High-mobility Group Box 1 Neutralizing Research Articles

HMGB1 Release Induced by EV71 Infection Exacerbates Blood-Brain Barrier Disruption via VE-cadherin Phosphorylation

PurposeEV71 (Enterovirus 71) is a major causative agent of the outbreaks of HFMD (hand, foot, and mouth disease), which is associated with neurological damage caused by permeability disruption of BBB (blood-brain barrier). HMGB1 (high-mobility group box 1) is a widely expressed nuclear protein that triggers host inflammatory responses. Our work aimed to explore the function of HMGB1 in EV71 infection and its contributions to EV71-related BBB damage. MethodsHeLa cells, HT-29 cells and AG6 mice were used to explore the translocation of HMGB1 in EV71 infection in vitro and in vivo. The roles of released HMGB1 on EV71 replication and associated inflammatory cytokines were investigated using recombinant HMGB1 in HeLa cells. The mechanisms of released HMGB1 in EV71-induced BBB injury were explored using recombinant HMGB1 and anti-HMGB1 neutralizing antibodies in monolayer HCMECs (immortalized human brain microvascular endothelial cells) and AG6 mice brain. ResultsEV71 induced HMGB1 nucleocytoplasmic translocation and extracellular release in vitro and in vivo. Released HMGB1 acted as an inflammatory mediator in EV71 infection rather than affecting viral replication in vitro. Released HMGB1 disrupted BBB integrity by enhancing VE-cadherin phosphorylation at tyrosine 685 in HCMECs, and reducing total VE-cadherin levels in HCMECs and AG6 mice in EV71 infection. And released HMGB1 induced an increase in activated astrocytes. Neutralization of HMGB1 reversed the increased endothelial hyperpermeability and phosphorylation of VE-cadherin in HCMECs. ConclusionThe inflammatory mediator HMGB1 released by EV71 exacerbated BBB disruption by enhancing VE-cadherin phosphorylation, which in turn aggravated EV71-induced neuroinflammation.

Acute muscle mass loss was alleviated with HMGB1 neutralizing antibody treatment in severe burned rats

Burn injury is associated with muscle wasting, though the involved signaling mechanisms are not well understood. In this study, we aimed to examine the role of high mobility group box 1 (HMGB1) in signaling hyper-inflammation and consequent skeletal muscle impairment after burn. Sprague Dawley rats were randomly assigned into three groups: (1) sham burn, (2) burn, (3) burn/treatment. Animals in group 2 and group 3 received scald burn on 30% of total body surface area (TBSA) and immediately treated with chicken IgY and anti-HMGB1 antibody, respectively. Muscle tissues and other samples were collected at 3-days after burn. Body mass and wet/dry weights of the hind limb muscles (total and individually) were substantially decreased in burn rats. Acute burn provoked the mitochondrial stress and cell death and enhanced the protein ubiquitination and LC3A/B levels that are involved in protein degradation in muscle tissues. Further, an increase in muscle inflammatory infiltrate associated with increased differentiation, maturation and proinflammatory activation of bone marrow myeloid cells and αβ CD4+ T and γδ T lymphocytes was noted in in circulation and spleen of burn rats. Treatment with one dose of HMGB1 neutralizing antibody reduced the burn wound size and preserved the wet/dry weights of the hind limb muscles associated with a control in the markers of cell death and autophagy pathways in burn rats. Further, anti-HMGB1 antibody inhibited the myeloid and T cells inflammatory activation and subsequent dysregulated inflammatory infiltrate in the muscle tissues of burn rats. We conclude that neutralization of HMGB1-dependent proteolytic and inflammatory responses has potential beneficial effects in preventing the muscle loss after severe burn injury.

Neutralization of HMGB1 improves fracture healing and \u03b3\u03b4 T lymphocyte counts at the fracture site in a polytrauma rat model

PurposeDelayed fracture healing is a common consequence of polytrauma (PT) occurring in patients with multiple injuries. We believe that when early release of high mobility group box 1 (HMGB1) molecules from necrotic tissues exceed their normal levels in blood, they dysregulate immune responses associated with normal healing. This study investigates the detrimental effect of such dysregulate immune responses by targeting HMGB1 in a PT rat model with debilitating injuries. We hypothesized that neutralization of extracellular HMGB1 immediately post-trauma would ameliorate local immune dysregulation and improve fracture healing in a PT rat model.MethodsPT rats received a single dose of either anti-rat HMGB1 polyclonal antibody (PT-Ab HMGB1) or IgY isotype (PT-IgY), were left untreated (PT-C), or had a single injury/osteotomy only (OST). Fracture healing was evaluated by micro-computed tomography (µCT) and histology at 5 weeks; and macrophages and T cell counts within the fracture site were determined with flow cytometry at 1 week.ResultsNotably, bone regeneration within the fracture site in PT-Ab HMGB1 rats was improved with comparable connective tissue organization than PT-C rats. Further, only γδTCR+ T cells, but not macrophages and CD4+ and CD8+ T cells, were diminished at the fracture site in PT-C and PT-IgY rats. Interestingly, the PT-Ab HMGB1 rats had increased γδTCR+ T cells compared to PT-C and PT-IgY, suggesting their potential role in regulating fracture healing.ConclusionsTherefore, the initial burst of systemic HMGB1 following trauma may have a role in regulating bone regeneration via the modulation of a subclass of T cells within the fracture site, suggesting it’s importance as a therapeutic target in PT to combat immune dysregulation and delayed fracture healing.

DOP49 Context-dependent roles of High-mobility group box 1 (HMGB1) during intestinal inflammation and carcinogenesis

Abstract Background HMGB1 is a ubiquitously expressed nucleoprotein with proinflammatory functions following cellular release. The protein is passively released during tissue necrosis, acting as a damage-associated molecular pattern, but can also be actively secreted by immune cells. Stool and serum HMGB1 levels have been suggested as markers of both inflammatory bowel disease (IBD) activity and colorectal cancer (CRC) invasiveness, and antibody-mediated HMGB1 neutralization was beneficial in animal models of IBD. We explored context-dependent functions of HMGB1 in the injured intestine using novel experimental mice with cell-specific genetic HMGB1 deficiency. Methods To circumvent the postnatal lethality of global HMGB1 deficiency in animals, we used the Cre-lox system to generate enterocyte-specific (Hmgb1ΔIEC , Villin-Cre) and myeloid cell-specific (Hmgb1ΔLysM , LysM-Cre) HMGB1-knockout mice. Animals were subjected to well-established models of acute (DSS, Citrobacter rodentium) and chronic (AOM+DSS, CD45RBhigh T cell transfer colitis, Apc+/min) intestinal injury, followed by clinical, endoscopic, histological and molecular analysis. HMGB1 expression was assessed in human IBD and CRC specimens. Results IBD and CRC biopsies exhibited high levels of HMGB1 expression in epithelia, immune cells, tumor cells and the peritumoral stroma. HMGB1 deficiency from enterocytes and myeloid cells did not alter Citrobacter- or T cell transfer-induced enterocolitis, when epithelial injury was comparably low. In contrast, Hmgb1ΔIEC mice exhibited aggravated DSS-induced colitis, as evidenced by severe weight loss as well as exacerbated neutrophil- and monocyte-driven mucosal inflammation compared to Hmgb1f/f . Whole tissue RNA sequencing indicated defective cellular proliferation in injured Hmgb1ΔIEC intestines. In the AOM+DSS-model, Hmgb1ΔIEC had a comparable tumor burden to Hmgb1f/f , whereas Hmgb1ΔLysM had significantly fewer and smaller tumors, potentially linked to metabolic alterations in the tumor micromilieu. In the Apc+/min model, enterocyte HMGB1 deficiency effectuated more and larger tumors, whereas leukocyte HMGB1 did not affect tumor load. Conclusion Contrasting antibody-mediated HMGB1 neutralization in animal models of IBD, our findings from genetic HMGB1 deletion studies reveal a critical role of enterocyte HMGB1 in the maintenance of the intestinal barrier during severe colitis. Impaired epithelial regeneration or inefficient local immune cell expansion in Hmgb1ΔIEC may account for the aggravated phenotype. HMGB1 from enterocytes and immune cells context-dependently affect maladaptive intestinal would healing, potentially mediated by cell-intrinsic and -extrinsic mechanisms that warrant further investigation.

Role of neuron-derived ATP in paclitaxel-induced HMGB1 release from macrophages and peripheral neuropathy

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

Autophagic secretion of HMGB1 from cancer-associated fibroblasts promotes metastatic potential of non-small cell lung cancer cells via NF\u03baB signaling

Tumor progression requires the communication between tumor cells and tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major components of stromal cells. CAFs contribute to metastasis process through direct or indirect interaction with tumor cells; however, the underlying mechanism is largely unknown. Here, we reported that autophagy was upregulated in lung cancer-associated CAFs compared to normal fibroblasts (NFs), and autophagy was responsible for the promoting effect of CAFs on non-small cell lung cancer (NSCLC) cell migration and invasion. Inhibition of CAFs autophagy attenuated their regulation on epithelial–mesenchymal transition (EMT) and metastasis-related genes of NSCLC cells. High mobility group box 1 (HMGB1) secreted by CAFs mediated CAFs’ effect on lung cancer cell invasion, demonstrated by using recombinant HMGB1, HMGB1 neutralizing antibody, and HMGB1 inhibitor glycyrrhizin (GA). Importantly, the autophagy blockade of CAFs revealed that HMGB1 release was dependent on autophagy. We also found HMGB1 was responsible, at least in part, for autophagy activation of CAFs, suggesting CAFs remain active through an autocrine HMGB1 loop. Further study demonstrated that HMGB1 facilitated lung cancer cell invasion by activating the NFκB pathway. In a mouse xenograft model, the autophagy specific inhibitor chloroquine abolished the stimulating effect of CAFs on tumor growth. These results elucidated an oncogenic function for secretory autophagy in lung cancer-associated CAFs that promotes metastasis potential, and suggested HMGB1 as a novel therapeutic target.

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.

Leukocyte-Derived High-Mobility Group Box 1 Governs Hepatic Immune Responses to Listeria monocytogenes.

High‐mobility group box 1 (HMGB1) is a nucleoprotein with proinflammatory functions following cellular release during tissue damage. Moreover, antibody‐mediated HMGB1 neutralization alleviates lipopolysaccharide (LPS)‐induced shock, suggesting a role for HMGB1 as a superordinate therapeutic target for inflammatory and infectious diseases. Recent genetic studies have indicated cell‐intrinsic functions of HMGB1 in phagocytes as critical elements of immune responses to infections, yet the role of extracellular HMGB1 signaling in this context remains elusive. We performed antibody‐mediated and genetic HMGB1 deletion studies accompanied by in vitro experiments to discern context‐dependent cellular sources and functions of extracellular HMGB1 during murine bloodstream infection with Listeria monocytogenes. Antibody‐mediated neutralization of extracellular HMGB1 favors bacterial dissemination and hepatic inflammation in mice. Hepatocyte HMGB1, a key driver of postnecrotic inflammation in the liver, does not affect Listeria‐induced inflammation or mortality. While we confirm that leukocyte HMGB1 deficiency effectuates disseminated listeriosis, we observed no evidence of dysfunctional autophagy, xenophagy, intracellular bacterial degradation, or inflammatory gene induction in primary HMGB1‐deficient phagocytes or altered immune responses to LPS administration. Instead, we demonstrate that mice devoid of leukocyte HMGB1 exhibit impaired hepatic recruitment of inflammatory monocytes early during listeriosis, resulting in alterations of the transcriptional hepatic immune response and insufficient control of bacterial dissemination. Bone marrow chimera indicate that HMGB1 from both liver‐resident and circulating immune cells contributes to effective pathogen control. Conclusion: Leukocyte‐derived extracellular HMGB1 is a critical cofactor in the immunologic control of bloodstream listeriosis. HMGB1 neutralization strategies preclude an efficient host immune response against Listeria.

Glycyrrhizic acid, as an inhibitor of HMGB1, alleviates bleomycin-induced pulmonary toxicity in mice through the MAPK and Smad3 pathways

Aim High-mobility group box 1 (HMGB1) protein has been noticed particularly for its pivotal role in several pathologies. However, the relevance between HMGB1 and pathological progress in lung toxicity still remains unclear. In the study, we evaluated the effect of glycyrrhizic acid as an HMGB1 inhibitor on the early inflammation and late fibrosis in bleomycin-induced pulmonary toxicity in mice. Methods We established a bleomycin-induced pulmonary toxicity model to detect the relevance between HMGB1 and pathological changes in the early inflammatory and late fibrotic stages. Results We found that bleomycin-induced increase in inflammatory cytokines interleukin (IL)-β1, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and inflammatory lesions in lung tissue in the early stage of the model. However, markers of fibrosis such as transforming growth factor (TGF)-β1 and α-smooth muscle actin (α-SMA) were significantly elevated on day 7 after bleomycin instillation. Interestingly, HMGB1 also began to rise on day 7, rather than in the early inflammatory phase. However, early (from day 0 to 14 after bleomycin instillation) or late (from day 14 to 28) intervention with HMGB1 neutralizing antibody or glycyrrhizic acid alleviated inflammation and fibrosis through down-regulating the inflammatory signaling mitogen-activated protein kinase (MAPK) and fibrotic signaling Smad3 pathway. Conclusion Our results suggested that HMGB1 mediates both inflammation and fibrosis in this model. The development of high-potency and low-toxicity HMGB1 inhibitors may be a class of potential drugs for the treatment of pulmonary fibrosis.

Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling.

Cardiac allograft vasculopathy (CAV) charactered with aberrant remodeling and fibrosis usually leads to the loss of graft after heart transplantation. Our previous work has reported that extracellular high-mobility group box 1 (HMGB1) participated in the CAV progression via promoting inflammatory cells infiltration and immune damage. The aim of this study was to investigate the involvement of HMGB1 in the pathogenesis of CAV/fibrosis and potential mechanisms using a chronic cardiac rejection model in mice. We found high levels of transforming growth factor (TGF)-β1 in cardiac allografts after transplantation. Treatment with HMGB1 neutralizing antibody markedly prolonged the allograft survival accompanied by attenuated fibrosis of cardiac allograft, decreased fibroblasts-to-myofibroblasts conversion, and reduced synthesis and release of TGF-β1. In addition, recombinant HMGB1 stimulation promoted release of active TGF-β1 from cardiac fibroblasts and macrophages in vitro, and subsequent phosphorylation of Smad2 and Smad3 which were downstream of TGF-β1 signaling. These data indicate that HMGB1 contributes to the CAV/fibrosis via promoting the activation of TGF-β1/Smad signaling. Targeting HMGB1 might become a new therapeutic strategy for inhibiting cardiac allograft fibrosis and dysfunction.

High Mobility Group Box-1 and Blood-Brain Barrier Disruption.

Increasing evidence suggests that inflammatory responses are involved in the progression of brain injuries induced by a diverse range of insults, including ischemia, hemorrhage, trauma, epilepsy, and degenerative diseases. During the processes of inflammation, disruption of the blood–brain barrier (BBB) may play a critical role in the enhancement of inflammatory responses and may initiate brain damage because the BBB constitutes an interface between the brain parenchyma and the bloodstream containing blood cells and plasma. The BBB has a distinct structure compared with those in peripheral tissues: it is composed of vascular endothelial cells with tight junctions, numerous pericytes surrounding endothelial cells, astrocytic endfeet, and a basement membrane structure. Under physiological conditions, the BBB should function as an important element in the neurovascular unit (NVU). High mobility group box-1 (HMGB1), a nonhistone nuclear protein, is ubiquitously expressed in almost all kinds of cells. HMGB1 plays important roles in the maintenance of chromatin structure, the regulation of transcription activity, and DNA repair in nuclei. On the other hand, HMGB1 is considered to be a representative damage-associated molecular pattern (DAMP) because it is translocated and released extracellularly from different types of brain cells, including neurons and glia, contributing to the pathophysiology of many diseases in the central nervous system (CNS). The regulation of HMGB1 release or the neutralization of extracellular HMGB1 produces beneficial effects on brain injuries induced by ischemia, hemorrhage, trauma, epilepsy, and Alzheimer’s amyloidpathy in animal models and is associated with improvement of the neurological symptoms. In the present review, we focus on the dynamics of HMGB1 translocation in different disease conditions in the CNS and discuss the functional roles of extracellular HMGB1 in BBB disruption and brain inflammation. There might be common as well as distinct inflammatory processes for each CNS disease. This review will provide novel insights toward an improved understanding of a common pathophysiological process of CNS diseases, namely, BBB disruption mediated by HMGB1. It is proposed that HMGB1 might be an excellent target for the treatment of CNS diseases with BBB disruption.

High mobility group box 1 induces bone pain associated with bone invasion in a mouse model of advanced head and neck cancer.

Advanced head and neck cancer (HNC) can invade facial bone and cause bone pain, thus posing a significant challenge to the quality of life of patients presenting with advanced HNC. The present study was designed to investigate HNC bone pain (HNC-BP) in an intratibial mouse xenograft model that utilized an HNC cell line (SAS cells). These mice develop HNC-BP that is associated with an expression of phosphorylated ERK1/2 (pERK1/2), which is a molecular indicator of neuron excitation in dorsal root ganglia (DRG) sensory neurons. Our experiments demonstrated that the inhibition of high mobility group box 1 (HMGB1) by short hairpin (shRNA) transduction, HMGB1 neutralizing antibody, and HMGB1 receptor antagonist suppressed the HNC-BP and the pERK1/2 expression in DRG. It was also observed that HNC-derived HMGB1 increased the expression of the acid-sensing nociceptor, transient receptor potential vanilloid 1 (TRPV1), which is a major cause of osteoclastic HNC-BP in DRG. Collectively, our results demonstrated that HMGB1 originating in HNC evokes HNC-BP via direct HMGB1 signaling and hypersensitization for the acid environment in sensory neurons.

High-mobility group box 1 promotes epithelial-to-mesenchymal transition in crystalline silica induced pulmonary inflammation and fibrosis

Silicosis is an inflammatory and fibrotic lung disease caused by prolonged inhalation of silica. The potential role of high-mobility group box-1 (HMGB-1) and its underlying mechanisms in silicosis remain unclear. In this study, intratracheal instillation of a silica suspension was used to establish silicosis in male C57BL/6 mice. To elucidate the effects of HMGB-1 on the pathogenesis of silicosis, we used HMGB-1 neutralizing antibody (anti-HMGB-1) and recombinant HMGB-1 (rmHMGB-1) to abrogate or increase the HMGB-1 levels, respectively. At days 7, 28, and 84, the accumulation of macrophages and neutrophils decreased by anti-HMGB-1 treatment. The expression levels of interleukin-6 and tumor necrosis factor-α in lung increased in response to silica exposure across three time points; anti-HMGB-1 could alleviate those expressions at day 28 and 84. In contrast, rmHMGB-1 aggravated this process. At days 28 and 84, the protein expression of fibronectin and col1a1 decreased in the silica + anti-HMGB-1 groups but increased in silica + rmHMGB-1 groups compared to mice with silica alone. Further study suggested that HMGB-1-mediated epithelial-mesenchymal transition participated in the development of silicosis. In conclusion, the findings demonstrate that HMGB-1 participates in the pathogenesis of silicosis and may represent a potential therapeutic target for the treatment of silicosis.

The nuclear sirtuin SIRT6 protects mice from polymicrobial sepsis by suppressing the host immune response

IntroductionSepsis is the leading cause of death in intensive care unit and refers to the host’s deleterious and non‐resolving systemic inflammatory response to infection. Despite advances in medical care and antibiotic therapy, the overall mortality rate of sepsis in United States is 28.3% highlighting the inadequacies of current therapies. Sirtuins are key regulators of inflammation in both immune and non‐immune cells. SIRT6 is known to exert an anti‐inflammatory action that promotes hypo‐inflammation and endotoxin tolerance in immune cells. The pathogenesis of sepsis is also mediated by an alarmin molecule, the high mobility group box 1 (HMGB1). HMGB1 neutralizing antibodies were found to have therapeutic potential in the treatment of sepsis. Hence, by using the rat cardiomyocytes and SIRT6 transgenic (SIRT6.Tg) mice, we tested the hypothesis that SIRT6 plays a role in HMGB1 secretion, further hypothesized that SIRT6.Tg mice are protected from bacteria‐induced sepsis in mice.MethodsInteractions between SIRT6 and HMGB1 were determined by immunoprecipitation followed by immunoblotting. The presence of HMGB1 in the serum was detected using HMGB1 ELISA kit. Sepsis was induced in wild type and SIRT6.Tg mice by intra‐peritonea injection of a four‐pathogen community (MDR S. marcescens, C. albicans, MDR K. oxytoca, and E. faecalis) that was isolated from an ICU patient suffering from poly‐microbial sepsis and previously characterized. Immune response of mice was determined by performing FACS analysis on the splenocytes obtained from infected mice.ResultsTo test whether SIRT6 can bind to HMGB1 in cardiomyocytes, we infected rat cardiomyocytes with adenovirus expressing SIRT6 or empty adenovirus vector for 24 h. Cell lysates were immunoprecipitated with an SIRT6‐antibody, and subjected to immunoblotting with the use of an anti‐HMGB1 antibody. We found that HMGB1 co‐precipitated with SIRT6 in cardiomyocytes. In order to confirm that SIRT6 binds to HMGB1 endogenously, we performed a reverse immunoprecipitation experiment. Endogenous HMGB1 was immunoprecipitated from rat cardiomyocytes and subjected to immunoblotting with the anti‐SIRT6 antibody. Our results confirmed that HMGB1 binds to SIRT6 endogenously. In order to understand the physiological significance of the interaction between SIRT6 and HMGB1, we tested the HMGB1 levels in 2 year old wild‐type and SIRT6.Tg mice. We found that SIRT6.Tg mice have 5 fold reduced levels of HMGB1 in the serum, suggesting that SIRT6 expression interferes with the secretion of HMGB1. Next we investigated whether SIRT6 is protective against polymicrobial sepsis by injecting a lethal dose of the pathogen community into wild type and SIRT6.Tg mice. SIRT6.Tg mice showed an increased survival of 25%, compared to wild type mice. We also compared the immune response of wild‐type with SIRT6.Tg mice. Both CD4 and CD8 T lymphocytes of SIRT6.Tg mice showed reduced interferon gamma and interleukin 17 response, compared to wild‐type mice, suggesting that SIRT6 can regulate the sepsis induced immune response.ConclusionsSIRT6 can regulate HMGB1 secretion and protect mice from polymicrobial sepsis by suppressing the host immune response.Support or Funding InformationNIH RO1 HL136712; NIH RO1 HL143488

Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

ABSTRACT The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; GORASP2 shRNA: lentivirus harboring shRNA against GORASP2; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; HMGB1 shRNA: lentivirus harboring shRNA against HMGB1; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; Krt14+/+-Atg5f/f mice: mice bearing an Atg5 flox allele, in which exon 3 of the Atg5 gene is flanked by two loxP sites; Krt14+/+-Hmgb1f/f : mice bearing an Hmgb1 flox allele, in which exon 2 to 4 of the Hmgb1 gene is flanked by two loxP sites; Krt14Cre/+-atg5f/f mice: keratinocyte-specific atg5 knockout mice generated by mating Atg5-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt4; Krt14Cre/+-hmgb1f/f mice: keratinocyte-specific hmgb1 knockout mice generated by mating Hmgb1-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt14; Krt14-Vegfa mice: mice expressing 164-amino acid Vegfa splice variant recombinase under the control of promoter of Krt14; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; tcrd KO mice: tcrd (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.

Potential Neuroprotective Effect of the HMGB1 Inhibitor Glycyrrhizin in Neurological Disorders.

Glycyrrhizin (glycyrrhizic acid), a bioactive triterpenoid saponin constituent of Glycyrrhiza glabra, is a traditional medicine possessing a plethora of pharmacological anti-inflammatory, antioxidant, antimicrobial, and antiaging properties. It is a known pharmacological inhibitor of high mobility group box 1 (HMGB1), a ubiquitous protein with proinflammatory cytokine-like activity. HMGB1 has been implicated in an array of inflammatory diseases when released extracellularly, mainly by activating intracellular signaling upon binding to the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). HMGB1 neutralization strategies have demonstrated disease-modifying outcomes in several preclinical models of neurological disorders. Herein, we reveal the potential neuroprotective effects of glycyrrhizin against several neurological disorders. Emerging findings demonstrate the therapeutic potential of glycyrrhizin against several HMGB1-mediated pathological conditions including traumatic brain injury, neuroinflammation and associated conditions, epileptic seizures, Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Glycyrrhizin's effects in neurological disorders are mainly attributed to the attenuation of neuronal damage by inhibiting HMGB1 expression and translocation as well as by downregulating the expression of inflammatory cytokines. A large number of preclinical findings supports the notion that glycyrrhizin might be a promising therapeutic alternative to overcome the shortcomings of the mainstream therapeutic strategies against neurological disorders, mainly by halting disease progression. However, future research is warranted for a deeper exploration of the precise underlying molecular mechanism as well as for clinical translation.

HMGB1 Is a Therapeutic Target and Biomarker in Diazepam-Refractory Status Epilepticus with Wide Time Window.

Status epilepticus (SE), a life-threatening neurologic emergency, is often poorly controlled by the current pharmacological therapeutics, which are limited to a narrow time window. Here, we investigated the proinflammatory cytokine high mobility group box-1 (HMGB1) as a candidate therapeutic target for diazepam (DZP)-refractory SE. We found that HMGB1 was upregulated and translocated rapidly during refractory SE period. Exogenous HMGB1 was sufficient to directly induce DZP-refractory SE in nonrefractory SE. Neutralization of HMGB1 with an anti-HMGB1 monoclonal antibody decreased the incidence of SE and alleviated the severity of seizure activity in DZP-refractory SE, which was mediated by a Toll-like receptor 4 (TLR4)-dependent pathway. Importantly, anti-HMGB1 mAb reversed DZP-refractory SE with a wide time window, extending the therapeutic window from 30 to 180 min. Furthermore, we found the upregulation of plasma HMGB1 level is closely correlated with the therapeutic response of anti-HMGB1 mAb in DZP-refractory SE. All these results indicated that HMGB1 is a potential therapeutic target and a useful predictive biomarker in DZP-refractory SE.

Entamoeba histolytica stimulates the alarmin molecule HMGB1 from macrophages to amplify innate host defenses.

Even though Entamoeba histolytica (Eh)-induced host pro-inflammatory responses play a critical role in disease, we know very little about the host factors that regulate this response. Direct contact between host cell and Eh signify the highest level of danger, and to eliminate this threat, the host immune system elicits an augmented immune response. To understand the mechanisms of this response, we investigated the induction and release of the endogenous alarmin molecule high-mobility group box 1 (HMGB1) that act as a pro-inflammatory cytokine and chemoattractant during Eh infection. Eh in contact with macrophage induced a dose- and time-dependent secretion of HMGB1 in the absence of cell death. Secretion of HMGB1 was facilitated by Eh surface Gal-lectin-activated phosphoinositide 3-kinase and nuclear factor-κB signaling and up-regulation of histone acetyltransferase activity to trigger acetylated HMGB1 translocation from the nucleus. Unlike lipopolysaccharide, Eh-induced HMGB1 release was independent of caspase-1-mediated inflammasome and gasdermin D pores. In vivo, Eh inoculation in specific pathogen-free but not germ-free mice was associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and keratinocyte-derived chemokine, which was suppressed with HMGB1 neutralization. This study reveals that Eh-induced active secretion of the HMGB1 plays a key role in shaping the pro-inflammatory landscape critical in innate host defense against amebiasis.

Evidence for SIRT1 Mediated HMGB1 Release From Kidney Cells in the Early Stages of Hemorrhagic Shock.

BackgroundThis study is to explore the effect of SIRT1 deacetylating inactivation on organ-derived high mobility group box 1 (HMGB1) during the development of severe hemorrhagic shock (HS).MethodsHemorrhagic shock model was reproduced in rats by blood shedding and mimicked in HK-2 cells by hypoxia-reoxygenation (H/R) treatment. The level and acetylation of HMGB1 and the expression and activity of SIRT1 were detected in tissue, serum and cultured cells and supernatant. The deacetylated sites of HMGB1 was determined by Co-IP.ResultsSerum HMGB1 in HS rats was increased but were reduced in multiple organs, especially in kidney tissue, with enhanced HMGB1 acetylation, and reduced deacetylase SIRT1 activity in isolated RTECs. HMGB1 in suspension of H/R-treated HK-2 cells was increased, accompanying with enhanced HMGB1 acetylation, and nuclear-plasma translocation. SIRT1 down-regulated by siRNA aggravated acetylation of HMGB1 and nucleus-to-cytoplasm translocation and resulted in increased HMGB1 in cultured HK-2 suspension. Immunoprecipitation data suggested that SIRT1-indcuced deacetylated sites of HMGB1 were K90 and K177 following H/R. SIRT1 overexpression inhibited the acetylation of HMGB1 and reduced the content of extracellular HMGB1 in H/R-treated HK-2 cells. Inhibiting mutation of SIRT1 restored the acetylation of HMGB1 and HMGB1 content in extracellular suspension. In HS rat model, the neutralization of HMGB1 with antibody could reduce serum HMGB1 and pro-inflammatory cytokine contents, but had no effect on SIRT1 protein expression and activity. Polydatin (PD), a potential SIRT1 agonist, up-regulated SIRT1 activity and inhibited nucleus-to-cytoplasm translocation of HMGB1 in RTECs. Moreover, PD also attenuated RTEC apoptosis, protected renal function, and prolonged survival in HS rats. These beneficial effect of PD is largely blocked by specific inhibition of SIRT1 with Ex527.ConclusionThe acetylation of HMGB1 in K99 and K177 is enhanced during HS due to the downregulation of SIRT1. The nucleus-to-cytoplasm translocation and the release of acetylated HMGB1 from RTECs of kidney might exacerbate the pro-inflammatory effects of HMGB1 during the development of HS.

High mobility group box 1 enables bacterial lipids to trigger receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis and apoptosis in mice

Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of programmed cell death and inflammation during viral infection or sterile tissue injury. Whether and how bacterial infection also activates RIPK3-dependent immune responses remains poorly understood. Here we show that bacterial lipids (lipid IVa or lipid A) form a complex with high mobility group box 1 (HMGB1), released by activated immune cells or damaged tissue during bacterial infection, and that this complex triggers RIPK3- and TIR domain-containing adapter-inducing IFN-β (TRIF)-dependent immune responses. We found that these responses lead to macrophage death, interleukin (IL)-1α release, and IL-1β maturation. In an air-pouch inflammatory infiltration model, genetic deletion of Ripk3, Trif, or IL-1 receptor (Il-1R), or monoclonal antibody-mediated HMGB1 neutralization uniformly attenuated inflammatory responses induced by Gram-negative bacteria that release lipid IVa and lipid A. These findings uncover a previously unrecognized mechanism by which host factors and bacterial components work in concert to orchestrate immune responses.