High Mobility Group Box-1 Protein Research Articles

HMGB1 mediates lipopolysaccharide-induced macrophage autophagy and pyroptosis

Autophagy and pyroptosis of macrophages play important protective or detrimental roles in sepsis. However, the underlying mechanisms remain unclear. High mobility group box protein 1 (HMGB1) is associated with both pyroptosis and autophagy. lipopolysaccharide (LPS) is an important pathogenic factor involved in sepsis. Lentivirus-mediated HMGB1 shRNA was used to inhibit the expression of HMGB1. Macrophages were treated with acetylation inhibitor (AA) to suppress the translocation of HMGB1 from the nucleus to the cytosol. Autophagy and pyroptosis-related protein expressions were detected by Western blot. The levels of caspase-1 activity were detected and the rate of pyroptotic cells was detected by flow cytometry. LPS induced autophagy and pyroptosis of macrophages at different stages, and HMGB1 downregulation decreased LPS-induced autophagy and pyroptosis. Treatment with acetylation inhibitor (anacardic acid) significantly suppressed LPS-induced autophagy, an effect that was not reversed by exogenous HMGB1, suggesting that cytoplasmic HMGB1 mediates LPS-induced autophagy of macrophages. Anacardic acid or an anti-HMGB1 antibody inhibited LPS-induced pyroptosis of macrophages. HMGB1 alone induced pyroptosis of macrophages and this effect was inhibited by anti-HMGB1 antibody, suggesting that extracellular HMGB1 induces macrophage pyroptosis and mediates LPS-induced pyroptosis. In summary, HMGB1 plays different roles in mediating LPS-induced autophagy and triggering pyroptosis according to subcellular localization.

The DNA binding high mobility group box protein family functionally binds RNA.

Nucleic acid binding proteins regulate transcription, splicing, RNA stability, RNA localization, and translation, together tailoring gene expression in response to stimuli. Upon discovery, these proteins are typically classified as either DNA or RNA binding as defined by their in vivo functions; however, recent evidence suggests dual DNA and RNA binding by many of these proteins. High mobility group box (HMGB) proteins have a DNA binding HMGB domain, act as transcription factors and chromatin remodeling proteins, and are increasingly understood to interact with RNA as means to regulate gene expression. Herein, multiple layers of evidence that the HMGB family are dual DNA and RNA binding proteins is comprehensively reviewed. For example, HMGB proteins directly interact with RNA in vitro and in vivo, are localized to RNP granules involved in RNA processing, and their protein interactors are enriched in RNA binding proteins involved in RNA metabolism. Importantly, in cell-based systems, HMGB-RNA interactions facilitate protein-protein interactions, impact splicing outcomes, and modify HMGB protein genomic or cellular localization. Misregulation of these HMGB-RNA interactions are also likely involved in human disease. This review brings to light that as a family, HMGB proteins are likely to bind RNA which is essential to HMGB protein biology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

The Clinical and Laboratory Efficacy of HA 330 Treatment Combined with Continuous Renal Replacement Therapy in Septic Shock Patients: A Case Series

Introduction: Blood purification therapy is a method used to enable cytokine removal and to improve disturbed immune homeostasis in patients with sepsis or septic shock. This study aimed to evaluate the impact of HA 330 treatment on biochemical and hemodynamic parameters and cytokine levels in adult patients with septic shock. Methods: Critically ill patients with septic shock who received continuous veno-venous hemodiafiltration and HA 330 treatment were included in this prospective observational study. Biochemical and hemodynamic parameters were followed throughout HA 330 treatment. Serum interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, high-mobility group box1 (HMGB-1) protein, IL-10 levels were analyzed by ELISA method, before and after each HA 330 session. Results: A total of 18 critically ill patients were included in this study. The median APACHE 2 score was 22.2 ± 7.49 and median SOFA score 9.6 ± 5.44 on intensive care unit admission. SOFA scores were significantly decreased on the 3rd day of HA 330 treatment, compared to 2nd day scores (p = 0.017). Median leukocyte value was significantly decreased (p = 0.027 and p = 0.024), while hemodynamic parameters remained unchanged throughout the HA 330 treatment. Median CRP and procalcitonin levels were significantly reduced at day 3 of HA 330 treatment compared to the baseline (p = 0.015 and p = 0.033, respectively). Serum IL-1 β, IL-6, IL-8, TNF-a, HMGB-1, and IL-10 levels decreased insignificantly by 11.5%, 26.4%, 11.4%, 37.9%, 0.02%, and 35.5%, respectively, at the end of the hemoperfusion treatment compared to the pre-treatment. Conclusion: The administration of HA 330-based hemoperfusion in septic shock patients revealed improvements in SOFA scores, leukocyte count, and CRP and procalcitonin levels. However, there was no statistically significant change in concentrations of inflammatory cytokines and hemodynamic parameters during HA 330 treatment.

Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways.

To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo. Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01). EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

Glycyrrhizin regulates the HMGB1/P38MAPK signalling pathway in status epilepticus.

In recent decades, studies have reported that inflammation serves key roles in epilepsy and that high mobility group box protein‑1 (HMGB1) may be involved in status epilepticus. However, it has not been reported whether HMGB1 participates in the pathogenesis of status epilepticus through the regulation of the p38 mitogen‑activated protein kinase (p38MAPK) signalling pathway. In the present study, Sprague‑Dawley rats were randomly divided into four groups as follows: Control, status epilepticus (SE), dimethyl sulfoxide treatment (DMSO + SE), and glycyrrhizin treatment (GL + SE) groups. Behavioural changes were then evaluated using the Racine score. In the hippocampus, the protein expression levels of HMGB1 were assessed using western blotting, the neuronal damage was evaluated using haematoxylin and eosin staining and transmission electron microscopy, and the activation of microglia was assessed using immunochemistry and immunofluorescence. The results demonstrated that, in the hippocampal region, HMGB1 existed in neurons and astrocytes and the protein expression levels of HMGB1, p38MAPK and phosphorylated‑p38MAPK were significantly inhibited after treatment with GL. Furthermore, GL could alleviate neuronal injury in the CA1 region of the hippocampus and prevented HMGB1 translocation from the nucleus into the cytoplasm in these areas. These findings expand the understanding of how HMGB1 may participate in SE and lay a foundation for evaluation of HMGB1 as a drug target.

Targeting HMGB1: A Potential Therapeutic Strategy for Chronic Kidney Disease.

High-mobility group protein box 1 (HMGB1) is a member of a highly conserved high-mobility group protein present in all cell types. HMGB1 plays multiple roles both inside and outside the cell, depending on its subcellular localization, context, and post-translational modifications. HMGB1 is also associated with the progression of various diseases. Particularly, HMGB1 plays a critical role in CKD progression and prognosis. HMGB1 participates in multiple key events in CKD progression by activating downstream signals, including renal inflammation, the onset of persistent fibrosis, renal aging, AKI-to-CKD transition, and important cardiovascular complications. More importantly, HMGB1 plays a distinct role in the chronic pathophysiology of kidney disease, which differs from that in acute lesions. This review describes the regulatory role of HMGB1 in renal homeostasis and summarizes how HMGB1 affects CKD progression and prognosis. Finally, some promising therapeutic strategies for the targeted inhibition of HMGB1 in improving CKD are summarized. Although the application of HMGB1 as a therapeutic target in CKD faces some challenges, a more in-depth understanding of the intracellular and extracellular regulatory mechanisms of HMGB1 that underly the occurrence and progression of CKD might render HMGB1 an attractive therapeutic target for CKD.

Association of High Mobility Group Box-Protein 1 and Platelet Microparticles in Patients After Hematopoietic Stem Cell Transplantation.

Thrombotic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly impact transplant outcomes. We focused on high mobility group box-protein (HMGB)1, one causative agent of thrombotic lesions in allo-HSCT, and investigated its association with platelets. We statistically analyzed available data from 172 patients with hematopoietic malignancies receiving allo-HSCT. A significant enhancement of monocyte-chemotactant protein-1, HMGB1, and platelet-derived microparticle (PDMP) levels was observed at day 0 after transplantation as compared to pre-transplantation. Multivariate analysis of the association among HMGB1 and 16 factors on day 0 revealed a significant correlation of HMGB1 levels with thrombin-antithrombin complex, interleukin-6, and PDMPs. High mobility group box-protein 1-induced procoagulant platelet induction and PDMP generation were performed in vitro using healthy platelets. High mobility group box-protein 1-induced PDMP generation was suppressed by toll-like receptor inhibitors and recombinant thrombomodulin. These results suggest that HMGB1 contributes to platelet activation in patients after allo-HSCT and is associated with PDMP-related thrombotic complications.

Effect of Chemotherapeutics on In Vitro Immune Checkpoint Expression in Non-Small Cell Lung Cancer.

Objectives: Immune checkpoint (ICP) expression in tumor cells could directly or indirectly affect the results of immunotherapy. ICP ligands on tumor cells usually bind their immune cell receptors to inhibit the activity, resulting in tumor immune escape. Thus, the purpose of this study was to ascertain the impact of various chemotherapeutic drugs on ICP expression in non-small cell lung cancer (NSCLC) cell lines with different pathological subtypes to provide a basis for the development of a superior regimen of chemotherapy combined with ICP blockade. Methods: Several first-line chemotherapy agents (cisplatin, carboplatin, paclitaxel, gemcitabine, vinorelbine, and pemetrexed) were selected to treat different NSCLC cell lines (squamous carcinoma H1703, adenocarcinoma A549, and large cell cancer H460) for 72 hours, and then the changes in ICP expression in the tumor cells were observed through flow cytometry. Results: Cisplatin, carboplatin, and paclitaxel upregulated the expressions of programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2) in A549 and H460 cell lines. Meanwhile, vinorelbine and pemetrexed upregulated PD-L1 and PD-L2 in H1703, A549, and H460 cell lines. Paclitaxel, gemcitabine, vinorelbine, and pemetrexed significantly upregulated the expressions of both galectin-9 and high-mobility group box protein 1 (HMGB1) in the A549 cell line. Cisplatin and paclitaxel significantly upregulated the expressions of major histocompatibility complex-II (MHC-II), galectin-3, α-synuclein, and fibrinogen-like protein 1 (FGL1) in A549 and H460 cell lines. In addition, cisplatin and vinorelbine significantly upregulated the expressions of both CD155 and CD112 in the H460 cell line. Vinorelbine upregulated MHC-I in all three cell lines. Conclusion: Chemotherapy agents have different effects on the expression of ICP ligands in tumor cells with different pathological types, and this may affect the efficacy of combined immunotherapy. These results provide a theoretical basis for further selection and optimization of the combination of chemotherapy and immunotherapy.

Synergistic chemo-photo anticancer therapy by using reversible Diels-Alder dynamic covalent bond mediated polyprodrug amphiphiles and immunoactivation investigation.

Highly efficient endocytosis and multi-approach integrated therapeutic tactics are important factors in oncotherapy. With the aid of thermally reversible furan-maleimide dynamic covalent bonds and the "polyprodrug amphiphiles" concept, thermo- and reduction-responsive PEG(-COOH)Fu/MI(-SS-)CPT copolymers were fabricated by the Diels-Alder (D-A) coupling of hydrophilic Fu(-COOH)-PEG and hydrophobic MI(-SS-)-CPT building blocks. The copolymers could self-assemble to form composite nanoparticles with a photothermal conversion reagent (IR780) and maintain excellent stability. In the in vitro simulated environments, the composite nanoparticles could detach Fu(-COOH)-PEG chains by a retro-D-A reaction upon near-infrared light (NIR) irradiation and reduce the size to facilitate endocytosis. Once in the intracellular environment, glutathione (GSH) could trigger a cascade reaction to release active CPT drugs to achieve chemotherapy, which could be further promoted by NIR light induced photothermal therapy. The in vivo mouse tumor model experiments demonstrated that these nanoparticles had an excellent therapeutic effect on solid tumors and inhibited their recurrence. Not only that, the synergistic chemical and optical therapy induced body immune response was also systematically evaluated; the maturation of dendritic cells, the proliferation of T cells, the increase of high mobility group box protein 1, and the decrease of immunosuppressive regulatory T cells confirmed that such synergistic therapy could effectively provide immune protection to the body. We believe such in situ generation of small-sized therapeutic units brought by a dynamically reversible D-A reaction could expand the pathway to design next generation drug delivery systems possessing superior design philosophy and excellent practice effects compared to currently available ones.

Revealing the extracellular function of HMGB1 N-terminal region acetylation assisted by a protein semi-synthesis approach.

HMGB1 (high-mobility group box 1) is a non-histone chromatin-associated protein that has been widely reported as a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in the proinflammatory process once it is in an extracellular location. Accumulating evidence has shown that HMGB1 undergoes extensive post-translational modifications (PTMs) that actively regulate its conformation, localization, and intermolecular interactions. However, fully characterizing the functional implications of these PTMs has been challenging due to the difficulty in accessing homogeneous HMGB1 with site-specific PTMs of interest. In this study, we developed a streamlined protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology enabled us to generate a series of N-terminal region acetylated HMGB1 proteins. Further studies revealed that acetylation regulates HMGB1-heparin interaction and modulates HMGB1's stability against thrombin, representing a regulatory switch to control HMGB1's extracellular activity.

Upregulation of HMGB1 promotes vascular dysfunction in the soft palate of patients with obstructive sleep apnea via the TLR4/NF-κB/VEGF pathway.

Obstructive sleep apnea (OSA) is characterized by the collapse of the soft palate in the upper airway, resulting in chronic intermittent hypoxia during sleep. Therefore, an understanding of the molecular mechanisms underlying pathophysiological dysfunction of the soft palate in OSA is necessary for the development of new therapeutic strategies. In the present study, we observed that high mobility group protein box 1 (HMGB1) was released by a large infiltration of macrophages in the soft palate of OSA patients. The toll-like receptor 4/nuclear factor kappa B pathway was observed to be activated by the release of HMGB1, and this was accompanied by an increased expression of pro-inflammatory factors, including tumor necrosis factor-α and interleukin-6. Importantly, increased expression of toll-like receptor 4 was observed in endothelial cells, contributing to upregulation of the angiogenesis-related factors vascular endothelial-derived growth factor and matrix metalloproteinase 9. Moreover, we confirmed the effect of the HMGB1-mediated toll-like receptor 4/nuclear factor kappa B pathway on cell proliferation and angiogenesis in an in vitro cell model of human umbilical vein endothelial cells. We conclude that HMGB1 may be a potential therapeutic target for preventing angiogenesis and pathology in OSA.

Dengue virus induced autophagy is mediated by HMGB1 and promotes viral propagation

Dengue virus (DENV) exploits various cellular pathways including autophagy to assure enhanced virus propagation. The mechanisms of DENV mediated control of autophagy pathway are largely unknown. Our investigations have revealed a novel role for high-mobility group box1 protein (HMGB1) in regulation of cellular autophagy process in DENV-2 infected A549 cell line. While induction of autophagy by rapamycin treatment resulted in enhanced DENV-2 propagation, the blockade of autophagy flux with bafilomycin A1 suppressed viral replication. Furthermore, siRNA-mediated silencing of HMGB1 significantly abrogated dengue induced autophagy, while LPS induced HMGB1 expression counteracted these effects. Interestingly, silencing of HMGB1 showed reduction of BECN1 and stabilization of BCL-2 protein. On the contrary, LPS induction of HMGB1 resulted in enhanced BECN1 and reduction in BCL-2 levels. This study shows that the modulation of autophagy by DENV-2 is HMGB1/BECN1 dependent. In addition, glycyrrhizic acid (GA), a potent HMGB1 inhibitor suppressed autophagy as well as DENV-2 replication. Altogether, our data suggests that HMGB1 induces BECN1 dependent autophagy to promote DENV-2 replication.

Jujuboside B Inhibited High Mobility Group Box Protein 1-Mediated Severe Inflammatory Responses in Human Endothelial Cells and Mice.

High mobility group box protein 1 (HMGB1) is a biomolecule that acts as an alerting signal of late sepsis by accelerating the production of proinflammatory cytokines, and eventually leads to various inflammation-related symptoms. When released into plasma at high concentration, it disrupts precise diagnosis and prognosis and worsens the survival of patients with systemic inflammatory conditions. Jujuboside B (JB) is a natural compound pressed from the seed of Zizyphi Spinosi Semen, which is known for its medical efficacies in treating various conditions such as hyperlipidemia, hypoxia, and platelet aggregation. Nevertheless, the medicinal activity of JB on HMGB1-involved inflammatory response in vascular cells in the human body is still ambiguous. Therefore, we hypothesized that JB could regulate the lipopolysaccharide (LPS)-induced dynamics of HMGB1 and its mediated cascade in inflammatory responses in human umbilical vein endothelial cells (HUVECs). In this experiment, JB and HMGB1 were administered in that order. In vitro and in vivo permeability, and cell viability, adhesion, and excavation of leukocytes, development of cell adhesion molecules, and lastly production of proinflammatory substances were investigated on human endothelial cells and mouse disease models to investigate the efficacy of JB in inflammatory condition. JB substantially blocked the translocation of HMGB1 from HUVECs and controlled HMGB1-induced adhesion and extravasation of the neutrophils through LPS-treated HUVECs. Moreover, JB decreased the formation of HMGB1 receptors and continually prevented HMGB1-induced proinflammatory mechanisms by blocking transcription of nuclear factor-κB and synthesis of tumor necrosis factor-α. In conclusion, JB demonstrated preventive effects against inflammatory pathologies and showed the potential to be a candidate substance for various inflammatory diseases by regulating HMGB1-mediated cellular signaling.

Fecal HBD-2 and Claudin-3 may be potential biomarkers to predict the deterioration of necrotizing enterocolitis: A prospective study.

Necrotizing enterocolitis (NEC) is a critical gastrointestinal disease. We aim to explore the value of fecal human β-defensin 2 (HBD-2), Claudin-3, high-mobility group box-1 protein (HMGB-1), and resistin-like molecule β (Relmβ) as well as some laboratory metrics to predict the deterioration of NEC. Infants diagnosed with NEC at Stage II were enrolled in our study. Those who progressed to Stage III were included in the Stage III group and the rest were included in the Stage II group. Clinical data and laboratory metrics of the infants were collected. Fecal samples of HBD2, HMGB-1, Claudin-3, and Relmβ collected during their enrollment were determined by using enzyme-linked immunosorbent assay (ELISA) kits. Student's t-test, the Mann-Whitney U test, the chi-square test, receiver operating characteristic (ROC), and logistic regression analysis were performed. Sixty infants diagnosed with NEC at Stage II were enrolled in our study, with 27 in the Stage III group (n = 27) and 33 in the Stage II group (n = 33). Although many of these NEC cases were late preterm and term infants, the infants in the Stage III group had a lower gestational age (P < 0.05). The incidence of gestational diabetes mellitus, peritonitis, intestinal adhesion, and sepsis was higher and more infants in the Stage III group underwent surgeries (P < 0.05). The levels of HBD-2 and Claudin-3 were higher and neutrophil count was lower in the Stage III group than in the Stage II Group, and the area under the curve (AUC) was 0.754, 0,755, and 0.666, respectively (P < 0.05). HBD-2 ≥ 1649.02 ng/g and Claudin-3 ≥ 2488.71 pg/g were included in the multivariate stepwise logistic regression analysis (P < 0.05), and the AUC of the model was 0.805 (95% CI: 0.688-0.922). Fecal HBD-2 and Claudin-3 may be potential biomarkers to predict the deterioration of NEC from Stage II to Stage III.

Regulatory mechanisms of HMGB1 and its receptors in polycystic ovary syndrome-driven gravid uterine inflammation.

High-mobility group box 1 (HMGB1) is critical for inflammatory homeostasis and successful pregnancy, and there is a strong association among elevated levels of HMGB1, polycystic ovary syndrome (PCOS), chronic inflammation and pregnancy loss. However, the mechanisms responsible for PCOS-driven regulation of uterine HMGB1 and its candidate receptors [toll-like receptor (TLR) 2 and 4] and inflammatory responses during pregnancy remain unclear. In this study, we found a gestational stage-dependent decrease in uterine HMGB1 and TLR4 protein abundance in rats during normal pregnancy. We demonstrated that increased expression of HMGB1, TLR2 and TLR4 proteins was associated with activation of inflammation-related signalling pathways in the gravid uterus exposed to 5α-dihydrotestosterone and insulin, mimicking the clinical features (hyperandrogenism and insulin resistance) of PCOS and this elevation was completely inhibited by treatment with the androgen receptor (AR) antagonist flutamide. Interestingly, acute exposure to lipopolysaccharide suppressed HMGB1, TLR4 and inflammation-related protein abundance but did not affect androgen levels or AR expression in the gravid uterus with viable fetuses. Furthermore, immunohistochemical analysis revealed that, in addition to being localized predominately in the nuclear compartment, HMGB1 immunoreactivity was also detected in the cytoplasm in the PCOS-like rat uterus, PCOS endometrium and pregnant rat uterus with haemorrhagic and resorbed fetuses, possibly via activation of nuclear factor κB signalling. These results suggest that both AR-dependent and AR-independent mechanisms contribute to the modulation of HMGB1/TLR2/TLR4-mediated uterine inflammation. We propose that the elevation of HMGB1 and its receptors and disruption of the pro-/anti-inflammatory balance in the gravid uterus may participate in the pathophysiology of PCOS-associated pregnancy loss.

HMGB1 Promotes In Vitro and In Vivo Skeletal Muscle Atrophy through an IL-18-Dependent Mechanism

Skeletal muscle atrophy occurs due to muscle wasting or reductions in protein associated with aging, injury, and inflammatory processes. High-mobility group box-1 (HMGB1) protein is passively released from necrotic cells and actively secreted by inflammatory cells, and is implicated in the pathogenesis of various inflammatory and immune diseases. HMGB1 is upregulated in muscle inflammation, and circulating levels of the proinflammatory cytokine interleukin-18 (IL-18) are upregulated in patients with sarcopenia, a muscle-wasting disease. We examined whether an association exists between HMGB1 and IL-18 signaling in skeletal muscle atrophy. HMGB1-induced increases of IL-18 levels enhanced the expression of muscle atrophy markers and inhibited myogenic marker expression in C2C12 and G7 myoblast cell lines. HMGB1-induced increases of IL-18 production in C2C12 cells involved the RAGE/p85/Akt/mTOR/c-Jun signaling pathway. HMGB1 short hairpin RNA (shRNA) treatment rescued the expression of muscle-specific differentiation markers in murine C2C12 myotubes and in mice with glycerol-induced muscle atrophy. HMGB1 and IL-18 signaling was suppressed in the mice after HMGB1 shRNA treatment. These findings suggest that the HMGB1/IL-18 axis is worth targeting for the treatment of skeletal muscle atrophy.

Wogonin attenuates neutrophilic inflammation and airway smooth muscle proliferation through inducing caspase-dependent apoptosis and inhibiting MAPK/Akt signaling in allergic airways.

Severe neutrophilic asthma is often characterized by persistent airway inflammation and irreversible airway remodeling, which are overstimulated by the high-mobility group box protein 1 (HMGB1). Although wogonin, an O-methylated flavone, has been widely used to treat inflammatory and allergic diseases, its therapeutic effects and potential mechanisms on severe neutrophilic asthma remain elusive. To evaluate whether wogonin alleviates airway neutrophilia through inducing neutrophil apoptosis and attenuates airway smooth muscle cells (ASMCs) proliferation and migration. The effect of wogonin on reducing neutrophilic airway inflammation, including neutrophil infiltration and inflammatory mediators, was examined in a mouse model of severe neutrophilic asthma sensitized with ovalbumin and lipopolysaccharide. Also, the effect of wogonin on inducing human neutrophil apoptosis was manifested using cellular morphology, flow cytometry, and caspase inhibition assays. Furthermore, the effect of wogonin on inhibiting HMGB1-mediated ASMCs proliferation and migration was determined. Wogonin reduced the frequency of neutrophils and inhibited the production of multiple inflammatory mediators, including ovalbumin-specific IgE, tumor necrosis factor-α, interleukin-6, and HMGB1, in bronchoalveolar lavage fluid and lung tissues of the neutrophilic asthmatic mouse model. These data strongly support a significantly suppressed neutrophilic airway inflammation, functionally consistent to the relieved airway hyperresponsiveness by wogonin in vivo. Wogonin induced human neutrophil apoptosis in a dose-dependent manner by activating caspase-8 and caspase-3 in vitro. Wogonin pretreatment abolished HMGB1-induced ASMCs proliferation and migration, which can be explained by the inhibition of phosphorylation in the mitogen-activated protein kinase (MAPK) /Akt singling pathways. Our findings demonstrate that wogonin augments caspase-dependent apoptosis in neutrophils to alleviate neutrophilic inflammatory responses and regulates intracellular signaling to inhibit HMGB1-mediated ASMCs activation, providing a promising therapeutic agent for severe neutrophilic asthma.

Keratinocyte TLR2 and TLR7 contribute to chronic itch through pruritic cytokines and chemokines in mice.

Although neuronal Toll-like receptors (TLRs) (e.g.,TLR2, TLR3, and TLR7) have been implicated in itch sensation, the roles of keratinocyte TLRs in chronic itch are elusive. Herein, we evaluated the roles of keratinocyte TLR2 and TLR7 in chronic itch under dry skin and psoriasis conditions, which was induced by either acetone-ether-water treatment or 5% imiquimod cream in mice, respectively. We found that TLR2 and TLR7 signaling were significantly upregulated in dry skin and psoriatic skin in mice. Chronic itch and epidermal hyperplasia induced by dry skin or psoriasis were comparably reduced in TLR2 and TLR7 knockout mice. In the dry skin model, the enhanced messenger RNA(mRNA) expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, TNF-α, and IFN-γ were inhibited in TLR2-/- mice, while CXCL2, IL-31, and IL-6 were inhibited in TLR7-/- mice. In psoriasis model, the enhanced mRNA expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, and TNF-α were inhibited in TLR2-/- mice, while CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, and TNF-α were inhibited in TLR7-/- mice. Incubation with Staphylococcus aureus (S. aureus) peptidoglycan (PGN-SA) (a TLR2 agonist), imiquimod (a TLR7 agonist), and miR142-3p (a putative TLR7 agonist) were sufficient to upregulate the expression of pruritic cytokines or chemokines in cultured keratinocyte HaCaT cells. Finally, pharmacological blockade of C-X-C Motif Chemokine Receptor 1/2 and high mobility group box protein 1 dose-dependently attenuated acute and chronic itch in mice. Together, these results indicate that keratinocyte TLR2 and TLR7 signaling pathways are distinctly involved in the pathogenesis of chronic itch.

PfHMGB2 has a role in malaria parasite mosquito infection.

Differentiation of asexually replicating parasites into gametocytes is critical for successful completion of the sexual phase of the malaria parasite life cycle. Gametes generated from gametocytes fuse to form a zygote which differentiates into ookinetes and oocysts. The sporozoites are formed inside oocysts which migrate to the salivary glands for next cycle of human infection. These morphologically and functionally distinct stages require stage-specific gene expression via specific transcriptional regulators. The capacity of high mobility group box (HMGB) proteins to interact with DNA in a sequence independent manner enables them to regulate higher order chromosome organization and regulation of gene expression. Plasmodium falciparum HMGB2 (PfHMGB2) shows a typical L- shaped predicted structure which is similar to mammalian HMG box proteins and shows very high protein sequence similarity to PyHMGB2 and PbHMGB2. Functional characterization of PfHMGB2 by gene deletion (Pfhmgb2¯) showed that knockout parasites develop normally as asexual stages and undergo gametocytogenesis. Transmission experiments revealed that Pfhmgb2¯ can infect mosquitoes and develop as oocyst stages. However, transmission was reduced compared to wild type (WT) parasites and as a consequence, the salivary gland sporozoites were reduced in number. In summary, we demonstrate that PfHMGB2 has no role in asexual growth and a modest role in sexual phase development and parasite transmission to the mosquito.

Immunostimulatory oncolytic activity of coxsackievirus A11 in human malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive solid cancer with a poor prognosis, whereas coxsackievirus A11 (CVA11) is a potential oncolytic virus for cancer treatment. We here investigated the oncolytic activity of CVA11 with human MPM cell lines. CVA11 infection was cytotoxic in all six MPM cell lines examined and showed no or minimal cytotoxicity toward normal human normal cell lines. MPM cells with a higher surface level of intercellular adhesion molecule-1 (ICAM-1) expression tended to be more susceptible to CVA11-induced cytotoxicity, and a neutralizing antibody to ICAM-1 attenuated such cytotoxicity. CVA11 infection activated signaling by Akt and extracellular signal-regulated kinase (ERK) pathways, and inhibitors of such signaling also abrogated CVA11-mediated cytotoxicity. Furthermore, CVA11 infection-triggered multiple modes of tumor cell death including apoptosis, pyroptosis, and necroptosis, and such death was accompanied by the release or exposure of the proinflammatory cytokine interleukin-1β and damage-associated molecular patterns such as calreticulin, high-mobility group box-1, annexin A1, and heat shock protein 70, which are hallmarks of immunogenic cell death. Notably, in vivo treatment of human MPM xenografts with intratumoral CVA11 injection resulted in significant suppression of tumor growth in SCID mice, and all mice infected with CVA11 showed no significant change in body weight. Our findings collectively suggest that the oncolytic activity of CVA11 for MPM is dependent on ICAM-1 as a virus receptor, as well as on Akt and ERK signaling, and that oncolytic virotherapy with CVA11 is a promising treatment modality with immunostimulatory activity for human MPM.