the regulation of the intestinal microcirculation in NEC is not well understood. Therefore, we investigated the mechanism(s) by which HMGB1 regulates the generation of the following vasodilatory signals during the development of NEC: endothelial nitric oxide synthase (eNOS) and nitric oxide (NO). Experimental NEC was induced in full-term C57BL/6 mouse pups through the formula gavage and hypoxia technique. The blockade of HMGB1 was achieved with a subcutaneous injection of anti-HMGB1 antibody. Intestinal tissues and blood samples were collected at predetermined time points for the assessment of intestinal microcirculation, lipid peroxidation levels, and evaluation of eNOS activation. We found elevations in HMGB1 expression as early as 12 h after induction of NEC stress, which preceded intestinal injury. Treatment of mouse pups with HMGB1 neutralizing antibody attenuated the intestinal microvascular features and symptoms of NEC, but this improvement was not found in the eNOS knockout mice, suggesting that HMGB1 inhibition increased intestinal microcirculatory perfusion in an eNOS-dependent manner. Moreover, HMGB1 inhibition rescued NO production and eliminated O2•- production in experimental NEC mice through eNOS activation. These data indicate that excessive HMGB1 signaling is associated with the pathogenesis of NEC, suggesting that HMGB1 inhibition might be a promising strategy for NEC treatment.

High mobility group box-1 protects against Aflatoxin G1-induced pulmonary epithelial cell damage in the lung inflammatory environment

Aflatoxin G1 (AFG1) is a member of the carcinogenic aflatoxin family. Our previous studies indicated that oral administration of AFG1 caused tumor necrosis factor (TNF)-α-dependent inflammation that enhanced oxidative DNA damage in alveolar epithelial cells, which may be related to AFG1-induced lung carcinogenesis. High mobility group box-1 (HMGB1) is a nuclear DNA-binding protein; the intracellular and extracellular roles of HMGB1 have been shown to contribute to DNA repair and sterile inflammation. The role of HMGB1 in DNA damage in an aflatoxin-induced lung inflammatory environment was investigated in this study. Upregulation of HMGB1, TLR2, and RAGE was observed in AFG1-induced lung inflamed tissues and adenocarcinoma. Blocking AFG1-induced inflammation by neutralization of TNF-α inhibited the upregulation of HMGB1 in mouse lung tissues, suggesting that AFG1-induced TNF-α-dependent inflammation regulated HMGB1 expression. In the in vitro human pulmonary epithelial cell line model, Beas-2b, AFG1 directly enhanced the cytosolic translocation of HMGB1 and its extracellular secretion. The addition of extracellular soluble HMGB1 protected AFG1-induced DNA damage through the TLR2/NF-κB pathway in Beas-2b cells. In addition, blockade of endogenous HMGB1 by siRNA significantly enhanced AFG1-induced damage. Thus, our findings showed that both extracellularly-released and nuclear and cytosolic HMGB1 could protect the cell from AFG1-induced cell damage in a TNF-α-dependent lung inflammatory environment.

HMGB1 upregulates NF-kB by inhibiting IKB-α and associates with diabetic retinopathy

AimsDiabetic retinopathy (DR) is the main cause of blindness in adults and investigating new therapeutic targets for DR is necessary. This study aimed to investigate the effect of high-mobility group box 1 (HMGB1) protein and its mechanism in diabetic retinopathy (DR) were investigated. Main methodsHuman retinal endothelial cells (HREC) were uesd for chip-seq. Sprague Dawley (SD) rats were randomly divided into control group, HMGB1 group, diabetes mellitus (DM) combined with HMGB1 siRNA group, and DM group. Next, eyeballs were removed and retinas were detached for western blot. The DM model of cell was built by increasing the glucose concentration in cell culture medium. The regulation of HMGB1 was achieved by short hairpin (sh)-HMGB1 transfection, then, the transfected cells were harvested for luciferase assay, western blot and qRT-PCR analyses as well as proliferation and apoptosis detection. Key findingsChip-seq and luciferase assay showed the possible transcription factor functions of HMGB1 and IKB-α was one of the HMGB1 binding sites. In vivo and in vitro results indicated high expression of HMGB1 and NF-kB and low expression of IKB-α in DR and the expression of IKB-α and NF-kB was regulated by HMGB1. Moreover, cell assays showed that HMGB1 inhibited cell proliferation and promoted apoptosis. SignificanceThe results from the present study showed that HMGB1 may be involved in the pathogenesis of DR as a transcription factor through NF-kB pathway. Therefore, blockade of HMGB1 may be a new method for the treatment of DR.

Cigarette smoke-induced HMGB1 translocation and release contribute to migration and NF-κB activation through inducing autophagy in lung macrophages.

High‐mobility group box 1 (HMGB1) shows pro‐inflammatory activity in various inflammatory diseases and has been found up‐regulated in chronic obstructive pulmonary disease (COPD). Lung macrophages play an important role in airway inflammation and lung destruction in COPD, yet whether HMGB1 is involved in cigarette smoke (CS)‐induced lung macrophage dysfunction is unknown. We sought to evaluate the intracellular localization and release of HMGB1 in lung macrophages from COPD patients and CS‐exposed mice, and to investigate the role of HMGB1 in regulating autophagy in CS extract (CSE)‐treated lung macrophages (MH‐S cells). Our results showed that HMGB1 was highly expressed in lung tissues and sera of COPD patients and CS‐exposed mice, along with predominantly cytoplasmic exporting from nuclei in lung macrophages. In vitro experiments revealed that CSE promoted the expression, nucleocytoplasmic translocation and release of HMGB1 partly via the nicotinic acetylcholine receptor (nAChR). Blockade of HMGB1 with chicken anti‐HMGB1 polyclonal antibody (anti‐HMGB1) or glycyrrhizin (Gly) attenuated the increase of LC3B‐II and Beclin1, migration and p65 phosphorylation, suggesting the involvement of HMGB1 in autophagy, migration and NF‐κB activation of lung macrophages. Hydroxychloroquine (CQ), an autophagy inhibitor, enhanced the increase of LC3B‐II but not Beclin1 in CSE or rHMGB1‐treated MH‐S cells, and inhibition of autophagy by CQ and 3‐methyladenine (3‐MA) abrogated the migration and p65 phosphorylation of CSE‐treated cells. These results indicate that CS‐induced HMGB1 translocation and release contribute to migration and NF‐κB activation through inducing autophagy in lung macrophages, providing novel evidence for HMGB1 as a potential target of intervention in COPD.

High-mobility group box-1 inhibition stabilizes intestinal permeability through tight junctions in experimental acute necrotizing pancreatitis.

In acute necrotizing pancreatitis (ANP), bacterial translocation (BT) from the gastrointestinal tract is the essential pathogenesis in the development of septic complications. Although high-mobility group box-1 (HMGB1) is associated with BT and organ dysfunction in ANP, the mechanism of HMGB1 in the intestinal barrier dysfunction and BT has not been well addressed. In this study, we intend to address the role of HMGB1 in ANP involving BT and intestinal barrier dysfunction. Experimental ANP was achieved in male Sprague-Dawley rats through a retrograde injection of taurocholate into the common biliopancreatic duct following a laparotomy operation. HMGB1 blockade intervention was conducted with a subcutaneous injection of anti-HMGB1 antibody immediately before the laparotomy procedure. Twenty-four hours after ANP induction, pancreatic and intestinal tissues and blood samples were collected for a histopathological assessment and lipid peroxidation or glutathione (GSH) evaluation. AP-induced barrier dysfunction was determined by an intestinal permeability assessment. Tight junction proteins and autophagy regulators were investigated by western blotting, immunohistological analysis and confocal immunofluorescence imaging. ANP developed as indicated by microscopic parenchymal necrosis and fat necrosis, which were associated with intestinal mucosal barrier dysfunction. HMGB1 inhibition played a protective role in intestinal mucosal barrier dysfunction, protected against microbiome changes in ANP, and relieved intestinal oxidative stress. Additionally, HMGB1 inhibition attenuated intestinal permeability; preserved the expression of TJs, such as claudin-2 and occludin; and decreased autophagy. Furthermore, the autophagy regulator LC3 and TJ protein claudin-2 were both upregulated in ANP according to dual immunofluorescence analysis. HMGB1 inhibition ameliorated the severity of experimental ANP though beneficial effects on BT, mainly involving in TJ function.

Therapeutic blockade of HMGB1 reduces early motor deficits, but not survival in the SOD1G93A mouse model of amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease without effective treatment. The receptor for advanced glycation end products (RAGE) and the toll-like receptor (TLR) system are major components of the innate immune system, which have been implicated in ALS pathology. Extracellularly released high-mobility group box 1 (HMGB1) is a pleiotropic danger-associated molecular pattern (DAMP), and is an endogenous ligand for both RAGE and TLR4.MethodsThe present study examined the effect of HMGB1 inhibition on disease progression in the preclinical SOD1G93A transgenic mouse model of ALS using a potent anti-HMGB1 antibody (2G7), which targets the extracellular DAMP form of HMGB1.ResultsWe found that chronic intraperitoneal dosing of the anti-HMGB1 antibody to SOD1G93A mice transiently improved hind-limb grip strength early in the disease, but did not extend survival. Anti-HMGB1 treatment also reduced tumour necrosis factor α and complement C5a receptor 1 gene expression in the spinal cord, but did not affect overall glial activation.ConclusionsIn summary, our results indicate that therapeutic targeting of an extracellular DAMP, HMGB1, improves early motor dysfunction, but overall has limited efficacy in the SOD1G93A mouse model of ALS.

Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis

Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here we show that high-mobility group box 1 (HMGB1), a “danger signal” that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and macrophage inflammatory protein (MIP)-1β, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor-kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM.

HMGB1 blockade significantly improves luminal fibrous obliteration in a murine model of bronchiolitis obliterans syndrome

BackgroundAlthough high-mobility group box-1 (HMGB1), which is a nuclear protein, was reported to enhance the allogeneic responses in transplantation, the effect of HMGB1 on bronchiolitis obliterans syndrome (BOS) is unknown. MethodsA murine heterotopic tracheal transplantation model was used. Protein concentrations of HMGB1, interferon-γ (IFN-γ), interleukin (IL)-10, and IL-17 were analyzed in the isografts, allografts, controls, and HMGB1-neutralizing antibody administered allografts (n = 6; Days 1, 3, 5, 7, 14, 21, and 28). The luminal fibrous occlusion was analyzed (n = 6; Days 7, 14, 21, and 28). Infiltrating CD8 and CD4 T lymphocytes around the allografts and serum levels of IFN-γ and IL-10 were evaluated (n = 6; Day 7). ResultsThe HMGB1 levels in the allografts were significantly increased compared with the isografts at Day 7. HMGB1 blockade did not change the IL-17 level, but decreased the IFN-γ/IL-10 ratio in the early phase (Days 5 and 7) and significantly improved the fibrous occlusion in the late phase (Days 14, 21, and 28). HMGB1 blockade significantly suppressed the CD8 T lymphocytes infiltration and decreased the serum IFN-γ/IL-10 ratio compared with the control at Day 7. ConclusionsHMGB1 may be a trigger of the BOS pathogenesis and candidate target for the treatment of the disease.

Abstract 465: Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis

Abstract Oral mucositis (OM) is a common complication in cancer patients undergoing anticancer treatment. Despite the clinical and economic consequences of OM, there are no drugs available for its fundamental control. Here, we show that high-mobility group box 1 (HMGB1), a “danger signal” that acts as a potent innate immune mediator, plays a critical role in the pathogenesis of OM. In addition, we investigated treatment of OM through HMGB1 blockade using NecroX-7 (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1Hindole-7-yl]amine). NecroX-7 ameliorated basal-layer epithelial cell death and ulcer size in OM induced by chemotherapy or radiotherapy. This protective effect of NecroX-7 was mediated by inhibition of HMGB1 release and downregulation of mitochondrial oxidative stress. Additionally, NecroX-7 inhibited the HMGB1-induced release of TNF-α, IL-1β and MIP-1β, as well as the expression of p53-upregulated modulator of apoptosis (PUMA) and the excessive inflammatory microenvironment, including nuclear factor kB (NF-kB) pathways. In conclusion, our findings suggest that HMGB1 plays a key role in the pathogenesis of OM; therefore, blockade of HMGB1 by NecroX-7 may be a novel therapeutic strategy for OM. Citation Format: Keon-Il Im, Young-Sun Nam, Nayoun Kim, YuneJin Song, Young-Woo Jeon, Jung-Yeon Lim, Seok-Goo Cho. Regulation of HMGB1 release protects chemoradiotherapy-associated mucositis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 465.

Blockade of HMGB1 Attenuates Diabetic Nephropathy in Mice

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2−/−, modest protection against podocyte damage only in TLR4−/− and no protection in RAGE−/− diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

High Mobility Group Box 1 Neutralization in the Brain Prevents Inflammation, Sympathoexcitation and Hypertension

High mobility group box 1 (HMGB1) is a nuclear protein, that once translocated to cytoplasm, can act as a chemoattractant and a proinflammatory mediator. Post‐translationally modified HMGB1 can interact with different receptors, such as toll‐like receptor 4 (TLR4) and chemokine receptor CXC receptor 4 (CXCR4), to induce acute and chronic inflammation. We have previously showed that central TLR4 blockade delayed progression of hypertension and improved cardiac function. However, the involvement of HMGB1 and its interaction with TLR4 in the pathogenesis of hypertension has not been fully explored. Therefore, in this study, we investigated the role of HMGB1 in mediating hypertension, inflammation and sympathoexcitation. We hypothesized that blockade of HMGB1 prevents the development of hypertension. Chronic angiotensin II (AngII) infusion (200 ng/kg/min, SC, 14 days) significantly increased mean arterial pressure (telemetry) in male Sprague‐Dawley rats compared to saline‐infused controls (163 ±5 vs. 105 ±4 mmHg, n=7, p<0.01). AngII‐infusion significantly increased HMGB1 levels in the plasma (21 ±3 vs.15 ±2 ng/ml, p<0.01) and cerebrospinal fluid (34±3 vs. 21±1 ng/ml, p<0.01), and elevated HMGB1 protein expression (2.5 fold, n=6, p<0.01 vs. Control) in the brain hypothalamic paraventricular nucleus (PVN). Intracerebroventricular infusion of neutralizing monoclonal anti‐HMGB1 antibody (10 μg/kg/day) attenuated AngII‐induced hypertension (120 ±2 mmHg, p<0.01 vs. AngII). Furthermore, AngII‐infusion significantly increased expression of TNF, IL‐1, IL‐6, TLR4, CXCR4, and NF‐kB in the PVN, which was prevented by HMGB1 neutralization. AngII infusion resulted in sympathoexcitation, as indicated by increased plasma norepinephrine levels (1602 ±53 vs. 549 ±25 pg/ml, P<0.01 vs. Control), but this increase was blunted by HMGB1 neutralization (720 ±25 pg/ml, P<0.05 vs. AngII). Taken together, these data suggest that neutralization of HMGB1 in the brain reduces inflammation and decreases sympathoexcitation, thereby preventing the development of AngII‐induced hypertension.Support or Funding InformationThe American Heart Association, 15SDG25720021 (SS), 16GRNT30510012 (JF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SRAGE alleviates neutrophilic asthma by blocking HMGB1/RAGE signalling in airway dendritic cells

Receptor for advanced glycation end products (RAGE) plays a role in inflammatory reactions. The soluble form of RAGE (sRAGE) acts as a decoy to inhibit interactions of RAGE with advanced glycation end products such as High mobility group box 1 (HMGB1). We have demonstrated that HMGB1 directs Th17 skewing by regulating dendritic cell (DC) functions in a previous study. However, the protective effects of HMGB1 blockade with sRAGE in the development of neutrophilic asthma remain unclear. Here, we showed that allergen challenge decreased expression of sRAGE in a murine model of neutrophilic asthma, correlating well with neutrophil counts and interleukin (IL)-17 production. When HMGB1 signalling was blocked by intratracheal administration of sRAGE before sensitisation, HMGB1 expression, neutrophilic inflammation, and Th17-type responses were reduced significantly. Anti-asthma effects of sRAGE were achieved by inhibition of RAGE and IL-23 expression in airway CD11c+ antigen-presenting cells. Finally, we showed that sRAGE inhibited Th17 polarisation induced by recombinant HMGB1 (rHMGB1)-activated dendritic cells (DCs) in vitro. Adoptive transfer of rHMGB1-activated DCs was sufficient to restore airway inflammation, whereas transfer of rHMGB1 plus sRAGE-activated DCs significantly reduced neutrophilic inflammation. Thus, sRAGE prevents Th17-mediated airway inflammation in neutrophilic asthma at least partly by blocking HMGB1/RAGE signalling in DCs.

Blockade of high-mobility group box 1 attenuates intestinal mucosal barrier dysfunction in experimental acute pancreatitis

The release of inflammatory cytokines, that plays a dominant role in local pancreatic inflammation and systemic complications in severe acute pancreatitis (SAP). High-mobility group box 1 (HMGB1) is implicated in the mechanism of organ dysfunction and bacterial translocation in SAP. This current study aims to investigate possible role of HMGB1 in the intestinal mucosal barrier dysfunction of SAP, and the effect of anti-HMGB1 antibody treatment in intestinal mucosal injury in SAP. Our data revealed that the HMGB1 expression was significantly increased in AP mice induced by caerulein and LPS, and the inhibition of HMGB1 played a protective role in intestinal mucosal barrier dysfunction, reduced the serum level of other proinflammatory cytokines include IL-1β, IL-6, TNF-α. Next we investigated the downstream receptors involving in HMGB1 signaling. We found that the expressions of toll-like receptor (TLR) 4 and TLR9 were elevated in ileum of AP mice, the administration of HMGB1 neutralizing antibody significantly reduced the TLR4 and TLR9 expression. It was concluded that HMGB1 contributed the mechanism to the intestinal mucosal barrier dysfunction during AP. Blockade of HMGB1 by administration of HMGB1 neutralizing antibody may be a beneficial therapeutic strategy in improving intestinal mucosal barrier dysfunction in SAP.

Bio-HMGB1 from breast cancer contributes to M-MDSC differentiation from bone marrow progenitor cells and facilitates conversion of monocytes into MDSC-like cells.

Myeloid-derived suppressor cells (MDSC) constitute the major cell population that regulates immune responses. They are known to accumulate in tumors, chronic inflammatory and autoimmune diseases. Previous data indicate that high mobility group box 1(HMGB1) facilitates MDSC differentiation from bone marrow, suppresses NK cells, CD4+ and CD8+ T cells and is involved in cancer development. However, it remains unclear what potential mechanisms of HMGB1 facilitate MDSC differentiation. In the present work, we clearly demonstrate that HMGB1 secreted by cancer cells is N-glycosylated at Asn37, which facilitates monocytic (M)-MDSC differentiation from bone marrow via the p38/NFκB/Erk1/2 pathway and also contributes to conversion of monocytes into MDSC-like cells; HMGB1 blockade by a monoclonal antibody against the HMGB1 B box obviously reduced the accumulation of M-MDSC in tumor-bearing mice, delaying tumor growth and development; additionally, MDSC expansion and HMGB1 up-regulation were also found in breast cancer patients. All these data indicate that HMGB1 might be a potential tumor immunotherapy target.

Hypothermia inhibits the propagation of acute ischemic injury by inhibiting HMGB1.

Acute ischemic stroke causes significant chronic disability worldwide. We designed this study to clarify the mechanism by which hypothermia helps alleviate acute ischemic stroke. In a middle cerebral artery occlusion model (4 h ischemia without reperfusion), hypothermia effectively reduces mean infarct volume. Hypothermia also prevents neurons in the infarct area from releasing high mobility group box 1 (HMGB1), the most well-studied damage-associated molecular pattern protein. By preventing its release, hypothermia also prevents the typical middle cerebral artery occlusion-induced increase in serum HMGB1. We also found that both glycyrrhizin-mediated inhibition of HMGB1 and intracerebroventricular neutralizing antibody treatments before middle cerebral artery occlusion onset diminish infarct volume. This suggests a clear neuroprotective effect of HMGB1 inhibition by hypothermia in the brain. We next used real-time polymerase chain reaction to measure the levels of pro-inflammatory cytokines in peri-infarct regions. Although middle cerebral artery occlusion increases the expression of interleukin-1β and tissue necrosis factor-α, this elevation is suppressed by both hypothermia and glycyrrhizin treatment. We show that hypothermia reduces the production of inflammatory cytokines and helps salvage peri-infarct regions from the propagation of ischemic injury via HMGB1 blockade. In addition to suggesting a potential mechanism for hypothermia’s therapeutic effects, our results suggest HMGB1 modulation may lengthen the therapeutic window for stroke treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0260-0) contains supplementary material, which is available to authorized users.