Recent discoveries in signal-transducing innate receptors have illustrated the close link between innate and adaptive immunity. These advances revisit a fundamental issue of immunology, the recognition of self and non-self molecules by the immune system. Indeed, mounting evidence has been provided that the sensing of self-derived molecules by the immune system is important for health and disease. The high-mobility group box (HMGB) proteins, particularly HMGB1, are self-derived immune activators that have multiple functions in the regulation of immunity and inflammation. In this review, we summarize current knowledge of the function of HMGB proteins, as a ligand that can evoke inflammatory responses, and as a sensor for nucleic-acid-mediated immune responses.

BACKGROUND

High mobility group box-1 (HMGB1) is a newly recognized factor regulating cancer cell tumorigenesis, expansion and invasion. We investigated the correlation between the serum HMGB1 levels and the clinical and pathologic features of gastric cancer and evaluated the validity of HMGB1 as a potential biomarker for the early diagnosis of gastric cancer.

METHODS

A total of 227 subjects were classified into 5 disease groups according to the 'gastritis-dysplasia-carcinoma' sequence of gastric carcinogenesis and their serum levels of HMGB1 were analyzed by an enzyme-linked immunosorbent assay (ELISA) method. Clinical parameters, International Union Against Cancer (UICC) TNM stage, cancer size, differentiation or lymphatic invasion, vascular or perineural invasion and prognosis were used as analysis variables.

RESULTS

The serum HMGB1 levels were significantly different among disease groups (ANOVA, p < 0.05) and HMGB1 levels tended to increase according to the progression of gastric carcinogenesis. Serum HMGB1 levels were significantly associated with depth of invasion, lymph node metastasis, tumor size, and poor prognosis (p < 0.05). However, HMGB1 levels were not associated with patient gender or age, differentiation of tumor cells, or lymphatic, vascular and perineural invasion, or the existence of distant metastasis in advanced cancer (p>> 0.05). The sensitivity and specificity of serum HMGB1 was 71% and 67% (cut-off value of 5 ng/ml) for the diagnosis of early gastric cancer, and 70% and 64% (cut-off value of 4 ng/ml) for the diagnosis of high-risk lesions, respectively. These values were greater than those for carcinoembryonic antigen (CEA) (30-40% of sensitivity).

CONCLUSIONS

HMGB1 appears to be a useful serological biomarker for early diagnosis as well as evaluating the tumorigenesis, stage, and prognosis of gastric cancer.

Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB transposase was found to interact with HMGB1 in vivo, suggesting that the transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.

High-mobility group box-1 (HMGB1) is a nuclear protein with cytokine-type functions upon its extracellular release. HMGB1 activates inflammatory pathways by stimulating multiple receptors, chiefly toll-like receptor 4 (TLR4) and Receptor for Advanced Glycation End Products (RAGE). TLR4 and RAGE activation has been implicated in memory impairments, although the endogenous ligand subserving these effects is unknown. We examined whether HMGB1 induced memory deficits using novel object recognition test, and which of the two receptor pathways was involved in these effects. Non-spatial long-term memory was examined in wild type, TLR4 knockout, and RAGE knockout mice. Recombinant HMGB1 (10μg, intracerebroventricularly, i.c.v.) disrupted memory encoding equipotently in wild type, TLR4 knockout and RAGE knockout animals, but affected neither memory consolidation, nor retrieval. Neither TLR4 knockout nor RAGE knockout mice per se, exhibited memory deficits. Blockade of TLR4 in RAGE knockout mice using Rhodobacter sphaeroides lipopolysaccharide (LPS-Rs; 20 μg, i.c.v.) prevented the detrimental effect of HMGB1 on memory. These data show that elevated brain levels of HMGB1 induce memory abnormalities which may be mediated by either TLR4, or RAGE. This mechanism may contribute to memory deficits under various neurological and psychiatric conditions associated with the increased HMGB1 levels, such as epilepsy, Alzheimer's disease and stroke.

High-mobility-group box 1 (HMGB1), a nuclear protein, has recently been identified as an important mediator of local and systemic inflammatory diseases when released into the extracellular milieu. Anti-inflammatory regulation by the stress response is an effective autoprotective mechanism when the host encounters harmful stimuli, but the mechanism of action remains incompletely delineated. In this study, we demonstrate that increases in levels of a major stress-inducible protein, heat shock protein 72 (Hsp72) by gene transfection attenuated LPS- or TNF-alpha-induced HMGB1 cytoplasmic translocation and release. The mechanisms involved inhibition of the chromosome region maintenance 1 (CRM1)-dependent nuclear export pathway. Overexpression of Hsp72 inhibited CRM1 translocation and interaction between HMGB1 and CRM1 in macrophages post-LPS and TNF-alpha treatment. In addition, overexpression of Hsp72 strongly inhibited HMGB1-induced cytokine (TNF-alpha, IL-1beta) expression and release, which correlated closely with: 1) inhibition of the MAP kinases (p38, JNK, and ERK); and 2) inhibition of the NF-kappaB pathway. Taken together, these experiments suggest that the anti-inflammatory activity of Hsp72 is achieved by interfering with both the release and proinflammatory function of HMGB1. Our experimental data provide important insights into the anti-inflammatory mechanisms of heat shock protein protection.

A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells, and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE) or toll-like receptor 4 (TLR4), HMGB1 activates vascular endothelial cells and macrophages/monocytes to express proinflammatory cytokines, chemokines and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of the pathogenic role of inflammation in cardiovascular diseases, we propose that HMGB1, a proinflammatory cytokine derived from both injured endothelium and activated macrophages/monocytes, could contribute to the progression of atherosclerosis and other cardiovascular diseases.

Sepsis, a life-threatening complication of infections and the most common cause of death in intensive care units, is characterized by a hyperactive and out-of-balance network of endogenous proinflammatory cytokines. None of the current therapies are entirely effective, illustrating the need for novel therapeutic approaches. Ghrelin (GHR) is an orexigenic peptide that has emerged as a potential endogenous anti-inflammatory factor. In this study, we show that the delayed administration of GHR protects against the mortality in various models of established endotoxemia and sepsis. The therapeutic effect of GHR is mainly mediated by decreasing the secretion of the high mobility box 1 (HMGB1), a DNA-binding factor that acts as a late inflammatory factor critical for sepsis progression. Macrophages seem to be the major cell targets in the inhibition of HMGB1 secretion, in which GHR blocked its cytoplasmic translocation. Interestingly, we also report that GHR shows a potent antibacterial activity in septic mice and in vitro. Remarkably, GHR also reduces the severity of experimental arthritis and the release of HMGB1 to serum. Therefore, by regulating crucial processes of sepsis, such as the production of early and late inflammatory mediators by macrophages and the microbial load, GHR represents a feasible therapeutic agent for this disease and other inflammatory disorders.

BACKGROUND

High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity.

METHODS

The study population consisted of 70 SLE patients and 35 age- and sex-matched HC. Thirty-three SLE patients had quiescent disease, the other 37 patients were selected for having active disease. Nineteen of these had lupus nephritis. HMGB1 levels were measured with both Western blot and ELISA. Anti-HMGB1 levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures.

RESULTS

HMGB1 levels in SLE patients could be measured reliably by Western blotting only, and were significantly increased compared to HC. During active disease HMGB1 levels increased, in particular in patients with renal involvement. Serum HMGB1 levels correlated with SLEDAI, proteinuria, and anti-dsDNA levels, and showed a negative correlation with complement C3. Anti-HMGB1 levels were significantly increased in SLE patients compared to HC, and positively correlated with HMGB1 levels.

CONCLUSIONS

Levels of HMGB1 in the sera of SLE patients, in particular in those with active renal disease, are increased. Serum HMGB1 levels are related to SLEDAI scores and proteinuria, as well as to levels of anti-HMGB1 antibodies. These findings suggest that besides HMGB1, HMGB1-anti-HMGB1 immune complexes play a role in the pathogenesis of SLE, in particular in patients with renal involvement.

OBJECTIVE

Due to its anatomic connection, the liver is constantly exposed to gut-derived bacterial products or metabolites. Disruption of gut homeostasis is associated with many human diseases. The aim of this study was to determine the role of gut homeostasis in initiation and progression of hepatocellular carcinoma (HCC).

METHODS

Disruption of intestinal homeostasis by penicillin or dextran sulfate sodium (DSS) and its restoration by probiotics were applied in a diethylnitrosamine (DEN) model of rat hepatocarcinogenesis.

RESULTS

Patients with liver cirrhosis and HCC had significantly increased serum endotoxin levels. Chronic DEN treatment of rats was associated with an imbalance of subpopulations of the gut microflora including a significant suppression of Lactobacillus species, Bifidobacterium species and Enterococcus species as well as intestinal inflammation. Induction of enteric dysbacteriosis or intestinal inflammation by penicillin or DSS, respectively, significantly promoted tumor formation. Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and most importantly, decreased liver tumor growth and multiplicity. Interestingly, probiotics not only inhibited the translocation of endotoxin, which bears pathogen-associated molecular patterns (PAMPs) but also the activation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1). As a result, the production of pro- and anti-inflammatory cytokines was skewed in favor of a reduced tumorigenic inflammation in the liver.

CONCLUSIONS

The data highlights the importance of gut homeostasis in the pathogenesis of HCC. Modulation of the gut microbiota by probiotics may represent a new avenue for therapeutic intervention to treat or prevent HCC development.

OBJECTIVE

We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8(+) T cells. Here, we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), which is derived from tumor cells after inhibition of protein degradation, and to provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy.

METHODS

DRibbles were characterized by Western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole-tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed, and the antitumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma.

RESULTS

The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiP), and damage-associated molecular pattern molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional and plasmacytoid dendritic cells (DC), and cross-presentation was partially CLEC9A dependent. Furthermore, this autophagy-assisted antigen cross-presentation pathway involved both caveolae- and clathrin-mediated endocytosis and endoplasmic reticulum-associated degradation machinery. It depends on proteasome and TAP1, but not lysosome functions of antigen-presenting cells. Importantly, DCs loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma.

CONCLUSIONS

These data documented the unique characteristics and potent antitumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials.

High mobility group box 1 (HMGB1) is a nonhistone architectural protein that is involved in many biological processes including chromatin remodeling, transcription, cell signaling of inflammation, DNA damage repair and others. Recent studies have identified the cross-link of HMGB1 with a DNA base excision repair intermediate indicating that this protein is involved in base excision repair (BER) pathway. Further characterization of the roles of HMGB1 in BER demonstrates that the protein acts as a cofactor to regulate BER sub-pathways by inhibiting single-nucleotide BER and stimulating long-patch BER through modulating the activities of base excision repair enzymes. Directing of base lesion repair to the long-patch sub-pathway can result in trinucleotide repeat instability suggesting an important role of HMGB1 in modulating genome stability.

The multiligand receptor RAGE (receptor for advanced glycation end-products) is emerging as a central mediator in the immune/inflammatory response. Epidemiological evidence accruing in the human suggests upregulation of RAGE's ligands (AGEs, S100/calgranulins, high mobility group box-1 (HMGB1), and amyloid beta-peptide and beta-sheet fibrils) and the receptor itself at sites of inflammation and in chronic diseases such as diabetes and neurodegeneration. The consequences of ligand-RAGE interaction include upregulation of molecules implicated in inflammatory responses and tissue damage, such as cytokines, adhesion molecules, and matrix metalloproteinases. In this review, we discuss the localization of RAGE and its ligand families and the biological impact of this axis in multiple cell types implicated in chronic diseases. Lastly, we consider findings from animal model studies suggesting that although tissue-damaging effects ensue from recruitment of the ligand-RAGE interaction, in distinct settings, adaptive and repair/regeneration outcomes appear to override detrimental effects of RAGE. As RAGE blockade moves further into clinical development, clarifying the biology of RAGE garners ever-increasing importance.

Inflammatory processes in brain tissue have been described in human epilepsy of various aetiologies and in experimental models of seizures. This, together with the anticonvulsant properties of anti-inflammatory therapies both in clinical and in experimental settings, highlights the important role of brain inflammation in the aetiopathogenesis of seizures. Preclinical investigations in experimental models using pharmacological and genetic tools have identified a significant contribution of interleukin-1 (IL-1) type 1 receptor/Toll-like receptor (IL-1R/TLR) signalling to seizure activity. This signalling can be activated by ligands associated with infections (pathogen-associated molecular patterns) or by endogenous molecules, such as proinflammatory cytokines (e.g. IL-1beta) or danger signals [damage-associated molecular patterns, e.g. high-mobility group box 1 (HMGB1)]. IL-1beta and HMGB1 are synthesized and released by astrocytes and microglia in the rodent brain during seizures. Notably, a rapid release of HMGB1 from neurons appears to be triggered by proconvulsant drugs even before seizure occurrence and is involved in their precipitation of seizures. The activation of IL-1R/TLR signalling mediates rapid post-translational changes in N-methyl-d-aspartate-gated ion channels in neurons. A long-term decrease in seizure threshold has also been observed, possibly mediated by transcriptional activation of genes contributing to molecular and cellular plasticity. This emerging evidence identifies specific targets with potential anticonvulsant effects in drug-resistant forms of epilepsy.

We examined our hypothesis that heme-oxygenase-1 (HO-1)-derived carbon monoxide (CO) inhibits the release of high-mobility group box 1 (HMGB1) in RAW264.7 cells activated with lipopolysaccharide (LPS) in vitro and in LPS- or cecal ligation and puncture (CLP)-induced septic mice in vivo, so that HO-1 induction or CO improves survival of sepsis in rodents. We found that pretreatment with HO-1 inducers (hemin, cobalt protoporphyrin IX) or transfection of HO-1 significantly inhibited HMGB1 release, which was blocked by HO-1 small interfering RNA, in cells activated by LPS. Carbon monoxide-releasing molecule 2 (CORM-2) but not bilirubin or deferoxamine inhibited HMGB1 release in LPS-activated macrophages. Oxyhemoglobin reversed the effect of HO-1 inducers on HMGB1 release. Translocation of HMGB1 from nucleus to cytosol was significantly inhibited by HO-1 inducers, CORM-2, or HO-1 transfection. Neutralizing antibodies to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, interferon-beta, and N(omega)-nitro-L-arginine methyl ester hydrochloride but not N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398) significantly inhibited HMGB1 release in LPS-activated cells. Production of TNF-alpha, IL-1beta, and IFN-beta was significantly reduced by pretreatment of HO-1 inducers, CORM-2, or HO-1 transfection in LPS-activated cells. Plasma levels of HMGB1 in mice challenged with LPS or CLP were significantly reduced by the administration of HO-1 inducers or CORM-2, which was accompanied by either reduction (pretreatment) or no change (delayed administration) of serum TNF-alpha and IL-1beta levels. Regardless of pretreatment or delayed administration, CORM-2 and hemin rescued mice from lethal endotoxemia and sepsis induced by LPS or CLP. Taken together, we concluded that HO-1-derived CO reduces HMGB1 release in LPS-activated cells and LPS- or CLP-induced animal model of sepsis.

BACKGROUND

Despite advances in treatment of diabetes mellitus, its prevalence continues to rise globally. Medications available are unable to control the vascular complications. Proposals for new therapeutic targets must take into account the hyperglycemia-induced signaling pathways that give rise to the inflammatory profile of the disease.

METHODS

How high-mobility-group box-1 (HMGB1) protein, acting as an activator of Toll-like receptors (TLR) and receptors for advanced glycation end products (RAGE), creates a functional tripod that contributes to increased production of pro-inflammatory mediators, and sustains the chronic inflammatory state associated with diabetes. The interaction of TLR2 and TRL4 with host-derived ligands, which links diabetic complications with the innate immune response, and the activation of RAGE, which induces a cascade of metabolic responses, leading to the production and secretion of pro-inflammatory cytokines.

CONCLUSIONS

Considering the involvement of the innate immune system, in association with the role of HMGB1 as an activator of TLR and RAGE, diabetes should be considered and treated as a metabolic and immunological disease, triggered by hyperglycemia. HMGB1 plays a central role in mediating injury and inflammation, and interactions involving HMGB1-TLR-RAGE constitute a tripod that trigger NF-κB activation. Blockade or downregulation of HMGB1, and/or control of the inflammatory tripod, represent a promising therapeutic approach for the treatment of diabetes.

High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein present in the nuclei and cytoplasm of nearly all cell types. In response to infection or injury, HMGB1 is actively secreted by innate immune cells and/or released passively by injured or damaged cells. Thus, serum and tissue levels of HMGB1 are elevated during infection, and especially during sepsis. Sepsis is a systemic inflammatory response to disease and the most severe complication of infections, and HMGB1 acts as a potent proinflammatory cytokine and is involved in delayed endotoxin lethality and sepsis. Furthermore, the targeting of HMGB1 with antibodies or specific antagonists has been found to have protective effects in established preclinical inflammatory disease models, including models of lethal endotoxemia and sepsis. In the present study, emerging evidence supporting the notion that extracellular HMGB1 acts as a proinflammatory danger signal is reviewed, and the potential therapeutic effects of a wide array of HMGB1 inhibitors agents in sepsis and ischemic injury are discussed.

BACKGROUND

Exosomes are extracellular vesicles that mediate cellular communication in health and diseases. Neutrophils could be polarized to a pro-tumor phenotype by tumor. The function of tumor-derived exosomes in neutrophil regulation remains unclear.

METHODS

We investigated the effects of gastric cancer cell-derived exosomes (GC-Ex) on the pro-tumor activation of neutrophils and elucidated the underlying mechanisms.

RESULTS

GC-Ex prolonged neutrophil survival and induced expression of inflammatory factors in neutrophils. GC-Ex-activated neutrophils, in turn, promoted gastric cancer cell migration. GC-Ex transported high mobility group box-1 (HMGB1) that activated NF-κB pathway through interaction with TLR4, resulting in an increased autophagic response in neutrophils. Blocking HMGB1/TLR4 interaction, NF-κB pathway, and autophagy reversed GC-Ex-induced neutrophil activation. Silencing HMGB1 in gastric cancer cells confirmed HMGB1 as a key factor for GC-Ex-mediated neutrophil activation. Furthermore, HMGB1 expression was upregulated in gastric cancer tissues. Increased HMGB1 expression was associated with poor prognosis in patients with gastric cancer. Finally, gastric cancer tissue-derived exosomes acted similarly as exosomes derived from gastric cancer cell lines in neutrophil activation.

CONCLUSIONS

We demonstrate that gastric cancer cell-derived exosomes induce autophagy and pro-tumor activation of neutrophils via HMGB1/TLR4/NF-κB signaling, which provides new insights into mechanisms for neutrophil regulation in cancer and sheds lights on the multifaceted role of exosomes in reshaping tumor microenvironment.

Endogenous ligands released from damaged cells, so-called damage-associated molecular pattern molecules (DAMPs), activate innate signaling pathways including the TLRs. We have shown that hepatic, warm ischemia and reperfusion (I/R) injury, generating local, noninfectious DAMPs, promotes inflammation, which is largely TLR4-dependent. Here, we demonstrate that increasing dendritic cell (DC) numbers enhance inflammation and organ injury after hepatic I/R. High-mobility group box 1 (HMGB1), a NF released by necrotic cells or secreted by stimulated cells, is one of a number of ligands promoting TLR4 reactivity. Augmentation of DC numbers in the liver with GM-CSF hydrodynamic transfection significantly increased liver damage after I/R when compared with controls. TLR4 engagement on hepatic DC was required for the I/R-induced injury, as augmentation of DC numbers in TLR4 mutant (C3H/HeJ) mice did not worsen hepatic damage. It is interesting that TLR4 expression was increased in hepatic DC following HMGB1 stimulation in vitro, suggesting a mechanism for the increased liver injury following I/R. It thus appears that functional TLR4 on DC is required for I/R-induced injury. Furthermore, HMGB1 may direct the inflammatory responses mediated by DC, at least in part, by enhancing TLR4 expression and reactivity to it and other DAMPs.

BACKGROUND

Increasing evidence links systemic inflammation to neuroinflammation and neurodegeneration. We previously found that systemic endotoxin, a TLR4 agonist or TNFα, increased blood TNFα that entered the brain activating microglia and persistent neuroinflammation. Further, we found that models of ethanol binge drinking sensitized blood and brain proinflammatory responses. We hypothesized that blood cytokines contribute to the magnitude of neuroinflammation and that ethanol primes proinflammatory responses. Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.

METHODS

Polyinosine-polycytidylic acid (poly I:C) was used to induce inflammatory responses when sensitized with D-galactosamine (D-GalN). Male C57BL/6 mice were treated with water or ethanol (5 g/kg/day, i.g., 10 days) or poly I:C (250 μg/kg, i.p.) alone or sequentially 24 hours after ethanol exposure. Cytokines, chemokines, microglial morphology, NADPH oxidase (NOX), reactive oxygen species (ROS), high-mobility group box 1 (HMGB1), TLR3 and cell death markers were examined using real-time PCR, ELISA, immunohistochemistry and hydroethidine histochemistry.

RESULTS

Poly I:C increased blood and brain TNFα that peaked at three hours. Blood levels returned within one day, whereas brain levels remained elevated for at least three days. Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment. Ethanol pretreatment potentiated poly I:C-induced brain TNFα (345%), IL-1β (331%), IL-6 (255%), and MCP-1(190%). Increased levels of brain cytokines coincided with increased microglial activation, NOX gp91phox, superoxide and markers of neurodegeneration (activated caspase-3 and Fluoro-Jade B). Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain. Minocycline and naltrexone blocked microglial activation and neurodegeneration.

CONCLUSIONS

Chronic ethanol potentiates poly I:C blood and brain proinflammatory responses. Poly I:C neuroinflammation persists after systemic responses subside. Increases in blood TNFα, IL-1β, IL-6, and MCP-1 parallel brain responses consistent with blood cytokines contributing to the magnitude of neuroinflammation. Ethanol potentiation of TLR3 agonist responses is consistent with priming microglia-monocytes and increased NOX, ROS, HMGB1-TLR3 and markers of neurodegeneration. These studies indicate that TLR3 agonists increase blood cytokines that contribute to neurodegeneration and that ethanol binge drinking potentiates these responses.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors, is essential for inducing caspase-1 activation and the consequent secretion of interleukin-1β (IL-1β), which is associated with local inflammation during liver ischemia/reperfusion injury (IRI). However, little is known about the mechanisms by which the ASC/caspase-1/IL-1β axis exerts its function in hepatic IRI. This study was designed to explore the functional roles and molecular mechanisms of ASC/caspase-1/IL-1β signaling in the regulation of inflammatory responses in vitro and in vivo. With a partial lobar liver warm ischemia (90 minutes) model, ASC-deficient and wild-type mice (C57BL/6) were sacrificed at 6 hours of reperfusion. Separate animal cohorts were treated with an anti-IL-1β antibody or control immunoglobulin G (10 mg/kg/day intraperitoneally). We found that ASC deficiency inhibited caspase-1/IL-1β signaling and led to protection against liver ischemia/reperfusion (IR) damage, local enhancement of antiapoptotic functions, and down-regulation of high mobility group box 1 (HMGB1)-mediated, toll-like receptor 4 (TLR4)-driven inflammation. Interestingly, the treatment of ASC-deficient mice with recombinant HMGB1 re-created liver IRI. Moreover, neutralization of IL-1β ameliorated the hepatocellular damage by inhibiting nuclear factor kappa B (NF-κB)/cyclooxygenase 2 signaling in IR-stressed livers. In parallel in vitro studies, the knockout of ASC in lipopolysaccharide-stimulated bone marrow-derived macrophages depressed HMGB1 activity via the p38 mitogen-activated protein kinase pathway and led to the inhibition of TLR4/NF-κB and ultimately the depression of proinflammatory cytokine programs.

CONCLUSIONS

ASC-mediated caspase-1/IL-1β signaling promotes HMGB1 to produce a TLR4-dependent inflammatory phenotype and leads to hepatocellular injury. Hence, ASC/caspase-1/IL-1β signaling mediates the inflammatory response by triggering HMGB1 induction in hepatic IRI. Our findings provide a rationale for a novel therapeutic strategy for managing liver injury due to IR.

The inflammatory responses in sepsis and hemorrhage remain a major cause of death. Clinically, it is generally accepted that shock in sepsis or hemorrhage differs in its mechanisms. However, the recognition of inflammatory cytokines as a common lethal pathway has become consent. Proinflammatory cytokines such as tumor necrosis factor (TNF) or high-mobility group box1 (HMGB1) are fanatically released and cause lethal multiorgan dysfunction. Inhibition of these cytokines can prevent the inflammatory responses and organ damage. In seeking potential anti-inflammatory strategies, we reported that ethyl pyruvate and alpha7 nicotinic acetylcholine receptor (alpha7nAChR) agonists effectively restrained cytokine production to provide therapeutic benefits in both experimental sepsis and hemorrhage. Here, we review the inflammatory responses and the anti-inflammatory strategies in experimental models of sepsis and hemorrhage, as they may have a consistent inflammatory pathway in spite of their different pathophysiological processes.

Phagocytosis of apoptotic cells is critical to resolution of inflammation. High mobility group box 1 protein (HMGB1), a mediator of inflammation, has been shown to diminish phagocytosis through binding to phosphatidylserine (PS) exposed on the surface of apoptotic neutrophils. However, it is currently unknown whether HMGB1 also modulates the activity of receptors involved in PS recognition on the surface of phagocytes. In the present studies, we found that preincubation of macrophages with HMGB1 decreased their ability to engulf apoptotic neutrophils or thymocytes. Preincubation of macrophages with HMGB1 prevented the enhancement of efferocytosis resulting from exposure to milk fat globule EGF factor 8 (MFG-E8), an opsonin that bridges PS and α(v)β(3) as well as α(v)β(5)-integrins on the surface of phagocytes. The inhibitory effect of HMGB1 on the phagocytic activity of macrophages was prevented by preincubation of HMGB1 with soluble α(v)β(3), but not with soluble α(v)β(5). HMGB1 colocalized with the β(3)-integrin on the cell membrane of macrophages and bound to soluble α(v)β(3), but not to soluble α(v)β(5). HMGB1 suppressed the interaction between MFG-E8 and α(v)β(3). HMGB1 also inhibited intracellular signaling events, including ERK phosphorylation and Rac-1 activation, which are activated in macrophages during phagocytosis of apoptotic cells. These results demonstrate that HMGB1 blocks α(v)β(3)-dependent recognition and uptake of apoptotic cells.

We have shown that folate-induced kidney dysfunction and interstitial fibrosis predisposes mice to sepsis mortality. Agents that increase survival in normal septic mice were ineffective in a two-stage kidney disease model. Here we used the 5/6 nephrectomy mouse model of progressive chronic kidney disease (CKD) to study how CKD affects acute kidney injury (AKI) induced by sepsis. We induced sepsis using cecal ligation and puncture and found that the presence of CKD intensified the severity of kidney and liver injury, cytokine release, and splenic apoptosis. Accumulation of High Mobility Group Box Protein-1 (HMGB1; a late proinflammatory cytokine released from apoptotic cells), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, interleukin (IL)-6, or IL-10 was increased in CKD or sepsis alone and to a greater extent in CKD-sepsis. Only part of the increase was explained by decreased renal clearance. Surprisingly, we found splenic apoptosis in CKD, even in the absence of sepsis. Although VEGF neutralization with soluble fms-like tyrosine kinase 1 (sFLT-1) (a soluble VEGF receptor) effectively treated sepsis, it was ineffective against CKD-sepsis. A single dose of HMGB1-neutralizing antiserum administered 6 h after sepsis alone was ineffective; however, CKD-sepsis was attenuated by anti-HMGB1. Splenectomy transiently decreased circulating HMGB1 levels, reversing the effectiveness of anti-HMGB1 treatment on CKD-sepsis. Thus, progressive CKD increases the severity of sepsis, in part, by reducing the renal clearance of several cytokines. CKD-induced splenic apoptosis and HMGB1 release could be important common mediators for both CKD and sepsis.

The nonhistone chromosomal protein high-mobility group 1 protein (HMG-1/HMGB1) can serve as an activator of p53 sequence-specific DNA binding (L. Jayaraman, N. C. Moorthy, K. G. Murthy, J. L. Manley, M. Bustin, and C. Prives, Genes Dev. 12:462-472, 1998). HMGB1 is capable of interacting with DNA in a non-sequence-specific manner and causes a significant bend in the DNA helix. Since p53 requires a significant bend in the target site, we examined whether DNA bending by HMGB1 may be involved in its enhancement of p53 sequence-specific binding. Accordingly, a 66-bp oligonucleonucleotide containing a p53 binding site was locked in a bent conformation by ligating its ends to form a microcircle. Indeed, p53 had a dramatically greater affinity for the microcircle than for the linear 66-bp DNA. Moreover, HMGB1 augmented binding to the linear DNA but not to the microcircle, suggesting that HMGB1 works by providing prebent DNA to p53. p53 contains a central core sequence-specific DNA binding region and a C-terminal region that recognizes various forms of DNA non-sequence specifically. The p53 C terminus has also been shown to serve as an autoinhibitor of core-DNA interactions. Remarkably, although the p53 C terminus inhibited p53 binding to the linear DNA, it was required for the increased affinity of p53 for the microcircle. Thus, depending on the DNA structure, the p53 C terminus can serve as a negative or a positive regulator of p53 binding to the same sequence and length of DNA. We propose that both DNA binding domains of p53 cooperate to recognize sequence and structure in genomic DNA and that HMGB1 can help to provide the optimal DNA structure for p53.