HMGB1 Secretion Research Articles

HMGB1 impairs endothelium-dependent relaxation in diabetes through TLR4/eNOS pathway.

Endothelium-dependent relaxation (EDR) is an initial key step leading to various vascular complications in patients with diabetes. However, the underlying mechanism of EDR impairment in diabetes is not fully understood. Present study defined the role of high-mobility group protein (HMGB1) in EDR related to diabetes. Serum level of HMGB1 was increased in diabetic patients and in db/db mice. Serum HMGB1 level was also positively correlated with HbA1c and negatively correlated with nitric oxide (NO) in diabetic patients. Results from wire myograph showed that recombinant HMGB1 (rHMGB1) was capable of impairing EDR of aortas from wild-type (WT) mice by an eNOS-dependent mechanism. Consistently, HMGB1 inhibitor glycyrrhizin acid (GA) decreased the serum level of HMGB1 and rescued EDR impairment partly in db/db mice. Furthermore, rHMGB1 mediated EDR impairment was abolished in aortas of TLR4-/- mice. In addition, high-glucose-induced HMGB1 upregulation and secretion in endothelial cells. In conclusion, HMGB1 contributes to the EDR impairment through TLR4/eNOS pathway in the setting of diabetes. GA as the HMGB1 inhibitor could attenuate EDR impairment in an animal model of diabetes.

Sex-specific stress response and HMGB1 release in pulmonary endothelial cells.

Women are known to be associated with a higher susceptibility to pulmonary arterial hypertension (PAH). In contrast, male PAH patients have a worse survival prognosis. In this study, we investigated whether the contribution of sex goes beyond the effects of sex hormones by comparing the ability of isolated male and female pulmonary endothelial cells to respire, proliferate and tolerate the stress. Mouse lung endothelial cells (MLEC) were isolated from the lungs of male and female 3-week old mice. Male MLEC showed an increased basal mitochondrial respiration rate, elevated maximal respiration, a significantly greater level of mitochondrial polarization, and a higher rate of proliferation. Exposure of cells to hypoxia (2% of O2 for 24 hours) induced a strong apoptotic response in female but not male MLEC. In contrast, treatment with mitochondrial respiratory Complex III inhibitor Antimycin A (AA, 50μM) mediated severe necrosis specifically in male MLEC, while female cells again responded primarily by apoptosis. The same effect with female cells responding to the stress by apoptosis and male cells responding by necrosis was confirmed in starved pulmonary endothelial cells isolated from human donors. Elevated necrosis seen in male cells was associated with a significant release of damage-associated alarmin, HMGB1. No stimuli induced a significant elevation of HMGB1 secretion in females. We conclude that male cells appear to be protected against mild stress conditions, such as hypoxia, possibly due to increased mitochondrial respiration. In contrast, they are more sensitive to impaired mitochondrial function, to which they respond by necrotic death. Necrosis in male vascular cells releases a significant amount of HMGB1 that could contribute to the pro-inflammatory phenotype known to be associated with the male gender.

Imiquimod Exerts Antitumor Effects by Inducing Immunogenic Cell Death and Is Enhanced by the Glycolytic Inhibitor 2-Deoxyglucose

The induction of immunogenic cell death (ICD) in cancer cells triggers specific immune responses against the same cancer cells. Imiquimod (IMQ) is a synthetic ligand of toll-like receptor 7 that exerts antitumor activity by stimulating cell-mediated immunity or by directly inducing apoptosis. Whether IMQ causes tumors to undergo ICD and elicits a specific antitumor immune response is unknown. We demonstrated that IMQ-induced ICD-associated features, including the surface exposure of calreticulin and the secretion of adenosine triphosphate and HMGB1, were mediated by ROS and endoplasmic reticulum stress. In a B16F10 melanoma mouse model, vaccinating mice with IMQ-induced ICD cell lysate or directly injecting IMQ in situ reduced tumor growth that was mediated by inducing tumor-specific T-cell proliferation, promoting tumor-specific cytotoxic killing by CD8+ T cells, and increasing the infiltration of various immune cells into tumor lesions. The ICD-associated features were crucial in the induction of specific antitumor immunity invivo. The glycolytic inhibitor 2-deoxyglucose enhanced IMQ-induced ICD-associated features and strengthened the antitumor immunity mediated by IMQ-induced ICD cell lysate in p53-mutant cancer cells, which were IMQ-resistant invitro. We conclude that IMQ is an authentic ICD inducer and provide a concept connecting IMQ-induced cancer cell death and antitumor immune responses.

STAT3 inhibitory stattic enhances immunogenic cell death induced by chemotherapy in cancer cells.

Induction of immunogenic cell death (ICD) is considered a promising strategy for cancer immunotherapy. Stattic is an inhibitor of STAT3, which is found constitutively active in many cancers and plays a major role in cancer progression. In the present study, we proposed to evaluate whether stattic can enhance the effects of chemotherapy in the induction of ICD in cancer cells harboring hyperactive STAT3. The growth inhibitory effects of stattic and chemo agents including doxorubicin (DOX) and oxaliplatin (OXP) were evaluated using MTT assay in B16F10 and CT26 cell lines. Flow cytometry was applied to study cell apoptosis and calreticulin (CRT) surface exposure. The levels of high mobility group box1 (HGMB1), heat shock protein70 (HSP70) and interleukin-12 (IL-12) were measured using ELISA. Treatment of B16F10 and CT26 cells with stattic in combination with DOX resulted in synergistic antitumor effects with combination index being 0.82 and 0.87, respectively. Interestingly, we found a higher level of ICD markers including CRT expression as well as HMGB1 and HSP70 secretion in the cells received combination therapy of stattic and DOX as compared with monotherapies. Moreover, exposure of dendritic cells (DCs) to conditioned media (CM) from cancer cells treated with stattic and/or DOX resulted in secretion of IL-12, which is an indicator of DCs maturation and induction of Th1 response. OXP and stattic monotherapy induced ICD in CT26 cells and stimulated IL-12 secretion by DCs; however, we did not observe a significant increase in the level of ICD in CT26 cells and IL-12 secretion by DCs when CT26 cells were treated with stattic and OXP combination as compared with monotherapy groups. These findings indicate that STAT3 inhibitory stattic can increase ICD induced by DOX. Graphical abstract.

Adenovirus VA RNAI Blocks ASC Oligomerization and Inhibits NLRP3 Inflammasome Activation.

Virus infected immune cells can rapidly respond to the invader by activating the inflammasome and as a consequence release proinflammatory cytokines and eventually die by pyroptosis. In human adenovirus-5 (Ad5) infected THP-1 cells, inhibition of NLRP3 inflammasome activation was demonstrated by a decreased secretion of HMGB1 and matured forms of caspase-1and IL-1ß. An Ad5 mutant virus defective in expression of the non-coding VA RNAI failed to inhibit the NLRP3 inflammasome and in addition displayed formation of ASC specks and increased cell lysis. Importantly, in vitro synthesized VA RNAI was able to inhibit the NLRP3 inflammasome activity in THP-1 cells in the absence of an Ad5 infection, suggesting that VA RNAI binding to PKR and blocking its function is sufficient for inhibition of the NLRP3 inflammasome. Although the inhibition of NLRP3 inflammasome activation required the phylogenetically conserved base paired tetranucleotide sequence in the central stem of VA RNAI, we demonstrate that PKR binding to VA RNAI primarily protected the apical stem, but not the tetranucleotide sequence itself. VA RNAI did not influence the interaction between PKR and NLRP3. In contrast, we describe a novel interaction between PKR and ASC and further show that VA RNAI inhibited ASC phosphorylation and oligomerization. Collectively, our results indicate a novel role for Ad5 VA RNAI as an inhibitor of NLRP3 inflammasome activation by targeting the cellular pro-inflammatory protein PKR.

ATIM-08. TRIAL IN PROGRESS: CA209-9Y8 PHASE 2 TRIAL OF TUMOR TREATING FIELDS (TTFS), NIVOLUMAB PLUS/MINUS IPILIMUMAB FOR BEVACIZUMAB-NAÏVE, RECURRENT GLIOBLASTOMA

Abstract Recurrent glioblastoma has limited therapeutic options. The EF11 trial of TTFs vs. physician’s choice chemotherapy showed an objective response rate (ORR) of 14%, but no survival (OS) benefit. CheckMate 143 randomized phase 3, open-label trial of nivolumab vs. bevacizumab failed to show an OS benefit, though nivolumab led to ORR of 7.8% with more durable response than bevacizumab (11.1 vs. 5.3 months). Preclinical studies suggest synergism between TTFs and immunotherapy. TTFs expose calreticulin on cell surfaces, causing HMGB1 and ATP secretion—hallmarks of anticancer immunogenic cell death. Observed trends in a glioma cohort also suggest synergism: 77% of glioma patients treated with TTFs received concurrent temozolomide (16, 31%), nitrosourea (4,8%), bevacizumab (19,37%), or immunotherapy (28,54%). Only 2 (4%) experienced skin toxicity requiring TTFs hold. For TTFs plus immunotherapy vs. chemotherapy, there was a trend toward increased OS (8.3 vs. 6.5 months,p=0.32) and ORR (26% vs. 13%), with significant increase in PFS for glioblastomas (4.6 vs. 3.5 months,p=0.04). ORR was >14% in EF11 or < 5- 10% in meta-analyses of immunotherapy for recurrent glioblastoma. This open-label, phase 2 trial with 2 parallel arms uses a 2-stage Minimax design with stopping rules for futility. We will enroll 30 (15/Arm) to 60 (30/Arm) evaluable subjects. The primary endpoint is ORR by modified RANO. Secondary objectives are OS, PFS, safety, compliance, and biomarker correlations. Adults with glioblastoma at first or second recurrence and no prior CTLA inhibitor or bevacizumab are eligible. All receive TTFs with nivolumab for ≤24 months and ipilimumab for ≤4 doses. Arm A (no prior PD1/PDL1 inhibitor) receive nivolumab only and add ipiliumumab at progression. Arm B (prior PD1/PDL1 inhibitor) receive nivolumab and ipilumimab. Nivolumab is dosed IV q2weeks 240mg as monotherapy and 3mg/kg with ipilumimab IV 1mg/kg q6weeks. TTFs starts by 1 month and worn for ≥75% over ≤24 months.

EXTH-61. CELECOXIB IMPROVES OUTCOME OF PATIENTS TREATED WITH TUMOR TREATING FIELDS

Abstract Tumor Treating Fields (TTFields) are known to cause mitotic catastrophe resulting in aberrant mitotic exit. However, there is accumulating evidence that the clinical effectiveness of TTFields requires immune competence. Exposure of cells to TTFields induced signs of cellular stress including cytoskeletal changes, surface expression of calreticulin and secretion of HMGB1 suggesting that TTFields may promote anti-tumor immunity that leads to subsequent tumor regression. Many tumor-protective mechanisms require immunosuppressive cell types within the tumor microenvironment whose development and suppressive functions are supported by prostaglandin E2 (PGE2). The rate limiting enzyme for PGE2 production is Cyclooxygenases-2 (COX-2) whose upregulation has been correlated with poor survival in glioma patients. Therefore, we reasoned that reduction of PGE2 production within the tumor microenvironment might improve response to TTFields therapy. Previous data from our group indicated that dexamethasone use interfered with TTFields-efficacy. When our population having low dexamethasone usage (n=42) was dichotomized according to celecoxib use, those without celecoxib (n = 14) had a median overall survival (OS) of 8.1 months while those receiving celecoxib had a median OS of 12.9 months (chi-square 5.42, p=0.002, HR = 2.1, 95% CI = 1.19–5.59). Patients not receiving celecoxib were treated with TTFields for a median of 3.8 months while those receiving celecoxib were treated for a median of 8.2 months (Chi-square = 6.75, p = 0.01, HR = 2.3, CI = 1.33–6.47). These data suggest that the addition of celecoxib improved the efficacy of TTFields against glioblastoma in this cohort. However, celecoxib did not sensitize TTFields-induced killing of cells in vitro suggesting an indirect mechanism of action. Together with our in vitro data, the results support an immune mechanism of action underlying TTFields’ anti-tumor efficacy. These findings concerning the effects of celecoxib on patient outcomes need to be confirmed in a prospective clinical trial.

Resveratrol induces immunogenic cell death of human and murine ovarian carcinoma cells

PurposeThis study aimed to clarify whether immunogenic cell death (ICD) contributed to the anti-tumor action of resveratrol against ovarian carcinoma.MethodsResveratrol suppressed cell proliferation and induced apoptosis in ovarian carcinoma cells. In addition, resveratrol treatment stimulated cell surface exposure of calreticulin, HMGB1 secretion and ATP release.ResultsVaccination with resveratrol-pretreated ID8 cells significantly inhibited growth of subsequent inoculated xenograft tumor. Direct administration with resveratrol suppressed tumor progression accompanied with compromised cell proliferation and enhanced cell apoptosis. We further characterized increases of both mature dendritic cells and cytotoxic T cells in xenograft tumor in response to resveratrol treatment, which also inhibited TGF-β production and stimulated both IL12p7 and IFN-γ secretion. Most importantly, we demonstrated that combination with PD-1 antibody greatly inhibited tumor growth, while depletion of CD8+ T cells by neutralizing antibody restored xenograft progression.ConclusionOur data suggested resveratrol exerted anti-tumor action against ovarian cancer via both apoptosis and ICD pathways.

Abstract 221: Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro

Abstract Tisotumab vedotin is an antibody-drug conjugate composed of a human IgG targeting tissue factor (TF), a valine citruline linker, and the microtubule disrupting agent monomethyl auristatin E (MMAE). TF is aberrantly expressed in solid tumors and is thought to contribute to tumor progression by enhancing tumor growth, neo-angiogenesis and metastatic potential through local activation of coagulation and protease-activated receptor-2 (PAR-2) signaling. Tisotumab vedotin was previously shown to induce cytotoxicity through MMAE- and Fc-dependent mechanisms. In addition, tisotumab vedotin inhibited PAR-2 signaling in TF-positive tumor cells. Here we aimed to elucidate additional effector mechanisms by assessing the capacity of tisotumab vedotin to induce immunogenic cell death (ICD), bystander cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP) in vitro. ICD was assessed by incubating A431, MDA-MB-231, and HPAFII cells with tisotumab vedotin, and measuring ER stress by western blot, ATP secretion by reporter assay, and HMGB1 release by ELISA. Bystander cytotoxicity was assessed by incubating mixed cultures of TF+ (MDA-MB-231) and TF- (A549) tumor cells with tisotumab vedotin and quantifying viable cells by flow cytometry. To assess ADCP, PKH26-labeled BxPC-3 or A431 cells were incubated with human monocyte derived macrophages in presence of tisotumab vedotin. ADCP activity was analyzed by measuring the fraction of macrophages that double stained with PKH26 and anti-CD11c antibody using flow cytometry. Tisotumab vedotin induced key hallmarks of ICD including induction of ER stress, ATP secretion, and release of HMGB1, which have been shown to facilitate antitumor immunity. In co-cultures of TF+ and TF- tumor cells, tisotumab vedotin induced cytotoxicity in both cell types, even at a target-positive to target-negative cellular ratio of 1:15. This was mediated through bystander cytotoxicity, as demonstrated by the lack of cytotoxicity in monocultures of TF- cells. Finally, ADCP was observed with macrophages derived from multiple donors, with double-positive macrophages comprising up to 50% of total macrophages. In summary, ICD, bystander cytotoxicity, and ADCP were identified as novel effector mechanisms of tisotumab vedotin in vitro. Combining the present study with previous results, tisotumab vedotin induces TF-dependent anti-tumor activity through 1) MMAE-mediated effector mechanisms, including MMAE-mediated direct and bystander cytotoxicity, and induction of ICD; 2) Fc-mediated effector mechanisms, including ADCC and ADCP; and 3) Fab-mediated inhibition of PAR-2 dependent signaling. Tisotumab vedotin is currently being investigated in a variety of solid tumors (NCT03245736, NCT03485209, NCT03438396, NCT03657043). Citation Format: Stephen C. Alley, Jeffrey R. Harris, Anthony Cao, Elke Gresnigt-van den Heuvel, Jyoti Velayudhan, David Satijn, Sandra Verploegen, Teresa Dominguez, Esther C. Breij. Tisotumab vedotin induces anti-tumor activity through MMAE-mediated, Fc-mediated, and Fab-mediated effector functions in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 221.

Autophagy-based unconventional secretion of alarmin HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

Abstract The precise mechanism by which autophagy affects psoriasis is poorly understood. Here we show that keratinocytes (KCs) autophagy was activated in psoriatic lesions of patients and mice model, positively correlating with psoriatic severity, and could be inhibited by MAPK family (ERK, p38, JNK) inactivation, implying autophagy was associated with psoriatic inflammation. Indeed, impaired autophagy flux, caused by autophagy inhibitors or KC-specific deletion of Atg5, alleviated psoriasisform inflammation, demonstrating autophagy positively regulated psoriatic inflammation. We then found an autophagy-based unconventional secretory pathway (autosecretion), depended on Atg5 and GRASP55, promoted the psoriasiform KCs inflammation. Moreover, alarmin HMGB1 was more effective than other autosecretory proteins, IL-1β and IL-18, to regulate the psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs vanished the psoriasiform inflammation differences between autophagy-efficient KCs and autophagy-deficient KCs. Conversely, recombinant HMGB1 almost completely restored the psoriasiform inflammation in autophagy-deficient KCs in vivo. These results suggested that HMGB1-associated autosecretion, not other intracellular autophagy pathways, played a pivotal role in cutaneous inflammation. Finally, we demonstrated that KC-specific HMGB1 deficient mice, but not DC- or myeloid cell-specific HMGB1 deficient mice, displayed attenuated psoriatic inflammation, due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, we uncover a novel autophagy mechanism in psoriasis pathogenesis, and imply its clinical significance in psoriasis treatment.

Cold Atmospheric Plasma Induces HMGB1 Expression in Cancer Cells.

Plasma medicine is a new field that provides great potential for the treatment of human diseases including cancer in addition to sterilizing the surface of skin and facilitating wound healing. Recently, non-thermal atmospheric plasma (or cold atmospheric plasma, CAP) was introduced, not only for denaturing cells and tissues, but also for operating under the threshold of thermal damage and for chemically inducing a specific response or modification. Microwave-mediated CAP was used in this study. CAP increased high-mobility group box 1 protein (HMGB1) expression, thereby increasing HMGB-1 secretion. In addition, we observed that the calreticulin (CRT) protein was concentrated at the cellular membrane when plasma was treated, representing immunogenic cell death. Overall, plasma treatment induces apoptosis via immunogenic cell death in cancer cells, implying a potential application to human cancer therapy and for the treatment of other human diseases.

Antagonism of RIP1 using necrostatin-1 (Nec-1) ameliorated damage and inflammation of HBV X protein (HBx) in human normal hepatocytes

Hepatitis B virus X protein (HBx), encoded by the hepatitis B virus (HBV) genome, plays a pivotal in mediating pathogenicity in liver diseases. However, the underlying mechanisms are still needed to be elucidated. Receptor-interacting protein 1 (RIP1) has been identified as a serine/threonine kinase with various biological functions in cell fate, proliferation, and death. In the current study, we found that overexpression of HBx in LO2 human normal hepatocytes increased the expression of RIP1. Importantly, our results indicate that blockage of RIP1 using its specific antagonist necrostatin-1 (Nec-1) ameliorated HBx-induced oxidative stress by mitigating the production of ROS and Nox-4 expression. Also, the presence of Nec-1 improved HBx-induced mitochondrial dysfunction by increasing MMP. Importantly, we found that Nec-1 could inhibit the production of pro-inflammatory cytokines IL-6, IL-8, and CXCL2 as well as the secretion of HMGB1. Mechanistically, we found that Nec-1 treatment suppressed the activation of the JNK/AP-1 and NF-κB signalling pathways. Our findings implicated a novel biological function of RIP1 in HBx-induced cytotoxicity and inflammation in human normal hepatocytes. Antagonism of RIP1 might be a potential therapeutic approach for the treatment of HBV-associated liver diseases.

Cancer-cell-secreted CXCL11 promoted CD8+ T cells infiltration through docetaxel-induced-release of HMGB1 in NSCLC

BackgroundChemotherapy combined with immunotherapy becomes the main trend in lung cancer intervention; however, how chemotherapy promotes the immune function remains elusive. Therefore, we sought to determine how chemotherapy promotes the immune function.MethodsWe determined in 100 NSCLC patients the expression of CD8, functional markers (IFN-γ, Granzyme B, and Perforin) and specific chemokines by quantitative real-time reverse transcriptase-PCR. Functional experiments were carried out to check whether docetaxel (DOC), a chemotherapeutic agent, modifies the expression of HMGB1 and CXCL11, and influences the infiltration properties of CD8+ T cells to the tumor microenvironment. The mechanism of the release of HMGB1 and CXCL11 was determined by flow cytometry, immunofluorescence and western blotting. In in vivo experiment, we confirmed how DOC enhanced the recruitment of HER2-CAR T cells to tumor sites.ResultsWe found that DOC upregulated the expression of chemokine receptor ligand CXCL11 in tumor microenvironment and subsequently enhanced CD8+ T cell recruitment. DOC treatment significantly increased HMGB1 release in an ROS-dependent manner. Recombinant protein HMGB1 stimulated the secretion of CXCL11 via NF-κB activation in vitro. Tumors from DOC-treated mice exhibited higher expression of HMGB1 and CXCL11, more HER2-CAR T cell infiltration, and reduced progression, relative to control. Increased HMGB1 and CXCL11 expressions were positively correlated with prolonged overall survival of lung cancer patients.ConclusionsOur results demonstrate that DOC induces CD8+ T cell recruitment to the tumor microenvironment by enhancing the secretion of HMGB1 and CXCL11, thus improving the anti-tumor efficacy, indicating that modulating the HMGB1-CXCL11 axis might be helpful for NSCLC treatment.

Mesenchymal stromal cells-derived exosomes alleviate ischemia/reperfusion injury in mouse lung by transporting anti-apoptotic miR-21-5p

MiR-21-5p is an anti-apoptotic miRNA known to mediate the protective effect of mesenchymal stromal cell-secreted exosomes (MSC-Exo) against oxidative stress-induced cell death. In the present research we employed murine lung ischemia/reperfusion (I/R) model and in vitro hypoxia/reoxygenation (H/R) model using primary murine pulmonary endothelial cells to investigate whether MSC-Exo could alleviate lung IRI by transporting miR-21-5p. Our data suggested that intratracheal administration of MSC-Exo or miR-21-5p agomir significantly reduced lung edema and dysfunction, M1 polarization of alveolar macrophages as well as secretion of HMGB1, IL-8, IL-1β, IL-6, IL-17 and TNF-α. Pre-challenge of MSCs by H/R significant increased miR-21-5p expression level in exosomes they secreted and the anti-IRI effect of these MSC-Exo, while pre-treatment of MSCs with miR-21-5p antagomir showed opposite effect. We further demonstrated that MSC-Exo ameliorated IRI in vivo or H/R induced apoptosis in vitro by inhibiting both intrinsic and extrinsic apoptosis pathway via miR-21-5p targeting PTEN and PDCD4, while artificial overexpressing PTEN or PDCD4 significantly attenuated the anti-apoptotic effect of MSC-Exo in vitro. Treatment with miR-21-5p agomir mimicked the IRI-reducing and anti-apoptotic effect of MSC-Exo. Our data suggested that MSC-Exo alleviate IRI in lung in an exosomal miR-21-5p-dependent manner. Treatment with MSC-Exo or miR-21-5p agomir might ameliorate IRI in lung.

Proinflammatory Effects of Respiratory Syncytial Virus-Induced Epithelial HMGB1 on Human Innate Immune Cell Activation.

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein that translocates to the cytoplasm and is subsequently released to the extracellular space during infection and injury. Once released, it acts as a damage-associated molecular pattern and regulates immune and inflammatory responses. Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants and elderly, for which no effective treatment or vaccine is currently available. This study investigated the effects of HMGB1 on cytokine secretion, as well as the involvement of NF-κB and TLR4 pathways in RSV-induced HMGB1 release in human airway epithelial cells (AECs) and its proinflammatory effects on several human primary immune cells. Purified HMGB1 was incubated with AECs (A549 and small alveolar epithelial cells) and various immune cells and measured the release of proinflammatory mediators and the activation of NF-κB and P38 MAPK. HMGB1 treatment significantly increased the phosphorylation of NF-κB and P38 MAPK but did not induce the release of cytokines/chemokines from AECs. However, addition of HMGB1 to immune cells did significantly induce the release of cytokines/chemokines and activated the NF-κB and P38 MAPK pathways. We found that activation of NF-κB accounted for RSV-induced HMGB1 secretion in AECs in a TLR4-dependent manner. These results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis. Therefore, blocking the proinflammatory function of HMGB1 may be an effective approach for developing novel therapeutics.

Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1.

BackgroundThis study aimed at investigating whether NLRP3 (the Nod like receptor family, pyrin domain‐containing 3 protein) inflammasome activation induced HMGB1 (high mobility group box‐1 protein) secretion and foam cell formation in human vascular smooth muscle cells (VSMCs) and atherosclerosis in ApoE−/− mice.Methods and Results VSMCs or ApoE−/− mice were treated with lipopolysaccharides (LPS) and/or ATP or LPS and high‐fat diet to induce NLRP3 inflammasome activation. HMGB1 distribution and foam cell formation in VSMCs were characterized. Liver X receptor α and ATP‐binding cassette transporter expression were determined. The impact of NLRP3 or receptor for advanced glycation end product silencing, ZYVAD‐FMK (caspase‐1 inhibitor), glycyrrhizin (HMGB1 inhibitor) or receptor for advanced glycation end product antagonist peptide on HMGB1 secretion, foam cell formation, liver X receptor α and ATP‐binding cassette transporter expression was examined. Expression level of HMGB1 in human atherosclerosis obliterans arterial tissues was characterized. Our results found that NLRP3 inflammasome activation promoted foam cell formation and HMGB1 secretion in VSMCs. Extracellular HMGB1 was a key signal molecule in inflammasome activation‐mediated foam cell formation. Furthermore, inflammasome activation‐induced HMGB1 activity and foam cell formation were achieved by receptor for advanced glycation end product/liver X receptor α /ATP‐binding cassette transporter glycyrrhizin. Experiments in vivo found glycyrrhizin significantly attenuated the LPS/high‐fat diet‐induced atherosclerosis and serum HMGB1 levels in mice. Finally, levels of HMGB1 and NLRP3 were increased in tunica media adjacent to intima of atherosclerosis obliteran arteries.ConclusionsOur results revealed that HMGB1 is a key downstream signal molecule of NLRP3 inflammasome activation and plays an important role in VSMCs foam cell formation and atherogenesis by downregulating liver X receptor α and ATP‐binding cassette transporter expression through receptor for advanced glycation end product.

ALA-PDT promotes HPV-positive cervical cancer cells apoptosis and DCs maturation via miR-34a regulated HMGB1 exosomes secretion.

5-Aminolevulinic acid photodynamic therapy(ALA-PDT) has been widely used for cervical cancer treatment, but the mechanisms are still not fully delineated. Here we showed that ALA-PDT significantly upregulated HMGB1 while downregulated miR-34a expression levels in cervical cancer tissues, and the percentages of mature DCs(mDCs) were increased in ALA-PDT treated patients' peripheral blood. After treating HPV-positive Hela, SiHa, Caski and HPV-negative C33 A cervical cancer cell lines with ALA-PDT, HPV-positive cells' proliferative ability was significantly inhibited and apoptosis rates were elevated, while no significant changes were found in HPV-negative C33 A cell line. Most importantly, in HPV-positive cells, we found that miR-34a were downregulated in cytoplasm, and both cytoplasm and exosome HMGB1 were significantly elevated comparing to cancer cells without ALA-PDT treatment, and it could be reversed by miR-34a mimic transfection, which indicated that HPV infection and miR-34a downregulation might be vital for ALA-PDT treatment. Based on the HMGB1 is the potential target of miR-34a and an inverse correlation between miR-34a and HMGB1 in ALA-PDT treated cancer tissues, we verified that HMGB1 could be targeted and downregulated by miR-34a mimic, and ALA-PDT promotes HMGB1 secretion by inhibiting miR-34a expression. By co-culturing cervical cancer cell lines with immature DCs(imDCs) in the Transwell systems, we found that ALA-PDT induced HMGB1 exosomes could promote DCs maturation, which could be reversed by silencing HMGB1 in HPV-positive cervical cancer cells. In vivo animal experiments also proved that ALA-PDT inhibited tumor growth in tumor bearing mice, which was reversed by co-transfected with miR-34a mimic or silencing HMGB1 in HPV-positive cells. Hence we concluded that ALA-PDT treatment specifically inhibited HPV-positive cervical cancer cells' proliferative ability, promoted cell apoptosis and modulated DCs maturation by regulating miR-34a mediated HMGB1 exosomes secretion.

Epidermal HMGB1 Activates Dermal Fibroblasts and Causes Hypertrophic Scar Formation in Reduced Hydration.

HMGB1 protein is a multifunctional cytokine involved in inflammatory reactions and is known to play a key role in tissue repair and fibrosis. However, the function of HMGB1 in fibrotic skin diseases, such as hypertrophic scar formation, remains unclear. In this study, HMGB1 was detected in the nuclei of epidermal cells in normal skin and had accumulated in the cytoplasm in hypertrophic scars. By establishing a keratinocyte-fibroblast co-culture and conditional medium treatment models, we found that a reduced hydration condition increased the expression and secretion of HMGB1 in keratinocytes, subsequently activating dermal fibroblasts. HMGB1 secreted from keratinocytes activated fibroblasts by promoting the nuclear import of MRTF-A, increased the nuclear accumulation of MRTF-A/SRF complexes and consequently enhanced α-smooth muscle actin promoter activation. Moreover, blockade of advanced glycation end products or Toll-like receptor 2/4 inhibited the fibroblast activation induced by HMGB1. Finally, local delivery of HMGB1 resulted in marked hypertrophic scar formation in rabbit hypertrophic scar models, while HMGB1 blockade exerted a clear anti-scarring effect. Our results indicate that high HMGB1 levels induced by a reduced hydration status play an important role in hypertrophic scar formation, strongly suggesting that HMGB1 is a novel target for preventing scarring.

PKR and GCN2 stress kinases promote an ER stress-independent eIF2α phosphorylation responsible for calreticulin exposure in melanoma cells

ABSTRACTThe immunogenic cell death (ICD) process represents a novel therapeutic approach to treat tumours, in which cytotoxic compounds promote both cancer cell death and the emission of damage-associated molecular patterns (DAMPs) from dying cells, to activate the immune system against the malignancy. Therefore, we explored the possibility to stimulate the key molecular players with a pivotal role in the execution of the ICD program in melanoma cells. To this aim, we used the pro-ICD agents mitoxantrone and doxorubicin and found that both agents could induce cell death and stimulate the release/exposure of the strictly required DAMPs in melanoma cells: i) calreticulin (CRT) exposure on the cell membrane; ii) ATP secretion; iii) type I IFNs gene up-regulation and iv) HMGB1 secretion, highlighting no interference by oncogenic BRAF. Importantly, although the ER stress-related PERK activation has been linked to CRT externalization, through the phosphorylation of eIF2α, we found that this stress pathway together with PERK were not involved in melanoma cells. Notably, we identified PKR and GCN2 as key mediators of eIF2α phosphorylation, facilitating the translocation of CTR on melanoma cells surface, under pro-ICD drugs stimulation. Therefore, our data indicate that pro-ICD drugs are able to stimulate the production/release of DAMPs in melanoma cells at least in vitro, indicating in this approach a potential new valuable therapeutic strategy to treat human skin melanoma malignancy.

Antioxidant treatment ameliorates cigarette smoke and respiratory syncytial virus-induced inflammatory response by blocking HMGB1 release

Abstract Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants, and causes severe respiratory morbidity and mortality in the elderly. Second hand tobacco smoke is the major environmental risk factor for the development and severity of lower respiratory tract infections, however, the molecular mechanism of tobacco smoke-induced RSV severity is not fully understood. High mobility group box 1 protein (HMGB1) has been shown to functions in the regulation of cellular transcription and the activation of proinflammatory responses in many pathogenic conditions, but little is known about its molecular mechanism of action during its release in respiratory viral infections and tobacco smoke-induced lung inflammation. In this study we investigated the mechanism of HMGB1 release and associated cellular signaling in RSV and cigarette smoke (CS)-induced airway inflammation. A549 cells, a human lung carcinoma cell line and normal small alveolar epithelial (SAE) cells were pretreated with antioxidants and exposed to CS followed by RSV infection. CS exposure in combination with RSV infection synergistically enhanced HMGB1 secretion with concomitant decrease in the total cell lysates when compared to uninfected as well as each stimulus alone. NFκB and MAPK were significantly activated in CS exposed and RSV-infected cells. Treatment with antioxidant significantly inhibited RSV and CS-induced HMGB1 secretion, cytokine release, and NFκB and MAPK activation. The results of these studies will help to design new therapeutic strategies not only for RSV infection, but also for other respiratory diseases that have been shown to be caused or exacerbated by environmental toxicants such as tobacco smoke.