Human High Mobility Group Box 1 Research Articles

Altered expression of 15-hydroxyprostaglandin dehydrogenase in chronic rhinosinusitis with nasal polyps

Objective:To investigate the expression level and regulatory mechanism of 15-hydroxyprostaglandin dehydrogenase（HPGD） in chronic rhinosinusitis with nasal polyps（CRSwNP）. Methods:The expression pattern and level of HPGD in CRSwNP and control was observed using immunofluorescence, and western blot was used for analysis of HPGD expression in nasal polyp tissues. The effect of recombinant human high mobility group box-1（HMGB1） on HPGD expression in primary human nasal epithelial cells was observed, and the potential blocking effect of RAGE neutralizing antibody on HMGB1-induced HPGD expression was investigated. Results:The expression of HPGD was elevated in CRSwNP patients compared to the control, while the protein mainly localized at CD68-positive cells and epithelial cells. Recombinant human HMGB1 stimulated an increase in HPGD expression in primary human nasal mucosal epithelial cells at a time-dependent manner. Additionally, increased phosphorylation levels of MEK and elevated RAGE expression were also observed at 12 hours, but decreased at 24 hours after the incubation of HMGB1. The increase in the expression of HPGD induced by HMGB1 in primary human nasal epithelial cells was partly inhibited with RAGE neutralizing antibody. Conclusion:Elevated HPGD expression is observed in CRSwNP, predominantly in macrophages and epithelial cells. HMGB1 regulates HPGD expression through the RAGE-MEK signaling pathway, potentially providing a new target for future regulation of PGE2levels in CRSwNP.

The HMGB1-RAGE axis induces apoptosis in acute respiratory distress syndrome through PERK/eIF2α/ATF4-mediated endoplasmic reticulum stress.

Apoptosis plays a major role in the progression of acute respiratory distress syndrome (ARDS) that may involve the interaction of the high mobility group box 1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). However, the underlying mechanism remains unclear. Thus, we aimed to explore the mechanisms of HMGB1-RAGE axis-induced apoptosis in ARDS. Blood samples from ARDS patients and healthy volunteers were collected to investigate the correlation between serum HMGB1 levels and the severity of ARDS in patients. Mouse models of ARDS induced by caecal ligation and perforation and A549 cell models established by stimulation with recombinant human HMGB1 (rHMGB1) were designed to explore lung inflammatory injury and apoptosis. Serum HMGB1 levels were significantly increased in ARDS patients compared to controls, and HMGB1 levels in the Severe group and Nonsurvival group were significantly higher than those in the Mild and Moderate group and Survival group. In vivo, compared to sham mice, ARDS mice showed significant lung inflammatory injury and apoptosis as well as upregulation of HMGB1 and RAGE and endoplasmic reticulum stress (ERs) protein expression. All injury was attenuated by treatment with an HMGB1 inhibitor GA, a RAGE blocker FPS-ZM1, and an ERs inhibitor 4-PBA. In vitro, A549 cells challenged with rHMGB1 exhibited significant increases in the levels of proteins in the RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2alpha (eIF2α)/activating transcription factor 4 (ATF4) pathway and in apoptosis, all of which were significantly inhibited by pre-treatment with lenti-shPERK and an anti-RAGE antibody. The HMGB1-RAGE axis induces apoptotic injury during ARDS, possibly through PERK/eIF2α/ATF4-mediated ERs.

Indoprofen exerts a potent therapeutic effect against sepsis by alleviating high mobility group box 1-mediated inflammatory responses

Indoprofen is a non-steroidal anti-inflammatory drug, and has provided insights into treatment of spinal muscular atrophies; however, the treatment effect of indoprofen on sepsis and the precise underlying mechanism remain to be elucidated. This study was carried out to examine the inhibitory effect of indoprofen on high mobility group box 1 (HMGB1)-mediated inflammatory responses in vivo and in vitro. Intraperitoneal injection of indoprofen (20 or 40 mg/kg) at 8 h post-sepsis markedly improved the survival of BALB/c mice and ameliorated multiple-organ injury by blocking the inflammatory responses. In addition, indoprofen partially reduced the HMGB1 level in the serum and in the lung, as well as ameliorated pulmonary edema. Mechanistically, indoprofen potently inhibited the release of HMGB1 following stimulation by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C), and suppressed recombinant human HMGB1(rhHMGB1)-induced inflammatory responses. It was also found that indoprofen has both cyclooxygenase 2-dependent and -independent inhibitory effects on the proinflammatory effect of HMGB1 in THP-1 cells. Further, the drug reduced rhHMGB1-induced cell surface levels of toll-like receptor 2, toll-like receptor 4, and receptor of advanced glycation end-products in a concentration-dependent manner. Collectively, these data demonstrated that the anti-inflammatory effect of indoprofen in sepsis was associated with HMGB1-mediated inflammatory responses, thus offering a favorable mechanistic basis to support the therapeutic potential of indoprofen for the treatment of lethal sepsis or other inflammatory diseases.

Salicylic Acid, a Plant Hormone, Suppresses Phytophagous Insect Immune Response by Interrupting HMG-Like DSP1.

Salicylic acid is a plant hormone that can mediate various plant physiological processes. Salicylic acid can bind to human high mobility group box 1 (HMGB1) and interrupt its role in mediating immune responses. Dorsal switch protein 1 (DSP1) is an insect homolog of HMGB1. In this study, a DSP1 (Se-DSP1) encoded in Spodoptera exigua, a phytophagous insect, was characterized, and its potential role in immune response was explored. Upon bacterial challenge, Se-DSP1 was localized in the nucleus and released into the hemolymph. The released Se-DSP1 could mediate both cellular and humoral immune responses by activating eicosanoid biosynthesis. Salicylic acid could bind to Se-DSP1 with a high affinity. The immune responses of S. exigua were significantly interrupted by SA feeding. Larvae reared on tomatoes with high endogenous SA levels became more susceptible to entomopathogens. Taken together, these results suggest a tritrophic defensive role of plant SA against phytophagous insects.

STim-3 alleviates liver injury via regulation of the immunity microenvironment and autophagy

Liver failure (LF) is a monocyte/macrophage-mediated liver injury that has been associated with inflammatory mediators. However, the mechanism through which monocytes/macrophages regulate LF has not been fully elucidated. In this study, we investigated the role of soluble T-cell immunoglobulin domain and mucin domain-containing molecule-3 (sTim-3) in inhibition of release of inflammatory mediators. We further assess this role in protection against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver failure (ALF), via monocyte/macrophage regulation and autophagy induction in mice. Our findings indicate significantly higher plasma sTim-3 in acute-on-chronic liver failure (ACLF) group relative to other groups, with this trend associated with disease progression. Furthermore, infiltrated recombinant sTim-3 inhibited release of various inflammatory mediators, including cytokines and human high-mobility group box-1 (HMGB1), potentially via autophagy induction. Furthermore, H&E staining and the low levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ALF mice, supported that recombinant sTim-3 effectively alleviated liver injury. Moreover, sTim-3 induced changes in monocyte/macrophage population in mice’s liver or blood, which consequently caused a reduction in proinflammatory CD11bhiF4/80lo monocyte-derived macrophages and Ly-6C(+)CD11b(+) monocytes. Conversely, sTim-3 increased autophagy levels of hepatic CD11b(+) monocyte-derived macrophages and decreased apoptosis rate of CD11b (+) monocytes in the blood. Collectively, our findings demonstrated that sTim-3 alleviated inflammatory response and liver injury by promoting autophagy and regulating monocyte/macrophage function. This indicates its potential for future development of novel therapeutic strategies against LF.

High-mobility group box 1 protein induces epithelialmesenchymal transition in upper airway epithelial cells.

In the treatment of rhinosinusitis, nasal polyps are a major problem, and the epithelial-to-mesenchymal transition (EMT) process is considered pivotal in their development. Although various studies have addressed the role of high mobility group box 1 (HMGB1) nuclear protein in this setting, its impact on EMT has yet to be evaluated. Our aim was the pathogenic mechanism of HMGB1 in EMT and EMT-induced upper respiratory nasal polyps. We investigated the EMT-related effects of HMGB1 in human nasal epithelial (HNE) cells using western blot analysis, transepithelial-electrical resistance (TEER) testing, wound healing assay, and immunofluorescence. HNE cells were incubated in a low-oxygen environment to evaluate the role of HMGB1 in hypoxia-induced EMT. Further support for our in vitro findings was obtained through murine models. Human nasal polyps and nasal lavage fluid samples were collected for western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). HMGB1 increased mesenchymal markers and decreased epithelial markers in HNE cells. Hypoxia-induced HMGB1 in turn induced EMT, apparently through RAGE signaling. We verified HMGB1-induced EMT in the upper respiratory epithelium of mice by instilling intranasal HMGB1. In testing of human nasal polyps, HMGB1 and mesenchymal markers were heightened, whereas epithelial markers were reduced, compared with tissue controls. HMGB1 secretion in nasal epithelium may be a major pathogenic factor in upper respiratory EMT, contributing to nasal polyps.

The association between high mobility group box 1 chromatin protein and mitotic chromosomes in glioma cells.

High mobility group box 1 (HMGB1) is an abundant non-histone nuclear protein that functions as a structural protein of chromatin, regulating genome replication and recombination, mRNA transcription and DNA repair. HMGB1 has been implicated in the tumorigenesis of various cancer types, and the upregulation of HMGB1 has been demonstrated in glioma cells. However, the association between HMGB1 and the mitotic chromosomes in glioma remains uncharacterized. In the present study, the sub-cellular localization of HMGB1 in glioma tissues and cells was investigated. In addition, enhanced green fluorescent protein (EGFP)-tagging of the human HMGB1 protein and chromosome spreading were used to investigate the combination of HMGB1 with mitotic chromosomes. The results of the current study indicated that HMGB1 was localized to the nucleus and the cytoplasm, and it was determined to combine with the condensed chromosomes of proliferating cells in paraformaldehyde (PFA)-fixed glioma tissues. However, HMGB1 was also associated with interphase (but not mitotic chromosomes) when fixed with chilled methanol and 5% (v/v) acetic acid or PFA in vitro. Data from live cell imaging and chromosome spreading indicated the association of HMGB1 with mitotic chromosomes in glioma cells. The present results suggest that HMGB1 combines with mitotic chromosomes in glioma cells, and that the use of fixatives may result in the dissociation of the HMGB1-DNA interaction. Therefore, in live specimens and chromosome spreads, EGFP fusion proteins may represent an accurate indicator for the determination of the correct localization and interaction of HMGB1 in glioma cells.

ResolvinD1 attenuates high-mobility group box 1-induced epithelial-to-mesenchymal transition in nasopharyngeal carcinoma cells.

Epithelial-to-mesenchymal transition (EMT) process is prevalent during the progression of tumors. Nasopharyngeal carcinoma (NPC) is no exception. High-mobility group box 1 (HMGB1) was reported to have the effect of inducing EMT in malignancy. However, the impact of HMGB1-induced EMT in NPC is unclear. Resolvin D1 (RvD1) was reported to regress the progression of inflammation and apoptosis of phagocytes. The effect of RvD1 in the EMT is largely unknown. The current research explored the role of RvD1 on HMGB1-induced EMT in NPC. EMT markers were investigated in 10 NPC and 10 nasopharyngitis (NPG) patients using immunohistochemistry and Western blot. In vitro, expression of EMT markers and HMGB1 in CNE1 and CNE2 cells was assessed with immunohistochemical, Western blot, and confocal microscopy after treatment with recombinant human HMGB1 (rhHMGB1) or HMGB1 gene silencing or RvD1. The invasion and migration of NPC cells were detected by scratch test and transwell assay. Overexpression and gene silencing of lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) and G protein-coupled receptor 32 (GPR32) in CNE2 cells confirmed the effect of RvD1 using Western blots. N-cadherin, vimentin, and HMGB1 were found up-regulated in NPC samples compared with NPG samples, while ZO-1 and E-cadherin were down-regulated in NPC tissues. RhHMGB1-induced EMT in CNE1 and CNE2 cells in a dose-dependent way. CNE2 cell lines treated with rhHMGB1 possessed greater invasion and migration ability, which was confirmed by gene silencing. RvD1 suppressed HMGB1-induced EMT in NPC cells via ALX/FPR2 and GPR32 receptors. These results showed that EMT was obvious in NPC. HMGB1 played a key role in inducing EMT. RvD1 inhibited HMGB1-induced EMT and might have potential application in the area of NPC treatment. Impact statement Nasopharyngeal carcinoma has a high incidence in China. Discussing the molecular mechanism of nasopharyngeal carcinoma is important because of high recurrent rate and low quality of life after treatment. HMGB1, as an important inflammatory factor, promotes the process in many cancers. But little is known about how HMGB1 affects the progress of nasopharyngeal carcinoma cells. In our research, we assessed the role of HMGB1 on metastasis and invasion of nasopharyngeal carcinoma cells. The result of study indicates HMGB1-induced EMT in nasopharyngeal carcinoma cells. Furthermore, we observed that RvD1, which plays an actively protective role in many diseases, controls the migration and invasion of nasopharyngeal carcinoma cells by inhibiting the HMGB1-induced EMT. RvD1 can be further studied as a protective factor for nasopharyngeal carcinoma.

Molecular modelling and docking of Mus musculus HMGB1 inflammatory protein with CGA.

Recently, High Mobility Group Box1 (HMGB1) protein has been reported as an inflammatory cytokine present in all nucleated cells with crucial role in the genesis and promotion of cancer. No HMGB1 protein mice model and its active site details are available to validate mice in vivo experiments. Here, for the first time we have reported in silico mice HMGB1 model using human HMGB1 template. Prepared HMGB1 secondary structure showed 6-α helices, 5-β turns, 2-γ turns with 67% α-helices, 32% coil and 9% turn without β-sheet, and classified as α-class protein. Ramachandran plot analysis showed 98.2% and 92.3% residues lies in favoured region, verified by RAMPAGE and PDBsum server respectively. Cancer atlas of HMGB1 protein showed up-regulated expression of HMGB1 gene in different cancer, proved by CAB (CAB005873) and HPA-antibody (HPA003506) in silico. HMGB1 protein showed interaction with different biologically important inflammatory protein as depicted in STRING result.Prominent active site has residues Tyr78Ile79Pro80-81Lys82Gly83vGlu84Thr85Lys86-88Phe89Lys90Asp91Pro92Asn93Tyr162Lys165 with 310 Å3 site volume.Interacting residues of CGA-HMGB1 docked complex were ILE79PRO80-81LYS82GLY83GLU84LYS86-88PHE89Arg163Ala164LYS165Gly166 with docking score 3872 and surface area 412.6. CGA-conformer C3950 showed best docking than CGA and conformer-ZINC03947476, iso-chlorogenic acid and cischlorogenic acid. HMGB1 mice model could be a good therapeutic target for anti-cancerous drugs.

TLR9-HIF-1α-VEGF Axis Is Essential for HMGB1-Mediate Post-Myocardial Infarction Tissue Repair

Background: Toll-like receptor 9 (TLR9) as an essential part of the innate immune system plays a vital role in regulating cardiomyocyte survival and wound healing. During hypoxia, High Mobility Group Box 1 (HMGB1) is rapidly released extracellularly and translocates from the nucleus to bind with cytoplasmic TLR9. However, the mechanism by which TLR9 interacts with HMGB1 and regulates myocardial damage remains unclear. Our current study investigated the effects of TLR9 and HMGB1 on experimental MI. Methods: Experimental MI was induced by left anterior descending (LAD) ligation in wild type (WT) or TLR9 knockout (TLR9KO)mice, to delineate the functional effects of TLR9 on cardiac healing, apoptosis, angiogenesis. Left ventricular injection of recombination human HMGB1(rhHMGB1)was performed to explore the mechanism of TLR9 and HMGB1 in MI. Findings: The survival rate of TLR9KO mice with a higher rate of cardiac rupture was significantly lower than that in WT mice after 28-days postoperation. The effect of TLR9 knockout on insufficient wound healing in experimental MI is caused by a diminished number of myofibroblasts and defective matrix synthetic capability. Moreover, the increased myocardial apoptotic cells and decreased angiogenic capacity were found in TLR9 knockout mice after MI. The results showed contrary in rhHMGB1 (Recombinant Human High Mobility Group Box 1) treated WT mice and similarity after applying rhHMGB1 in TLR9KO mice. Interpretation: Our study demonstrates that TLR9 is essential for the repair of infarcted myocardium and interaction of HMGB1 and TLR9 is involved in the survival and wound healing of myocardial cells after myocardial infarction. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (No: 81470402, 81800216, 81700218, 81770399 and 81700254), the Key Project of the National Natural Science Foundation (No. 81530012) and the Fundamental Research Funds for the Central Universities (No. 2042017kf0085, 2042018kf1032 and 2042015kf0073). Declaration of Interests: The authors declare: None. Ethics Approval Statement: All human heart samples are dedicated for research purposes only, approved by the Renmin Hospital of Wuhan University Review Board, according to the Helsinki Declaration. All animal care and experimental procedures were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (approval no. 2016005) in accordance with the guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH).

HMGB1-Induced p62 Overexpression Promotes Snail-Mediated Epithelial-Mesenchymal Transition in Glioblastoma Cells via the Degradation of GSK-3β.

Rationale: Glioblastoma (GBM) is the most common and aggressive brain tumor, characterized by its propensity to invade the surrounding brain parenchyma. The effect of extracellular high-mobility group box 1 (HMGB1) protein on glioblastoma (GBM) progression is still controversial. p62 is overexpressed in glioma cells, and has been associated with the malignant features and poor prognosis of GBM patients. Hence, this study aimed to clarify the role of p62 in HMGB1-induced epithelial-mesenchymal transition (EMT) of GBM both in vitro and in vivo.Methods: Immunoblotting, immunofluorescence and qRT-PCR were performed to evaluate EMT progression in both human GBM cell line and primary GBM cells. Transwell and wound healing assays were used to assess the invasion and migration of GBM cells. shRNA technique was used to investigate the role of p62 in HMGB1-induced EMT both in vitro and in vivo orthotopic tumor model. Co-immunoprecipitation assay was used to reveal the interaction between p62 and GSK-3β (glycogen synthase kinase 3 beta). Immunohistochemistry was performed to detect the expression levels of proteins in human GBM tissues.Results: In this study, GBM cells treated with recombinant human HMGB1 (rhHMGB1) underwent spontaneous EMT through GSK-3β/Snail signaling pathway. In addition, our study revealed that rhHMGB1-induced EMT of GBM cells was accompanied by p62 overexpression, which was mediated by the activation of TLR4-p38-Nrf2 signaling pathway. Moreover, the results demonstrated that p62 knockdown impaired rhHMGB1-induced EMT both in vitro and in vivo. Subsequent mechanistic investigations showed that p62 served as a shuttling factor for the interaction of GSK-3β with proteasome, and ultimately activated GSK-3β/Snail signaling pathway by augmenting the degradation of GSK-3β. Furthermore, immunohistochemistry analysis revealed a significant inverse correlation between p62 and GSK-3β, and a combination of the both might serve as a more powerful predictor of poor survival in GBM patients.Conclusions: This study suggests that p62 is an effector for HMGB1-induced EMT, and may represent a novel therapeutic target in GBM.

Agonist of PPAR-γ Reduced Epithelial-Mesenchymal Transition in Eosinophilic Chronic Rhinosinusitis with Nasal Polyps via Inhibition of High Mobility Group Box1.

Epithelial-mesenchymal transition (EMT) has been reported to occur in eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP). Among the cytokines that cause EMT, high mobility group box 1 (HMGB1) has been shown to give rise to EMT in airway epithelial cells. However, the mechanism of HMGB1-induced EMT in ECRSwNP is unknown. We explored the mechanism and possible inhibitor. Immunohistochemistry (IHC), immunofluorescence (IF), and western blot assay were used to detect the expression and location of HMGB1, peroxisome proliferator-activated receptor-γ (PPAR-γ), and EMT markers in eighteen ECRSwNP and twelve normal nasal mucosa tissues. Epithelial cells isolated from ECRSwNP were cultured with various doses of recombinant human HMGB1 (rhHMGB1) to study the expression of PPAR-γ, and EMT markers. Additionally, the ligand of PPAR-γ was incubated with epithelial cells to interfere with the effects of lipopolysaccharide (LPS) or rhHMGB1 to explore the effect on expression of HMGB1 and EMT markers. These results suggest that HMGB1 was highly expressed in ECRSwNP compared with its expression in control tissues, and EMT was also found highly in ECRSwNP compared with control tissues. Moreover, the cytoplasmic accumulation of HMGB1 in ECRSwNP was obvious compared with normal tissues. We also found dose-dependent induction by rhHMGB1 of up-regulation of N-cadherin and vimentin and down-regulation of ZO-1 and E-cadherin in epithelial cells isolated from ECRSwNP. The agonist of PPAR-γ not only reduced release of HMGB1 induced by LPS, but also reversed the EMT. The protective role of PPAR-γ also appeared in cells that had been incubated with rhHMGB1. In the current study, we discovered that the agonist of PPAR-γ has a potential role in inhibited HMGB1-induced EMT in ECRSwNP. The agonist of PPAR-γ may contribute to inhabit epithelial cells to become mesenchymal-like cells which play an important role in the pathogenesis of ECRSwNP.

1640. Toward New Anti-Biofilm Therapies: High Mobility Group Box 1 (HMGB1) Protein and Its Structural Variants Can Be Used to Disrupt Bacterial Biofilms (BBs)

BackgroundBB-related infections are a major public health problem, as they are notoriously refractory to current treatments. One of the defining characteristics of BBs is the extracellular polymeric substance (EPS). Extracellular DNA and the bacterial DNABII family of proteins are key components of EPS and are crucial for BBs structural integrity. It is known that targeting DNABII proteins disrupts BBs. We hypothesized that HMGB1, a DNA-binding eukaryotic protein, could affect BBs as it binds to the same DNA structures as the DNABII proteins. HMGB1 is comprised of 3 domains, A Box, B Box, and C tail, all of which have different functions. We aimed to determine in vitro the effects of HMGB1 and its individual domains against BBs.Methods Klebsiella pneumoniae (KP), a common cause of nosocomial infections, was used for all BBs disruption assays. Human recombinant full-length HMGB1 (rHMGB1; 1–215), a C45S mutation variant (mHMGB1) and the HMGB1 domains A Box (1–89), B Box (90–176), AB Boxes (1–176), B-linker Box (80–179), and B-linker Box C106S were expressed (in E. coli) and purified to >95%. To evaluate the effect of rHMGB1 and the various domains on established BBs, each protein species (200 nM) was added to preformed BBs at 24 hours. At 40 hours the BBs were washed, stained with LIVE/DEAD®, visualized via confocal laser scanning microscopy and images were analyzed by COMSTAT to calculate average thickness and biomass.ResultsExogenous rHMGB1 and its individual domains, with the exception of A Box caused a significant reduction (P < 0.05) in average thickness (AT) and biomass (BM) of KP biofilms when compared with untreated KP biofilms (% reduction mean ± SE in AT: 44% ± 0.33, 75% ± 0.04, 63% ± 0.1, 77% ± 0.03, 64% ± 0.08, 54% ± 0.15 and in BM: 61% ± 0.01, 80% ± 0.01, 68% ± 0.02, 67% ± 0.01, 73% ± 0.02, 56% ± 0.02 induced by rHMGB1, mHMGB1, B-Box, B-linker Box, AB Boxes, and B-linker Box C106S, respectively).ConclusionFull-length recombinant HMGB1 was able to significantly disrupt established KP biofilms as were all truncated HMGB1 forms containing the B Box domain and could potentially be used as a therapeutic treatment for BB-related infections.Disclosures J. Buzzo, ProclaRx: Collaborator, Research support. S. Goodman, ProclaRx: Collaborator and Scientific Advisor, Research support.

HMGB1/RAGE Mediates UVB-Induced Secretory Inflammatory Response and Resistance to Apoptosis in Human Melanocytes

The major modifiable risk factor in melanomagenesis is UV exposure and mutagenesis of melanocytes. Other UV-induced events that contribute to early tumorigenesis are poorly understood. Herein we show that therepeated exposure of human primary melanocytes to UVB results in a sustained senescence response, increases in expression of signal transducer and activator of transcription 1, MX1, OAS2, and IRF7 proteins of up to 75-fold, and resistance to subsequent UVB-induced apoptosis. In the setting of UVB-induced DNA damage, we detected time-dependent increases in the release of damage-associated molecular patterns such as high-mobility group box 1 (HMGB1). After intermittent UVB exposure, melanocytes treated with the JAK inhibitor ruxolitinib reduced expression of HMGB1 and MX1 as well as activation of JAK1 (pJAK1) and signal transducer and activator of transcription 1 (pSTAT1). In addition, melanocytes expressing small hairpin RNA selective for the HMGB1 receptor, receptor for advanced glycosylation end product (RAGE), exhibited decreased expression of both HMGB1 and MX1 after UVB exposure. The response of small hairpin RAGE-infected cells to human recombinant HMGB1 was blunted with decreased MX1 expression and JAK activation. Finally, depletion of receptor for advanced glycosylation end product decreased UVB-induced resistance to apoptosis (P < 0.05). These findings highlight a cell autonomous response to UV damage, contribute to their resistance to apoptosis and cell death, and may have implications for early stages of melanoma development.

HMGB1 inhibits insulin signalling through TLR4 and RAGE in human retinal endothelial cells

In the past decade, the role of inflammation has been shown in diabetes and its complications. Little is available on high mobility group box 1 (HMGB1) actions on the proteins involved in insulin signal transduction, which may be altered to result in insulin resistance in the retina. Retinal endothelial cells (REC) were grown in normal or high glucose and treated with recombinant human HMGB1, an Epac1 agonist, or both. Additional cells were treated with advanced glycation end-products (RAGE) or toll-like receptor 4 (TLR4) siRNA prior to rhHMGB1. Proteins lysates were processed for Western blotting for TLR4, RAGE, insulin receptor, Akt, and IRS-1 phosphorylation. We found that rhHMGB1 blocked insulin and Akt phosphorylation through either RAGE or TLR4 actions. Epac1 overcame both endogenous and exogenous HMGB1 to maintain normal insulin signalling. Taken together, these data offer upstream targets to maintain proper insulin signal transduction in the retinal vasculature.

LrHMGB1 Shares Structural Similarities with Human HMGB1, and Its Expression Is Induced in Bacterial Infection, Antiviral Vaccination, and Pathogen-Associated Molecular Patterns Stimulation.

The high-mobility group box 1 (HMGB1) protein is a highly conserved nonhistone chromosomal protein ubiquitously present in almost all cell types. In the nucleus, it facilitates DNA repair and replication, V(D)J recombination, stabilization of nucleosome, and in the cytoplasm, it regulates autophagy and apoptosis. In addition to these intracellular functions, HMGB1 also facilitates activation of innate immune responses and plays key roles in host defense. To investigate its role in fish, we cloned and characterized HMGB1 in Labeo rohita (LrHMGB1), the most important freshwater fish species in the Indian subcontinent. The full-length cDNA sequence of LrHMGB1 consisted of 787 bp having an open reading frame of 618 bp encoding 205 amino acids (aa) polypeptide, with an estimated molecular mass of 23.61 kDa and isoelectric point (pI) of 5.96. Motif search of LrHMGB1 revealed two homologous DNA-binding domains, the A-box and B-box comprising 8-78 aa and 94-162 aa, respectively, and a negatively charged acidic C-terminal tail (179-204) that consisted of 26 consecutive aspartic and glutamic acid residues. The amino acids sequence of LrHMGB1 protein and the secondary structure having helix (H) and coils (C) in tandem in the A- and B-box region and only coils in the acidic tail region shared significant similarity with mouse and human HMGB1. In addition to the three prominent motifs (A-box, B-box, and the acidic tail), the site of acetylation (lys27-29), phosphorylation (serine38,41,45,52), methylation (lys43), and oxidation (cysteine44,105) in LrHMGB1 was also conserved across the fish species, mouse, and human. LrHMGB1 was expressed during embryogenesis and was widely expressed in various organs/tissues having highest expression in blood. In response to bacterial infection, antiviral vaccination, and pathogen-associated molecular patterns stimulations, LrHMGB1 gene expression was significantly (p < 0.05) induced in various organs and tissues. These results together suggest an evolutionary conserved structure and function of HMGB1 from fish to human.

High-Mobility Group Box 1 in Dry Eye Inflammation.

PurposeTo determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation.MethodsEDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining.ResultsEDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment.ConclusionsHMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.

Unfractionated Heparin Alleviates Human Lung Endothelial Barrier Dysfunction Induced by High Mobility Group Box 1 Through Regulation of P38–GSK3β–Snail Signaling Pathway

Background/Aims: The high mobility group box 1 (HMGB1) has been regarded as an important inflammatory mediator. Previous studies showed the involvement of HMGB1 protein in the dysfunction of endothelial barrier function during acute lung injury. However, the molecular mechanism remains unclear. Methods: In this study, we used recombinant human HMGB1 (rhHMGB1) and HMGB1 plasmid to treat human pulmonary microvascular endothelial cell (HPMECs). We examined endothelial permeability by measuring TEER value and HRP flux. Western blot and real-time PCR were used to examined change of endothelial-to-mesenchymal transition (EndoMT) markers and related pathways. Immunofluorescence was used to examine localization and expression of ZO-1 and VE-cadherin. SB203580.was used to block p38 pathway. Unfractionated heparin (UFH) and RAGE siRNA were also used to antagonize the effect of HMGB1. Results: We showed that HMGB1 induced EndoMT with downregulation of ZO-1 and VE-cadherin at both mRNA and protein levels in HPMECs. We also demonstrated that HMGB1 upregulated endothelial permeability by measuring TEER value and HRP flux. Moreover, HMGB1 activated p38/GSK3β/Snail signaling pathway and treatment with p38 inhibitor SB203580 abolished its biological effects. In addition, we found that UFH was able to reverse the effect of HMGB1 on EndoMT and endothelial permeability through inhibition of p38 signaling in a dose-dependent manner. We discovered that RAGE, a membrane receptor of HMGB1, transduced p38/Snail pathway to EndoMT. RAGE siRNA inhibited the effect of HMGB1 induced EndoMT in HPMECs. Conclusion: The present study demonstrated that HMGB1 induced EndoMT through RAGE receptor and p38/GSK3β/Snail pathway. While UFH antagonized HMGB1 and maintained the integrity of the endothelial barrier through p38 inhibition.

High mobility group box1 contributes to hypoxia-induced barrier dysfunction of nasal epithelial cells

Objective:To investigated the promotion of high mobility group box1 (HMGB1) under hypoxia, and determined the regulatory role of HMGB1 on the barrier function of nasal epithelial cells.Method:Primary nasal epithelial cells (NECs) collected from patients with septal deviation were cultured at air-liquid interface. The release of HMGB1 under hypoxia was detected by ELISA. The effect of HMGB1 on fluorescein isothiocyanatedextran 4 kDa (FD4) permeability of NECs was measured. Western blot analysis was utilized to examine the level of major junction proteins, namely E-cadherin, ZO-1, Occludin and Claudin-1.Result:The release of HMGB1 was significantly upregulated in NECs under hypoxia. Recombinant human HMGB1 increased FD4 permeability in a dose and time-dependent manner, indicating the impaired epithelial barrier function. HMGB1-mediated barrier hyperpermeability was accompanied by the selective downregulation of ZO-1, occludin and Claudin-1, but not E-cadherin.Conclusion:HMGB1 mediates hypoxia-induce barrier dysfunction of nasal epithelium, which may be a potential target for the treatment of chronic rhinosinusitis.

Short Thymic Stromal Lymphopoietin Attenuates Toluene Diisocyanate-induced Airway Inflammation and Inhibits High Mobility Group Box 1-Receptor for Advanced Glycation End Products and Long Thymic Stromal Lymphopoietin Expression.

Short thymic stromal lymphopoietin (short TSLP), one of TSLP variants, exerts anti-inflammatory activities in endotoxin shock and colitis mouse models. Our latest work reported that short TSLP prevented house dust mite-induced epithelial barrier disruption. Yet the role of short TSLP in toluene diisocyanate (TDI)-induced asthma is unknown. Male BALB/c mice were sensitized and challenged with TDI to generate a chemical-induced asthma model. Synthetic short TSLP peptides were given intranasally or intraperitoneally before each challenge. TDI significantly increased inflammation and hyperresponsiveness of airway, which were suppressed by short TSLP treatment. Levels of mouse TSLP, high mobility group box 1 (HMGB1), and receptor for advanced glycation end products (RAGE) in airway epithelium and whole lung tissues were markedly increased in TDI group compared with control mice, which were decreased after administration of short TSLP. Meanwhile, short TSLP also inhibited STAT5(Y694) phosphorylation, which was highly expressed in airways of TDI-exposure mice. In vitro, both TDI-human serum albumin (HSA) and recombinant human (rh) HMGB1 promoted long TSLP but not short TSLP gene production in human bronchial epithelial cells (16HBE). Cells pre-treated with short TSLP exhibited less expression of RAGE and long TSLP and lower phosphorylation of Akt(S473), p38 MAPK(T180/Y182), and STAT5(Y694) than stimulated with TDI-HSA or rhHMGB1 alone. Results suggest that short TSLP prevents airway inflammation in a chemical-induced asthma model, which might be associated with the inhibitions of HMGB1-RAGE and long TSLP expression and STAT5(Y694) phosphorylation.