Extracellular High Mobility Group Box 1 Research Articles

Clinicopathological Significance and Prognostic Role of High Mobility Group Box 1 (HMGB1), Toll-Like Receptor (TLR) 2 and TLR4 in Breast Cancer.

High-mobility group box 1 (HMGB1) functions as damage-associated molecular pattern (DAMPs), released into extracellular space during cellular stress. Extracellular HMGB1 act as signal molecules through Toll-like receptor (TLR) 2 or TLR4, exerting diverse functions in both normal cells and malignant cells including breast cancer. However, their comprehensive examination in breast cancer tissues is lacking. Thus, we immunolocalized them in 112 breast cancer tissues, correlating their immunoreactivity with clinicopathological parameters and clinical outcomes to clarify their significance in breast cancer. We demonstrated that nuclear HMGB1 immunoreactivity was correlated with tumor progression and longer disease-free survival. In contrast, TLR2 immunoreactivity was correlated with increased cell proliferation and shorter disease-free survival, dependent on cytoplasmic HMGB1 immunoreactivity. Additionally, TLR4 immunoreactivity correlated with chemoresistance, regardless of cytoplasmic HMGB1 immunoreactivity. It was therefore considered that TLR2 collaboratively contributed to breast cancer progression with HMGB1-DAMPs to become a worse prognostic factor. Meanwhile, TLR4 served as a worse prognostic factor associated with chemoresistance, irrespective of HMGB1.

GLUT1 mediates the release of HMGB1 from airway epithelial cells in mixed granulocytic asthma

Asthma is quite heterogenous and can be categorized as eosinophilic, mixed granulocytic (presence of both eosinophils and neutrophils in the airways) and neutrophilic. Clinically, mixed granulocytic asthma (MGA) often tends to be severe and requires large doses of corticosteroids. High mobility group box 1 (HMGB1) is one of the epithelium-derived alarmins that contributes to type 2 inflammation and asthma. This study was aimed to investigate the role of glucose transporter 1 (GLUT1) in modulation of airway epithelial HMGB1 production in MGA. Induced sputum and bronchial biopsy specimens were obtained from healthy subjects and asthma patients. BALB/c mice, the airway epithelial cell line BEAS-2B, or primary human bronchial epithelial cells (HBECs) were immunized with allergens. Intracellular and extracellular HMGB1 were both detected. The role of GLUT1 was assessed by using a pharmacological antagonist BAY876. MGA patients have a significant higher sputum HMGB1 level than the health and subjects with other inflammatory phenotypes. Nuclear-to-cytoplasmic translocation of HMGB1 was also observed in the bronchial epithelia. Allergen exposure markedly induced GLUT1 expression in murine lungs and cultured epithelial cells. Pharmacological antagonism of GLUT1 with BAY876 dramatically decreased airway hyperresponsiveness, neutrophil and eosinophil accumulation, as well as type 2 inflammation in murine models of MGA. Besides, the allergen-induced up-regulation of HMGB1 was also partly recovered by BAY876, accompanied by inhibited secretion into the airway lumen. In vitro, treatment with BAY876 relieved the allergen-induced over-expression and secretion of HMGB1 in airway epithelia. Taken together, our data indicated that GLUT1 mediates bronchial epithelial HMGB1 release in MGA.

Cisplatin Dependent Secretion of Immunomodulatory High Mobility Group Box 1 (HMGB1) Protein from Lung Cancer Cells.

High mobility group box 1 (HMGB1) is secreted from activated immune cells, necrotic cells, and certain cancers. Previous studies have reported that different patterns of post-translational modification, particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. Here we report that cisplatin but not carboplatin induces secretion of HMGB1 from human A549 non-small cell lung cancer (NSCLC) cells. Cisplatin-mediated HMGB1 secretion was dose-dependent and was regulated by nuclear exportin 1 (XPO1) also known as chromosomal maintenance 1 (CRM1) rather than adenosine diphosphate (ADP)-ribosylation, acetylation, or oxidation. HMGB1, as well as lysine acetylation and cysteine disulfide oxidation of secreted HMGB1, were monitored by sensitive and specific assays using immunoprecipitation, stable isotope dilution, differential alkylation, and nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nano-LC-PRM/HRMS). A major fraction of the HMGB1 secreted by low-dose cisplatin treatment of A549 NSCLC cells was found to be in the fully reduced form. In contrast, mainly oxidized forms of HMGB1 were secreted by dimethyl sulfoxide (DMSO)-mediated apoptosis. These findings suggest that inhibition of XPO1 could potentiate the anti-tumor activity of cisplatin by increasing the nuclear accumulation of HMGB1 protein, an inhibitor of cisplatin DNA-adduct repair. Furthermore, low-dose cisplatin therapy could modulate the immune response in NSCLC through the established chemokine activity of extracellular reduced HMGB1. This could potentially enhance the efficacy of subsequent immunotherapy treatment.

IL-33 and HMGB1 modulate the progression of EAE via oppositely regulating each other

Interleukin-33 (IL-33) and high mobility group box 1 (HMGB1) have been reported to play crucial and distinct roles in experimental autoimmune encephalomyelitis (EAE). However, little is known about their interaction in the progression of EAE. In this study, the dynamic expression and release of IL-33 and HMGB1 in different stages of EAE in vivo, and their interaction in vitro were explored. We found that HMGB1 was dominant in pre-onset stage of EAE, while IL-33 was dominant in peak stage. Moreover, both blockade of extracellular HMGB1 in the central nervous system (CNS) and conditional knockout of HMGB1 in astrocytes decreased IL-33 release. HMGB1 promoted the release of IL-33, while IL-33 reduced the release of HMGB1 from primary astrocytes in vitro. Taken together, IL-33 and HMGB1 in the CNS jointly participate in the EAE progression and the inhibitory effect of IL-33 on HMGB1 may be involved in the self-limiting of EAE.

Identification of Neutralizing Anti-HMGB1 IgM Autoantibody and Its B Cell Producers

Abstract Extracellular High Mobility Group Box 1 (HMGB1) is a prototypic damage-associated molecular pattern (DAMP). While a homeostatic level of extracellular HMGB1 may be beneficial for immune defense, tissue repair and tissue regeneration, excessive HMGB1 is linked to chronic inflammation and inflammatory diseases. This prompts an intriguing question: How does a healthy body control the level of extracellular HMGB1? Here we have identified, in the plasma of both healthy humans and healthy mice, an anti-HMGB1 IgM autoantibody that neutralizes extracellular HMGB1 via binding specifically to a 100% conserved epitope, namely HMW4 (HMGB1 98–112). In mice, this anti-HMGB1 IgM (i.e., anti-HMW4 IgM) is produced by peritoneal B-1 cells, and concomitant triggering of their B cell receptor and toll-like receptor 4 by extracellular HMGB1 stimulates the production of anti-HMW4 IgM. The ability of extracellular HMGB1 to induce its own neutralizing antibody suggests a feedback loop limiting the level of this DAMP in a healthy body. Currently, our studies are ongoing to identify and characterize the human counterpart B cells that produce the anti-HMGB1 IgM, by first amplifying HMW4-reactive B cells from human peripheral blood mononuclear cells (PBMCs) in NSG recipient mice, in the presence of B cell-stimulating adjuvants (such as B cell mitogens). These studies serve as the prelude to exploring human anti-HMGB1 IgM down the road for treating patients with inflammatory diseases and provide insight pertaining to the role and regulation of other DAMP molecules.

Inhibition of macrophages inflammasome activation via autophagic degradation of HMGB1 by EGCG ameliorates HBV-induced liver injury and fibrosis.

Liver fibrosis is a reversible wound-healing response that can lead to end-stage liver diseases without effective treatment, in which HBV infection is a major cause. However, the underlying mechanisms for the development of HBV-induced fibrosis remains elusive, and efficacious therapies for this disease are still lacking. In present investigation, we investigated the effect and mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) on HBV-induced liver injury and fibrosis. The effect of EGCG on liver fibrosis was examined in a recombinant cccDNA (rcccDNA) chronic HBV mouse model by immunohistochemical staining, Sirius red and Masson's trichrome staining. The functional relevance between high mobility group box 1 (HMGB1) and inflammasome activation and the role of EGCG in it were analyzed by Western blotting. The effect of EGCG on autophagic flux was determined by Western blotting and flow cytometric analysis. EGCG treatment efficiently was found to alleviate HBV-induced liver injury and fibrosis in a recombinant cccDNA (rcccDNA) chronic HBV mouse model, a proven suitable research platform for HBV-induced fibrosis. Mechanistically, EGCG was revealed to repress the activation of macrophage NLRP3 inflammasome, a critical trigger of HBV-induced liver fibrosis. Further study revealed that EGCG suppressed macrophage inflammasome through downregulating the level of extracellular HMGB1. Furthermore, our data demonstrated that EGCG treatment downregulated the levels of extracellular HMGB1 through activating autophagic degradation of cytoplasmic HMGB1 in hepatocytes. Accordingly, autophagy blockade was revealed to significantly reverse EGCG-mediated inhibition on extracellular HMGB1-activated macrophage inflammasome and thus suppress the therapeutic effect of EGCG on HBV-induced liver injury and fibrosis. EGCG ameliorates HBV-induced liver injury and fibrosis via autophagic degradation of cytoplasmic HMGB1 and the subsequent suppression of macrophage inflammasome activation. These data provided a new pathogenic mechanism for HBV-induced liver fibrosis involving the extracellular HMGB1-mediated macrophage inflammasome activation, and also suggested EGCG administration as a promising therapeutic strategy for this disease.

Deficiency of neutrophil high-mobility group box-1 in liver transplant recipients exacerbates early allograft injury in mice.

Early allograft dysfunction (EAD) is a severe event leading to graft failure after liver transplant (LT). Extracellular high-mobility group box-1 (HMGB1) is a damage-associated molecular pattern that contributes to hepatic ischemia-reperfusion injury (IRI). However, the contribution of intracellular HMGB1 to LT graft injury remains elusive. We hypothesized that intracellular neutrophil-derived HMGB1 from recipients protects from post-LT EAD. We generated mice with conditional ablation or overexpression of Hmgb1 in hepatocytes, myeloid cells, or both. We performed LTs and injected lipopolysaccharide (LPS) to evaluate the effect of intracellular HMGB1 in EAD. Ablation of Hmgb1 in hepatocytes and myeloid cells of donors and recipients exacerbated early allograft injury after LT. Ablation of Hmgb1 from liver grafts did not affect graft injury; however, lack of Hmgb1 from recipient myeloid cells increased reactive oxygen species (ROS) and inflammation in liver grafts and exacerbated injury. Neutrophils lacking HMGB1 were more activated, showed enhanced pro-oxidant and pro-inflammatory signatures, and reduced biosynthesis and metabolism of inositol polyphosphates (InsPs). On LT reperfusion or LPS treatment, there was significant neutrophil mobilization and infiltration into the liver and enhanced production of ROS and pro-inflammatory cytokines when intracellular Hmgb1 was absent. Depletion of neutrophils using anti-Ly6G antibody attenuated graft injury in recipients with myeloid cell Hmgb1 ablation. Neutrophil HMGB1 derived from recipients is central to regulate their activation, limits the production of ROS and pro-inflammatory cytokines, and protects from early liver allograft injury.

Abstract 1366: HMGB1 localization and its effect on the immune response in the lung tumor microenvironment

Abstract Lung adenocarcinoma is the leading cause of cancer mortality. High Mobility Group Box 1 (HMGB1) is a master regulator of innate immunity that is elevated in lung cancer tissues. Furthermore, HMGB1 has been previously demonstrated to suppress functional anti-tumor immune responses in this context. However, beyond demonstrating that HMGB1 is elevated and modulates inflammation, studies to date have not comprehensively investigated the influence of HMGB1 on the immune response to lung cancer. HMGB1 contributes to PD-L1 expression in melanoma and promotes secretion of inhibitory cytokines TGF-α and IL-10, which have roles in the differentiation of regulatory T cells. Therefore, HMGB1 sits atop an immunomodulatory signaling cascade in the tumor microenvironment. While treatment of lung cancers with immune checkpoint inhibitors (ICI) (anti-PD-1/PD-L1) has had success, there are limitations. One major limitation is the failure of patients with advanced or refractory disease to generate a strong anti-tumor immune response. Recently, we have identified HMGB1 as a protein that is functionally dependent on monounsaturated fatty acid (MUFA) availability. Utilizing a combination of molecular assays and 3-dimensional cultures, we now show that HMGB1 is sequestered intracellularly, rather than being secreted extracellularly in a MUFA-dependent fashion. We hypothesized that MUFAs promote an anti-tumor immune response via modulating HMGB1 release from cancer cells. Thus, by restricting extracellular HMGB1 levels, HMGB1-responsive proinflammatory/tumor signaling pathways (e.g., NF-kB) are suppressed. Taken together, these observations suggest a novel approach to decreasing maladaptive immune and inflammatory signaling from innate immune cells. Exploiting the MUFA/HMGB1 signaling axis could be a novel approach to improve the efficacy of therapies in populations that are not currently benefiting from current strategies for eliminating tumors. Citation Format: Kisa Jafri, Nicole M. Andre, Glenn E. Simmons. HMGB1 localization and its effect on the immune response in the lung tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1366.

Tubular Epithelial Cell HMGB1 Promotes AKI-CKD Transition by Sensitizing Cycling Tubular Cells to Oxidative Stress: A Rationale for Targeting HMGB1 during AKI Recovery.

Cells undergoing necrosis release extracellular high mobility group box (HMGB)-1, which triggers sterile inflammation upon AKI in mice. Neither deletion of HMGB1 from tubular epithelial cells, nor HMGB1 antagonism with small molecules, affects initial ischemic tubular necrosis and immediate GFR loss upon unilateral ischemia/reperfusion injury (IRI). On the contrary, tubular cell-specific HMGB1 deficiency, and even late-onset pharmacological HMGB1 inhibition, increased functional and structural recovery from AKI, indicating that intracellular HMGB1 partially counters the effects of extracellular HMGB1. In vitro studies indicate that intracellular HMGB1 decreases resilience of tubular cells from prolonged ischemic stress, as in unilateral IRI. Intracellular HMGB1 is a potential target to enhance kidney regeneration and to improve long-term prognosis in AKI. Late diagnosis is a hurdle for treatment of AKI, but targeting AKI-CKD transition may improve outcomes. High mobility group box-1 (HMGB1) is a nuclear regulator of transcription and a driver of necroinflammation in AKI. We hypothesized that HMGB1 would also modulate AKI-CKD transition in other ways. We conducted single-cell transcriptome analysis of human and mouse AKI and mouse in vivo and in vitro studies with tubular cell-specific depletion of Hmgb1 and HMGB1 antagonists. HMGB1 was ubiquitously expressed in kidney cells. Preemptive HMGB1 antagonism with glycyrrhizic acid (Gly) and ethyl pyruvate (EP) did not affect postischemic AKI but attenuated AKI-CKD transition in a model of persistent kidney hypoxia. Consistently, tubular Hmgb1 depletion in Pax8 rtTA, TetO Cre, Hmgb1fl/fl mice did not protect from AKI, but from AKI-CKD transition. In vitro studies confirmed that absence of HMGB1 or HMGB1 inhibition with Gly and EP does not affect ischemic necrosis of growth-arrested differentiated tubular cells but increased the resilience of cycling tubular cells that survived the acute injury to oxidative stress. This effect persisted when neutralizing extracellular HMGB1 with 2G7. Consistently, late-onset HMGB1 blockade with EP started after the peak of ischemic AKI in mice prevented AKI-CKD transition, even when 2G7 blocked extracellular HMGB1. Treatment of AKI could become feasible when ( 1 ) focusing on long-term outcomes of AKI; ( 2 ) targeting AKI-CKD transition with drugs initiated after the AKI peak; and ( 3 ) targeting with drugs that block HMGB1 in intracellular and extracellular compartments.

EsRAGE-expressing oHSV enhances anti-tumor efficacy by inhibition of endothelial cell activation

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) molecule that plays an important role in inflammation and tumorigenesis. Receptor for advanced glycation end products (RAGE) is one of the major receptors to which extracellular HMGB1 binds to mediate its activity. RAGE is highly expressed on the endothelial cells (ECs) and regulates endothelial permeability during inflammation. Here, we introduced the endogenous secretory form of RAGE (esRAGE) as a decoy receptor for RAGE ligands into an oncolytic herpes simplex virus 1 (oHSV) (OVesRAGE), which, upon release, can function to block RAGE signaling. OVesRAGE significantly decreased phosphorylation of MEK1/2 and Erk and increased cleaved PARP in glioblastoma (GBM) cells invitro and invivo. oHSV-infected GBM cells co-cultured with ECs were used to test OVesRAGE effect on EC activation, vessel leakiness, virus replication, and tumor cell killing. OVesRAGE could effectively secrete esRAGE and rescue virus-induced EC migration and activation. Reduced EC activation facilitated virus replication in tumor cells when co-cultured with ECs. Finally, OVesRAGE significantly enhanced therapeutic efficacy in GBM-bearing mice. Collectively, our data demonstrate that HMGB1-RAGE signaling could be a promising target and that its inhibition is a feasible approach to improve the efficacy of oHSV therapy.

HMGB1 accumulation in cytoplasm mediates noise-induced cochlear damage.

Damage-associated molecular pattern molecules (DAMPs) play a critical role in mediating cochlear cell death, which leads to noise-induced hearing loss (NIHL). High-mobility group box 1 (HMGB1), a prototypical DAMP released from cells, has been extensively studied in the context of various diseases. However, whether extracellular HMGB1 contributes to cochlear pathogenesis in NIHL and the potential signals initiating HMGB1 release from cochlear cells are not well understood. Here, through the transfection of the adeno-associated virus with HMGB1-HA-tag, we first investigated early cytoplasmic accumulation of HMGB1 in cochlear hair cells after noise exposure. We found that the cochlear administration of HMGB1-neutralizing antibody immediately after noise exposure significantly alleviated hearing loss and outer hair cells (OHCs) death induced by noise exposure. In addition, activation of signal transducer and activators of transcription 1 (STAT1) and cellular hyperacetylation were verified as potential canonical initiators of HMGB1 cytoplasmic accumulation. These findings reveal the adverse effects of extracellular HMGB1 on the cochlea and the potential signaling events mediating HMGB1 release in hair cells, indicating multiple potential pharmacotherapeutic targets for NIHL.

DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein

Neutrophil extracellular traps (NETs) are produced through ejection of genomic DNA by neutrophils into extracellular space and serve as a weapon to fight against pathogens. Neutrophil elastase, a serine protease loaded on NETs, attacks and kills pathogens, while extracellular high-mobility-group-box-1 (HMGB1) protein serves as a danger signal to other cells. How the action of these factors is coordinated as part of the innate immune response is not fully understood. In this article, using biochemical and biophysical approaches, we demonstrate that DNA mediates specific proteolysis of HMGB1 by neutrophil elastase and that the proteolytic processing remarkably enhances binding activities of extracellular HMGB1. Through the DNA-mediated proteolysis of HMGB1 by neutrophil elastase, the negatively charged segment containing D/E repeats is removed from HMGB1. This proteolytic removal of the C-terminal tail causes a substantial increase in binding activities of HMGB1 because the D/E repeats are crucial for dynamic autoinhibition via electrostatic interactions. Our data on the oxidized HMGB1 (i.e., 'disulfide HMGB1') protein show that the truncation substantially increases HMGB1's affinities for the toll-like receptor TLR4•MD-2 complex, DNA G-quadruplex, and the Holliday junction DNA structure. The DNA-mediated proteolysis of HMGB1 by neutrophil elastase in NETs may promote the function of extracellular HMGB1 as a damage-associated molecular pattern that triggers the innate immune response of nearby cells.

Extracellular HMGB1 as Inflammatory Mediator in the Progression of Mycoplasma Gallisepticum Infection.

High-mobility group box 1 (HMGB1), a member of damage-associated molecular patterns (DAMPs), is involved in the immune regulation of several infectious diseases. Mycoplasma gallisepticum (MG) infection is proved to cause an abnormal immune response, but the role of HMGB1 in MG-induced chronic respiratory disease (CRD) is unclear. In this study, we found that HMGB1 was released from the nucleus to the extracellular in macrophages upon infection with MG. Extracellular HMGB1 bound to TLR2 activating the NF-κB pathway triggering a severe inflammatory storm and promoting the progression of MG infection. More importantly, TLR4 could be activated by HMGB1 to trigger immune disorders after TLR2 was silenced. This disease process could be interrupted by ethyl pyruvate (EP) inhibition of HMGB1 release or glycyrrhizic acid (GA). Furthermore, treatment of MG-infected chickens with GA significantly alleviated immune organ damage. In conclusion, we demonstrate that HMGB1 is secreted extracellularly to form an inflammatory environment upon MG infection, triggering a further cellular inflammatory storm in a positive feedback approach. Blocking MG-induced HMGB1 release or suppression downstream of the HMGB1-TLR2/TLR4 axis may be a promising novel strategy for the treatment of CRD. Furthermore, this study may provide a theoretical reference for understanding non-LPS-activated TLR4 events.

Abstract 1112: Detection ofHMGB1 and extracellular ATP for the assessment of immunogenic cell death

Abstract Immunogenic cell death (ICD) is a form of apoptosis that kills susceptible populations of cancer cells while teaching the immune system to attack the remaining resistant cells. Chemotherapeutics which induce ICD elicit their immune response by inducing tumor cells to display or release damage-associated molecular patterns (DAMPs). There are two key biomarkers for ICD: 1) during the apoptotic process, tumor cells secrete ATP (extracellular ATP or eATP) and 2) during secondary necrosis, tumor cells release HMGB1 (high mobility group box 1). Because the dying tumor cells display these DAMP molecules, they stimulate the recruitment of dendritic cells (DCs) into the tumor bed and ultimately “teach” cytotoxic T-lymphocytes (CTLs) to respond to these tumor specific antigens. These primed CTLs will then kill additional tumor cells through a direct cytotoxic response. Therefore, therapeutics which provoke an ICD response offer a therapeutically desirable outcome for cancer therapy. Current assay methods such as ELISA assays and flow cytometry for measuring these ICD biomarkers are laborious and involve multiple transfer and wash steps. We have developed homogeneous (no wash), single addition assays for measuring the two principle ICD biomarkers. The eATP assay uses optimized luciferase detection chemistry that is applied directly to live cells to measure ATP release over 24 hours. The HMGB1 assay uses a complementary luciferase fragment-labeled antibody approach to measure protein concentration. We demonstrated the use of these two assays with model systems for inducing Immunogenic Cell Death with human and murine cell lines including U2OS, EL4, and U937 cells. The potency and response magnitude of ICD-inducing compounds including doxorubicin, idarubicin and mitoxantrone differs depending on the model cell line used. Further, the timing of extracellular ATP release and HMGB1 release differs between cell lines and inducers. Improved assays to measure two markers of Immunogenic Cell Death will expedite the discovery and development of inducers of Immunogenic Cell Death, including small molecule therapeutics, oncolytic viruses, radiation therapy and other novel therapeutics, by streamlining the workflow, increasing sample throughput and providing real time live cell data. Citation Format: Richard L. Somberg, Kevin Kupcho, Andrew Niles, James Cali. Detection ofHMGB1 and extracellular ATP for the assessment of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1112.

Targeting HMGB1-NFκb Axis and miR-21 by Glycyrrhizin: Role in Amelioration of Corneal Injury in a Mouse Model of Alkali Burn.

Corneal neovascularization (CNV) is a sight-threatening condition usually associated with various inflammatory settings including chemical injury. High mobility group box 1 (HMGB1) is identified as an inflammatory alarmin in diverse tissue damage. Here, we evaluate the expression of HMGB1 and the consequences of its inhibition through its selective inhibitor glycyrrhizin (GLY) in alkali burn-induced corneal inflammation and neovascularization. GLY effectively attenuated alkali burn-induced HMGB1 expression at both mRNA and protein levels. Furthermore, slit-lamp analysis, ink perfusion, H&E staining, and CD31 histochemical staining showed that GLY relieved corneal neovascularization, while GLY attenuated VEGF expression via inhibiting HMGB1/NF-κB/HIF-1α signal pathway. In addition, GLY treatment decreased the cytokine expression of CCL2 and CXCL5, accompanied by the reduction of their receptors of CCR2 and CXCR2. GLY diminished the inflammatory cell infiltration of the cornea, as well as reduced the expression of IL-1β, IL-6, and TNF-α. Moreover, treatment with GLY reduced the degree of cornea opacity through inactivating extracellular HMGB1 function, which otherwise induces TGF-β1 release and myofibroblast differentiation. Furthermore, we found that GLY treatment attenuated the upregulation of miR-21 levels in alkali burned cornea; while inhibition of miR-21in keratocytes in vitro, significantly inhibited TGF-β1-induced myofibroblast differentiation. Collectively, our results suggested that targeting HMGB1-NFκb axis and miR-21 by GLY could introduce a therapeutic approach to counter CNV.

Serum Level of High-Mobility Group Box Protein 1 as a Potential Treatment Target in Egyptian Sickle Cell Disease Patients

Background:
 During tissue injury, high mobility group box 1 (HMGB1) is passively released from necrotic cells and actively secreted by inflammatory cells. Extracellular HMGB1 acts as an amplifier of Toll-Like Receptor (TLR)-dependent inflammation rather than a primary trigger of inflammation. We studied HMGB1 quantitative trait locus reference sequence 2249825 (rs2249825) and its serum level in both sickle cell disease (SCD) patients and healthy subjects to explore its possible role in the pathogenesis of vaso-occlusive crises (VOCs).
 Methods:
 HMGB1 rs2249825 was assayed in peripheral blood samples using real-time polymerase chain reaction (RT-PCR). While the serum level was assayed using a two-site enzyme-linked immunosorbent technique (ELISA).
 Results:
 Both the SCD patients and the control group had comparable HMGB1 rs2249825 genotype frequencies (P-value >0.05). SCD patients at their steady-state showed statistically significantly higher serum HMGB1 levels than the healthy controls, a median of 0.6 ng/ml with a range of 0.1- 85 ng/ml versus a median of 0.3 ng/ml and a range of 0.1-3 ng/ml (P-value <0.001), respectively. Statistically significant skewed high serum HMGB1 in the VOC samples in contrast to the steady-state samples was observed in the SCD patients with a median of 3.2 ng/ml and a range of 0.3-76.4 ng/ml versus a median of 0.2 ng/ml and a range 0.2-7.4 ng/ml (P-value <0.0001), respectively.
 
 
 Conclusion:
 HMGB1 could have a role in the VOC pathogenesis, hence it is suggested as a potential additive therapeutic target in SCD in general and in vaso-occlusions in specific.
 Keywords:
 Sickle cell disease, HMGB1, Hemoglobin S

Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91.

High-mobility group box 1 (HMGB1) protein can impair phagocyte function by suppressing the macrophage-mediated clearance of apoptotic cells (ACs), thereby delaying inflammation resolution in the lungs and allowing the progression of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the precise mechanism underlying this HMGB1-mediated inhibition of efferocytosis remains unknown. The aim of this study was to determine the effect of HMGB1 on macrophage-mediated efferocytosis. We discovered that HMGB1 prevented efferocytosis by bone marrow-derived macrophages (BMDMs) and suppressed the expression of Ras-related GTP-binding protein 43 (Rab43), a member of the Ras-associated binding (Rab) family. The downregulation of Rab43 expression resulted in impaired clearance of apoptotic thymocytes by BMDMs. Subsequent analysis of HMGB1-treated and Rab43-deficient BMDMs revealed the inhibited transport of cluster of differentiation 91 (CD91), a phagocyte recognition receptor, from the cytoplasm to the cell surface. Notably, Rab43 directly interacted with CD91 to mediate its intercellular trafficking. Furthermore, Rab43 knockout delayed the inflammation resolution and aggravated the lung tissue damage in mice with ALI. Therefore, our results provide evidence that HMGB1 impairs macrophage-mediated efferocytosis and delays inflammation resolution by suppressing the Rab43-regulated anterograde transport of CD91, suggesting that the restoration of Rab43 levels is a promising strategy for attenuating ALI and ARDS in humans.

Role of Extracellular High-Mobility Group Box-1 as a Therapeutic Target of Gastric Cancer.

Background: High-mobility group box-1 (HMGB1) is involved in the tumorigenesis and metastasis of various cancers. The present study investigated the roles of extracellular HMGB1 in the progression of gastric cancer (GC) and the therapeutic effects of recombinant human soluble thrombomodulin (rTM) targeting HMGB1. Methods: The effects of extracellular HMGB1 and rTM on GC cells were assessed using proliferation and Transwell assays. Their effects on local tumor growth and metastasis were evaluated using subcutaneous tumor and liver metastasis mouse models, respectively. Plasma HMGB1 concentrations in GC patients were measured using ELISA. The relationships between plasma HMGB1 concentrations and the prognosis and clinicopathological factors of patients were also investigated. Results: GC proliferation, migration, and invasion abilities were promoted by increases in extracellular HMGB1 concentrations and alleviated by rTM. In the subcutaneous tumor model, local tumor growth was promoted by the addition of rhHMGB1 and alleviated by rTM. Similar changes occurred in the liver metastasis model. Recurrence-free survival (p < 0.01) and overall survival (p = 0.01) were significantly worse in patients with high plasma HMGB1 concentrations. Conclusion: Plasma HMGB1 concentrations are a prognostic marker in GC patients. Extracellular HMGB1 promotes cancer progression and has potential as a novel treatment target in GC cells for rTM.

Extracellular Release of HMGB1 as an Early Potential Biomarker for the Therapeutic Response in a Xenograft Model of Boron Neutron Capture Therapy.

Simple SummaryBoron neutron capture therapy (BNCT) is a non-invasive therapeutic technique for treating malignant tumors, however, there is a lack of methods to evaluate its therapeutic efficacy. Herein, we investigated whether high mobility group box 1 (HMGB1) is a potential biomarker of BNCT response in tumor cells and mice in combination with 10B-p-boronophenylalanine in the Kyoto University Nuclear Reactor. We observed an increased extracellular HMGB1 release in cancer cells 24 h post-BNCT irradiation, compared to that observed with an equivalent dose of gamma-ray irradiation. High levels of plasma HMGB1 were observed on day 3 post-BNCT irradiation in a xenograft mouse model. These levels were stably detected even when the size of the tumor decreased, suggesting that HMGB1 is a potential biomarker for the therapeutic efficacy of BNCT.Boron neutron capture therapy (BNCT) is a non-invasive therapeutic technique for treating malignant tumors, however, methods to evaluate its therapeutic efficacy and adverse reactions are lacking. High mobility group box 1 (HMGB1) is an inflammatory molecule released during cell death. Therefore, we aimed to investigate HMGB1 as a biomarker for BNCT response, by examining the early responses of tumor cells to 10B-boronophenylalanine (BPA)-based BNCT in the Kyoto University Nuclear Reactor. Extracellular HMGB1 release was significantly increased in human squamous carcinoma SAS and melanoma A375 cells 24 h after neutron irradiation but not after γ-irradiation. At 3 days post-BPA-based BNCT irradiation in a SAS xenograft mouse model, plasma HMGB1 levels were higher than those in the non-irradiation control, and HMGB1 was detected in both nuclei and cytoplasm in tumor cells. Additionally, increased plasma HMGB1 levels post-BNCT irradiation were detected even when tumors decreased in size. Collectively, these results indicate that the extracellular HMGB1 release occurs at an early stage and is persistent when tumors are reduced in size; therefore, it is a potential biomarker for evaluating the therapeutic response during BNCT.

Neutralization of HMGB1 improves fracture healing and \u03b3\u03b4 T lymphocyte counts at the fracture site in a polytrauma rat model

PurposeDelayed fracture healing is a common consequence of polytrauma (PT) occurring in patients with multiple injuries. We believe that when early release of high mobility group box 1 (HMGB1) molecules from necrotic tissues exceed their normal levels in blood, they dysregulate immune responses associated with normal healing. This study investigates the detrimental effect of such dysregulate immune responses by targeting HMGB1 in a PT rat model with debilitating injuries. We hypothesized that neutralization of extracellular HMGB1 immediately post-trauma would ameliorate local immune dysregulation and improve fracture healing in a PT rat model.MethodsPT rats received a single dose of either anti-rat HMGB1 polyclonal antibody (PT-Ab HMGB1) or IgY isotype (PT-IgY), were left untreated (PT-C), or had a single injury/osteotomy only (OST). Fracture healing was evaluated by micro-computed tomography (µCT) and histology at 5 weeks; and macrophages and T cell counts within the fracture site were determined with flow cytometry at 1 week.ResultsNotably, bone regeneration within the fracture site in PT-Ab HMGB1 rats was improved with comparable connective tissue organization than PT-C rats. Further, only γδTCR+ T cells, but not macrophages and CD4+ and CD8+ T cells, were diminished at the fracture site in PT-C and PT-IgY rats. Interestingly, the PT-Ab HMGB1 rats had increased γδTCR+ T cells compared to PT-C and PT-IgY, suggesting their potential role in regulating fracture healing.ConclusionsTherefore, the initial burst of systemic HMGB1 following trauma may have a role in regulating bone regeneration via the modulation of a subclass of T cells within the fracture site, suggesting it’s importance as a therapeutic target in PT to combat immune dysregulation and delayed fracture healing.