Hmgb1 binds with high affinity to Rage that triggers inflammation and damage. Our results suggest that an increased level of Hmgb1 and signaling via Rage contribute to neurotoxicity following retinal ischemia.
High-mobility group protein B1 (Hmgb1) is released from necrotic cells and induces an inflammatory response. Although Hmgb1 has been implicated in ischemia/reperfusion (IR) injury of the brain, its role in IR injury of the retina remains unclear. Here, the authors provide evidence that Hmgb1 contributes to retinal damage after IR.
Retinal IR injury was induced by unilateral elevation of intraocular pressure and the level of Hmgb1 in vitreous humor was analyzed 24 hours after reperfusion. To test the functional significance of Hmgb1 release, ischemic or normal retinas were treated with the neutralizing anti-Hmgb1 antibody or recombinant Hmgb1 protein respectively. To elucidate in which cell type Hmgb1 exerts its effect, primary retinal ganglion cell (RGC) cultures and glia RGC cocultures were treated with Hmgb1. To clarify the downstream signaling pathways involved in Hmgb1-induced effects in the ischemic retina, receptor for advanced glycation end products (Rage)-deficient mice (RageKO) were used.
Hmgb1 is accumulated in the vitreous humor 24 hours after IR. Inhibition of Hmgb1 activity with neutralizing antibody significantly decreased retinal damage after IR, whereas treatment of retinas or retinal cells with Hmgb1 induced a loss of RGCs. The analysis of RageKO versus wild-type mice showed significantly reduced expression of proinflammatory genes 24 hours after reperfusion and significantly increased survival of ganglion cell layer neurons 7 days after IR injury.
These results suggest that an increased level of Hmgb1 and signaling via the Rage contribute to neurotoxicity after retinal IR injury.
High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells.
Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data.
These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection.
The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain–containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain–containing adaptor-inducing interferon-β [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.
High-mobility group box-1 (HMGB1) protein is an alarmin, a normal cell constituent, which is released into the extracellular environment upon cellular stress/damage, and is capable of activating inflammation and tissue repair. The receptor for advanced glycation end products (RAGE) can bind HMGB1. RAGE, in turn, can induce the production of pro-inflammatory cytokines; this may be modulated the soluble truncated forms of RAGE, including soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE). The objective of this study was to determine: 1) if clinical chorioamnionitis at term is associated with changes in amniotic fluid concentrations of HMGB1, sRAGE and esRAGE; and 2) if the amniotic fluid concentration of HMGB1 changes with labor or as a function of gestational age.
Amniotic fluid samples were collected from the following groups: 1) mid-trimester (MT) (n=45); 2) term with (n=48) and without labor (n=22) without intra-amniotic infection; and 3) term with clinical chorioamnionitis (n=46). Amniotic fluid concentrations of HMGB1, sRAGE and esRAGE concentrations were determined by ELISA.
1) the median amniotic fluid HMGB1 concentration was higher in patients at term with clinical chorioamnionitis than that of those without this condition (clinical chorioamnionitis: median 3.8 ng/mL vs. term in labor: median 1.8 ng/mL, p=0.007; and vs. term not in labor median 1.1 ng/mL, p=0.003); 2) in contrast, patients with clinical chorioamnionitis had a lower median sRAGE concentration than those without this condition (clinical chorioamnionitis: median 9.3 ng/mL vs. term in labor: median 18.6 ng/mL, p=0.001; and vs. term not in labor median 28.4 ng/mL, p<0.001); 3) amniotic fluid concentrations of esRAGE did not significantly change in patients with clinical chorioamnionitis at term (clinical chorioamnionitis: median 5.4 ng/mL vs. term in labor: median 6.1 ng/mL, p=0.9; and vs. term not in labor median 9.5 ng/mL, p=0.06); and 4) there was no significant difference in the median AF HMGB1 concentration between women at term in labor and those not in labor (p=0.4) and between women in the mid-trimester and those at term not in labor (mid-trimester: median 1.5 ng/mL; p=0.2).
An increase in the amniotic fluid HMGB1 concentration and a decrease in sRAGE were observed in clinical chorioamnionitis at term. This finding provides evidence that an alarmin, HMGB1, and one of its receptors, sRAGE, are engaged in the process of clinical chorioamnionitis at term. These changes are quite different from those observed in cases of intra-amniotic infection/inflammation in preterm gestations.
danger signal; intra-amniotic inflammation; sterile inflammation; pregnancy; neuroinflammation; neuro-immune reflects; amniotic fluid; DAMPs; damage-associated molecular patterns; intra-amniotic infection; term labor
High-mobility group box 1 (HMGB1) was initially discovered as a nuclear protein that interacts with DNA as a chromatin-associated non-histone protein to stabilize nucleosomes and to regulate the transcription of many genes in the nucleus. Once leaked or actively secreted into the extracellular environment, HMGB1 activates inflammatory pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 2, TLR4, and receptor for advanced glycation end products (RAGE), leading to tissue injury. Although HMGB1’s ability to induce inflammation has been well documented, no studies have examined the role of HMGB1 in wound healing in the gastrointestinal field. The aim of this study was to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1’s effects on ulcer healing. Gastric ulcers were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation. The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and the expression of tumor necrosis factor α (TNFα) mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity and TNFα expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO activity and TNFα expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNFα, whereas ulcer healing in TLR2 knockout (KO) mice was similar to that in wild-type mice. In TLR4 KO and RAGE KO mice, exogenous HMGB1 did not affect ulcer healing and TNFα expression. Thus, we showed that HMGB1 is a complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive inflammatory responses.
A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE), or Toll-like receptor 4 (TLR4), HMGB1 activates macrophages/monocytes to express proinflammatory cytokines, chemokines, and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of observations that many viruses (e.g., West Nile virus, Salmon anemia virus) can induce passive HMGB1 release, we propose a potential pathogenic role of HMGB1 in viral infectious diseases.
Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1–nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1β, IL-6, IL-10, and tumor necrosis factor (TNF) α and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2–dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1–nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.
Necrotic cells release inflammatory mediators that activate cytokine production from innate immune cells. One mediator of this activation is high mobility group box 1 protein (HMGB1). HMGB1 is normally a chromatin-associated protein and is sequestered at condensed chromatin during apoptosis. How it is released from chromatin during necrotic cell death is not known. Here we show that after DNA-alkylating damage, the activation of poly(ADP)-ribose polymerase (PARP) regulates the translocation of HMGB1 from the nucleus to the cytosol. This displaced HMGB1 is subject to release if the cell then loses plasma membrane integrity as a result of necrosis. Both full-length HMGB1 and a truncated form of HMGB1 lacking the highly conserved glutamate-rich C-terminal tail can induce macrophage activation and tumor necrosis factor-α production. However, displacement of HMGB1 from the nucleus following PARP activation requires the presence of the glutamate-rich C-terminal tail. Although the C-terminal tail is not the sole substrate for PARP modification of HMGB1, it appears to be required to destabilize HMGB1 association with chromatin following PARP-dependent chromatin modifications. These data suggest that PARP-dependent nuclear-to-cytosolic translocation of HMGB1 serves to establish the ability of cells to release this potent inflammatory mediator upon subsequent necrotic death.
A nuclear protein, high mobility group box 1 (HMGB1), is released passively by necrotic cells, and actively by macrophages/monocytes in response to exogenous and endogenous inflammatory stimuli. After binding to the receptor for advanced glycation end products (RAGE) or toll-like receptor 4 (TLR4), HMGB1 activates vascular endothelial cells and macrophages/monocytes to express proinflammatory cytokines, chemokines and adhesion molecules. Pharmacological suppression of its activities or release is protective against lethal endotoxemia and sepsis, establishing HMGB1 as a critical mediator of lethal systemic inflammation. In light of the pathogenic role of inflammation in cardiovascular diseases, we propose that HMGB1, a proinflammatory cytokine derived from both injured endothelium and activated macrophages/monocytes, could contribute to the progression of atherosclerosis and other cardiovascular diseases.
High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant, nonhistone DNA-binding protein. It has well-established functions in the maintenance of nuclear homeostasis. The HMGB1 can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatory signals. Extracellular HMGB1 interacts with receptors, including those for advanced glycation endproducts (RAGEs) as well as Toll-like receptor 2 (TLR2) and TLR4. The HMGB1 functions in a synergistic manner with other proinflammatory mediators and acts as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Numerous reports point to HMGB1 as a novel player in the ischemic brain. This review provides an appraisal of the emerging roles of HMGB1 in cerebral ischemia injury, highlighting the relevance of HMGB1-blocking agents as potent therapeutic tools for neuroprotection.
cerebral ischemia; HMGB1; inflammation; RAGE; TLR
High mobility group box 1 (HMGB1), an evolutionarily highly conserved and abundant nuclear protein also has roles within the cytoplasm and as an extracellular damage-associated molecular pattern (DAMP) molecule. Extracellular HMGB1 is the prototypic endogenous ‘danger signal’ that triggers inflammation and immunity. Recent findings suggest that posttranslational modifications dictate the cellular localization and secretion of HMGB1. HMGB1 is actively secreted from immune cells and stressed cancer cells, or passively released from necrotic cells. During cancer development or administration of therapeutic agents including chemotherapy, radiation, epigenetic drugs, oncolytic viruses, or immunotherapy, the released HMGB1 may either promote or limit cancer growth, depending on the state of progression and vascularization of the tumor. Extracellular HMGB1 enhances autophagy and promotes persistence of surviving cancer cells following initial activation. When oxidized, it chronically suppresses the immune system to promote cancer growth and progression, thereby enhancing resistance to cancer therapeutics. In its reduced form, it can facilitate and elicit innate and adaptive anti-tumor immunity, recruiting and activating immune cells, in conjunction with cytotoxic agents, particularly in early transplantable tumor models. We hypothesize that HMGB1 also functions as an epigenetic modifier, mainly through regulation of NF-kB-dependent signaling pathways, to modulate the behavior of surviving cancer cells as well as the immune cells found within the tumor microenvironment. This has significant implications for developing novel cancer therapeutics.
Cancer; HMGB1; NF-kB signaling; activation; innate immunity; dendritic cells; CD8+ T cells; epigenetic pathways
High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. To function as an alarmin, HMGB1 translocates from the nucleus of the cell to the extra-cellular milieu, a process that can take place with cell activation as well as cell death. HMGB1 can interact with receptors that include RAGE (receptor for advanced glycation endproducts) as well as Toll-like receptor-2 (TLR-2) and TLR-4 and function in a synergistic fashion with other proinflammatory mediators to induce responses. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. New approaches to therapy for these diseases may involve strategies to inhibit HMGB1 release from cells, its interaction with receptors, and downstream signaling.
Background and Purpose
The signals that initiate the post-stroke inflammatory response are unknown. High mobility group box (HMGB) 1 protein is a nuclear protein that is passively released from necrotic tissue and is able to activate leukocytes, which in turn secrete HMGB1. HMGB1 is also able to activate antigen presenting cells and therefore stands at the crossroads of innate and adaptive immunity.
Plasma HMGB1 concentrations were determined at multiple time points after ischemic stroke (N=110) and correlated to stroke severity and biomarkers of inflammation. The relationships between HMGB1, stroke outcome and autoimmune responses to brain antigens were also assessed.
Stroke resulted in an increase in HMGB1 that persisted for 30 days. Plasma HMGB1 was correlated with the number of circulating leukocytes but not predictive of stroke outcome nor the development of autoimmune responses to brain antigens. Patients with a TH1(+) response to myelin basic protein at 90 days after stroke, however, had higher plasma HMGB1.
HMGB1 appears to be involved in the post-ischemic inflammatory response, but it remains unclear whether HMGB1 initiates this response or merely reflects activation of leukocytes by another signal.
stroke; HMGB1; alarmin; DAMP; inflammation; monocytes
We explored the role of a chromatin-associated nuclear protein HMGB1 in apoptotic response to widely used anticancer drugs. A murine fibroblast model system generated from Hmgb1+/+ and Hmgb1−/− mice was used to assess the role of HMGB1 protein in cellular response to anticancer nucleoside analogs and precursors which act without destroying integrity of DNA. Chemosensitivity experiments with 5-fluorouracil (FU), cytosine arabinoside (araC), and mercaptopurine (MP) demonstrated that Hmgb1−/− MEFs were 3–10 times more resistant to these drugs compared with Hmgb1+/+ MEFs. Hmgb1-deficient cells showed compromised cell cycle arrest and reduced caspase activation after treatment with MP and araC. Phosphorylation of p53 at Ser12 (corresponding to Ser 9 in human p53) and Ser18 (corresponding to Ser 15 in human p53), as well as phosphorylation of H2AX after drug treatment was reduced in Hmgb1-deficient cells. Trans-activation experiments demonstrated diminished activation of pro-apoptotic promoters Bax, Puma, and Noxa in Hmgb1-deficient cells after treatment with MP or araC, consistent with reduced transcriptional activity of p53. For the first time, we demonstrated that Hmgb1 is an essential activator of cellular response to genotoxic stress caused by chemotherapeutic agents (thiopurines, cytarabine and 5-fluorouracil), which acts at early steps of antimetabolite-induced stress by stimulating phosphorylation of two DNA damage markers p53 and H2AX. This finding makes HMGB1 a potential target for modulating activity of chemotherapeutic antimetabolites. Identification of proteins sensitive to DNA lesions which occur without the loss of DNA integrity provides new insights into the determinants of drug sensitivity in cancer cells.
High mobility group protein B1 (HMGB1) binds to the internucleosomal linker DNA in chromatin and abuts the nucleosome. Bending and untwisting of the linker DNA results in transmission of strain to the nucleosome core, disrupting histone/DNA contacts. An interaction between H3 and HMGB1 has been reported. Here we confirm and characterize the interaction of HMGB1 with H3, which lies close to the DNA entry/exit points around the nucleosome dyad, and may be responsible for positioning of HMGB1 on the linker DNA. We show that the interaction is between the N-terminal unstructured tail of H3 and the C-terminal unstructured acidic tail of HMGB1, which are presumably displaced from DNA and the HMG boxes, respectively, in the HMGB1-nucleosome complex. We have characterized the interaction by nuclear magnetic resonance spectroscopy and show that it is extensive for both peptides, and appears not to result in the acquisition of significant secondary structure by either partner.
Type 1 diabetes mellitus is caused by the autoimmune destruction of β cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were β cells; few α cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on β cells and that HMGB1 may signal via TLR4 to selectively damage β cells rather than α cells during the development of type 1 diabetes mellitus.
diabetes mellitus, type 1; HMGB1 protein; islets of Langerhans; mice, inbred NOD; toll-like receptor 4
High mobility group box 1 (HMGB1) is a molecular alarm signal that triggers an immune response when released. It was assumed that the receptor for advanced glycation end-products (RAGE) would mediate the signal to the immune system. Recently pattern recognition receptors that are triggered by molecules of bacterial origin (the Toll-like receptor (TLR) family) were shown to also respond to HMGB1. Now two papers establish the TLR4–HMGB1 axis as proinflammatory, eventually leading to disparate conditions like seizures or skin cancer. These reports add a new twist to our understanding of the mode of action of the alarm signal HMGB1.
HMGB1; inflammation; TLR4
The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.
CXCL12 forms a complex with HMGB1 that binds to the chemokine receptor CXCR4 and increases inflammatory cell migration.
After tissue damage, inflammatory cells infiltrate the tissue and release proinflammatory cytokines. HMGB1 (high mobility group box 1), a nuclear protein released by necrotic and severely stressed cells, promotes cytokine release via its interaction with the TLR4 (Toll-like receptor 4) receptor and cell migration via an unknown mechanism. We show that HMGB1-induced recruitment of inflammatory cells depends on CXCL12. HMGB1 and CXCL12 form a heterocomplex, which we characterized by nuclear magnetic resonance and surface plasmon resonance, that acts exclusively through CXCR4 and not through other HMGB1 receptors. Fluorescence resonance energy transfer data show that the HMGB1–CXCL12 heterocomplex promotes different conformational rearrangements of CXCR4 from that of CXCL12 alone. Mononuclear cell recruitment in vivo into air pouches and injured muscles depends on the heterocomplex and is inhibited by AMD3100 and glycyrrhizin. Thus, inflammatory cell recruitment and activation both depend on HMGB1 via different mechanisms.
In response to infection or injury, a ubiquitous nucleosomal protein, HMGB1 is secreted actively by innate immune cells, and / or released passively by injured/damaged cells. Subsequently, extracellular HMGB1 alerts, recruits, and activates various innate immune cells to sustain a rigorous inflammatory response. A growing number of HMGB1 inhibitors ranging from neutralizing antibodies, endogenous hormones, to medicinal herb-derived small molecule HMGB1 inhibitors (such as nicotine, glycyrrhizin, tanshinones, and EGCG) are proven protective against lethal infection and ischemic injury. Here we review emerging evidence that support extracellular HMGB1 as a proinflammatory alarmin(g) danger signal, and discuss a wide array of HMGB1 inhibitors as potential therapeutic agents for sepsis and ischemic injury.
innate immune cells; phagocytes; inflammation; cytokines; sepsis; antibodies; HMGB1; tanshinones
Plasmacytoid dendritic cells (pDC) are potent antigen-presenting cells known to regulate immune responses to self-antigens, particularly DNA. The mitochondrial fraction of necrotic cells was found to most potently promote human pDC activation, as reflected by Type I interferon release, which was dependent upon the presence of mitochondrial DNA and involved TLR9 and receptors for advanced glycation endproducts (RAGE). Mitochondrial transcription factor A (TFAM), a highly abundant mitochondrial protein that is functionally and structurally homologous to high-mobility group box protein 1 (HMGB1), was observed to synergize with CpGA DNA to promote human pDC activation. pDC Type I interferon responses to TFAM and CpGA DNA indicated their engagement with RAGE and TLR9, respectively, and were dependent upon endosomal processing and PI3K, ERK and NF-κB signaling. Together, these results indicate that pDC contribute to sterile immune responses by recognizing the mitochondrial component of necrotic cells and further incriminate TFAM and mitochondrial DNA as likely mediators of pDC activation under these circumstances.
Cell necrosis; DAMP; IFNα; IRF-7; Sterile Inflammation
Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. HMGB1, a nuclear non-histone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and FRET demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage (BAL) fluid from Scnn+ mice, a murine model of cystic fibrosis lung disease, which contains elevated concentrations of HMGB1 inhibits neutrophil efferocytosis. Anti-HMGB1 antibodies reverse the inhibitory effect of Scnn+ BAL on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses.
HMGB1; Phagocytosis; Neutrophils; Cystic fibrosis
In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1β, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1β has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes.
Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1β. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-β, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA.
Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1β enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1β increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively.
HMGB1 in complex with LPS, IL-1α or IL-1β boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from RA and OA patients. A mechanism for the pathogenic role of HMGB1 in arthritis could thus be through enhancement of inflammatory and destructive mechanisms induced by other proinflammatory mediators present in the arthritic joint.
High mobility group box chromosomal protein 1 (HMGB1) is a DNA binding protein that exhibits pro-inflammatory properties when present in the extracellular compartment. Putative receptors for HMGB1 include Toll-like receptor (TLR) 4, TLR2 and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce tolerance to the bacterial product, lipoteichoic acid (LTA). Pretreatment of human monocyte-like THP-1 cells with 1 μg/ml HMGB1 18 h before exposure to LTA (10 μg/ml) decreased secretion of TNF, NF-κB DNA-binding, and degradation of IκBα. Denaturation of HMGB1 with boiling water or co-incubation with anti-HMGB1 antibody abrogated the induction of tolerance to LTA. In contrast, co-incubation with polymyxin B failed to diminish HMGB1-induced tolerance to LTA. These findings support the view that the induction of LTA tolerance by HMGB1 was not due to LPS contamination. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type and RAGE-deficient mice became LTA-tolerant following HMGB1 exposure ex vivo. We were unable to demonstrate LTA tolerance in TLR2 and TLR4-deficient macrophages, as they are hyporesponsive to LTA. These findings suggest that extracellular HMGB1 induces LTA tolerance, and RAGE receptor is not required for this induction.
High mobility group box chromosomal protein 1 (HMGB1); Lipoteichoic acid; tolerance; signal transduction; Toll-like receptor -2
The functional relationship and cross-regulation between autophagy and apoptosis is complex. Here we show that high-mobility group box 1 protein (HMGB1) is a redox-sensitive regulator of the balance between autophagy and apoptosis. In cancer cells, anti-cancer agents enhanced autophagy and apoptosis as well as HMGB1 release. HMGB1 release may be a pro-survival signal for residual cells following various cytotoxic cancer treatments. Diminished HMGB1 by shRNA transfection or inhibition of HMGB1 release by ethyl pyruvate or other small molecules led to predominantly apoptosis and decreased autophagy in stressed cancer cells. In this setting, reducible HMGB1 binds to the receptor for advanced glycation end products (RAGE) but not Toll-like receptor 4 (TLR4), induces Beclin1-dependent autophagy, and promotes tumor resistance to alkylators (melphalan), tubulin disrupting agents (paclitaxel), DNA crosslinkers (ultraviolet light) and DNA-intercalators (oxaliplatin or adriamycin). Oxidized HMGB1 conversely increases the cytotoxicity of these agents and induces apoptosis mediated by the caspase-9/-3 intrinsic pathway. HMGB1 release as well as its redox state thus link autophagy and apoptosis, representing a suitable target when coupled with conventional tumor treatments.