[The plasma protein HRG is an important factor for preventing sepsis and maintaining homeostatic response].

Acute phase proteins such as CRP, amyloid protein A, and α1-antitrypsin are produced in the liver and their plasma levels are increased during the acute inflammatory response. In contrast, there are plasma proteins whose dynamics are opposite to acute phase proteins. This group includes histidine-rich glycoprotein (HRG), inter-α-inhibitor proteins, albumin, and transthyretin. HRG binds to a variety of factors and regulates the fundamental processes; the blood coagulation, the clearance of apoptotic cells, and tumor growth. In the present review, we focus on the anti-septic effects of HRG in mice model, the actions of HRG on human blood cells/vascular endothelial cells, and the identification of a novel receptor CLEC1A for HRG, based on our recent findings. HRG appears to maintain the quiescence of neutrophils; a round shape, the low levels of spontaneous release of ROS, the ease passage through artificial microcapillaries, and prevention of adhesion to vascular endothelial cells. HRG also inhibited activation of vascular endothelial cells; the suppression of adhesion molecules and the inhibition of HMGB1 mobilization and cytokine secretion. It was shown that plasma HRG level was an excellent biomarker of septic patients in ICU for the evaluation of severity and prognosis. So far little attention has been paid to HRG in terms of a functional role in sepsis and ARDS, however, it is strongly suggested that HRG may be an important plasma factor that prevents a progress in the septic cascade and maintains the homeostasis of blood cells and vascular endothelial cells.

Perineural Treatment with High Mobility Group Box-1 Monoclonal Antibody Prevents Initiation of Pain-Like Behaviors in Female Mice with Trigeminal Neuropathy.

Post-traumatic trigeminal neuropathy (PTTN) is a type of chronic pain caused by damage to the trigeminal nerve. A previous study reported that pretreatment with anti-high mobility group box-1 (HMGB1) neutralizing antibodies (nAb) prevented the onset of PTTN following distal infraorbital nerve chronic constriction injury (dIoN-CCI) in male mice. Clinical evidence indicates a high incidence of PTTN in females. Although our previous study found that perineural HMGB1 is crucial in initiation of PTTN in male mice, it is currently unknown whether HMGB1 is also involved in the pathogenesis of PTTN in female mice. Therefore, in the current study, we examined the effect of anti-HMGB1 nAb on pain-like behavior in female mice following dIoN-CCI surgery. We found that dIoN-CCI surgery enhanced reactivity to mechanical and cold stimuli in female mice, which was suppressed by treatment with anti-HMGB1 nAb. Moreover, the increase in macrophages after dIoN-CCI was significantly attenuated by pretreatment with anti-HMGB1 nAb. Furthermore, anti-HMGB1 nAb treatment inhibited microglial activation in the trigeminal spinal tract nucleus. These data suggest that HMGB1 also plays a crucial role in the onset of PTTN after nerve injury in female mice. Thus, anti-HMGB1 nAb could be a novel therapeutic agent for inhibiting the onset of PTTN in female and male mice.

Cationic ribosomal proteins can inhibit pro-inflammatory action stimulated by LPS+HMGB1 and are hindered by advanced glycation end products.

We previously found that ribosomal protein L9 (RPL9) is a novel advanced glycation end product (AGE)-binding protein that can decrease pro-inflammatory TNF-α expression stimulated by lipopolysaccharide (LPS) plus high-mobility group box 1 (HMGB1), suggesting that RPL9 has a role in regulating LPS+HMGB1-stimulated inflammatory reactions. Among the various ribosomal proteins, it was found that RPS5 reproduced the regulatory activity of RPL9 on LPS+HMGB1-stimulated TNF-α expression in macrophage-like RAW264.7 cells. RPL9 and RPS5 share a common feature as cationic proteins. Polylysine, a cationic polypeptide, and a synthetic peptide of the cationic region from RPL9 also exhibited reducing activity on LPS+HMGB1-induced TNF-α expression. By pull-down assay, RPL9 and RPS5 were confirmed to interact with AGEs. When AGEs coexisted with LPS, HMGB1, plus RPL9 or RPS5, the reducing effect of TNF-α expression by these cationic ribosomal proteins was shown to be abrogated. The results suggest that cationic ribosomal proteins have a regulatory role in the pro-inflammatory response induced by LPS+HMGB1, and in the pathophysiological condition of accumulating AGEs, this regulatory effect is abolished, which exacerbates inflammation.

Different modulation of STING/TBK1/IRF3 signaling by advanced glycation end products.

Advanced glycation end products (AGEs) are a heterogeneous group of compounds that are non-enzymatically produced by reactions between carbonyl compounds and proteins. Many types of AGEs are produced according to the type or concentration of the reacting carbonyl compound. We have previously demonstrated that a glycolaldehyde-derived AGE suppresses stimulator of interferon gene (STING)/TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3), which is a component of the innate immune system. In this report, we investigated the effects of AGEs prepared by several carbonyl compounds on STING/TBK1/IRF3 signaling. AGEs used in the present study were numbered based on the carbonyl compound type: AGE1, derived from glucose; AGE2, derived from glyceraldehyde; AGE3, derived from glycolaldehyde; AGE4, derived from methylglyoxal; and AGE5, derived from glyoxal. AGEs derived from aldehyde (AGE2 and AGE3) and dicarbonyl compounds (AGE4 and AGE5) suppressed cyclic GMP-AMP (cGAMP)-induced activation of STING/TBK1/IRF3 signaling, with different suppression efficiencies observed. Lysine modification by carbonyl compounds was related to the efficiency of the suppressive effect on STING/TBK1/IRF3 signaling. Among the AGEs used, only AGE1 enhanced cGAMP-induced activation of STING/TBK1/IRF3 signaling. Enhancing the modulation of STING/TBK1/IRF3 signaling by AGE1 was mediated by toll-like receptor 4. These results indicated that modulation of STING/TBK1/IRF3 signaling by prepared AGEs is dependent on the type and concentration of the carbonyl compound present. Modulating STING/TBK1/IRF3 signaling by AGEs may involve modification of lysine residues in proteins.

Repeated Social Defeat Stress Induces HMGB1 Nuclear Export in Prefrontal Neurons, Leading to Social Avoidance in Mice.

Inflammation has been associated with depression, and innate immune receptors, such as the Toll-like receptor (TLR) 2/4 in the medial prefrontal cortex (mPFC), are crucial for chronic stress-induced depression-related behaviors in mice. HMGB1, a putative ligand for TLR2/4, has been suggested to promote depression-related behaviors under acute stress. However, the roles of endogenous HMGB1 under chronic stress remain to be investigated. Here, we found that the cerebroventricular infusion of HMGB1 proteins blocked stress-induced social avoidance and that HMGB1-neutralizing antibodies augmented repeated social defeat stress-induced social avoidance in mice, suggesting the antidepressive-like effect of HMGB1 in the brain. By contrast, the infusion of HMGB1-neutralizing antibodies to the mPFC and HMGB1 knockout in α-CaMKII-positive forebrain neurons attenuated the social avoidance, suggesting the pro-depressive-like effect of HMGB1 released from prefrontal neurons under chronic stress. In addition, repeated social defeat stress induced HMGB1 nuclear export selectively in mPFC neurons, which was abolished in the mice lacking RAGE, one of HMGB1 receptors, suggesting the positive feedback loop of HMGB1-RAGE signaling under chronic stress. These findings pave the way for identifying multiple roles of HMGB1 in the brain for chronic stress and depression.

Glycolaldehyde-derived advanced glycation end products promote macrophage proliferation via the JAK-STAT signaling pathway.

BACKGROUND: Advanced glycation end products (AGEs) are heterogeneous proinflammatory molecules produced by a non-enzymatic glycation reaction between reducing sugars (and their metabolites) and biomolecules with amino groups, such as proteins. Although increases in and the accumulation of AGEs have been implicated in the onset and exacerbation of lifestyle- or age-related diseases, including diabetes, their physiological functions have not yet been elucidated in detail. METHODS AND RESULTS: The present study investigated the cellular responses of the macrophage cell line RAW264.7 stimulated by glycolaldehyde-derived AGEs (Glycol-AGEs) known as representative toxic AGEs. The results obtained showed that Glycol-AGEs significantly promoted the proliferation of RAW264.7 cells at a low concentration range (1-10 µg/mL) in a concentration-dependent manner. On the other hand, neither TNF-α production nor cytotoxicity were induced by the same concentrations of Glycol-AGEs. The increases observed in cell proliferation by low concentrations of Glycol-AGEs were also detected in receptor triple knockout (RAGE-TLR4-TLR2 KO) cells as well as in wild-type cells. Increases in cell proliferation were not affected by various kinase inhibitors, including MAP kinase inhibitors, but were significantly suppressed by JAK2 and STAT5 inhibitors. In addition, the expression of some cell cycle-related genes was up-regulated by the stimulation with Glycol-AGEs. CONCLUSIONS: These results suggest a novel physiological role for AGEs in the promotion of cell proliferation via the JAK-STAT pathway.

Protective effects of an anti-4-HNE monoclonal antibody against liver injury and lethality of endotoxemia in mice.

4-hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product that is known to be elevated during oxidative stress. During systemic inflammation and endotoxemia, plasma levels of 4-HNE are elevated in response to lipopolysaccharide (LPS) stimulation. 4-HNE is a highly reactive molecule due to its generation of both Schiff bases and Michael adducts with proteins, which may result in modulation of inflammatory signaling pathways. In this study, we report the production of a 4-HNE adduct-specific monoclonal antibody (mAb) and the effectiveness of the intravenous injection of this mAb (1 mg/kg) in ameliorating LPS (10 mg/kg, i.v.)-induced endotoxemia and liver injury in mice. Endotoxic lethality in control mAb-treated group was suppressed by the administration of anti-4-HNE mAb (75 vs. 27%). After LPS injection, we observed a significant increase in the plasma levels of AST, ALT, IL-6, TNF-α and MCP-1, and elevated expressions of IL-6, IL-10 and TNF-α in the liver. All these elevations were inhibited by anti-4-HNE mAb treatment. As to the underlining mechanism, anti-4-HNE mAb inhibited the elevation of plasma high mobility group box-1 (HMGB1) levels, the translocation and release of HMGB1 in the liver and the formation of 4-HNE adducts themselves, suggesting a functional role of extracellular 4-HNE adducts in hypercytokinemia and liver injury associated with HMGB1 mobilization. In summary, this study reveals a novel therapeutic application of anti-4-HNE mAb for endotoxemia.

Consistently low levels of histidine-rich glycoprotein as a new prognostic biomarker for sepsis: A multicenter prospective observational study.

BACKGROUND: Few sepsis biomarkers accurately predict severity and mortality. Previously, we had reported that first-day histidine-rich glycoprotein (HRG) levels were significantly lower in patients with sepsis and were associated with mortality. Since the time trends of HRG are unknown, this study focused on the time course of HRG in patients with sepsis and evaluated the differences between survivors and non-survivors. METHODS: A multicenter prospective observational study was conducted involving 200 patients with sepsis in 16 Japanese hospitals. Blood samples were collected on days 1, 3, 5, and 7, and 28-day mortality was used for survival analysis. Plasma HRG levels were determined using a modified quantitative sandwich enzyme-linked immunosorbent assay. RESULTS: First-day HRG levels in non-survivors were significantly lower than those in survivors (mean, 15.7 [95% confidence interval (CI), 13.4-18.1] vs 20.7 [19.5-21.9] µg/mL; P = 0.006). Although there was no time × survivors/non-survivors interaction in the time courses of HRG (P = 0.34), the main effect of generalized linear mixed models was significant (P < 0.001). In a univariate Cox proportional hazards model with each variable as a time-dependent covariate, higher HRG levels were significantly associated with a lower risk of mortality (hazard ratio, 0.85 [95% CI, 0.78-0.92]; P < 0.001). Furthermore, presepsin levels (P = 0.02) and Sequential Organ Function Assessment scores (P < 0.001) were significantly associated with mortality. Harrell's C-index values for the 28-day mortality effect of HRG, presepsin, procalcitonin, and C-reactive protein were 0.72, 0.70, 0.63, and 0.59, respectively. CONCLUSIONS: HRG levels in non-survivors were consistently lower than those in survivors during the first seven days of sepsis. Repeatedly measured HRG levels were significantly associated with mortality. Furthermore, the predictive power of HRG for mortality may be superior to that of other singular biomarkers, including presepsin, procalcitonin, and C-reactive protein.

Histamine promotes angiogenesis through a histamine H1 receptor-PKC-VEGF-mediated pathway in human endothelial cells.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

[Role of histidine-rich glycoprotein as anti-DAMPs and therapeutic effects on DAMPs-related diseases].

Histidine-rich glycoprotein (HRG) is a plasma glycoprotein produced mainly in the liver. We have shown that HRG replacement therapy has a marked therapeutic effect on sepsis, in which high mobility group box 1 (HMGB1), one of the representative damage-associated molecular patterns (DAMPs), is known to play an important role in the disease progression. The mechanisms of action are diverse, including inhibition of immune thrombus formation and inhibition of ROS production. In addition, HRG has been shown to neutralize the toxicity of heme, a type of DAMPs, and neutralize the activity of LPS, a type of pathogen-associated molecular patterns (PAMPs), and to inhibit the translocation of HMGB1 from the nucleus of vascular endothelial cells to the extracellular space. Since DAMPs/PAMPs are known to play a central role in the pathogenesis of not only sepsis but also many inflammatory diseases, HRG has wide therapeutic applications and is considered to be a very promising seed for drug discovery.

Glycolaldehyde-derived advanced glycation end products suppress STING/TBK1/IRF3 signaling via CD36.

AIMS: We have previously reported that advanced glycation end products derived from incubation of albumin with glycolaldehyde (glycol-AGE), lead to suppression of the toll-like receptor 4 (TLR4) signaling response to lipopolysaccharide. Glycol-AGE-induced suppression of TLR4 signaling is involved in the downregulation of CD14, which is an adaptor protein necessary for transferring lipopolysaccharide to TLR4. Therefore, glycol-AGEs impair the innate immune response through suppression of the upstream process in TLR4 signaling. However, the effect of glycol-AGEs on intracellular signaling related to the innate immune response remains unclear. This study aimed to examined the effect of glycol-AGEs on stimulator of interferon gene (STING) signaling in macrophages. MAIN METHODS: In differentiated THP-1 cells, which are a human monocytic leukemia cell line, cyclic GMP-AMP (cGAMP) transfection was used to activate STING signaling. The phosphorylation levels of TANK-binding kinase 1 (TBK1)/interferon regulatory transcription factor 3 (IRF3) were evaluated by western blot analysis. Downstream cytokine levels were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays. KEY FINDINGS: Glycol-AGEs suppressed cGAMP-induced phosphorylation of TBK1 and IRF3, as well as the production of cytokines regulated by IRF3. There was no effect of glycol-AGEs on the efficacy of cGAMP transfection. Treatment of a neutralizing antibody against CD36 prevented cGAMP-induced phosphorylation of TBK1 and IRF3, and also upregulation of interferon-ß and C-X-C motif chemokine ligand 10 in glycol-AGE-treated cells. SIGNIFICANCE: Glycol-AGEs negatively regulate cGAMP-induced activation of STING/TBK1/IRF3 signaling via CD36. Our findings suggest that glycol-AGEs lead to impairment of the innate immune response by suppressing intracellular signaling.

Histamine induced high mobility group box-1 release from vascular endothelial cells through H1 receptor.

Background: Systemic allergic reaction is characterized by vasodilation and vascular leakage, which causes a rapid, precipitous and sustained decrease in arterial blood pressure with a concomitant decrease of cardiac output. Histamine is a major mediator released by mast cells in allergic inflammation and response. It causes a cascade of inflammation and strongly increases vascular permeability within minutes through its four G-protein-coupled receptors (GPCRs) on endothelial cells. High mobility group box-1 (HMGB1), a nonhistone chromatin-binding nuclear protein, can be actively secreted into the extracellular space by endothelial cells. HMGB1 has been reported to exert pro-inflammatory effects on endothelial cells and to increase vascular endothelial permeability. However, the relationship between histamine and HMGB1-mediated signaling in vascular endothelial cells and the role of HMGB1 in anaphylactic-induced hypotension have never been studied. Methods and results: EA.hy 926 cells were treated with different concentrations of histamine for the indicated periods. The results showed that histamine induced HMGB1 translocation and release from the endothelial cells in a concentration- and time-dependent manner. These effects of histamine were concentration-dependently inhibited by d-chlorpheniramine, a specific H1 receptor antagonist, but not by H2 or H3/4 receptor antagonists. Moreover, an H1-specific agonist, 2-pyridylethylamine, mimicked the effects of histamine, whereas an H2-receptor agonist, 4-methylhistamine, did not. Adrenaline and noradrenaline, which are commonly used in the clinical treatment of anaphylactic shock, also inhibited the histamine-induced HMGB1 translocation in endothelial cells. We therefore established a rat model of allergic shock by i.v. injection of compound 48/80, a potent histamine-releasing agent. The plasma HMGB1 levels in compound 48/80-injected rats were higher than those in controls. Moreover, the treatment with anti-HMGB1 antibody successfully facilitated the recovery from compound 48/80-induced hypotension. Conclusion: Histamine induces HMGB1 release from vascular endothelial cells solely through H1 receptor stimulation. Anti-HMGB1 therapy may provide a novel treatment for life-threatening systemic anaphylaxis.

Nordihydroguaiaretic acid inhibits glyoxalase I, and causes the accumulation of methylglyoxal followed by cell-growth inhibition.

BACKGROUND: Methylglyoxal (MGO) is a known toxic byproduct of glycolysis, with MGO-induced cytotoxicity believed to contribute to the pathogenesis of several diseases. Glyoxalase I (GLO1) is a key enzyme for eliminating MGO in mammalian cells, therefore, compounds affecting GLO1 activity are potential therapeutic agents for MGO-induced disorders. Previously, we found nordihydroguaiaretic acid (NDGA) as a potent GLO1 inhibitor. METHODS: The inhibitory characteristics of NDGA were determined spectrophotometrically with recombinant GLO1. NDGA-induced growth-inhibition and accumulation of MGO-derived advanced glycation end products (AGEs) were examined in EA.hy926 cells. RESULTS: NDGA showed significant inhibition of GLO1 enzymatic activity in a dose-dependent manner. Its Ki value was estimated to be 146-fold lower than that of myricetin, a known GLO1 inhibitor. The co-addition of MGO with NDGA to the cells resulted in significant growth inhibition, suggesting that MGO accumulation, sufficient to affect cell growth, was caused by NDGA inhibiting GLO1. These findings were supported by the observations that the addition of aminoguanidine, a typical MGO scavenger, significantly reversed cell-growth inhibition by co-addition of MGO with NDGA, and that an increase in intracellular MGO-derived AGEs was observed during incubation with the co-addition of MGO with NDGA. CONCLUSION: NDGA was found to be a novel and potent inhibitor of GLO1. The co-addition of NDGA with MGO to the cells resulted in increased intracellular MGO accumulation followed by enhanced cell-growth inhibition.

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

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

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

Identification of ribosomal protein L9 as a novel regulator of proinflammatory damage-associated molecular pattern molecules.

BACKGROUND: We previously reported that advanced glycation endproducts (AGEs) increase the proinflammatory activity of high mobility group box-1 (HMGB1), a representative damage-associated molecular pattern molecule (DAMP), through their direct interaction. This suggested that AGEs activate other DAMPs and led us to search for novel DAMPs capable of interacting with AGEs. METHODS AND RESULTS: The chromatographic analysis using AGE-immobilized gel revealed the ribosomal protein family to be a factor with binding activity to AGEs. Ribosomal protein L9 (RPL9), a member of the ribosomal protein family, was found in the centrifugal supernatant of ruptured cells and in the serum of lipopolysaccharide (LPS)-stimulated sepsis model mice, exhibiting similar characteristic properties to HMGB1. Although HMGB1 potentiated LPS-stimulated TNF-α expression in macrophage-like RAW264.7 cells, RPL9 hardly exhibited this activity. Of note, RPL9 significantly suppressed the potentiated mRNA expression and protein production of TNF-α by HMGB1 plus LPS stimulation, suggesting its regulatory roles in DAMP-induced proinflammatory activity. Based on the differential scanning fluorimetric analysis, the direct interaction between RPL9 and HMGB1 may play a role in the suppressive effects of RPL9. CONCLUSIONS: This study suggested that RPL9 is a novel type of DAMP with a regulatory role in the proinflammatory response and provided insight into the pathophysiology of inflammatory diseases.

Central high mobility group box-1 induces mechanical hypersensitivity with spinal microglial activation in a mouse model of hemi-Parkinson's disease.

Parkinson's disease (PD) patients often complain of pain, but this problem has been neglected and is poorly understood. High mobility group box-1 (HMGB1), an alarmin/damage-associated molecular patterns protein, is increased in the cerebrospinal fluid in PD patients. However, little is known of the relationship between HMGB1 and pain associated with PD. Here, we investigated the role of central HMGB1 in the regulation of nociceptive hypersensitivity in a mouse model of PD. Male ddY mice were microinjected unilaterally with 6-hydroxydopamine (6OHDA) into the striatum. These hemi-PD mice were treated with anti-HMGB1 neutralizing antibody (nAb; 10 µg in 10 µL) by intranasal (i.n.) administration. The mechanical hypersensitivity of the hind paws was evaluated with the von Frey test. Spinal microglial activity was analyzed by immunostaining for ionized calcium-binding adapter molecule 1. The 6OHDA-administered mice displayed unilateral loss of dopamine neurons in the substantia nigra and mechanical hypersensitivity in both hind paws. Moreover, spinal microglia were activated in these hemi-PD mice. Twenty-eight days after the 6OHDA injections, repeated i.n., but not systemic, treatment with anti-HMGB1 nAb inhibited the bilateral mechanical hypersensitivity and spinal microglial activation. However, the anti-HMGB1 nAb did not ameliorate the dopamine neuron loss. Moreover, intracerebroventricular injection with recombinant HMGB1 induced mechanical hypersensitivity. These findings indicate that HMGB1 is involved in the maintenance of nociceptive symptoms in hemi-PD mice via spinal microglial activation. Therefore, central HMGB1 may have potential as a therapeutic target for pain associated with PD.

Involvement of multiple scavenger receptors in advanced glycation end product-induced vessel tube formation in endothelial cells.

Toxic advanced glycation end products (toxic AGEs) derived from glycolaldehyde (AGE3) have been implicated in the development of diabetic vascular complications such as retinopathy characterised by excessive angiogenesis. Different receptor types, such as receptor for AGEs (RAGE), Toll like receptor-4 and scavenger receptors, are expressed in endothelial cells and contribute to AGE-elicited alteration of cell function. In the present study, we examined the involvement of AGE-related receptors on AGE-induced angiogenesis in endothelial cells. The effects of pharmacological inhibitors or receptor neutralizing antibodies on AGE3-induced tube formation were investigated using the in vitro Matrigel tube formation assay in b.End5 cells (mouse endothelial cells). AGE3-induced signalling pathways and receptor expression changes were analysed by Western blot analysis and flow cytometry, respectively. Both FPS-ZM1, a RAGE inhibitor, and fucoidan, a ligand for scavenger receptors, suppressed AGE3-induced tube formation. Cocktails of neutralizing antibodies against the scavenger receptors CD36, CD163 and LOX-1 prevented AGE3-induced tube formation. AGE3 activated mTOR signalling, resulting in facilitation of tube formation. Activation of the AGE-RAGE pathway also led to the upregulation of scavenger receptors. Taken together, our findings suggest that the scavenger receptors CD36, CD163 and LOX-1 in conjunction with the RAGE receptor work together to mediate toxic AGE-induced facilitation of angiogenesis.

Osteopontin silencing attenuates bleomycin-induced murine pulmonary fibrosis by regulating epithelial-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-ß1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-ß1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-ß1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

Histidine-rich glycoprotein as a prognostic biomarker for sepsis.

Various biomarkers have been proposed for sepsis; however, only a few become the standard. We previously reported that plasma histidine-rich glycoprotein (HRG) levels decreased in septic mice, and supplemental infusion of HRG improved survival in mice model of sepsis. Moreover, our previous clinical study demonstrated that HRG levels in septic patients were lower than those in noninfective systemic inflammatory response syndrome patients, and it could be a biomarker for sepsis. In this study, we focused on septic patients and assessed the differences in HRG levels between the non-survivors and survivors. We studied ICU patients newly diagnosed with sepsis. Blood samples were collected within 24 h of ICU admission, and HRG levels were determined using an enzyme-linked immunosorbent assay. Ninety-nine septic patients from 11 institutes in Japan were included. HRG levels were significantly lower in non-survivors (n = 16) than in survivors (n = 83) (median, 15.1 [interquartile ranges, 12.7-16.6] vs. 30.6 [22.1-39.6] µg/ml; p < 0.01). Survival analysis revealed that HRG levels were associated with mortality (hazard ratio 0.79, p < 0.01), and the Harrell C-index (predictive power) for HRG was 0.90. These results suggested that HRG could be a novel prognostic biomarker for sepsis.