High Mobility Group Box 1 Release Research Articles

Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7144.

Hypochlorous acid derived from microglial myeloperoxidase could mediate high-mobility group box 1 release from neurons to amplify brain damage in cerebral ischemia–reperfusion injury

Myeloperoxidase (MPO) plays critical role in the pathology of cerebral ischemia–reperfusion (I/R) injury via producing hypochlorous acid (HOCl) and inducing oxidative modification of proteins. High-mobility group box 1 (HMGB1) oxidation, particularly disulfide HMGB1 formation, facilitates the secretion and release of HMGB1 and activates neuroinflammation, aggravating cerebral I/R injury. However, the cellular sources of MPO/HOCl in ischemic brain injury are unclear yet. Whether HOCl could promote HMGB1 secretion and release remains unknown. In the present study, we investigated the roles of microglia-derived MPO/HOCl in mediating HMGB1 translocation and secretion, and aggravating the brain damage and blood-brain barrier (BBB) disruption in cerebral I/R injury. In vitro, under the co-culture conditions with microglia BV cells but not the single culture conditions, oxygen–glucose deprivation/reoxygenation (OGD/R) significantly increased MPO/HOCl expression in PC12 cells. After the cells were exposed to OGD/R, MPO-containing exosomes derived from BV2 cells were released and transferred to PC12 cells, increasing MPO/HOCl in the PC12 cells. The HOCl promoted disulfide HMGB1 translocation and secretion and aggravated OGD/R-induced apoptosis. In vivo, SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus different periods of reperfusion. Increased MPO/HOCl production was observed at the reperfusion stage, accomplished with enlarged infarct volume, aggravated BBB disruption and neurological dysfunctions. Treatment of MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) and HOCl scavenger taurine reversed those changes. HOCl was colocalized with cytoplasm transferred HMGB1, which was blocked by taurine in rat I/R-injured brain. We finally performed a clinical investigation and found that plasma HOCl concentration was positively correlated with infarct volume and neurological deficit scores in ischemic stroke patients. Taken together, we conclude that ischemia/hypoxia could activate microglia to release MPO-containing exosomes that transfer MPO to adjacent cells for HOCl production; Subsequently, the production of HOCl could mediate the translocation and secretion of disulfide HMGB1 that aggravates cerebral I/R injury. Furthermore, plasma HOCl level could be a novel biomarker for indexing brain damage in ischemic stroke patients.

Antiseptic Functions of CGK012 against HMGB1-Mediated Septic Responses.

High mobility group box 1 (HMGB1), a protein with important functions, has been recognized as a potential therapeutic target for the treatment of sepsis. One possible mechanism for this is that inhibiting HMGB1 secretion can exert antiseptic effects, which can restore the integrity of the vascular barrier. (7S)-(+)-cyclopentyl carbamic acid 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester (CGK012) is a newly synthesized pyranocoumarin compound that could function as a novel small-molecule inhibitor of the Wnt/β-catenin signaling pathway. However, no studies have yet determined the effects of CGK012 on sepsis. We investigated the potential of CGK012 to attenuate the excessive permeability induced by HMGB1 and enhance survival rates in a mouse model of sepsis with reduced HMGB1 levels following lipopolysaccharide (LPS) treatment. In both LPS-stimulated human endothelial cells and a mouse model exhibiting septic symptoms due to cecal ligation and puncture (CLP), we assessed proinflammatory protein levels and tissue damage biomarkers as indicators of reduced vascular permeability. CGK012 was applied after induction in human endothelial cells exposed to LPS and the CLP-induced mouse model of sepsis. CGK012 effectively mitigated excessive permeability and suppressed HMGB1 release, resulting in improved vascular stability, decreased mortality, and enhanced histological conditions in the mouse model of CLP-induced sepsis. In conclusion, our findings indicate that CGK012 treatment in mice with CLP-induced sepsis diminished HMGB1 release and increased the survival rate, suggesting its potential as a pharmaceutical intervention for sepsis.

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.

Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an “inflammatory signaling cascade” in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Sirtuin 1 alleviates alcoholic liver disease by inhibiting HMGB1 acetylation and translocation.

Alcoholic liver disease (ALD) encompasses a spectrum of liver disorders resulting from prolonged alcohol consumption and is influenced by factors such as oxidative stress, inflammation, and apoptosis. High Mobility Group Box 1 (HMGB1) plays a pivotal role in ALD due to its involvement in inflammation and immune responses. Another key factor, Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is known for its roles in cellular stress responses and metabolic regulation. Despite individual studies on HMGB1 and SIRT1 in ALD, their specific molecular interactions and combined effects on disease advancement remain incompletely understood. Alcohol-induced liver injury (ALI) models were established using HepG2 cells and male C57BL/6 mice. HMGB1 and SIRT1 expressions were assessed at the mRNA and protein levels usingreverse transcription-quantitative polymerase chain reaction, western blot, and immunofluorescence staining. The physical interaction between HMGB1 and SIRT1 was investigated using co-immunoprecipitation and immunofluorescence co-expression analyses. Cellular viability was evaluated using the CCK-8 assay. In patients with clinical ALI, HMGB1 mRNA levels were elevated, while SIRT1 expression was reduced, indicating a negative correlation between the two. ALI models were successfully established in cells and mice, as evidenced by increased markers of cellular and liver damage. HMGB1 acetylation and translocation were observed in both ALI cells and mouse models. Treatment with the SIRT1 agonist, SRT1720, reversed the upregulation of HMGB1 acetylation, nuclear translocation, and release in the ethyl alcohol (EtOH) group. Furthermore, SIRT1 significantly attenuated ALI. Importantly, in vivo binding was confirmed between SIRT1 and HMGB1. SIRT1 alleviates HMGB1 acetylation and translocation, thereby ameliorating ALI.

Lactate promotes HMGB1 phosphorylation and release via Akt signaling pathway in gastric cancer cells HGC-27

Objective: To investigate the molecular mechanism of how lactate induces high mobility group box 1 (HMGB1) release. Methods: Gastric cancer HGC-27 cells were divided into the control group and the lactate group (The cells were treated with lactate for 6 h). The level of HMGB1 in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA), the localization of HMGB1 was detected using laser confocal microscopy, and the nuclear translocation of HMGB1 was detected using the nucleoplasmic separation assay. The phosphorylation and acetylation levels of HMGB1 were determined by co-immunoprecipitation, and Western blot was used to measure the phosphorylation of Akt and protein kinase C (PKC). HGC-27 cells were first treated with lactate and LY294002, the inhibitor of Akt, and then the phosphorylation of HMGB1 and Akt was analyzed by co-immunoprecipitation and Western blot, respectively. The localization of HMGB1 in cells was detected by laser confocal microscopy. EdU and Transwell assays were used to detect the proliferation and migration abilities of HGC-27 cells, respectively. HGC-27 cells were then injected into the BALB/C null mice for subcutaneous tumor implantation. Mice in the lactate group were intraperitoneally injected with lactate (0.2 g/kg/2 d), while those in the control group were intraperitoneally injected with an equal amount of PBS for 20 consecutive days. ELISA was used to detect the HMGB1 levels in the blood samples taken from the medial canthus vein of the mice, while co-immunoprecipitation and Western blot were used to detect the phosphorylation of HMGB1 and Akt in tumor tissue proteins, respectively. Results: The release levels of HMGB1 in the lactate group were (2 995.00±660.91) pg/ml and (696.33±22.03) pg/ml, after lactate treatment for 6 h and 12 h, respectively, both higher than those in the control group (485.00±105.83) pg/ml (P<0.001 and P=0.028, respectively). After lactate treatment for 6 h, the relative expression of HMGB1 protein in the cytoplasm of HGC-27 cells was 1.13±0.09, higher than that of the control group (0.83±0.07, P=0.001), while the relative expression of HMGB1 in the nucleus was 0.79±0.06, lower than that of the control group (1.07±0.06, P=0.007). The phosphorylation level of HMGB1 reached 1.41±0.09, which was higher than that of the control group (0.97±0.10, P=0.031). The phosphorylation level of Akt was 11.16±0.06, higher than that of the control group (0.91±0.022, P=0.002). The phosphorylation level and nuclear translocation of HMGB1 induced by lactate decreased obviously after Akt inhibition; the proliferation and migration abilities induced by lactate were also obviously inhibited after Akt inhibition. In vivo, the HMGB1 level in the peripheral blood was (1 280.70±389.66) pg/ml in the lactate group, which was obviously higher than that in the control group (595.11±44.75) pg/ml (P=0.008), and the phosphorylation levels of HMGB1 and Akt in tumor tissues in the lactate group were obviously enhanced compared with the control group. Conclusion: Lactate induces HMGB1 release through enhancing HMGB1 phosphorylation via the Akt signaling pathway.

HMGB1 Release Induced by EV71 Infection Exacerbates Blood-Brain Barrier Disruption via VE-cadherin Phosphorylation

PurposeEV71 (Enterovirus 71) is a major causative agent of the outbreaks of HFMD (hand, foot, and mouth disease), which is associated with neurological damage caused by permeability disruption of BBB (blood-brain barrier). HMGB1 (high-mobility group box 1) is a widely expressed nuclear protein that triggers host inflammatory responses. Our work aimed to explore the function of HMGB1 in EV71 infection and its contributions to EV71-related BBB damage. MethodsHeLa cells, HT-29 cells and AG6 mice were used to explore the translocation of HMGB1 in EV71 infection in vitro and in vivo. The roles of released HMGB1 on EV71 replication and associated inflammatory cytokines were investigated using recombinant HMGB1 in HeLa cells. The mechanisms of released HMGB1 in EV71-induced BBB injury were explored using recombinant HMGB1 and anti-HMGB1 neutralizing antibodies in monolayer HCMECs (immortalized human brain microvascular endothelial cells) and AG6 mice brain. ResultsEV71 induced HMGB1 nucleocytoplasmic translocation and extracellular release in vitro and in vivo. Released HMGB1 acted as an inflammatory mediator in EV71 infection rather than affecting viral replication in vitro. Released HMGB1 disrupted BBB integrity by enhancing VE-cadherin phosphorylation at tyrosine 685 in HCMECs, and reducing total VE-cadherin levels in HCMECs and AG6 mice in EV71 infection. And released HMGB1 induced an increase in activated astrocytes. Neutralization of HMGB1 reversed the increased endothelial hyperpermeability and phosphorylation of VE-cadherin in HCMECs. ConclusionThe inflammatory mediator HMGB1 released by EV71 exacerbated BBB disruption by enhancing VE-cadherin phosphorylation, which in turn aggravated EV71-induced neuroinflammation.

APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release

Apolipoprotein E4 (APOE4) is an important driver of Tau pathology, gliosis, and degeneration in Alzheimer's disease (AD). Still, the mechanisms underlying these APOE4-driven pathological effects remain elusive. Here, we report in a tauopathy mouse model that APOE4 promoted the nucleocytoplasmic translocation and release of high-mobility group box 1 (HMGB1) from hippocampal neurons, which correlated with the severity of hippocampal microgliosis and degeneration. Injection of HMGB1 into the hippocampus of young APOE4-tauopathy mice induced considerable and persistent gliosis. Selective removal of neuronal APOE4 reduced HMGB1 translocation and release. Treatment of APOE4-tauopathy mice with HMGB1 inhibitors effectively blocked the intraneuronal translocation and release of HMGB1 and ameliorated the development of APOE4-driven gliosis, Tau pathology, neurodegeneration, and myelin deficits. Single-nucleus RNA sequencing revealed that treatment with HMGB1 inhibitors diminished disease-associated and enriched disease-protective subpopulations of neurons, microglia, and astrocytes in APOE4-tauopathy mice. Thus, HMGB1 inhibitors represent a promising approach for treating APOE4-related AD.

Transcriptomic Profiling Identifies Ferroptosis-Related Gene Signatures in Ischemic Muscle Satellite Cells Affected by Peripheral Artery Disease-Brief Report.

We hypothesized that transcriptomic profiling of muscle satellite cells in peripheral artery disease (PAD) would identify damage-related pathways contributing to skeletal muscle myopathy. We identified a potential role for ferroptosis-a form of programmed lytic cell death by iron-mediated lipid peroxidation-as one such pathway. Ferroptosis promotes myopathy in ischemic cardiac muscle but has an unknown role in PAD. Muscle satellite cells from donors with PAD were obtained during surgery. cDNA libraries were processed for single-cell RNA sequencing using the 10X Genomics platform. Protein expression was confirmed based on pathways inferred by transcriptomic analysis. Unsupervised cluster analysis of over 25 000 cells aggregated from 8 donor samples yielded distinct cell populations grouped by a shared unique transcriptional fingerprint. Quiescent cells were diminished in ischemic muscle while myofibroblasts and apoptotic cells were prominent. Differential gene expression demonstrated a surprising increase in genes associated with iron transport and oxidative stress and a decrease in GPX4 (glutathione peroxidase 4) in ischemic PAD-derived cells. Release of the danger signal HMGB1 (high mobility group box-1) correlated with ferroptotic markers including surface transferrin receptor and were higher in ischemia. Furthermore, lipid peroxidation in muscle satellite cells was modulated by ferrostatin, a ferroptosis inhibitor. Histology confirmed iron deposition and lipofuscin, an inducer of ferroptosis in PAD-affected muscle. This report presents a novel finding that genes known to be involved in ferroptosis are differentially expressed in human skeletal muscle affected by PAD. Targeting ferroptosis may be a novel therapeutic strategy to reduce PAD myopathy.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway.

Opioids, such as morphine, are the most potent drugs used to treat pain. Long-term use results in high tolerance to morphine. High mobility group box-1 (HMGB1) has been shown to participate in neuropathic or inflammatory pain, but its role in morphine tolerance is unclear. In this study, we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days. We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1. HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4 receptor expression in microglia, thereby inducing morphine tolerance. Glycyrrhizin, an HMGB1 inhibitor, markedly attenuated chronic morphine tolerance in the mouse model. Finally, compound C (adenosine 5'-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin (heme oxygenase-1 inhibitor) alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tolerance, and alleviated morphine tolerance in the mouse model. These findings suggest that morphine induces HMGB1 release via the adenosine 5'-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway, and that inhibiting this signaling pathway can effectively reduce morphine tolerance.

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.

Inducing a Proinflammatory Response with Bioengineered Yeast Vacuoles with TLR2-Binding Peptides (VacT2BP) as a Drug Carrier for Daunorubicin Delivery.

Immune adjuvants have roles in immune activation for cancer therapy, and adjuvants derived from microbes have been applied. In this study, we propose the use of bioengineered vacuoles, derived from recombinant yeast with acute myeloid leukemia (AML) specificity and having a TLR-2-binding peptide (VacT2BP) on their surface, to induce a proinflammatory response as a dual-function nanomaterial for daunorubicin (DNR) delivery. Our results demonstrate that nanosized, isolated VacT2BP induced HL-60 cell-specific DNR delivery and apoptosis. Furthermore, we observed the selective release of high-mobility group box 1 from apoptotic HL-60 cells by DNR@VacT2BP. We concluded that DNR@VacT2BP exhibited target selectivity, and the indiscriminate occurrence of damage-associated molecular patterns (DAMPs) was inhibited by the VacT2BP carrier. The therapeutic efficacy of DNR@VacT2BP was confirmed in AML xenograft mice, with about 82% tumor growth inhibition. Following drug delivery, apoptotic cells and DAMPs with residual VacT2BP (apopDNR@VacT2BP) upregulated the proinflammatory immune response of macrophages. In addition, apopDNR@VacT2BP enhanced phagocytosis activity. Macrophages stimulated by apopDNR@VacT2BP suppressed cancer proliferation by about 40%. In summary, our results suggest that dual-functional vacuoles with a target-specific peptide can be a potential strategy for selective drug delivery and construction of an immune environment to fight cancer, thereby improving prognosis.

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.

QHRD106 ameliorates ischemic stroke injury as a long-acting tissue kallikrein preparation

Ischemic stroke is the second leading cause of death worldwide, and there are limited effective treatment strategies. QHRD106, a polyethyleneglycol (PEG)-modified long-acting tissue kallikrein preparation, has not been reported previously. In this study, we aimed to investigate the therapeutic effect of QHRD106 in ischemic stroke and its possible mechanism. We found that QHRD106 treatment alleviated brain injury after stroke via bradykinin (BK) receptor B2 (B2R) instead of BK receptor B1 (B1R). Mechanistically, QHRD106 reduced high-mobility group box 1 (HMGB1)-induced apoptosis and inflammation after ischemic stroke invivo and invitro. Moreover, we confirmed that QHRD106 reduced the level of acetylated HMGB1 and reduced the binding between heat shock protein 90 alpha family class A member 1 (HSP90AA1) and HMGB1, thus inhibiting the translocation and release of HMGB1. In summary, these findings indicate that QHRD106 treatment has therapeutic potential for cerebral ischemic stroke.

TNF-α induced extracellular release of keratinocyte high-mobility group box 1 in Stevens-Johnson syndrome/toxic epidermal necrolysis: Biomarker and putative mechanism of pathogenesis.

Decreased epidermal high-mobility group box 1 (HMGB1) expression is an early marker of epidermal injury in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Etanercept, an anti-tumor necrosis factor therapeutic, is effective in the treatment of SJS/TEN. The objective was to characterize antitumor necrosis factor-alpha (TNF-α)-mediated HMGB1 keratinocyte/epidermal release and etanercept modulation. HMGB1 release from TNF-α treated (± etanercept), or doxycycline-inducible RIPK3 or Bak-expressing human keratinocyte cells (HaCaTs) was determined by western blot/ELISA. Healthy skin explants were treated with TNF-α or serum (1:10 dilution) from immune checkpoint inhibitor-tolerant, lichenoid dermatitis or SJS/TEN patients ± etanercept. Histological and immunohistochemical analysis of HMGB1 was undertaken. TNF-α induced HMGB1 release in vitro via both necroptosis and apoptosis. Exposure of skin explants to TNF-α or SJS/TEN serum resulted in significant epidermal toxicity/detachment with substantial HMGB1 release which was attenuated by etanercept. Whole-slide image analysis of biopsies demonstrated significantly lower epidermal HMGB1 in pre-blistered SJS/TEN versus control (P < 0.05). Keratinocyte HMGB1 release, predominantly caused by necroptosis, can be attenuated by etanercept. Although TNF-α is a key mediator of epidermal HMGB1 release, other cytokines/cytotoxic proteins also contribute. Skin explant models represent a potential model of SJS/TEN that could be utilized for further mechanistic studies and targeted therapy screening.

The effect of lipopolysaccharide on liver homeostasis and diseases based on the mutual interaction of macrophages, autophagy, and damage-associated molecular patterns in male F344/DuCrlCrlj rats.

Lipopolysaccharide (LPS) has dose-dependent biphasic functions (cell protective versus cell toxic). To clarify the different effects of LPS on liver homeostasis or liver diseases, comparisons were made between low and high doses of LPS, in terms of the mutual relation of hepatic macrophages, autophagy, and damage-associated molecular patterns (DAMPs) in male F344/DuCrlCrlj rats. Rats injected with low dose (0.1 mg/kg) or high dose (2.0 mg/kg) of LPS were examined at 6, 10, and 24 hours following single injections. Histologically, focal hepatocellular necrosis was occasionally present in high-dose animals, whereas there were no significant changes in low-dose animals. In low-dose animals, Kupffer cells reacting to CD163 and CD204 were hypertrophic and regarded as M2 macrophages, which promote resolution of inflammation and tissue repair, whereas in high-dose animals, infiltration of M1 macrophages expressing CD68 and major histocompatibility complex class II, which enhance cell injury, was seen. Hepatocytes with high-mobility-group box-1 (HMGB1) (one of DAMPs)-positive cytoplasmic granules appeared more frequently in high-dose animals than in low-dose animals, indicating the translocation of nuclear HMGB1 into the cytoplasm. However, although light-chain 3 beta-positive autophagosomes in hepatocytes increased in both doses, abnormally vacuolated autophagosomes were only seen in injured hepatocytes in the high-dose group, indicating possible extracellular release of HMGB1, which might result in cell injury and inflammation. These findings suggested that low-dose LPS induced a favorable mutual relationship among hepatic macrophages, autophagy, and DAMPs leading to cytoprotection of hepatocytes, whereas failures of the relationship in high-dose LPS caused hepatocyte injury.

Induced ischemia-reperfusion and oxidative stress in patients with systemic sclerosis lead to increased release of High-Mobility Group Box-1 which potentially activate skin fibroblast to produce interferon-γ Induced Protein-10: a translational study

Induced ischemia-reperfusion and oxidative stress in patients with systemic sclerosis lead to increased release of High-Mobility Group Box-1 which potentially activate skin fibroblast to produce interferon-γ Induced Protein-10: a translational study

Role of HMGB1 in the activation of innate immune responses during SARS-CoV-2 infection

Abstract The ongoing COVID-19 pandemic caused by SARS-CoV-2 has resulted in overwhelming number of deaths worldwide, affecting all age groups. Due to the emergence and rapid spread of SARS-CoV-2 variants and waning immunity in humans to current vaccines, it is imperative that new treatment modalities must continue to be developed for combating this deadly disease. The molecular mechanisms by which SARS-CoV-2 increases risk of serious respiratory disease is largely unknown but exuberant lung inflammatory and immune responses are key. Understanding of the cascades of events that initiate and sustain inflammatory processes contributing to more severe disease is crucial for developing effective therapies for COVID-19. In this study, we found that the release of high-mobility group box 1 (HMGB1), a proinflammatory mediator that alerts the immune system, was increased in the medium of human lung epithelial cells (A549-ACE2 and Calu-3/2B4) in vitro infected with SARS-CoV-2, and in the serum of SARS-CoV-2 infected mice and humans. Our studies showed that SARS-CoV-2 induced acute inflammation by downregulating the retinoic acid inducible gene-I (RIG-I)-dependent interferon (IFN)-mediated innate antiviral immune response and nuclear factor erythroid 2-related factor (NRF2)-dependent antioxidant mechanism. Treatment with rHMGB1 significantly activated the innate antiviral and antioxidant pathways and inhibited the viral replication. Our studies demonstrated an important role of HMGB1 in the regulation of viral replication and cellular protection as well as the activation of innate immune signaling cascades to promote inflammation. Modulation of HMGB1’s proinflammatory signaling may attenuate SARS-CoV-2-induced lung inflammation. This work was supported by COVID-19 and Emerging Respiratory Viruses Research Award from American Lung Association (COVID-920427), COVID-19 Bridging grant from Sealy Institute for Vaccine Sciences (C28455), Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (FAMRI) (grant I.D. number 123385), NIH/NIAID R21 AI35619, Short-Term Provost’s Scholar Award by CTSA Mentored Career Development Award, CTSA KL2 COVID Funds (NIH/NCATS KL2TR001441) to YMH.