High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Glycyrrhizin regulates rat TMJOA progression by inhibiting the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway

To investigate the role of glycyrrhizin on the progression of temporomandibular joint osteoarthritis (TMJOA) and the underlying mechanism by regulation of the high‐mobility group box 1 (HMGB1) receptor for advanced glycation end products (RAGE)/toll‐like receptor 4 (TLR4)‐nuclear factor kappa B (NF‐κB)/protein kinase B (AKT) pathway. After a rat model of TMJOA was built by intra‐articular injection of monosodium iodoacetate, glycyrrhizin was intragastrically administered at low concentration (20 mg/kg) or high concentration (50 mg/kg). Micro‐computed tomography, histological and immunohistochemical analysis were used to reveal the progression of TMJOA. Rat TMJ chondrocytes and disc cells were cultured in inflammatory condition with different doses of glycyrrhizin. Western blot was used to evaluate the effect of glycyrrhizin on the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway. Administration of glycyrrhizin alleviated cartilage degeneration, lowered the levels of inflammatory and catabolic mediators and reduced the production of HMGB1, RAGE and TLR4 in TMJOA animal model. Increased production of RAGE and TLR4, and activated intracellular NF‐κB and/or AKT signalling pathways in chondrocytes and disc cells were found in inflammatory condition. Upon activation, matrix metalloprotease‐3 and interleukin‐6 were upregulated. Glycyrrhizin inhibited not only HMGB1 release but also RAGE and TLR4 in inflammatory condition. Glycyrrhizin alleviated the pathological changes of TMJOA by regulating the HMGB1‐RAGE/TLR4‐NF‐kB/AKT signalling pathway. This study revealed the potential of glycyrrhizin as a novel therapeutic drug to suppress TMJ cartilage degradation.     

Physiologic Concentrations of HMGB1 Have No Impact on Cytokine-Mediated Eosinophil Survival or Chemotaxis in Response to Eotaxin-2 (CCL24)

HMGB1 is an alarmin that can stimulate the innate immune system alone or in a complex with other inflammatory mediators. Given the recent interest in HMGB1 with respect to the pathogenesis of eosinophil-associated disorders, including asthmatic inflammation and chronic rhinosinusitis, we have explored the role of this mediator and in promoting eosinophil activation. HMGB1 receptors RAGE and TLR4 but not TLR2 were detected on freshly isolated human eosinophils from healthy donors. Physiologic and relevant pathophysiologic levels of biologically-active HMGB1 had no effect on survival of human eosinophils alone or in combination with pro-survival cytokines IL-5, IL-3, or GM-CSF, and increasing concentrations of HMGB1 had no impact on surface expression of RAGE, TLR2 or TLR4. Similarly, HMGB1 did not elicit chemotaxis of human eosinophils alone and had no effect in combination with the eosinophil chemotactic agent, eotaxin-2 (CCL24). However, surface expression of TLR2 and TLR4 increased in response to cell stress, notably on eosinophils that remain viable after 48 hours without IL-5. As such, HMGB1 signaling on eosinophils may be substantially more detailed, and may involve complex immunostimulatory pathways other than or in addition to those evaluated here.     

高速泳动族蛋白1的致炎症作用机制

高速泳动族蛋白1(high-mobility group box 1,HMGB1)是一种高度保守的DNA结合蛋白,具有维持核小体结构和调节基因转录的功能,近来发现它是炎性反应强有力的促炎因子。在大多炎性疾病,特别是脓毒症病例中,HMGB1的血清和组织水平均显著升高,而且它与其受体如糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、Toll样受体4(toll-like receptor,TLR4)、Toll样受体2(TLR2)等相互作用促进炎性疾病的发展。为了进一步了解HMGB1,本文就HMGB1的结构、生物学活性、与免疫细胞相互作用、细胞表面受体、以及拮抗HMGB1的药物等进行综述。     

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

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

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.     

Role of HMGB1 signaling in the inflammatory process in diabetic retinopathy

High mobility group box 1 (HMGB1) is a key player in retinal inflammation. HMGB1 is a danger associated protein pattern receptor which can sense high glucose as a stressor. Increased HMGB1 levels have been found in patients with late stage diabetic retinopathy. HMGB1 can bind toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE), leading to increased inflammation commonly through nuclear factor kappa beta (NFkB). Because diabetic patients have been found to have increased HMGB1 and RAGE levels, as well as polymorphisms of TLR4, a number of investigations have focused on inhibition of these pathways in the diabetic retina. Work in diabetic animal models and cell culture have demonstrated a number of factors that can inhibit HMGB1/TLR4/RAGE signaling. This regulation offers potential new avenues for therapeutic development. This review is focused on HMGB1 signaling and downstream pathways leading to inflammation in the diabetic retina.     

A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin

High-mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac-1-dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1-mediated recruitment was prevented in mice deficient in the beta2-integrin Mac-1 but not in those deficient in LFA-1. As observed by bone marrow chimera experiments, Mac-1-dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of nuclear factor-kappaB in neutrophils required both Mac-1 and RAGE. Together, a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE is described here.     

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

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.     

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

IntroductionHigh 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.MethodsThe various effects of HMGB1 in ANCA-induced neutrophil activation were measured. Antagonists for relevant receptors and signaling molecules were employed.ResultsANCA 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.ConclusionsHMGB1 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.     

High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor

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.     

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

High mobility group protein box1 (HMGB1) and its receptor—receptor for advanced glycation end products (RAGE) are pivotal factors in the development and progression of many types of tumor, but the role of HMGB1-RAGE axis in hepatocellular carcinoma (HCC) especially its effects on metastasis and recurrence remains obscure. Here, we report the role of HMGB1-RAGE axis in the biological behaviors of HCC cell lines and the underlying molecular mechanism. We show that the expressions of HMGB1, RAGE, and extracellular HMGB1 increase consistently according to cell metastasis potentials, while the concentration of soluble form of RAGE (sRAGE) is inversely related to metastasis potential of HCC cells. Furthermore, our data show that rhHMGB1 promotes cellular proliferation, migration, and invasion, and increases the level of nuclear factor kappa B (NF-κB), while administrations of HMGB1-siRNA, RAGE-siRNA, anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody, and sRAGE inhibit cellular proliferation, migration, and invasion. Moreover, we also demonstrate that the expression of NF-кB is inhibited by knockdown of HMGB1 or RAGE. Collectively, these data demonstrate that HMGB1 activates RAGE signaling pathways and induces NF-кB activation to promote cellular proliferation, invasion, and metastasis, in HCC cell lines. Taken together, HMGB1-RAGE axis may become a potential target in HCC therapy.     

The role of hyperglycemia in mechanisms of exacerbated inflammatory responses within the oral cavity

Immune modulating factors are necessary for pathogen clearance, but also contribute to host tissues damage, as those seen in periodontal diseases. Many of these responses can be exacerbated by host conditions including type 2 diabetes [T2D], where toll-like receptor 4 [TLR4] and the receptor for advanced glycated end products [RAGE] play a significant role. Here we investigate causality associated with the increase in inflammatory markers observed in periodontally diseased patients with T2D using multi-variant correlation analysis. Inflammation associated with periodontal diseases, characterized by elevated pro-inflammatory cytokines, innate immune receptor expression, and cellular infiltrate was exacerbated in patients with T2D. In addition, a feed forward loop regulated by poor glycemic control was associated with a loss of mucosal barrier integrity and accumulation of innate immune receptor ligands resulting in an exacerbation of ongoing inflammation, where RAGE and TLR4 cooperated to induce responses in oral epithelial cells, which were exacerbated by hyperglycemia.     

低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响*

目的:探讨低氧时人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC)的高迁移率族蛋白1(HMGB1)及相关受体和炎症因子表达,并检测HMGB1对两种细胞增殖、迁移活性的影响。方法:低氧(1%氧浓度,Hypoxia组)及常氧(Control组)条件下培养HPASMC和HPAEC,RealTime-PCR检测两种细胞HMGB1、TLR2、TLR4、TLR9、RAGE、CD24、IL-6 、TNF-a和CXCL8 mRNA等受体和炎性因子的表达。MTS法观察不同浓度HMGB1对HPASMC和HPAEC增殖的影响;划痕法观察HMGB1对HPASMC和HPAEC迁移的影响。结果:Hypoxia组HPASMC、HPAEC中HMGB1及RAGE mRNA表达量较Control 组明显升高(P＜0.05及0.01);Hypoxia组HPAEC中CD24及HPASMC中IL-6 mRNA表达明显增高(P均＜0.05)。MTS结果显示在345 pmol/L 剂量下 HMGB1明显抑制HPAEC的增殖(P＜0.01),而对HPASMC增殖无影响。划痕实验示HMGB1对HPASMC和HPAEC迁移无明显影响。结论:低氧诱导HPAEC、HPASMC 产生HMGB1;HMGB1通过抑制HPAEC增殖引起内皮屏障功能障碍;而低氧进一步刺激HPASMC产生炎症因子。     

High mobility group box-1 induces migration of vascular smooth muscle cells via TLR4-dependent PI3K/Akt pathway activation

High mobility group box-1 (HMGB1), a potent mediator of inflammation, is known to regulate cellular events through binding to the multiple cell-surface receptors, including RAGE and TLRs. However, the role of TLR4 and details of HMGB1 signaling in vascular smooth muscle cells (VSMCs) migration has not been reported so far. The present study was designed to investigate the hypothesis that HMGB1-induced VSMCs migration is mediated via activation of phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathway through TLR4. VSMCs from rat thoracic aorta were studied. HMGB1 (0.1–1000 ng/ml) stimulated VSMCs migration in a dose-dependent manner, with the highest value (about 3.5-fold increase). Incubation of VSMCs with 100 ng/ml caused a rapid increase in PI3K activity and Akt phosphorylation. Migration of VSMCs toward HMGB1 was significantly inhibited by silencing of TLR4 (P < 0.05). We also found pretreated cells with TLR4 siRNA or the PI3 K inhibitor LY294002 could markedly block PI3K/Akt pathway activation and VSMCs migration mediated by HMGB1 (P both <0.05). In conclusion, HMGB1 induces migration of VSMCs through a TLR4-dependent PI3 K/Akt signaling pathway, which suggests a possible molecular mechanism for HMGB1 may contribute to neointima formation in restenosis after vascular damage.     

A murine cellular model of necroinflammation displays RAGE‐dependent cytokine induction that connects to hepatoma cell injury

Unresolved inflammation maintained by release of danger‐associated molecular patterns, particularly high‐mobility group box‐1 (HMGB1), is crucial for hepatocellular carcinoma (HCC) pathogenesis. To further characterize interactions between leucocytes and necrotic cancerous tissue, a cellular model of necroinflammation was studied in which murine Raw 264.7 macrophages or primary splenocytes were exposed to necrotic lysates (N‐lys) of murine hepatoma cells or primary hepatocytes. In comparison to those derived from primary hepatocytes, N‐lys from hepatoma cells were highly active—inducing in macrophages efficient expression of inflammatory cytokines like C‐X‐C motif ligand‐2 , tumor necrosis factor‐α, interleukin (IL)‐6 and IL‐23‐p19. This activity associated with higher levels of HMGB1 in hepatoma cells and was curbed by pharmacological blockage of the receptor for advanced glycation end product (RAGE)/HMGB1 axis or the mitogen‐activated protein kinases ERK1/2 pathway. Analysis of murine splenocytes furthermore demonstrated that N‐lys did not comprise of functionally relevant amounts of TLR4 agonists. Finally, N‐lys derived from hepatoma cells supported inflammatory splenic Th17 and Th1 polarization as detected by IL‐17, IL‐22 or interferon‐γ production. Altogether, a straightforward applicable model was established which allows for biochemical characterization of immunoregulation by HCC necrosis in cell culture. Data presented indicate a remarkably inflammatory capacity of necrotic hepatoma cells that, at least partly, depends on the RAGE/HMGB1 axis and may shape immunological properties of the HCC microenvironment.