High mobility group (HMG) box proteins are abundant and ubiquitous DNA binding proteins with a remarkable array of functions throughout the cell. The structure of the HMG box DNA binding domain and general mechanisms of DNA binding and bending have been known for more than a decade. However, new mechanisms that regulate HMG box protein intracellular translocation, and by which HMG box proteins recognize DNA with and without sequence specificity, have only recently been uncovered. This review focuses primarily on the Sry-like HMG box family, HMGB1, and mitochondrial transcription factor A. For these proteins, structural and biochemical studies have shown that HMG box protein modularity, interactions with other DNA binding proteins and cellular receptors, and post-translational modifications are key regulators of their diverse functions.

Matrine is one of the main active components of Chinese herb Sophora flavescens Ait (Kushen), which has been demonstrated to be effective in suppressing inflammation. The aim of the present study is to investigate the effect of matrine on LPS-induced lung injury. Lung injury was assessed by histological study and wet to dry weight ratios, as well as cell count and protein content in bronchoalveolar lavage fluid. We also detected MPO activity reflecting neutrophil infiltration and MDA activity examining oxidative stress in lung tissues. Cytokines and ROS production in cells were monitored by ELISA and flow cytometry, respectively. The results showed that high dose of matrine significantly reduced the mortality rate of mice with LPS administration. Treatment with matrine improved LPS-induced lung histopathologic changes, alleviated pulmonary edema and lung vascular leak, inhibited MPO and MDA activity,and reduced the production of inflammatory mediators including TNF-α, IL-6 and HMGB1. In vitro, matrine administration reduced the production of ROS and inflammatory factors, which was possibly associated with inhibition of NF-κB. In conclusion, the current study demonstrated that matrine exhibited a protective effect on LPS-induced acute lung injury by inhibiting of the inflammatory response, which may involve the suppression of ROS and tissue oxidative stress.

A pathogenic role for high-mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Activated protein C (APC) is the only drug approved by the Food and Drug Administration for severe sepsis; however, its effect on HMGB1 signaling has never been investigated. Here, we monitored the effect of APC on the lipopolysaccharide-mediated release of HMGB1 and the HMGB1-mediated modulation of proinflammatory responses in HUVECs. APC potently inhibited the release of HMGB1 and down-regulated the adhesion of the monocytic cell line, THP-1, to HMGB1-activated endothelial cells. HMGB1 up-regulated proinflammatory responses by interacting with 3 pathogen-related pattern recognition receptors: TLR2 and TLR4 and the receptor for advanced glycation end products. APC not only inhibited HMGB1 release but also down-regulated the cell surface expression of all 3 HMGB1 receptors in endothelial cells. The protective effects of APC were mediated through endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR-1). Interestingly, a thrombin derivative containing the Gla-domain of APC recapitulated all protective effects of APC with a 20- to 50-fold higher efficacy. These results suggest that the EPCR- and PAR-1-dependent protective effects of APC in severe sepsis may partially be mediated through the inhibition of HMGB1 signaling and that the chimeric thrombin mutant has potential therapeutic utility for severe sepsis.

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.

The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. The inflammatory response is partly mediated by innate immune cells (such as macrophages, monocytes, and neutrophils), which not only ingest and eliminate invading pathogens but also initiate an inflammatory response by producing early (e.g., TNF and IFN-gamma) and late (e.g., high-mobility group box [HMGB1]) proinflammatory cytokines. Here, we briefly review emerging evidence that support extracellular HMGB1 as a late mediator of experimental sepsis and discuss therapeutic potential of several HMGB1-inhibiting agents (including neutralizing antibodies and steroid-like tanshinones) in experimental sepsis.

OBJECTIVE

High mobility group box chromosomal protein 1 (HMGB1) is a lately discovered candidate molecule identified as an important extracellular mediator in systemic inflammation. Systemic inflammation results in endothelial cell activation and microvascular injury. In the present study, we investigated the effects of HMGB1 on the activation of human umbilical vein endothelial cells (HUVECs) and defined pathways activated by HMGB1.

METHODS

HUVECs obtained by collagenase treatment of umbilical cord veins were stimulated in vitro with HMGB1. The activation of HUVECs was studied regarding (i) the kinetics of tumor necrosis factor-α (TNF-α) production in HUVECs, (ii) HMGB1-induced up-regulation of receptor for advanced glycation end products (RAGE), (iii) HMGB1-induced nuclear translocation of nuclear factor kappa B (NF-κB) in HUVECs, (iv) the activation of signalling transduction pathways.

RESULTS

HUVECs activation was stimulated by HMGB1 partially in a RAGE-dependent manner. Additionally, the HMGB1-induced activation of HUVECs was significantly inhibited by anti-RAGE monoclonal antibody and Ethyl pyruvate (EP) that had been shown to be an effective anti-inflammatory agent. Short-term prestimulation of HUVECs with HMGB1 caused a time-dependent increase in the secretion of TNF-α and expression of RAGE. Furthermore, HMGB1 stimulation resulted in nuclear translocation of transcription factor NF-κB. Most importantly, pretreatment with anti-RAGE monoclonal antibody significantly decreased the amounts of TNF-α and inhibited the nuclear translocation of NF-κB. Additionally in HUVECs cultures, EP specifically inhibited activation of NF-κB signaling pathway that are critical for TNF-α release.

CONCLUSIONS

In conclusion, Our data present a link between HMGB1and RAGE function of endothelial cells and demonstrate the pathway activated by HMGB1. These findings may provide a novel therapeutic strategy to improve the endothelial cells function.

The nuclear protein high-mobility group box chromosomal protein 1 (HMGB1) was recently described to act as a pro-inflammatory cytokine and as a late mediator of severe sepsis and septic shock. The protein is released from monocytes in response to endotoxin and activates monocytes and endothelial cells through nuclear factor kappa B. We have previously demonstrated that the B-box of HMGB1 mediates a pro-inflammatory effect on endothelial cells including the upregulation of cell-adhesion molecules and release of interleukin (IL)-8 and granulocyte colony-stimulating factor. Here, we report that HMGB1 is released from human umbilical vein endothelial cells (HUVEC) in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-alpha. A nuclear relocation of HMGB1 to the cytoplasm was seen at 4 h. Subsequently, high amounts of HMGB1 could be seen in the supernatants from stimulated cells after 16 h. It was also observed that the pro-inflammatory activity of HMGB1 is sensitive to dexamethasone. Interestingly, the HMGB1-induced TNF-alpha release from monocytes could be inhibited by either the A-box of the protein or the p38 inhibitor CNI-1493, but neither had any inhibitory effects on the HMGB1-dependent upregulation of cell-adhesion molecules on HUVEC. Altogether, these results suggest that HUVEC may be an important source of HMGB1 secretion in response to systemic infection and that endothelial cells and monocytes may use different signalling pathways.

The high-mobility group protein HMGB1 contains two tandem DNA-binding HMG box domains, A and B, linked by a short flexible linker that allows the two domains to behave independently in the free protein. There is no structural information on how the linked domains and linker behave when bound to DNA, mainly due to the lack of any DNA-sequence preference of HMGB1. We report the structure determination, by NMR spectroscopy, of a well-defined complex of two tandem HMG boxes bound to a 16 bp oligonucleotide. The protein is an engineered version of the AB di-domain of HMGB1, in which the A box has been replaced by the HMG box of the sequence-specific transcription factor SRY, to give SRY.B. In the SRY.B/DNA complex, both HMG boxes bind in the minor groove and contribute to the overall DNA bending by intercalation of bulky hydrophobic residues between base-pairs; the bends reinforce each other, and the basic linker lies partly in the minor groove. As well as being the first structure of an HMG-box di-domain bound to DNA, this provides the first structure of the B domain of HMGB1 bound to DNA.

Several estrogen-tethered platinum(IV) complexes were prepared and characterized by ESI-MS and (1)H NMR spectroscopy. Their design was inspired by the observation that estrogen receptor-positive cells exposed to the hormone are sensitized to cisplatin. Intracellular reduction of bis-estrogen-cis-diamminedichloroplatinum(IV), BEP(n) (where n = 1-5 methylene groups between Pt and estrogen), occurs to afford cisplatin and two equivalents of the linker-modified estrogen. The ability of BEP(n) to induce overexpression of HMGB1 was established by immunofluorescence microscopy. The cytotoxicity of the compounds was evaluated in ER(+) MCF-7 and ER(-) HCC-1937 human breast cancer cell lines. BEP3 selectively induces overexpression of HMGB1 in MCF-7 cells, compared to HCC-1937 cells, and enhances their sensitivity (IC(50) = 2.1 +/- 0.4 microM versus 3.7 +/- 0.9 microM, respectively) to the compound. The difference in compound activities and the potential of compounds of this class for treating breast and ovarian cancer are discussed.

OBJECTIVE

To measure levels of high-mobility group box -1 (HMGB1) and soluble receptor for advanced glycation end products (sRAGE) in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and to correlate their levels with clinical disease activity and the levels of the inflammatory biomarkers monocyte chemoattractant protein-1 (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-1β (IL-1β), and granulocyte macrophage colony-stimulating factor (GM-CSF). In addition, we examined the expression of HMGB1 in the retinas of diabetic mice.

METHODS

Vitreous samples from 29 PDR and 17 nondiabetic patients were studied by enzyme-linked immunosorbent assay. Retinas of mice were examined by immunofluorescence analysis and western blotting.

RESULTS

HMGB1 was detected in all vitreous samples and sRAGE was detected in 5 PDR samples. IL-1β was detected in 3PDR samples and GM-CSF was not detected. Mean HMGB1 levels in PDR with active neovascularization were twofold and threefold higher than that in inactive PDR and nondiabetic patients, respectively. Mean HMGB1 levels in PDR patients with hemorrhage were significantly higher than those in PDR patients without hemorrhage and nondiabetic patients (p=0.0111). There were significant correlations between levels of HMGB1 and levels of MCP-1 (r=0.333, p=0.025) and sICAM-1 (r=0.548, p<0.001). HMGB1 expression was also upregulated in the retinas of diabetic mice.

CONCLUSIONS

Subclinical chronic inflammation might contribute to the progression of PDR.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our and other studies have found that hydrogen gas (H₂) treatment can ameliorate the lung injury induced by sepsis, ventilator, hyperoxia, and ischemia-reperfusion. However, the molecular mechanisms by which H₂ ameliorates lung injury remain unclear. In the current study, we investigated whether H₂ or hydrogen-rich saline (HS) could exert protective effects in a mouse model of ALI induced by intratracheal administration of lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway-mediated inflammation and apoptosis. Two percent of H₂ was inhaled for 1 h beginning at 1 and 6 h after LPS administration, respectively. We found that LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology and histologic scores, wet-to-dry weight ratio, and oxygenation index (PaO₂/FIO₂), as well as total protein in the bronchoalveolar lavage fluid (BALF), which was attenuated by H₂ treatment. Hydrogen gas treatment inhibited LPS-induced pulmonary early and late NF-κB activation. Moreover, H₂ treatment dramatically prevented the LPS-induced pulmonary cell apoptosis in LPS-challenged mice, as reflected by the decrease in TUNEL (deoxynucleotidyl transferase dUTP nick end labeling) staining-positive cells and caspase 3 activity. Furthermore, H₂ treatment markedly attenuated LPS-induced lung neutrophil recruitment and inflammation, as evidenced by downregulation of lung myeloperoxidase activity, total cells, and polymorphonuclear neutrophils in BALF, as well as proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, interleukin 6, and high-mobility group box 1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein [MIP] 1α, MIP-2, and monocyte chemoattractant protein 1) in BALF. In addition, i.p. injection of 10 mL/kg hydrogen-rich saline also significantly attenuated the LPS-induced ALI. Collectively, these results demonstrate that molecular hydrogen treatment ameliorates LPS-induced ALI through reducing lung inflammation and apoptosis, which may be associated with the decreased NF-κB activity. Hydrogen gas may be useful as a novel therapy to treat ALI. munosorbent assay; H₂-hydrogen gas; HMGB1-high-mobility group box 1; HS-hydrogen-rich saline; i.t.-intratracheal; KC-keratinocyte-derived chemokine; LPS-lipopolysaccharide; MCP-1-monocyte chemoattractant protein 1; MIP-1α-macrophage inflammatory protein 1α; MIP-2-macrophage inflammatory protein 2; MPO-myeloperoxidase; PBS-phosphate-buffered saline; PMNs-polymorphonuclear neutrophils; TUNEL-deoxynucleotidyl transferase dUTP nick end labeling; W/D-wet-to-dry.

Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.

OBJECTIVE

Minocycline, a semisynthetic tetracycline antibiotic, has been reported to ameliorate brain injury and inhibit microglial activation after focal cerebral ischemia. However, the cerebroprotective mechanism of minocycline remains unclear. In the present study, we investigated that mechanism of minocycline in a murine model of 4-hour middle cerebral artery (MCA) occlusion.

METHODS

One day after 4-hour MCA occlusion, minocycline was administered intraperitoneally for 14 days. Neurologic scores were measured 1, 7, and 14 days after cerebral ischemia. Motor coordination was evaluated at 14 days by the rota-rod test at 10 rpm. Activated microglia and high-mobility group box1 (HMGB1), a cytokine-like mediator, were also evaluated by immunostaining and Western blotting. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling immunostaining was carried out 14 days after cerebral ischemia.

RESULTS

Repeated treatment with minocycline (1, 5, and 10 mg/kg) for 14 days improved neurologic score, motor coordination on the rota-rod test, and survival in a dose-dependent manner. Minocycline decreased the expression of Iba1, a marker of activated microglia, as assessed by both immunostaining and Western blotting. Moreover, minocycline decreased the activation of microglia expressing HMGB1 within the brain and also decreased both brain and plasma HMGB1 levels. Additionally, minocycline significantly decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells and prevented ischemic brain atrophy 14 days after cerebral ischemia.

CONCLUSIONS

Our results suggest that minocycline inhibits activated microglia expressing HMGB1 and decreases neurologic impairment induced by cerebral ischemia. Minocycline will have a palliative action and open new therapeutic possibilities for treatment of postischemic injury via an HMGB1-inhibiting mechanism.

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage-associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue-healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti-tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

Most nuclear proteins reside on a specific chromatin site only for seconds or less. The hit-and-run model of transcriptional control maintains that transcription complexes are assembled in a stochastic fashion from freely diffusible proteins; this contrasts to models involving stepwise assembly of stable holo complexes. However, the chances of forming a productive complex improve if the binding of one factor promotes the binding of its interactors. We prove here that in living cells, the glucocorticoid receptor and HMGB1 interact only within chromatin and not in the nucleoplasm and decrease each other's mobility. Thus, the formation of a GR-HMGB1-chromatin complex is more likely than one would expect from independent binding to chromatin of GR and HMGB1. Remarkably, this complex is potentially stable, and its disassembly is effected by active, ATP-consuming processes. We propose that kinetic cooperativity among transcription factors in chromatin binding may be a common feature in transcription and DNA transactions.

Cells of the innate immune system sense tissue damage recognizing in the extracellular environment bona fide intracellular moieties, like high mobility group box 1 (HMGB1). In the case of tumors, HMGB1 recognition has a paradoxical dual effect: it promotes tumor neoangiogenesis and triggers protective anti-neoplastic T-cell responses. Recent advances in the study of HMGB1 have identified candidate molecular mechanisms underlying these apparently contrasting outcomes. A surprising role for innate receptors, including toll like receptor 4 (TLR4), in the response to conventional cancer radio and chemotherapy has also recently emerged, providing new insight into the mechanisms by which these treatments actually work.

The pathogenesis of sepsis is partly attributable to dysregulated inflammatory response mediated by pathogen-associated molecular patterns (PAMPs) (for example, endotoxin) and damage-associated molecular patterns (DAMPs) (for example, high-mobility group box 1 [HMGB1]). An endogenous ubiquitous polyamine, spermine, inhibits endotoxin-induced cytokine release in vitro, but its capacities to attenuate sepsis- and HMGB1-induced inflammatory responses was previously unknown. We thus tested the hypothesis that spermine protects mice against lethal sepsis by attenuating sepsis-induced local and systemic inflammatory responses. Intraperitoneal (i.p.) administration of spermine (10 mg/kg, twice daily, for 3 d) conferred a significant protection against lethal sepsis. The protective effects were associated with a significant reduction in peritoneal and serum levels of several surrogate markers of sepsis (for example, Interleukin-6 [IL-6], keratinocyte-derived chemokine [KC], monocytes chemoattractant protein-1 [MCP-1], macrophage inflammatory protein-2 [MIP-2], tissue inhibitor of metalloproteinase-1 [TIMP-1], soluble tumor necrosis factor-alpha receptor I [sTNFRI], and soluble tumor necrosis factor-alpha receptor II [sTNFRII]) during a late stage of sepsis. In vitro, spermine effectively inhibited HMGB1-induced release of the above surrogate markers in peritoneal macrophages. Thus, spermine confers protection against lethal sepsis partly by attenuating sepsis- and HMGB1-induced inflammatory responses.

OBJECTIVE

Recently, metformin, a well-known anti-diabetic drug, has been shown to possess anti-inflammatory activities. This study investigated the effect of metformin on the expression of pro-inflammatory cytokines including high mobility group box 1 (HMGB1) in lipopolysaccharide (LPS)-treated animals and cells.

METHODS

We investigated whether metformin inhibits the release of HMGB1 in LPS-treated RAW 264.7 cells and increases survival rate in endotoxaemic mice (lethal endotoxaemia was induced by an i.p. injection of LPS). This was achieved by a range of techniques including Western blotting, enzyme-linked immunosorbent assay, specific pharmacological inhibitors, knock out of α(1) -subunit of AMP-activated protein kinase (AMPK) and recombinant HMGB1.

RESULTS

Both pre- and post-treatment with metformin significantly improved survival of animals during lethal endotoxaemia (survival rate was monitored up to 2 weeks), decreased serum levels of tumour necrosis factor-alpha (TNF-α), interleukin-1β, HMGB1 expression and myeloperoxidase activity in lungs. However, metformin failed to improve survival in endotoxaemic animals that had additionally been treated with recombinant HMGB1. In an in vitro study, metformin dose-dependently inhibited production of pro-inflammatory cytokines and HMGB1 release. Metformin activated AMPK by its phosphorylation. Compound C (pharmacological inhibitor of AMPK) and siAMPKα1 reversed the anti-inflammatory effect of metformin in LPS-treated cells.

CONCLUSIONS

Our data indicate that metformin significantly attenuates the pro-inflammatory response induced by LPS both in vivo and in vitro. Metformin improved survival in a mouse model of lethal endotoxaemia by inhibiting HMGB1 release. AMPK activation was implicated as one of the mechanisms contributing to this inhibition of HMGB1 secretion.

Pattern recognition receptors (PRR), immune sensors that discriminate self from non-self, link innate to adaptive immunity. PRR are involved in microbe internalization by phagocytes (soluble PRR and endocytic receptors) and/or cell activation (signaling PRR). PRR also recognize dying cells (i.e. modified self). Apoptotic cell recognition involves soluble bridging molecules (e.g. pentraxins) and endocytic receptors (e.g. scavenger receptors, the CD91-calreticulin complex). Apoptotic cells induce an immunosuppressive signal, avoiding the initiation of an autoimmune response. By contrast, necrotic cells, via the release of stimulatory molecules [heat shock protein (HSP), high-mobility group box 1 protein (HMGB1)], activate immune cells. This review summarizes the PRR involved in the recognition of dying cells and the consequences on the outcome of the immune response directed against dying cell antigens.

BACKGROUND

High-mobility group box-1 protein (HMGB1) is a nuclear protein that regulates gene expression throughout the body. It can also become cytoplasmic and function as a neuromodulatory cytokine after tissue damage or injury. The manner in which HMGB1 influences the peripheral nervous system following nerve injury is unclear. The present study investigated the degree to which HMGB1 signaling contributes to the maintenance of neuropathic pain behavior in the rodent.

RESULTS

Redistribution of HMGB1 from the nucleus to the cytoplasm occurred in both sensory neurons derived from a tibial nerve injured (TNI) rat and in a sensory neuron-like cell line following exposure to a depolarizing stimulus. We also observe that exogenous administration of HMGB1 to acutely dissociated sensory neurons derived from naïve or TNI rodents elicit increased excitability. Furthermore systemic injection of glycyrrhizin (50 mg/kg; i.p.), a known inhibitor of HMGB1, reversed TNI-induced mechanical hyperalgesia at fourteen days and three months following nerve injury.

CONCLUSIONS

We have identified that a persistent endogenous release of HMGB1 by sensory neurons may be a potent, physiologically relevant modulator of neuronal excitability. More importantly, the use of the anti-inflammatory compound and known inhibitor of HMGB1, glycyrrhizin, has the ability to diminish persistent pain behavior in a model of peripheral neuropathy, presumably through its ability to neutralize the cyotkine. The identification of HMGB1 as a potential therapeutic target may contribute to a better understanding of mechanisms associated with chronic pain syndromes.

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

Recent and compelling investigation has expanded our view of the biological settings in which the products of nonenzymatic glycation and oxidation of proteins and lipids - the advanced glycation endproducts (AGEs) - form and accumulate. Beyond diabetes, natural ageing and renal failure, AGEs form in inflammation, oxidative stress and in ischaemia-reperfusion. The chief signal transduction receptor for AGEs - the receptor for AGEs (RAGE) - is a multiligand-binding member of the immunoglobulin superfamily. In addition to AGEs, RAGE binds certain members of the S100/calgranulin family, high-mobility group box 1 (HMGB1), and beta-amyloid peptide and beta-sheet fibrils. Recent studies demonstrate beneficial effects of RAGE antagonism and genetic deletion in rodent models of atherosclerosis and ischaemia-reperfusion injury in the heart and great vessels. Experimental evidence is accruing that RAGE ligand generation and release during ischaemia-reperfusion may signal through RAGE, thus suggesting that antagonism of this receptor might provide a novel form of therapeutic intervention in heart disease. However, it is plausible that innate, tissue-regenerative roles for these RAGE ligands may also impact the failing heart - perhaps through RAGE and/or distinct receptors. In this review, we focus on RAGE and the consequences of its activation in the cardiovasculature.

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models.