Endogenous High Mobility Group Box 1 Research Articles

Knockdown of HMGB1 inhibits the crosstalk between oral squamous cell carcinoma cells and tumor-associated macrophages.

Tumor-associated macrophages (TAMs), the major component of the tumor microenvironment (TME), play distinctly different roles in different tumors. High mobility group box 1 (HMGB1), a nonhistone protein in the nucleus, can perform functions during inflammation and cancers. However, the role of HMGB1 in the crosstalk between oral squamous cell carcinoma (OSCC) cells and TAMs remains unclear. Here, we established a coculture system of TAMs and OSCC cells to explore the bidirectional effect and potential mechanism of HMGB1 in OSCC cell-TAM interactions. Our results showed that HMGB1 was significantly upregulated in OSCC tissues and positively associated with tumor progression, immune cell infiltration and macrophage polarization. Then, knocking down HMGB1 in OSCC cells inhibited the recruitment and polarization of cocultured TAMs. Moreover, the knockdown of HMGB1 in macrophages not only suppressed polarization, but also inhibited cocultured OSCC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, macrophages secreted higher levels of HMGB1 than OSCC cells, and dampening endogenous HMGB1 reduced HMGB1 secretion. Both OSCC cell-generated and macrophage-endogenous HMGB1 may regulate TAM polarization by promoting receptor TLR4 expression and NF-κB/p65 activation and enhancing IL-10/TGF-β expression. HMGB1 in OSCC cells may regulate macrophage recruitment via IL-6/STAT3. In addition, TAM-derived HMGB1 may affect aggressive phenotypes of cocultured OSCC cells by regulating the immunosuppressive microenvironment through the IL-6/STAT3/PD-L1 and IL-6/NF-κB/MMP-9 pathways. In conclusion, HMGB1 may regulate the crosstalk between OSCC cells and TAMs, including modulating macrophage polarization and attraction, enhancing cytokine secretion, and remodeling and creating an immunosuppressive TME to further affect OSCC progression.

Selective TLR4 Antagonism Prevents and Reverses Morphine-Induced Persistent Postoperative Cognitive Dysfunction, Dysregulation of Synaptic Elements, and Impaired BDNF Signaling in Aged Male Rats.

Perioperative neurocognitive disorders (PNDs) are characterized by confusion, difficulty with executive function, and episodic memory impairment in the hours to months following a surgical procedure. Postoperative cognitive dysfunction (POCD) represents such impairments that last beyond 30 d postsurgery and is associated with increased risk of comorbidities, progression to dementia, and higher mortality. While it is clear that neuroinflammation plays a key role in PND development, what factors underlie shorter self-resolving versus persistent PNDs remains unclear. We have previously shown that postoperative morphine treatment extends POCD from 4 d (without morphine) to at least 8 weeks (with morphine) in aged male rats, and that this effect is likely dependent on the proinflammatory capabilities of morphine via activation of toll-like receptor 4 (TLR4). Here, we extend these findings to show that TLR4 blockade, using the selective TLR4 antagonist lipopolysaccharide from the bacterium Rhodobacter sphaeroides (LPS-RS Ultrapure), ameliorates morphine-induced POCD in aged male rats. Using either a single central preoperative treatment or a 1 week postoperative central treatment regimen, we demonstrate that TLR4 antagonism (1) prevents and reverses the long-term memory impairment associated with surgery and morphine treatment, (2) ameliorates morphine-induced dysregulation of the postsynaptic proteins postsynaptic density 95 and synaptopodin, (3) mitigates reductions in mature BDNF, and (4) prevents decreased activation of the BDNF receptor TrkB (tropomyosin-related kinase B), all at 4 weeks postsurgery. We also reveal that LPS-RS Ultrapure likely exerts its beneficial effects by preventing endogenous danger signal HMGB1 (high-mobility group box 1) from activating TLR4, rather than by blocking continuous activation by morphine or its metabolites. These findings suggest TLR4 as a promising therapeutic target to prevent or treat PNDs.SIGNIFICANCE STATEMENT With humans living longer than ever, it is crucial that we identify mechanisms that contribute to aging-related vulnerability to cognitive impairment. Here, we show that the innate immune receptor toll-like receptor 4 (TLR4) is a key mediator of cognitive dysfunction in aged rodents following surgery and postoperative morphine treatment. Inhibition of TLR4 both prevented and reversed surgery plus morphine-associated memory impairment, dysregulation of synaptic elements, and reduced BDNF signaling. Together, these findings implicate TLR4 in the development of postoperative cognitive dysfunction, providing mechanistic insight and novel therapeutic targets for the treatment of cognitive impairments following immune challenges such as surgery in older individuals.

The association between mediators of the receptor for advanced glycation end product (RAGE) axis and immune checkpoint inhibitor (ICI)–induced colitis in patients with melanoma.

9587 Background: Colitis and other gastrointestinal (GI) toxicity are a frequent and occasionally severe form of immune-related adverse events (irAEs) in patients treated with ICIs. To date, no definitive mechanism has been identified, and this area remains an active field of investigation. We hypothesized that activation of the RAGE axis, known to be implicated in inflammatory bowel disease through stimulation of signal transduction targeted by pro-inflammatory RAGE ligands, members of the S100 family and High Mobility Group Box 1 (HMGB1), might be associated with irAE- colitis. Methods: We examined sera from 111 advanced melanoma patients prospectively accrued and followed up at NYULH (treated with anti-PD-L1, n = 44; antiCTL4, n = 23; and combination, n = 44). 24 (22%) developed GI toxicity grade >2. Serum biomarkers of the ligand-RAGE pathways, soluble (s)RAGE, endogenous secretory (es)RAGE, S100B, and HMGB1, were measured in the patients’ sera during ICI treatment. Multivariable ordinal logistic regression analyses with all grades of GI toxicity as the primary outcome for all the recorded covariates (including serum biomarkers, clinical covariates) were performed. We then used ordinal multivariable logistic regression with stepwise variable selection. Similar analyses with GI toxicity as a binary outcome ((≥grade 1 vs no toxicity) were also conducted. Only those variables that jointly contributed to the odds of developing toxicity were included in the final stepwise model. No adjustments for multiplicity were included. As sRAGE and esRAGE are highly correlated (r = 0.86), esRAGE concentrations were not used in the joint models. Results: A significant association between GI toxicity and concentrations of sRAGE and S100B was identified. The final stepwise multivariable logistic model includes only sRAGE and S100B. The odds of having a one level increase in GI toxicity grade increase 1.100 times (95% CI: 1.008, 1.199; p = 0.029) for each unit decrease of sRAGE ( = sRAGE/100). The odds of a one level increase in GI toxicity increase 1.059 times (95% CI: 1.004, 1.116; p = 0.035) for each unit increase of S100B ( = s100B/100). All other analyses yielded comparable results. In contrast, concentrations of HMGB1 and other clinical covariates, including response and treatment category, were not associated with GI toxicity. Conclusions: Mediators of the RAGE axis, specifically sRAGE and S100B, might have a role in GI toxicity in patients receiving ICIs. The ligand-RAGE axis may be a novel target for irAE therapies for patients receiving ICIs to mitigate the severity of GI toxicity.

Increased Toll-like Receptor-MyD88-NFκB-Proinflammatory neuroimmune signaling in the orbitofrontal cortex of humans with alcohol use disorder.

BackgroundMany brain disorders, including alcohol use disorder (AUD), are associated with induction of multiple proinflammatory genes. One aspect of proinflammatory signaling is progressive increases in expression across cells and induction of other innate immune genes. High‐mobility group box 1 (HMGB1) heteromers contribute to amplification by potentiating multiple proinflammatory responses, including Toll‐like receptors (TLRs). TLR signaling recruits coupling proteins linked to nuclear transcription factors that induce proinflammatory cytokines and chemokines and their respective receptors. We tested the hypothesis that AUD induction of TLR expression increases levels of proinflammatory genes and cellular signaling cascades in association with neurodegeneration in the orbitofrontal cortex (OFC).MethodsPostmortem human OFC tissue samples (n = 10) from males diagnosed with AUD were compared to age‐matched moderate drinking controls (CON). Neuroimmune signaling molecules were assessed using immunohistochemistry for protein and reverse transcription polymerase chain reaction for messenger RNA (mRNA).ResultsIn the AUD OFC, we report induction of the endogenous TLR agonist HMGB1 as well as all TLRs assessed (i.e., TLR2‐TLR9) except TLR1. This was accompanied by increased expression of the TLR adaptor protein myeloid differentiation primary response 88 (MyD88), activation of the proinflammatory nuclear transcription factor nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB), and downstream induction of proinflammatory cytokines, chemokines, and their corresponding receptors. Several of these proinflammatory signaling markers are expressed in glia and neurons. The induction of HMGB1‐TLR‐MyD88‐NFκB proinflammatory signaling pathways correlates with neurodegeneration (i.e., Fluoro‐Jade B), lifetime alcohol consumption, and age of drinking onset.ConclusionThese data implicate the induction of HMGB1‐TLR‐MyD88‐NFκB cascades through coordinated glial and neuronal signaling as contributors to the neurodegeneration seen in the postmortem human OFC of individuals with AUD.

Conditional Knock out of High-Mobility Group Box 1 (HMGB1) in Rods Reduces Autophagy Activation after Retinal Detachment.

After retinal detachment (RD), the induction of autophagy protects photoreceptors (PR) from apoptotic cell death. The cytoplasmic high-mobility group box 1 (HMGB1) promotes autophagy. We previously demonstrated that the deletion of HMGB1 from rod PRs results in a more rapid death of these cells after RD. In this work, we tested the hypothesis that the lack of HMGB1 accelerates PR death after RD due to the reduced activation of protective autophagy in the retina after RD. The injection of 1% hyaluronic acid into the subretinal space was used to create acute RD in mice with a rhodopsin-Cre-mediated conditional knockout (cKO) of HMGB1 in rods (HMGB1Δrod) and littermate controls. RD sharply increased the number of apoptotic cells in the outer nuclear layer (ONL), and this number was further increased in HMGB1Δrod mouse retinas. The activation of autophagy after RD was reduced in the HMGB1Δrod mouse retinas compared to controls, as evidenced by diminished levels of autophagy regulatory proteins LC3-II, Beclin1, ATG5/12, and phospho-ATG16L1. The cKO of HMGB1 in rods increased the expression of Fas and the Bax/Bcl-2 ratio in detached retinas, promoting apoptotic cell death. In conclusion, endogenous HMGB1 facilitates autophagy activation in PR cells following RD to promote PR cell survival and reduce programmed apoptotic cell death.

Intrinsic Activation of the NLRP3 Inflammasome in Primary Human ALL Cells Is Regulated By Endogenous HMGB1 and Bone Marrow Macrophages

Inflammasomes are cytosolic signaling complexes that assemble in response to damage-associated molecular patterns (DAMPs). Intrinsic expression of the NLRP3 has been identified in acute lymphoblastic leukemia (ALL) cell lines and implicated in glucocorticoid resistance (Paugh W et al., 2015). The endogenous function of NLRP3 in ALL remains unknown. In this study, we use primary human ALL cells to demonstrate that NLRP3 is activated in primary ALL cells in response to chemotherapy stress, is regulated by the endogenously expressed “danger protein” HMGB1 and can be silenced through direct contact with bone marrow macrophages.To determine whether chemotherapy treatment of primary ALL cells induces NLRP3 expression, we treated CD19-selected cells from n=3 pediatric patients with ALL and lymphocytes from healthy donors with either 2nM doxorubicin or vehicle and compared NLRP3 expression by qPCR and Western blot at 24 hours. NLRP3 mRNA expression was increased by 4.5 + 0.8- fold over baseline in doxorubicin-treated ALL cells compared to no change in vehicle-treated ALL and undetectable levels in healthy lymphocytes. This corresponded to a 2-fold increase in NLRP3 protein expression was measured by Western Blot. Neither doxorubicin-treated nor vehicle-treated ALL expressed other NOD-family inflammasome proteins, including NLRP1 and NLRC4.In healthy monocytes, NLRP3 activates caspase-1 to induce transcription of inflammatory cytokines. To determine whether NLRP3 up-regulation has a similar function in primary ALL cells, we measured pro- and cleaved-caspase-1 in doxorubicin-treated primary ALL, vehicle-treated ALL and healthy lymphocytes. Primary ALL cells treated with doxorubicin expressed the transcriptionally active p20 subunit of caspase-1 by Western blot; vehicle-treated ALL cells and healthy lymphocytes did not. This corresponded to a 3.7 + 0.61-fold increase in global caspase-1 transcription by mRNA in doxorubicin-treated ALL cells and a significant increase in soluble IL1B production by ELISA (p<0.02). Treatment of primary ALL with staurosporin (an autophagy-inducer) and serum starvation up-regulated NLRP3 but not caspase 1/p20. Taken together, these data established proof-of-principle that primary ALL cells activate the NLRP3 inflammasome in response to clinically relevant cellular stress.The activating signal for NLRP3 assembly is unknown in leukemia. We have previously observed that DNA-binding DAMP high mobility group-box 1 (HMGB1) is endogenously expressed in ALL cells. To determine whether HMGB1 activates NLRP in leukemia, we knocked down HMGB1 by siRNA in primary ALL cells (n=3 patients) and measured NLRP3 and caspase-1/p20 at baseline and after treatment with doxorubicin vs. vehicle. HMGB1 siRNA ALL cells displayed at least a 2-fold decrease in NLRP3 protein expression 48 hours after knockdown compared to control siRNA cells that could not be rescued with doxorubicin treatment. Co-immunoprecipitation studies indicate that HMGB1 forms a binding complex in primary ALL cells.To determine whether NLRP3 expression in primary ALL is impacted by cells in the nonmalignant field, we co-cultured primary ALL cells with bone marrow-derived monocyte/macrophage, either directly or separated by 0.6 uM transwell. Unexpectedly, NLRP3 expression was completely abrogated in primary ALL cells when they were placed in direct, but not transwell, co-culture with macrophages. Direct co-culture of ALL with peripheral monocytes, however, did not abrogate NLRP3. These data indicate that direct interactions of ALL with macrophages in the nonmalignant bone marrow microenvironment have a negative regulatory effect on NLRP3 inflammasome activation.Taken together these data show, for the first time in primary pediatric ALL cells, that NLRP3 inflammasome is activated in response to chemotherapy cell stress that is regulated by endogenous HMGB1 expression and direct interactions with bone marrow macrophages. Studies are underway to define these interactions and their physiologic consequences for ALL cell survival in tne marrow niche. DisclosuresNo relevant conflicts of interest to declare.

HMGB1 enhances chemotherapy resistance in multiple myeloma cells by activating the nuclear factor-κB pathway.

Chemotherapy resistance is a main obstacle in the clinical chemotherapeutic treatment of multiple myeloma (MM). High-mobility group box 1 (HMGB1) has been revealed to be associated with the sensitivity of MM cells to chemotherapy, but how HMGB1 regulates chemotherapy resistance in MM has yet to be fully elucidated. In the present study, the exact molecular mechanism underlying HMGB1-mediated drug resistance in MM was explored using three chemotherapy-resistant MM cells (RPMI8226/ADR, RPMI8226/BOR and RPMI8226/DEX) that were successfully established. Reverse transcription-quantitative polymerase chain reaction revealed that the three chemotherapy-resistant MM cells exhibited a higher release of HMGB1 compared with the parental RPMI8226 cells. Interference with endogenous HMGB1 increased the sensitivity of drug-resistant MM cells to chemotherapy, which was supported by the low IC50 value and the enlargement of cell apoptosis. Furthermore, short hairpin (sh)RNA-transfected MM cells showed an obvious elevation in phosphorylated (p)-IKKα/β, p-IκBα and p-p65 in whole cell lysate and/or nucleus, and treatment of nuclear factor (NF)-κB activator reversed the effect of shHMGB1-mediated cell viability and apoptosis in MM cells. In conclusion, HMGB1 regulates drug resistance in MM cells by regulating NF-κB signaling pathway, suggesting that HMGB1 has the potential to serve as a target for MM treatment.

Role of high-mobility group box 1 and its modulation by thrombomodulin/thrombin axis in neuropathic and inflammatory pain.

High-mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, facilitates pain signals as well as inflammation. Intraplantar or intraspinal application of HMGB1 elicits hyperalgesia/allodynia in rodents by activating the advanced glycosylation end-product specific receptor (receptor for advanced glycation end-products; RAGE) or Toll-like receptor 4 (TLR4). Endogenous HMGB1 derived from neurons, perineuronal cells or immune cells accumulating in the dorsal root ganglion or sensory nerves participates in somatic and visceral pain consisting of neuropathic and/or inflammatory components. Endothelial thrombomodulin (TM) and recombinant human soluble TM, TMα, markedly increase thrombin-dependent degradation of HMGB1, and systemic administration of TMα prevents and reverses various HMGB1-dependent pathological pain. Low MW compounds that directly inactivate HMGB1 or antagonize HMGB1-targeted receptors would be useful to reduce various forms of intractable pain. Thus, HMGB1 and its receptors are considered to serve as promising targets in developing novel agents to prevent or treat pathological pain. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.4/issuetoc.

High mobility group box-1 protects against Aflatoxin G1-induced pulmonary epithelial cell damage in the lung inflammatory environment

Aflatoxin G1 (AFG1) is a member of the carcinogenic aflatoxin family. Our previous studies indicated that oral administration of AFG1 caused tumor necrosis factor (TNF)-α-dependent inflammation that enhanced oxidative DNA damage in alveolar epithelial cells, which may be related to AFG1-induced lung carcinogenesis. High mobility group box-1 (HMGB1) is a nuclear DNA-binding protein; the intracellular and extracellular roles of HMGB1 have been shown to contribute to DNA repair and sterile inflammation. The role of HMGB1 in DNA damage in an aflatoxin-induced lung inflammatory environment was investigated in this study. Upregulation of HMGB1, TLR2, and RAGE was observed in AFG1-induced lung inflamed tissues and adenocarcinoma. Blocking AFG1-induced inflammation by neutralization of TNF-α inhibited the upregulation of HMGB1 in mouse lung tissues, suggesting that AFG1-induced TNF-α-dependent inflammation regulated HMGB1 expression. In the in vitro human pulmonary epithelial cell line model, Beas-2b, AFG1 directly enhanced the cytosolic translocation of HMGB1 and its extracellular secretion. The addition of extracellular soluble HMGB1 protected AFG1-induced DNA damage through the TLR2/NF-κB pathway in Beas-2b cells. In addition, blockade of endogenous HMGB1 by siRNA significantly enhanced AFG1-induced damage. Thus, our findings showed that both extracellularly-released and nuclear and cytosolic HMGB1 could protect the cell from AFG1-induced cell damage in a TNF-α-dependent lung inflammatory environment.

The nuclear sirtuin SIRT6 protects mice from polymicrobial sepsis by suppressing the host immune response

IntroductionSepsis is the leading cause of death in intensive care unit and refers to the host’s deleterious and non‐resolving systemic inflammatory response to infection. Despite advances in medical care and antibiotic therapy, the overall mortality rate of sepsis in United States is 28.3% highlighting the inadequacies of current therapies. Sirtuins are key regulators of inflammation in both immune and non‐immune cells. SIRT6 is known to exert an anti‐inflammatory action that promotes hypo‐inflammation and endotoxin tolerance in immune cells. The pathogenesis of sepsis is also mediated by an alarmin molecule, the high mobility group box 1 (HMGB1). HMGB1 neutralizing antibodies were found to have therapeutic potential in the treatment of sepsis. Hence, by using the rat cardiomyocytes and SIRT6 transgenic (SIRT6.Tg) mice, we tested the hypothesis that SIRT6 plays a role in HMGB1 secretion, further hypothesized that SIRT6.Tg mice are protected from bacteria‐induced sepsis in mice.MethodsInteractions between SIRT6 and HMGB1 were determined by immunoprecipitation followed by immunoblotting. The presence of HMGB1 in the serum was detected using HMGB1 ELISA kit. Sepsis was induced in wild type and SIRT6.Tg mice by intra‐peritonea injection of a four‐pathogen community (MDR S. marcescens, C. albicans, MDR K. oxytoca, and E. faecalis) that was isolated from an ICU patient suffering from poly‐microbial sepsis and previously characterized. Immune response of mice was determined by performing FACS analysis on the splenocytes obtained from infected mice.ResultsTo test whether SIRT6 can bind to HMGB1 in cardiomyocytes, we infected rat cardiomyocytes with adenovirus expressing SIRT6 or empty adenovirus vector for 24 h. Cell lysates were immunoprecipitated with an SIRT6‐antibody, and subjected to immunoblotting with the use of an anti‐HMGB1 antibody. We found that HMGB1 co‐precipitated with SIRT6 in cardiomyocytes. In order to confirm that SIRT6 binds to HMGB1 endogenously, we performed a reverse immunoprecipitation experiment. Endogenous HMGB1 was immunoprecipitated from rat cardiomyocytes and subjected to immunoblotting with the anti‐SIRT6 antibody. Our results confirmed that HMGB1 binds to SIRT6 endogenously. In order to understand the physiological significance of the interaction between SIRT6 and HMGB1, we tested the HMGB1 levels in 2 year old wild‐type and SIRT6.Tg mice. We found that SIRT6.Tg mice have 5 fold reduced levels of HMGB1 in the serum, suggesting that SIRT6 expression interferes with the secretion of HMGB1. Next we investigated whether SIRT6 is protective against polymicrobial sepsis by injecting a lethal dose of the pathogen community into wild type and SIRT6.Tg mice. SIRT6.Tg mice showed an increased survival of 25%, compared to wild type mice. We also compared the immune response of wild‐type with SIRT6.Tg mice. Both CD4 and CD8 T lymphocytes of SIRT6.Tg mice showed reduced interferon gamma and interleukin 17 response, compared to wild‐type mice, suggesting that SIRT6 can regulate the sepsis induced immune response.ConclusionsSIRT6 can regulate HMGB1 secretion and protect mice from polymicrobial sepsis by suppressing the host immune response.Support or Funding InformationNIH RO1 HL136712; NIH RO1 HL143488

Glycyrrhizin attenuates hepatic ischemia-reperfusion injury by suppressing HMGB1-dependent GSDMD-mediated kupffer cells pyroptosis

Gasdermin D (GSDMD), a genetic substrate for inflammatory caspases, plays a central role in pyroptosis of macrophages and release of interleukin‑1β (IL-1β), but was mainly referred to microbial infection. High mobility group box-1 (HMGB1), served as an alarm molecule during various pathological process, has been widely recognized to be involved in liver ischemia-reperfusion (I/R). Glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, was recently referred to have ability to prevent I/R induced liver injury by inhibiting HMGB1 expression and activity. However, the mechanisms responsible for damage amelioration subsequently to HMGB1 inhibition during liver I/R remain enigmatic. This study was designed to explore the functional role and molecular mechanism of glycyrrhizin in the regulation of I/R induced liver injury. We found that liver I/R promotes GSDMD-mediated pyroptotic cell death of Kupffer cells, which was inhibited by glycyrrhizin. Interestingly, endogenous HMGB1, not exogenous one, was involved in hypoxia-reoxygenation (H/R) induced pyroptosis. Moreover, GSDMD knockdown protects kupffer cells against H/R induced pyroptosis in vitro. Here, we report, for the first time, that glycyrrhizin attenuated tissue damage and kupffer cells pyroptosis during liver ischemia-reperfusion injury (LIRI) and identify a previously unrecognized HMGB1- dependent GSDMD- mediated signaling pathway in the mechanism of kupffer cells pyroptosis induced by H/R. Our findings provide the first demonstration of GSDMD-determined pyroptotic cell death responsible for I/R induced release of IL-1β and this would provide a mandate to better understand the unconventional mechanisms of cytokine release in the sterile innate immune system.

HMGB1-induced autophagy facilitates hepatic stellate cells activation: a new pathway in liver fibrosis

High-mobility group box-1 (HMGB1) plays a context-dependent role in autophagy, which is required for hepatic stellate cells (HSCs) activation. However, the significance of HMGB1-induced HSCs autophagy in liver fibrosis has not been elucidated. Here, we first documented an enrichment of peripheral and intrahepatic HMGB1 signal in hepatitis B virus (HBV)-related liver fibrosis progression, and presented a direct evidence of anatomic proximity of HMGB1 with a-SMA (a marker for HSCs activation) in cirrhotic liver specimens. Then, we demonstrated the autophagy-inducing effects by serum-sourced HMGB1 in both primary murine HSCs and human HSCs cell line (LX-2), reflected by increased number of autophagic vacuoles (AVs) under the transmission electron microscope (TEM) and up-regulated protein expression of lipidated microtubule-associated light chain 3 (LC3-II) (a marker for autophagosome) in Western blot analysis. Intriguingly, there is a possible translocation of endogenous HMGB1 from the nucleus to cytoplasm to extracellular space, during exogenous HMGB1-induced HSCs autophagy. Meanwhile, the dose- and time-dependent effects by recombinant HMGB1 (rHMGB1) in enhancing LX-2 autophagy and fibrogenesis have been revealed with activated extracellular regulated protein kinase (ERK)/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) and restrained mammalian target of rapamycin (mTOR)/STAT3 signaling pathways. Additionally, the ERK or JNK inhibitor could not only inhibit rHMGB1-induced autophagy and fibrogenesis in LX-2 cells, but also restore the suppressed mTOR and STAT3 pathways. Furthermore, using LC3-siRNA transfected LX-2, we found HMGB1-induced fibrogenesis is dependent on its autophagy-inducing effects. Finally, we elucidated the involvement of extracellular HMGB1-receptor for advenced glycation end product (RAGE) axis and endogenous HMGB1 in exogenous HMGB1-induced effects. Our findings could open new perspectives in developing an antifibrotic therapy by targetting the HSCs autophagy.

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

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

Quantification of Serum High Mobility Group Box 1 by Liquid Chromatography/High-Resolution Mass Spectrometry: Implications for Its Role in Immunity, Inflammation, and Cancer.

High mobility group box 1 (HMGB1) is a non-histone chromosomal protein, which can be secreted through a variety of pathways and bind to pattern recognition receptors to release pro-inflammatory cytokines. Previous studies have suggested that HMGB1 is upregulated in numerous inflammatory diseases and that it could be a biomarker for such diseases. However, these studies used immunoassay-based methods to analyze serum HMGB1. Autoantibodies to HMGB1 in serum are found in healthy control subjects as well as in patients with different diseases. HMGB1 also binds to haptoglobin, a highly abundant plasma protein. This means that antibodies used in immunoassays must compete with binding of HMGB1 to endogenous serum HMGB1 autoantibodies and haptoglobin. To overcome these potential problems, we developed and validated a specific and sensitive assay based on stable isotope dilution and immunopurification to quantify HMGB1 in plasma and serum using two-dimensional nano-ultra-high-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry. Using this assay, we found that serum HMGB1 in 24 healthy control subjects (6.0 ± 2.1 ng/mL) was above the mean concentration reported for 18 different diseases (5.4 ± 2.8 ng/mL) where the analyses were conducted with immunoassay methodology. In light of our finding, the role of HMGB1 in these diseases will have to be re-evaluated. The concentration of HMGB1 in citrated and EDTA-treated plasma from the same healthy control subjects was below the limit of detection of our assay (1 ng/mL), confirming that HMGB1 in serum arises when blood is allowed to clot. This means that future studies on the role of HMGB1 in vivo should be conducted on plasma rather than serum.

Human soluble thrombomodulin-induced blockade of peripheral HMGB1-dependent allodynia in mice requires both the lectin-like and EGF-like domains

Thrombomodulin (TM), an endothelial protein with anti-coagulant activity, is composed of 5 domains, D1-D5. Recombinant human soluble TM (TMα) consisting of D1-D3, which is generated in CHO cells, suppresses inflammatory and nociceptive signals by inactivating high mobility group box 1 (HMGB1), one of damage-associated molecular patterns. TMα sequesters HMGB1 with the lectin-like D1 and promotes its degradation by thrombin binding to the EGF-like D2. We prepared TM's D123, D1 and D2 by the protein expression system of yeast, and evaluated their effects on HMGB1 degradation in vitro and on the allodynia caused by HMGB1 in distinct redox forms in mice in vivo. TMα and TM's D123, but not D1, promoted the thrombin-dependent degradation of all-thiol (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), an effect mimicked by TM's D2, though to a lesser extent. Intraplantar administration of TMα and TM's D123, but not D1, D2 or D1 plus D2, strongly prevented the mechanical allodynia caused by intraplantar at-HMGB1, ds-HMGB1 or lipopolysaccharide in mice. Our data suggest that, apart from the role of D3, TMα and TM's D123 require both lectin-like D1 capable of sequestering HMGB1 and EGF-like D2 responsible for thrombin-dependent degradation of HMGB1, in abolishing the allodynia caused by exogenous or endogenous HMGB1.

Toll-like Receptor 2: A Novel Therapeutic Target for Ischemic White Matter Injury and Oligodendrocyte Death.

Despite paramount clinical significance of white matter stroke, there is a paucity of researches on the pathomechanism of ischemic white matter damage and accompanying oligodendrocyte (OL) death. Therefore, a large gap exists between clinical needs and laboratory researches in this disease entity. Recent works have started to elucidate cellular and molecular basis of white matter injury under ischemic stress. In this paper, we briefly introduce white matter stroke from a clinical point of view and review pathophysiology of ischemic white matter injury characterized by OL death and demyelination. We present a series of evidence that Toll-like receptor 2 (TLR2), one of the membranous pattern recognition receptors, plays a cell-autonomous protective role in ischemic OL death and ensuing demyelination. Moreover, we also discuss our recent findings that its endogenous ligand, high-mobility group box 1 (HMGB1), is released from dying OLs and exerts autocrine trophic effects on OLs and myelin sheath under ischemic condition. We propose that modulation of TLR2 and its endogenous ligand HMGB1 can be a novel therapeutic target for ischemic white matter disease.

Specific Monoclonal Antibodies Recognizing the Endogenous Chicken High Mobility Group Box 1 Protein.

High mobility group box 1 (HMGB1) is a key member of the "danger associated molecular patterns" (DAMPs), which can localize in various compartments of the cell, and plays important roles in systemic inflammation. In the present study, monoclonal antibodies (MAbs) specifically against chicken HMGB1 were generated. The open reading frame of chicken HMGB1 was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-chHMGB1. The recombinant chicken HMGB1 protein was expressed in Escherichia coli Rosetta under IPTG induction and then purified by Ni-NTA Purification System. BALB/c mice were immunized with the purified recombinant HMGB1 protein, and three strains of hybridoma cells named 1F10, 8C11, and 4D8 secreting MAbs of chicken HMGB1 were obtained by hybridoma technique. Western blot and indirect immunofluorescence assays showed that the endogenous HMGB1 in various cell lines and glycosylated HMGB1 could both be specifically recognized by the prepared MAbs. This work indicated that the MAbs against chicken HMGB1 would be a valuable tool for further studies of HMGB1-mediated signaling in virus-infected cells and investigates the role of HMGB1 in avian virus pathogenesis.

HMGB1 Mediates Autophagy Dysfunction via Perturbing Beclin1-Vps34 Complex in Dopaminergic Cell Model.

Parkinson’s disease (PD), a progressive neurodegenerative disorder, is characterized by irreversible dopaminergic neuron loss and intra-neuronal α-synuclein aggregation. High mobility group box 1 (HMGB1) has been proven to be involved in autophagy dysfunction induced by α-synuclein accumulation, and the Beclin1-vacuolar protein sorting 34 (Vps34) complex is of great importance to the initiation of autophagy. Nevertheless, the concrete interaction mechanism between HMGB1, α-synuclein and autophagy remains elusive, especially in the context of PD. Here in this study, we investigated the interaction between HMGB1 and α-synuclein in rotenone-induced PD cell models and their roles in autophagy flux. Results revealed elevated expression and cytosolic translocation of endogenous HMGB1 upon rotenone exposure. Besides, HMGB1 was found to be able to co-localize and interact with α-synuclein. Moreover, it had also been proven that HMGB1 could aggravate α-synuclein aggregation induced autophagy dysfunction via perturbing Beclin1-Vps34 complex formation. Based on these findings, we propose that HMGB1 is involved in rotenone-induced dopaminergic cell death via interacting with α-synuclein, perturbing the autophagy process, aggravating protein aggregation and finally propelling dopaminergic neurons to move from morbidity to mortality.

HMGB1 modulation in pancreatic islets using a cell-permeable A-box fragment

Although pancreatic islet implantation is an attractive strategy for curing diabetes mellitus, implanted cells are immunologically eliminated due to early islet graft loss. One of main issues in early islet graft loss is the secretion of high-mobility group-box-1 (HMGB1) protein from the damaged islet cells, which is known as a cytokine-like factor. Therefore, regulating the activity of HMGB1 protein offers an alternative strategy for improving outcomes of islet cell therapy. To this end, we first demonstrated that HMGB1 protein could be bound to its A-box fragment (HMGB1 A-box) with higher binding affinity, resembling anti-HMGB1 antibody. To be used as a pharmaceutical protein ex vivo, TAT-labeled HMGB1 A-box-His6 (TAT-HMGB1A) was structurally modified for cellular membrane penetration. TAT-HMGB1A significantly reduced secretion of endogenous HMGB1 protein through interaction in the cytosol without any damage to the viability or functionality of the islets. When TAT-HMGB1A-treated islets were implanted into diabetic nude mice, they completely cured diabetes, as evidenced by stable blood glucose level. TAT-HMGB1A treatment could also reduce the marginal islet mass needed to cure diabetes. Furthermore, TAT-HMGB1A positively protected xenotransplanted islets from xenogeneic immune reactions. Collectively, cell-penetrable TAT-HMGB1A could be used to modulate HMGB1 activity to increase successful outcomes of ex vivo pancreatic islet cell therapy.

Inhibiting the cytoplasmic location of HMGB1 reverses cisplatin resistance in human cervical cancer cells.

Cervical cancer is the fourth most common malignancy in women worldwide, and resistance to chemotherapy drugs is the biggest obstacle in the treatment of cervical cancers. In the present study, the molecular mechanisms underlying cisplatin resistance in human cervical cancer cells were investigated. When human cervical cancer cells were treated with 10µg/ml of cisplatin for 24 and 48h, high mobility group box1 (HMGB1) protein expression levels significantly increased in a time‑dependent manner. Comparisons between cisplatin‑sensitive HeLa cells and cisplatin‑resistant HeLa/DDP cells revealed higher levels of HMGB1 in HeLa/DDP cells than in HeLa cells. Additionally, the half maximal inhibitory concentration (IC50) value for cisplatin in HeLa/DDP cells was 5.3‑fold that in HeLa cells. Analysis of the distribution of cellular components revealed that HMGB1 translocation from the nucleus to cytoplasm contributed to cisplatin resistance. This was further confirmed by demonstration that ethyl pyruvate treatment suppressed the cytoplasmic translocation of HMGB1, resulting in inhibition of HeLa cell proliferation. Furthermore, endogenous HMGB1 was inhibited with HMGB1‑specific short hairpin (sh)RNA, and MTT assay results showed that interference with HMGB1 expression reduced cell viability and potentially reversed cisplatin resistance in HeLa cells. Transfection with HMGB1 shRNA was demonstrated to induce cell apoptosis in HeLa cells, as detected by FACS analysis. In addition, administration of recombinant HMGB1 protein in HeLa cells promoted cell autophagy, mediated by the phosphorylation of extracellular signal‑regulated kinase1/2. Thus, cytoplasmic HMGB1 translocation and HMGB1‑induced cell autophagy are proposed to contribute to cisplatin resistance by inhibiting apoptosis of cervical cancer cells. HMGB1 could, therefore, represent a novel therapeutic target for, and a diagnostic marker of, chemotherapy resistant cervical cancers.