Presence Of High Mobility Group Box 1 Research Articles

Inappropriate activation of invariant natural killer T cells and antigen-presenting cells with the elevation of HMGB1 in preterm births without acute chorioamnionitis.

Acute chorioamnionitis (aCAM) associated with microbial infection is a primary cause of preterm birth (PB). However, recent studies have demonstrated that innate immunity and sterile inflammation are causes of PB in the absence of aCAM. Therefore, we analyzed immune cells in the decidua of early to moderate PB without aCAM. Deciduas were obtained from patients with PB at a gestational age of 24+0 to 33+6 weeks without aCAM in pathological diagnosis. The patients were divided into two groups as follows: patients with labor and/or rupture of membrane (ROM) (no aCAM with labor and/or ROM: nCAM-w-LR), and patients without labor and/or ROM (no aCAM without labor and/or ROM: nCAM-w/o-LR). The immune cells and high mobility group box 1 (HMGB1) levels in the decidua were analyzed using flow cytometry. Co-culture of CD56+ cells with dendritic cells (DCs) and macrophages obtained from the decidua was also performed in the presence of HMGB1. The nCAM-w-LR group demonstrated an accumulation of iNKT cells, and increased expression of HMGB1, TLR4, receptors for advanced glycation end products, and CD1d on DCs and macrophages. HMGB1 facilitated the proliferation of iNKT cells co-cultured with DCs and macrophages, which was found to be inhibited by heparin. Inappropriate activation of innate immune cells and increased HMGB1 expression may represent parturition signs in human pregnancy. Therefore, control of these cells and HMGB1 antigenicity may be represent a potential therapeutic target for the prevention of PB.

Investigation of HMGB1 binding interactions with cell clearance molecules

High Mobility Group Box 1 (HMGB1) is a highly abundant DNA‐binding protein. HMGB1 was initially classified as an architectural nuclear protein that facilitates transcription factor binding, chromatin remodeling as well as genetic recombination and repair. It is now clear that HMGB1 also functions in the extracellular matrix (ECM) as a proinflammatory cytokine. More specifically, HMGB1 acts as a damage‐associated molecular pattern molecule. In this capacity, HMGB1 binds immune receptors (i.e. CXR4,RAGE and TLRs) to promote leukocyte migration and amplify proinflammatory gene expression. Ideally, HMGB1 would operate in the ECM to expunge infectious pathogens. However, this does not seem to always be the case. Studies show that the presence of HMGB1 in the ECM is also linked with diseases associated with defective cell clearance, such as lupus (SLE) and cystic fibrosis (CF). The investigation of HMGB1 in cell clearance is an understudied area of research. We hypothesize that HMGB1 exacerbates SLE and CF by binding to cell clearance molecules in the ECM to prevent the proper removal of apoptotic cells. In SLE, failed cell clearance can lead to breaking immune tolerance. In CF, chronic tissue damage is mediated by neutrophils that are not effectively engulfed by macrophages. We will evaluate HMGB1 binding interactions with four categories of cell clearance molecules: i)“eat‐me” signals, ii)“find‐me” signals, iii) phagocyte receptors and iv)adaptor molecules and opsonins. We will use circular dichroism (CD), far‐western blots and surface plasmon resonance to investigate HMGB1 binding interactions with each class of cell clearance biomolecule.Support or Funding InformationUniversity of San Diego

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

Increased dermal expression of chromatin-associated protein HMGB1 and concomitant T-cell expression of the DNA RAGE in patients with psoriasis vulgaris.

PurposePsoriasis vulgaris (PV) is an autoimmune-related chronic inflammatory disease of the skin, with both vascular and metabolic effects. Aggravating factors have been identified that initiate and maintain inflammation, including expression of Th1-, Th17-, and Th22-cell derived cytokines. Recently, we showed that the evolutionarily ancient and highly conserved damage-associated molecular pattern molecule “high mobility group box 1 (HMGB1)” is significantly increased in the serum of PV patients with disease progression and is decreased under standard therapies.Materials and methodsTo better understand the role of HMGB1 in the pathogenesis of PV, we recruited 22 untreated psoriatic patients with either mild or severe disease, defined by the Psoriasis Area Severity Index. We assessed HMGB1 and receptor for advanced glycation end products (RAGE) expression in the skin by immunohistochemistry and analyzed the immune-phenotype of Treg and Th17 cells by flow cytometry.ResultsWe found increased staining for HMGB1 in the dermis of psoriatic plaques in comparison to uninvolved skin of patients with PV. In addition, the major histocompatibility complex class III-encoded DNA and HMGB1 RAGE, induced by HMGB1, were highly expressed on psoriatic CD8+ T cells and CD4+ Treg. High expression of HMGB1 in the lesional skin was associated with even higher expression of its receptor, RAGE, on the cell surface of keratino-cytes in patients with severe PV.ConclusionThe presence of HMGB1 and RAGE signaling may impact orchestration of chronic inflammation in PV which might have implications for Treg and Th17 cells.

The Immunosuppressive Function of Myeloid-derived Suppressor Cells Is Regulated by the HMGB1-TLR4 Axis

Myeloid-derived suppressor cells (MDSCs) accumulate under pathological conditions, including cancer and chronic inflammation, and they suppress various immune responses such as T cell proliferation. Although several inflammatory signals enhance the differentiation and/or function of MDSCs, it is not clear which factors regulate their differentiation and immunosuppressive function. It has been highlighted that damage-associated molecular patterns (DAMPs) play important roles in the induction of inflammation. One of the DAMPs, the high mobility group box 1 (HMGB1), is released from necrotic cells and secreted by macrophages. It has been shown that HMGB1 level is elevated in tumors and tumor-bearing hosts. It has also been reported that HMGB1 transduces intracellular signaling via several receptors, including the receptor for advanced glycation end-products (RAGE) and the toll-like receptor (TLR)4, both of which enhance the differentiation and/or function of MDSCs. However, the effects of HMGB1 on MDSCs remain unclear. In the present study, we examined the effect of HMGB1 on in vitro MDSC differentiation and immunosuppressive functions. Since murine bone marrow (BM) cells can differentiate into MDSCs upon granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation for 4 d in vitro, we cultured murine BM cells in the presence of HMGB1 and GM-CSF. The results demonstrated that HMGB1 enhanced the suppressive activity of in vitro MDSCs, depending on TLR4, whereas lipopolysaccharide (LPS), one of the TLR4 ligands, interfered with this differentiation and immunosuppressive activity of in vitro MDSCs, depending on TLR4. Our findings thus suggest that the HMGB1-TLR4 axis enhances the immunosuppressive function of MDSCs.

Glycyrrhizin ameliorates experimental colitis through attenuating interleukin-17-producing T cell responses via regulating antigen-presenting cells.

Glycyrrhizin, a component of Chinese medicine licorice root, has the ability to inhibit the functions of high-mobility group box 1 (HMGB1). While glycyrrhizin is known to have anti-inflammatory activities, the underlying mechanisms by which glycyrrhizin inhibits inflammation during the development of trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis are not well understood. This study systemically examined the regulatory effects of glycyrrhizin on inflammatory response in TNBS-induced murine colitis and explored the potential mechanisms involved in this process. We reported that glycyrrhizin treatment ameliorated colitis and decreased the production of inflammatory mediators HMGB1, IFN-γ, IL-6, TNF-α, and IL-17. In addition, glycyrrhizin regulated responses of dendritic cells (DCs) and macrophages during the development of colitis. Furthermore, administration of glycyrrhizin suppressed the proliferation of Th17 cells in colitis. Moreover, the ability of DCs and macrophages to induce the differentiation of Th17 cells was enhanced in presence of HMGB1, which was inhibited by glycyrrhizin. These results demonstrated that glycyrrhizin alleviated colitis by inhibiting the promotive effect of HMGB1 on DC/macrophage-mediated Th17 proliferation. In conclusion, HMGB1 plays an important role in the development of colitis. As an inhibitor of HMGB1, glycyrrhizin might be a novel therapy for colitis.

High-mobility group box 1 protein, angiotensins, ACE2, and target organ damage.

High-mobility group box 1 (HMGB1) protein is an important chromatin protein. In the nucleus, HMGB1 interacts with nucleosomes, transcription factors, and histones. The nuclear protein organizes the DNA and regulates transcription [1]. After binding to DNA, HMGB1 bends DNA, thereby facilitating the binding of other proteins. HMGB1 supports transcription of many genes in interactions with numerous transcription factors. HMGB1 also interacts with nucleosomes to loosen packed DNA and remodel the chromatin. Contact with core histones changes the structure of nucleosomes. The presence of HMGB1 in the nucleus depends on posttranslational modifications. When the protein is not acetylated, it remains within the nucleus; however, hyper acetylation on lysine residues causes HMGB1 to translocate into the cytosol [2]. Here, HMGB1 assumes quite different roles. Immune cells can secrete the protein. For instance, dendritic cells, macrophages, and monocytes secrete HMGB1 in response to cytokines to mediate inflammation. The mechanism of inflammation and damage is HMGB1 binding to receptor for advanced glycation end products (RAGE), Toll-like receptor 2 (TLR2), and TLR4 to mediate nuclear factor kappaB (NF-κB) activation [3]. This state-of-affairs positions HMGB1 at the intersection of sterile and infectious inflammatory responses [4, 5]. The multifaceted activities of HMGB1 (Fig. 1) have been reviewed (5). Evidence has been presented that angiotensinconverting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) can reduce secretion of HMGB1 [6, 7]. This observation would be consistent with possible interactions between HMGB1 and the renin-angiotensin system. The angiotensin-converting enzyme (ACE) is a matrix metalloproteinase cleaving decapeptide angiotensin (Ang) I to the octapeptide Ang II. ACE, and its orthologs possess 2 peptidase M2 domains. ACE2, discovered more recently but probably a teleological older enzyme, and its orthologs possess a single peptidase M2 domain. Ang I and II can be cleaved by ACE2 into nonapeptides (Ang1–9) and septapeptides (Ang1–7), respectively. The ACE2 protein also serves as a receptor for corona viruses. The ACE metalloproteinases have been around on the planet for a long time. Similar sequences are found in flies and even in bacteria, while renin first enters the scene at the bony-fish level [8]. The generation of Ang1–7 by ACE2 is said to have a vascular protective function. Nevertheless, ACE2 targeting in mice resulted in mixed messages. There have been at least three lines of ACE2 knockout mice reported by various investigators [9]. Interestingly, there are significant differences in the phenotypes of these distinct lines, especially with regard to their cardiovascular physiology. ACE2 appears to have only modest effects on baseline cardiovascular functions and blood pressure control. These effects can be substantially modulated by genetic and, perhaps through environmental factors. In contrast, the activity of ACE2 may have more profound effects on susceptibility to pathological states, such as hypertension and cardiac hypertrophy. In this issue of J Mol Med, Qi et al. [10] studied mice harboring a FLAG-tagged mouse ACE2 gene * Friedrich C. Luft luft@charite.de

HMGB1 Binds to Lipoteichoic Acid and Enhances TNF-a and IL-6 Production through HMGB1-Mediated Transfer of Lipoteichoic Acid to CD14 and TLR2

Lipoteichoic acid (LTA) is a component of the cell wall of Gram-positive bacteria and induces a toll-like receptor 2 (TLR2)-mediated inflammatory response upon initial binding to lipopolysaccharide-binding protein (LBP) and subsequent transfer to CD14. In this study, we identified a novel role for the nuclear protein high-mobility group box 1 (HMGB1) in LTA-mediated inflammation. Results of ELISA, surface plasmon resonance and native PAGE electrophoretic mobility shift analyses indicated that HMGB1 binds to LTA in a concentration-dependent manner and that this binding is inhibited by LBP. Native PAGE, fluorescence-based transfer and confocal imaging analyses indicated that HMGB1 catalytically disaggregates LTA and transfers LTA to CD14. NF-κB p65 nuclear transmigration, degradation of IκBa and reporter assay results demonstrated that NF-κB activity in HEK293-hTLR2/6 cells is significantly upregulated by a mixture of LTA and soluble CD14 in the presence of HMGB1. Furthermore, the production of TNF-a and IL-6 in J774A.1 and RAW264.7 cells increased significantly following treatment with a mixture of LTA and HMGB1 compared with treatment with LTA or HMGB1 alone. Thus, we propose that HMGB1 plays an important role in LTA-mediated inflammation by binding to and transferring LTA to CD14, which is subsequently transferred to TLR2 to induce an inflammatory response.

All-Thiol HMGB1 Is a Critical Inducer of Anemia in Sepsis Survivors through CXCR4 Signaling

Introduction: Each year, at least 500,000 patients will survive acute sepsis. Anemia is extremely common in sepsis survivors, further interfering with quality of life and activities of daily living. The pathogenesis of anemia of sepsis is unclear. High mobility group box 1 (HMGB1) is a necessary and sufficient inductor of persistent inflammation in sepsis survivors. After translocation form the nucleus to the cytoplasm, HMGB1 undergoes epigenetic changes in response to environmental queues. Redox state is the best well understood inducer of those modifications. In experimental sepsis, the reduced all-thiol form is the predominant form throughout the first four weeks of sepsis. We recently found that HMGB1 is crucial for the development and maintenance of anemia in sepsis in part by inducing a block in differentiation of late erythropoiesis. Here we present evidence indicating a critical role of the all-thiol HMGB1 in anemia of sepsis survivors through a CXCR4-dependent mechanism.Methods and results: In order to investigate the kinetics of anemia of sepsis survivors, we first induced severe polymicrobial abdominal sepsis by surgical cecal ligation and puncture (CLP) in BALB/c mice. Sham-operated mice were used as controls. Within three days after CLP, hematocrit decreased significantly in CLP survivors three days after CLP (p≤0.001), and hemoglobin (Hb) decreased significantly within 5-7 days. Initially normocytic and normochromic, mean corpuscular hemoglobin (MCH) level was reduced significantly 15 days after CLP, while MCV dropped significantly 20 days after CLP. Sepsis-induced anemia persisted for at least 30 days. Administration of an anti-HMGB1 neutralizing monoclonal antibody (nmAb) reversed anemia. Daily administration of recombinant HMGB1 for one week was sufficient to significantly reduce Hb(p≤0.05), and MCH (p≤0.01).To better understand the role of HMGB1 during erythropoiesis, we isolated CD34+ cells from cord blood and derived them in a methylcellulose-based culture, as well as in three phase liquid culture systems. In methylcellulose, all-thiol HMGB1 (the inflammatory form secreted during necrosis) significantly reduced BFU-E formation (P≤0.0001) in a concentration-dependent way, while sulphonyl-HMGB1 (the fully oxidized form released during apoptosis) had no effect. Moreover, all-thiol HMGB1 disrupted the architecture of BFU-E colonies. In liquid culture, HMGB1 significantly reduced CD34+ cell proliferation (Fig. 1A), and reduced the proportion of GPA+ cells by flow cytometry. In the liquid culture system, all-thiol HMGB1 reduced the proportion of GPA+, CD71+ cells by D16. Recently, CXCR4 was recognized as the receptor needed for all-thiol HMGB1 signaling, after forming a heterodimer with CXCL12. In the three-phase culture system, all-thiol HMGB1 significantly reduced the proportion of GPA+, CD71+ cells, and a similar level of inhibition was found when interfering with an anti-CXCR4 nmAb, CXCR4 by adding CXCL14 (a competitive inhibitor of CXCL12), or a small molecule (AMD3100) (Fig. 1B). In the presence of HMGB1, adding CXCL12 failed to rescue erythropoiesis.Conclusions: Our findings suggest that all-thiol HMGB1, the redox form present during the first weeks after sepsis onset, has a critical role in initiating and sustaining anemia in sepsis survivors. Further, all-thiol HMGB1 interferes with late erythropoiesis through a CXCR4-dependent mechanism. This has potential therapeutic implications for a persistent disorder for which the usual support –including erythropoietin and transfusions- fails to improve outcome, survival, or both. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extracellular HMGB1 promotes the migration of cord Blood CD34⁺ cells via SDF-1/CXCR-4 axis

This study was aimed to investigate the effect of high mobility group box1(HMGB1) and/or stromal cell derived factor-1(SDF-1) on the migration of cord blood CD34⁺ cells, and to explore whether HMGB1 promotes cord blood CD34⁺ cell migration via SDF-1/CXCR4 axis. Cord blood mononuclear cells were isolated by Ficoll-Paque density centrifugation, CD34⁺ cells were collected by a positive immunoselection procedure (CD34 MicroBeads) according to the manufacturer's instructions, the purity of the isolated CD34⁺ cells was detected by flow cytometry. In vitro chemotaxis assays were performed using Transwell cell chambers to detect cells migration. 1 × 10⁵ cells/well cord blood CD34⁺ cells were added into the upper chambers. Different concentrations of HMGB1 and/or SDF-1 (0, 10, 25, 50, 100, 200 ng/ml) were used to detect the optimal concentrations of HMGB1 and/or SDF-1 for inducing migration of cord blood CD34⁺ cells. Freshly isolated cord blood CD34⁺ cells express CXCR4 (SDF-1 receptor), and HMGB1 receptor TLR-2,TLR-4 and RAGE. To explore which receptors were required for the synergy of HGMB1 and/or SDF-1 on cells migration, the anti-SDF-1, anti-CXCR4 and anti-RAGE antibodies were used to detect the effect of HGMB1 alone or with SDF-1 on cord blood CD34⁺ cells migration. The results showed that the purity of CD34⁺ cells isolated from cord blood mononuclear cells by magnetic cell sorting was 97.40 ± 1.26%, the 25 ng/ml SDF-1 did not induce migration of cord blood CD34⁺ cells, whereas the optimal migration was observed at 100 ng/ml. HMGB1 alone did not induce migration up to 100 ng/ml. The dose test found that the the best synergistic concentrations for cells migration were 100 ng/ml HMGB1 combined with 50 ng/ml SDF-1. The blocking test showed that both the anti-SDF-1 (4 µg/ml) and anti-CXCR4 (5 µg/ml) antibodies could block cell migration induced by HMGB1 or combined with SDF-1, but the cord blood CD34⁺ cells in the presence of anti-RAGE, anti-TLR-2 and anti-TLR-4 antibodies did not modify the response to SDF-1 in the presence of HMGB1. It is concluded that both HMGB1 and SDF-1 can induce cord blood CD34⁺ cells migration, HMGB1 enhances SDF-1-induced migration exclusively via CXCR4 and in a RAGE and TLR receptors-independent manner, the exact mechanism needs to be further explored.

High mobility group box 1 (HMGB1) protein in human uterine fluid and its relevance in implantation

Does a differential abundance of high mobility group box 1 (HMGB1) protein in uterine fluid (UF) have a functional significance? In rats, an excess of HMGB1 in UF during the receptive phase is detrimental to pregnancy. The identification of constituents of the human uterine secretome has been a subject of renewed interest, due to the advent of high throughput proteomic technologies. Proteomic-based investigations of human UF have revealed the presence of several proteins such as mucins, host defense proteins S100, heat shock protein 27 and haptoglobin, etc. The present study reports on the presence of HMGB1, a nuclear protein, in human UF. Activated macrophages/monocytes, natural killer cells, mature dendritic cells, pituicytes and erythroleukemic cells are also known to secrete HMGB1. Existing data suggest that extracellular HMGB1 plays a role in inflammation. The human part of this study was cross-sectional in design. UF and endometrial tissues were collected from regularly cycling women in the early secretory (i.e. pre-receptive phase, Day 2 post-ovulation, n = 7) or secretory phase (i.e. receptive phase, Day 6 post-ovulation, n = 7) of their menstrual cycles. Samples were also collected from cycling rats in the proestrous (n = 8) or metestrous (n = 8) phase of their estrous cycles. Uteri were also collected from HMGB1-treated pregnant (n = 7) and untreated pseudo-pregnant (n = 7) rats and from pregnant rats at Day 3-5 post-coitum (p.c.) (n = 18, 3 each for six-time points). In each group of human samples, four samples were used for isobaric tag for relative and absolute quantification (iTRAQ) analysis and three samples were used for immunoblotting experiments to determine the abundance of HMGB1 in pre-receptive and receptive phase UF samples. HMGB1 levels in rat UF and endometrial tissue samples were estimated by ELISA and immunohistochemical studies, respectively. The expression of inflammation-associated molecules, such as nuclear factor kappa B (NFκB), receptor for advanced glycation end products (RAGEs), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was analyzed by immunohistochemistry in HMGB1-treated and pseudo-pregnant rats. HMGB1 was identified as one of the differentially abundant proteins in the list generated by 8-plex iTRAQ analysis of receptive and pre-receptive phase UF samples. In both humans and rats, secreted and cellular levels of HMGB1 showed a similar pattern, i.e. significantly (P < 0.05) lower abundance in the receptive phase compared with that in the pre-receptive phase. A significant (P < 0.05) decline was also observed in the endometrial expression of HMGB1 on the day of implantation in pregnant rats. Exogenous administration of recombinant HMGB1, on Day 3 p.c., led to pregnancy failure, whereas administration of recombinant leukemia inhibitory factor or saline had no effect on pregnant rats. Further investigations revealed morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκB and significantly (P < 0.05) higher expression levels of endometrial RAGE, TNF-α and IL-6 in HMGB1-treated rats, compared with untreated pseudo-pregnant rats. The mechanisms, contributing to a decline in the cellular and extracellular levels of HMGB1 during the receptive phase, remain to be ascertained. An excess of HMGB1 in the UF may be associated with infertility in women.

HMGB1 Is a Key Modulator Of Stress Erythropoiesis During Sepsis

▪Severe sepsis is a leading cause of death and disability. Anemia in sepsis survivors affects close to 100% of patients after the third day of in-hospital stay, regardless of blood levels on admission. Circulating levels of Erythropoietin (Epo) are low; paradoxically, administration of recombinant Epo is ineffective, and related to increased morbidity. During sepsis, bone Marrow is hypoproliferative. While transfusions can improve outcome in the short term, its use increases the risk of infection and mortality without any sustained beneficial effect. The pathogenesis of anemia during sepsis is unclear. High mobility group box 1 (HMGB1), a cytokine that is a critical mediator of sepsis, is released into circulation a few days after sepsis onset, remaining increased for 8 weeks after severe sepsis. HMGB1 levels are increased for at least 8 weeks in murine models of sepsis survival.To induce severe sepsis, cecal ligation and puncture (CLP) was performed in BALB/c mice. Three days after CLP, mice developed persistent anemia, represented by a significant reduction in hematocrit (Sham=49.8±3.2 vs. CLP=29.7±6.7%; p≤0.001), hemoglobin (16.7±1.2 vs. 9.9±2.4mg/dL; p≤0.001), and red blood cells mass (10.2±0.7 vs. 5.4±1.7 x106/µL; p≤0.001). Anemia persisted for at least 25 days after CLP. In CLP survivors, reticulocyte counts were erratic, and insufficient to the degree and duration of anemia (8.2±0.8 vs. 6.6±2.1%; p=ns). Analysis of terminal erythroid differentiation using CD44 and Ter119 or CD44 and FSC as markers demonstrated a significant decrease in all erythroid progenitors, from proerythroblast to orthochromatic erythroblast. Concomitantly, mice surviving CLP developed splenomegaly. Splenic architecture was disrupted after CLP, with expansion of the red pulp, characteristic of stress erythropoiesis. Analysis of terminal erythroid differentiation demonstrated an increase in the quantity of erythroid progenitors.An anti-HMGB1 mAb (2G7) was administered after CLP. Strikingly, 2G7-treated septic mice were significantly protected from developing anemia, and had levels of hemoglobin and hematocrit similar to sham-operated mice. These results highlight a critical role for HMGB1 as key modulator of stress erythropoiesis in a murine model of sepsis survivors.To get further insight into the function of HMGB1 and translate our findings to the pathophysiology of human erythropoiesis, we used CD34+ cells derived from cord blood.Cord blood-derived CD34+ cells were incubated in MethoCult in the presence or not of HMGB1. HMGB1 induced a dose dependent decrease in CFU-E.In murine sepsis, there is a stepwise elevation of different redox forms of HMGB1, with an early increase in all-thiol (inflammatory), followed by a partially oxidized before a fully oxidized (with no known inflammatory activity) appears. At day 7, all-thiol HMGB1 reduced significantly the number of CFU-E, while the fully oxidized had no significant effect. At day 14, the number of BFU-E was reduced in the presence of HMGB1, and further decreased with all-thiol HMGB1.In conclusion, our findings suggest that CLP is a reproducible model to study anemia of sepsis. In mice surviving sepsis, stress erythropoiesis is consistently found. Administration of anti-HMGB1 monoclonal antibody reverses anemia of murine sepsis, demonstrating that HMGB1 can be a potential target in the anemia of sepsis survivors. Translating the findings to the human system, we found that HMGB1 impairs differentiation of CD34+ cells towards the BFU-E and CFU-E stages in colony formation assays, implying that HMGB1 might play a role early during differentiation. The redox status of HMGB1 is critical for its biological function, since its effects are not retrieved when HMGB1 is fully oxidized. Disclosures:No relevant conflicts of interest to declare.

Effects of high‐mobility group box 1 on the proliferation and odontoblastic differentiation of human dental pulp cells

To investigate the expression of high-mobility group box 1 (HMGB1) in human dental pulp tissues and the effects of HMGB1 on proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs). Immunohistochemical assay, immunofluorescence staining and flow cytometric analysis were used to detect the expression of HMGB1 in the human dental pulp and hDPCs, respectively. The proliferation of hDPCs was examined by CCK-8 after culturing human primary hDPCs in the presence of HMGB1 with different doses. Odontoblastic differentiation of hDPCs was determined using alkaline phosphatase (ALPase) activity assay and mineralized nodule formation. Important mineralization-related genes such as ALP, dental sialophosphoprotein (DSPP) and dental matrix protein-1 (DMP-1) were determined by real-time polymerase chain reaction. Western blot analysis was performed to determine the difference in expressions of DMP-1 and DSP with or without the presence of exogenous HMGB1. Simultaneously, messenger RNA and protein levels of HMGB1 and RAGE were also detected. The protein level of HMGB1 in the supernatants was quantified using ELISA analysis. HMGB1 was found in human dental pulp tissue and in the nuclei of hDPCs. During hDPC odontoblastic differentiation, HMGB1 translocated from the nuclei to the cytoplasm and then secreted out from hDPCs. Exogenous HMGB1 promoted hDPC proliferation and mineralized nodule formation. It up-regulated the activity of ALPase and the mRNA and protein levels of dentine matrix protein-1 (DMP-1), alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP) and receptor for advance glycation end (RAGE) of hDPCs. HMGB1 promoted the proliferation and odontoblastic differentiation of hDPCs.

Fecal HMGB1 Is a Novel Marker of Intestinal Mucosal Inflammation in Pediatric Inflammatory Bowel Disease

High-mobility group box 1 (HMGB1) is a nuclear protein with functions in the regulation of transcription. In inflammatory conditions, HMGB1 is actively secreted from immune cells in the extracellular matrix, where it behaves as a proinflammatory cytokine. The aim of the present study was to investigate the role of HMGB1 in pediatric inflammatory bowel disease (IBD). We analyzed the stools of 19 children with Crohn's disease (CD), 21 with ulcerative colitis (UC), and 13 controls. The gene/protein expression levels of HMGB1 were assessed in bioptic specimens of all children using real-time PCR and western blot assay. Finally, intracellular localization of the protein was analyzed by western blot, after separation of nuclear and cytoplasmic extracts, and by immunohistochemistry. HMGB1 protein levels were significantly increased (P<0.001) in the stools of patients, but were undetectable in the controls; fecal HMGB1 correlated well with fecal calprotectin levels (r: 0.77 in CD, r: 0.70 in UC; P<0.01); and mRNA and protein expression were unchanged in inflamed bioptic tissues compared with controls. However, by separately analyzing the nuclear and cytoplasmic fraction, we detected the cytoplasmic HMGB1 expression to be significantly enhanced (P<0.01) in the inflamed tissues of the patients. In addition, HMGB1 was significantly detected in 16 patients with inactive disease, whose endoscopic scores showed persisting inflammation, suggesting that it may be a sensitive marker of mucosal inflammation, although the disease is clinically inactive. It was shown for the first time in our study that HMGB1 is secreted by human inflamed intestinal tissues and abundantly found in the stools of IBD patients. Hence, it can be considered as a novel marker for intestinal inflammation. We can also suggest that the presence of HMGB1 in large amounts in the fecal stream of IBD patients is mainly due to active secretion of the protein stored in the nucleus rather than a "de novo" synthesis.

Increased concentration of high-mobility group box 1 protein in milk is related to the severity of bovine mastitis

High-mobility group box 1 (HMGB1) protein is the major component of the nonhistone nuclear protein group and is involved in nucleosome stabilization and transcription regulation. HMGB1 has recently been focused on as a proinflammatory cytokine associated with various inflammatory diseases and as a target of anti-inflammatory therapy. Mastitis, a serious inflammatory disease of dairy cows, is caused by infection of the mammary gland and has detrimental effects on the quantity and quality of milk. By detecting the presence of HMGB1 in milk, we investigated the correlation between HMGB1 concentration and the severity of bovine mastitis, which was determined using the California Mastitis Test and somatic cell count (SCC). We detected a substantial amount of HMGB1 in mastitic milk but not in the milk from normal cows. We used the Spearman rank correlation coefficient to assess the relationship between HMGB1 concentration and SCC and found a significant correlation (n = 12, r = 0.975). Thus, we confirmed the positive correlation between HMGB1 concentration and SCC in milk, i.e., the severity of mastitis, which suggested that HMGB1 in milk is a new indicator of bovine mastitis.

The Role of HMGB1 and HSP72 in Ischemia and Reperfusion Injury

High-mobility group box 1 (HMGB1) is a nuclear protein that is released from endogenous origins upon cellular injury. Specifically, the presence of HMGB1 has been demonstrated in several animal models undergoing lipopolysaccharide (LPS)-induced sepsis, as shown in the work of Hasegawa et al. [ 1 Hasegawa A. Iwasaka H. Hagiwara S. et al. Relationship between HMGB1 and tissue protective effects of HSP72 in a LPS-induced systemic inflammation model. J Surg Res. 2010; (in press) Google Scholar ] recently reviewed in this journal. Known to be present in all eukaryotic cells [ 2 Van Zoelen M.A. Yang H. Florquin S. et al. Role of Toll-like receptors 2 and 4, and the receptor for advanced glycation end products in high-mobility group box 1-induced inflammation in vivo. Shock. 2009; 31: 280 Crossref PubMed Scopus (218) Google Scholar ], HMGB1's role as a mediator of inflammation and further tissue damage extends to the realm of ischemia and reperfusion as its absence has been known to reduce liver injury in other studies [ 3 Yu M. Wang H. Ding A. et al. HMGB1 signals through Toll-like receptor (TLR)4 and TLR2. Shock. 2006; 26: 174 Crossref PubMed Scopus (648) Google Scholar , 4 Schwabe R.F. Seki E. Brenner D.A. Toll-like receptor signaling in liver. Gastroenterology. 2006; 130: 1886 Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar , 5 Lu Y.C. Yeh W.C. Ohashi P.S. LPS/TLR4 signal transduction pathway. Cytokine. 2008; 42: 145 Crossref PubMed Scopus (1919) Google Scholar , 6 Tsan M.F. Gao B. Endogenous ligands of Toll-like receptors. J Leukoc Biol. 2004; 76: 514 Crossref PubMed Scopus (595) Google Scholar , 7 Mollen K.P. Rahul J.A. Tsung A. et al. Emerging paradigm: Toll-like receptor 4—sentinel for the detection of tissue damage. Shock. 2006; 5: 430 Crossref Scopus (265) Google Scholar , 8 Tsung A. Klune J.R. Zhang Z. et al. HMGB1 release induced by liver ischemia involves Toll-like receptor 4-dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med. 2007; 12: 2913 Crossref Scopus (491) Google Scholar , 9 Ellett J.D. Evans Z.P. Atkinson C. et al. Toll-like receptor 4 is a key mediator of murine steatotic liver warm ischemia/reperfusion injury. Liver Transpl. 2009; 15: 1101 Crossref PubMed Scopus (51) Google Scholar , 10 Tsung A. Sahai R. Tanaka H. et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J Exp Med. 2005; 201: 1135 Crossref PubMed Scopus (498) Google Scholar ]. This becomes particularly important in the setting of organ preservation and procurement, for there is no single best method of prolonging currently used preservation times.

Abstract 5688: HMGB1 is a Novel Substrate of Dypeptidyl Peptidase IV

Introduction: High Mobility Group Box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. HMGB1 levels are reduced in diabetic skin and topical application of HMGB1 to skin wounds of diabetic mice enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. CD26 is a protease which selectively removes the N-terminal X-Pro or X-Ala containing motifs from a number of chemokines modulating their activity. High levels of CD26 have been detected in plasma of type II diabetic patients and its inhibition has proved effective in the treatment of diabetes. Since HMGB1 contains potential CD26 cleavage sites we determined whether HMGB1 may be a substrates for CD26 and, eventually, whether CD26-mediated cleavage may alter its biologic activity. Methods and results: Recombinant HMGB1 (5 uM) was incubated overnight with CD26 (2.2 uM). RP-HPLC analysis on Aquapore C18 column yielded a peptide with a molecular weigh lower than 2000 kDa. The CD26-mediated deletion of the N-terminal region of HMGB1 was confirmed by western blot analysis: time-dependent disappearance of the molecular weigh band corresponding to HMGB1 was detected with an anti-HMGB1 antibody which recognizes its first 16 N-terminal amino-acids of HMGB1. It was previously found that HMGB1 promoted endothelial cell migration through the activation of c-Jun N-terminal kinase (JNK) and extracellular regulated kinase (ERK) signaling pathways. To evaluate whether CD26 altered HMGB1 functions, endothelial cells were induced to migrate in presence of HMGB1 and CD26. HMGB1-mediated chemotactic activity was reduced by CD26 in a dose-dependent manner. Similarly, CD26 abolished HMGB1-induced JNK and ERK activation. Since diabetes is associated with the increased CD26 activity, both CD26 activity and HMGB1 levels were measured in serum of diabetic patients. Although HMGB1 serum levels did not significantly differ between diabetic and normoglycemic patients, increased N-terminal deleted HMGB1 was detected in serum from diabetic patients. Conclusions: CD26 cleaves HMGB1 and, via this mechanism, it inhibits its functions. These findings suggest that CD26 may account for the inhibition of HMGB1 effects found in diabetes.

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

V(D)J recombination is a process integral to lymphocyte development. However, this process is not always benign, since certain lymphoid malignancies exhibit recurrent chromosomal abnormalities, such as translocations and deletions, that harbor molecular signatures suggesting an origin from aberrant V(D)J recombination. Translocations involving LMO2, TAL1, Ttg-1, and Hox11, as well as a recurrent interstitial deletion at 1p32 involving SIL/SCL, are cited examples of illegitimate V(D)J recombination. Previous studies using extrachromosomal substrates reveal that cryptic recombination signal sequences (cRSSs) identified near the translocation breakpoint in these examples support V(D)J recombination with efficiencies ranging from about 30- to 20,000-fold less than bona fide V(D)J recombination signals. To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture.