HMGB1 and RAGE in inflammation and cancer.

The immune system has evolved to respond not only to pathogens, but also to signals released from dying cells. Cell death through necrosis induces inflammation, whereas apoptotic cell death provides an important signal for tolerance induction. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death; it is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 can associate with other molecules, including TLR ligands and cytokines, and activates cells through the differential engagement of multiple surface receptors including TLR2, TLR4, and RAGE. RAGE is a multiligand receptor that binds structurally diverse molecules, including not only HMGB1, but also S100 family members and amyloid-beta. RAGE activation has been implicated in sterile inflammation as well as in cancer, diabetes, and Alzheimer's disease. While HMGB1 through interactions with TLRs may also be important, this review focuses on the role of the HMGB1-RAGE axis in inflammation and cancer.

The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1.

The mechanisms by which tumor microenvironments modulate nucleic acid-mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.

HMGB1 promotes CXCL12-dependent egress of murine B cells from Peyer's patches in homeostasis.

High mobility group box-1 protein (HMGB1) is an alarmin that, once released, promotes inflammatory responses, alone and as a complex with the chemokine CXCL12. Here, we report that the HMGB1-CXCL12 complex plays an essential role also in homeostasis by controlling the migration of B lymphocytes. We show that extracellular HMGB1 is critical for the CXCL12-dependent egress of B cells from the Peyer's patches (PP). This promigratory function of the complex was restricted to the PPs, since HMGB1 was not required for B-cell migratory processes in other locations. Accordingly, we detected higher constitutive levels of the HMGB1-CXCL12 complex in PPs than in other lymphoid organs. HMGB1-CXCL12 in vivo inhibition was associated with a reduced basal IgA production in the gut. Collectively, our results demonstrate a role for the HMGB1-CXCL12 complex in orchestrating B-cell trafficking in homeostasis, and provide a novel target to control lymphocyte migration in mucosal immunity.

HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling.

Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling.

The role of type 1 interferons in coagulation induced by gram-negative bacteria.

Bacterial infection not only stimulates innate immune responses but also activates coagulation cascades. Overactivation of the coagulation system in bacterial sepsis leads to disseminated intravascular coagulation (DIC), a life-threatening condition. However, the mechanisms by which bacterial infection activates the coagulation cascade are not fully understood. Here we show that type 1 interferons (IFNs), a widely expressed family of cytokines that orchestrate innate antiviral and antibacterial immunity, mediate bacterial infection-induced DIC by amplifying the release of high-mobility group box 1 (HMGB1) into the bloodstream. Inhibition of the expression of type 1 IFNs and disruption of their receptor IFN-α/ßR or downstream effector (eg, HMGB1) uniformly decreased gram-negative bacteria-induced DIC. Mechanistically, extracellular HMGB1 markedly increased the procoagulant activity of tissue factor by promoting the externalization of phosphatidylserine to the outer cell surface, where phosphatidylserine assembles a complex of cofactor-proteases of the coagulation cascades. These findings not only provide novel insights into the link between innate immune responses and coagulation, but they also open a new avenue for developing novel therapeutic strategies to prevent DIC in sepsis.

HMGB1-Neutralizing IgM Antibody Is a Normal Component of Blood Plasma.

Extracellular high-mobility group box 1 (HMGB1) is a prototypic damage-associated molecular pattern. Although a homeostatic level of extracellular HMGB1 may be beneficial for immune defense, tissue repair, and tissue regeneration, excessive HMGB1 is linked to inflammatory diseases. This prompts an intriguing question: how does a healthy body control the level of extracellular HMGB1? In this study, in the plasma of both healthy humans and healthy mice, we have identified an anti-HMGB1 IgM autoantibody that neutralizes extracellular HMGB1 via binding specifically to a 100% conserved epitope, namely HMW4 (HMGB198-112). In mice, this anti-HMW4 IgM is produced by peritoneal B-1 cells, and concomitant triggering of their BCR and TLR4 by extracellular HMGB1 stimulates the production of anti-HMW4 IgM. The ability of extracellular HMGB1 to induce its own neutralizing Ab suggests a feedback loop limiting the level of this damage-associated molecular pattern in a healthy body.

Interplay between RAGE and TLR4 Regulates HMGB1-Induced Inflammation by Promoting Cell Surface Expression of RAGE and TLR4.

Receptor for advanced glycation end-products (RAGE) and TLR4 play an important role in the inflammatory response against High-mobility group box 1 protein (HMGB1), a late proinflammatory cytokine and a damage-associated molecular pattern. As cell surface receptors, both RAGE and TLR4 are constantly trafficking between the cytoplasm and plasma membrane. However, whether TLR4 is related to the intracellular transport of RAGE in HMGB1-induced inflammation remains unknown. In this study, we demonstrated that HMGB1 not only increased RAGE expression in both the cytoplasm and plasma membrane but also upregulated the expression of TLR4 in the plasma membrane. Knocking out of RAGE led to decreased MAPK activation, TLR4 cellular membrane expression, and corresponding inflammatory cytokine generation. Meanwhile, inhibiting MAPK activation also decreased TLR4 surface expression. These results indicated that HMGB1 may bind to cell surface RAGE receptors on the cell surface, leading to MAPK activation, thus promoting TLR4 translocation on the cell surface, but does not regulate its transcription and translation. In contrast, TLR4 can increase the transcription and translation of RAGE, which translocates to the cell surface and is able to bind to more HMGB1. The cell surface receptors TLR4 and RAGE bind to HMGB1, leading to the transcription and secretion of inflammatory cytokines. Finally, we also observed these results in the mice pseudofracture model, which is closely related to HMGB1-induced inflammatory response. All these results demonstrated that the interplay between RAGE and TLR4 are critical for HMGB1-induced inflammatory response.

High-mobility group box 1 protein (HMGB1) operates as an alarmin outside as well as inside cells.

Alarmins are preformed, endogenous molecules that can be promptly released to signal cell or tissue stress or damage. The ubiquitous nuclear molecule high-mobility group box 1 protein (HMGB1) is a prototypical alarmin activating innate immunity. HMGB1 serves a dual alarmin function. The protein can be emitted to alert adjacent cells about endangered homeostasis of the HMGB1-releasing cell. In addition to this expected path of an alarmin, extracellular HMGB1 can be internalized via RAGE-receptor mediated endocytosis to the endolysosomal compartment while attached to other extracellular proinflammatory molecules. The endocytosed HMGB1 may subsequently destabilize lysosomal membranes. The HMGB1-bound partner molecules depend on the HMGB1-mediated transport and the induced lysosomal leakage to obtain access to endosomal and cytosolic reciprocal sensors to communicate extracellular threat and to initiate the proper activation pathways.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

Evaluation of autophagy mediators in myeloid-derived suppressor cells during human tuberculosis.

Myeloid-derived suppressor cells (MDSC) are induced during active TB disease to restore immune homeostasis but instead exacerbate disease outcome due to chronic inflammation. Autophagy, in conventional phagocytes, ensures successful clearance of M.tb. However, autophagy has been demonstrated to induce prolonged MDSC survival. Here we investigate the relationship between autophagy mediators and MDSC in the context of active TB disease and during anti-TB therapy. We demonstrate a significant increase in MDSC frequencies in untreated active TB cases with these MDSC expressing TLR4 and significantly more mTOR and IL-6 than healthy controls, with mTOR levels decreasing during anti-TB therapy. Finally, we show that HMGB1 serum concentrations decrease in parallel with mTOR. These findings suggest a complex interplay between MDSC and autophagic mediators, potentially dependent on cellular localisation and M.tb infection state.

A novel RAG1 mutation reveals a critical in vivo role for HMGB1/2 during V(D)J recombination.

The Recombination Activating Genes, RAG1 and RAG2, are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo.

Anti-high mobility group box 1 monoclonal antibody suppressed hyper-permeability and cytokine production in human pulmonary endothelial cells infected with influenza A virus.

OBJECTIVE: High mobility group box-1 (HMGB1) has been reported to be involved in influenza A virus-induced acute respiratory distress syndrome (ARDS). We studied the efficacy of an anti-HMGB1 mAb using an in vitro model of TNF-α stimulation or influenza A virus infection in human pulmonary microvascular endothelial cells (HMVECs). METHODS: Vascular permeability of HMVECs was quantified using the Boyden chamber assay under tumor necrosis factor-α (TNF-α) stimulation or influenza A virus infection in the presence of anti-HMGB1 mAb or control mAb. The intracellular localization of HMGB1 was assessed by immunostaining. Extracellular cytokine concentrations and intracellular viral mRNA expression were quantified by the enzyme-linked immunosorbent assay and quantitative reverse transcription PCR, respectively. RESULTS: Vascular permeability was increased by TNF-α stimulation or influenza A infection; HMVECs became elongated and the intercellular gaps were extended. Anti-HMGB1 mAb suppressed both the increase in permeability and the cell morphology changes. Translocation of HMGB1 to the cytoplasm was observed in the non-infected cells. Although anti-HMGB1 mAb did not suppress viral replication, it did suppress cytokine production in HMVECs. CONCLUSION: Anti-HMGB1 mAb might be an effective therapy for severe influenza ARDS.

Expatiating the molecular approaches of HMGB1 in diabetes mellitus: Highlighting signalling pathways via RAGE and TLRs.

Diabetes mellitus (DM) has become one of the major healthcare challenges worldwide in the recent times and inflammation being one of its key pathogenic process/mechanism affect several body parts including the peripheral and central nervous system. High-mobility group box 1 (HMGB1) is one of the major non-histone proteins that plays a key role in triggering the inflammatory response. Upon its release into the extracellular milieu, HMGB1 acts as an "alarmin" for the immune system to initiate tissue repair as a component of the host defense system. Furthermore, HMGB1 along with its downstream receptors like Toll-like receptors (TLRs) and receptors for advanced glycation end products (RAGE) serve as the suitable target for DM. The forthcoming research in the field of diabetes would potentially focus on the development of alternative approaches to target the centre of inflammation that is primarily mediated by HMGB1 to improve diabetic-related complications. This review covers the therapeutic actions of HMGB1 protein, which acts by activating the RAGE and TLR molecules to constitute a functional tripod system, in turn activating NF-κB pathway that contributes to the production of mediators for pro-inflammatory cytokines associated with DM. The interaction between TLR2 and TLR4 with ligands present in the host and the activation of RAGE stimulates various immune and metabolic responses that contribute to diabetes. This review emphasizes to elucidate the role of HMGB1 in the initiation and progression of DM and control over the inflammatory tripod as a promising therapeutic approach in the management of DM.

Attenuating Effects of Dieckol on High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease by Decreasing the NLRP3 Inflammasome and Pyroptosis.

Nonalcoholic fatty liver disease (NAFLD), which promotes serious health problems, is related to the increase in the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and pyroptosis by a high-fat diet (HFD). Whether dieckol (DK), a component of Ecklonia cava extracts (ECE), attenuated NAFLD in an HFD-induced NAFLD animal model was evaluated. The expression of high mobility group box 1/Toll-like receptor 4/nuclear factor-κB, which initiated the NLRP3 inflammasome, was increased in the liver of HFD-fed animals and significantly decreased with ECE or DK administration. The expression of NLRP3/ASC/caspase-1, which are components of the NLRP3 inflammasome, and the number of pyroptotic cells were increased by HFD and decreased with ECE or DK administration. The accumulation of triglycerides and free fatty acids in the liver was increased by HFD and decreased with ECE or DK administration. The histological NAFLD score was increased by HFD and decreased with ECE or DK administration. The expression of lipogenic genes (FASN, SREBP-2, PPARγ, and FABP4) increased and that of lipolytic genes (PPARα, CPT1A, ATGL, and HSL) was decreased by HFD and attenuated with ECE or DK administration. In conclusion, ECE or DK attenuated NAFLD by decreasing the NLRP3 inflammasome and pyroptosis.

CD52 glycan binds the proinflammatory B box of HMGB1 to engage the Siglec-10 receptor and suppress human T cell function.

CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.

Pretreatment with High Mobility Group Box-1 Monoclonal Antibody Prevents the Onset of Trigeminal Neuropathy in Mice with a Distal Infraorbital Nerve Chronic Constriction Injury.

Persistent pain following orofacial surgery is not uncommon. High mobility group box 1 (HMGB1), an alarmin, is released by peripheral immune cells following nerve injury and could be related to pain associated with trigeminal nerve injury. Distal infraorbital nerve chronic constriction injury (dIoN-CCI) evokes pain-related behaviors including increased facial grooming and hyper-responsiveness to acetone (cutaneous cooling) after dIoN-CCI surgery in mice. In addition, dIoN-CCI mice developed conditioned place preference to mirogabalin, suggesting increased neuropathic pain-related aversion. Treatment of the infraorbital nerve with neutralizing antibody HMGB1 (anti-HMGB1 nAb) before dIoN-CCI prevented both facial grooming and hyper-responsiveness to cooling. Pretreatment with anti-HMGB1 nAb also blocked immune cell activation associated with trigeminal nerve injury including the accumulation of macrophage around the injured IoN and increased microglia activation in the ipsilateral spinal trigeminal nucleus caudalis. The current findings demonstrated that blocking of HMGB1 prior to nerve injury prevents the onset of pain-related behaviors, possibly through blocking the activation of immune cells associated with the nerve injury, both within the CNS and on peripheral nerves. The current findings further suggest that blocking HMGB1 before tissue injury could be a novel strategy to prevent the induction of chronic pain following orofacial surgeries.

High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair.

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage-associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue-healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti-tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

Danger signals: Chemotherapy enhancers?

Endogenous danger signals are molecules normally present in a given cell compartment that are rapidly released following cell stress and induce immune responses. We and others have shown that dying tumor cells treated with some chemotherapies are able to induce anticancer immune responses, which rely on their release of danger signals such as the nuclear protein HMGB1. DNA can also be released from chemotherapy-treated tumor cells, act as a danger signal, and boost anticancer immunity. While the immunostimulatory properties of DNA have been identified for decades, the recent discovery of a novel family of receptors, cytosolic DNA sensors, has provided a novel impetus not only to understand how chemotherapy can trigger anticancer immune responses but also to exploit DNA-derived molecules for therapeutic use. We will here discuss the molecular characteristics of endogenous danger signals released from chemotherapy-treated tumor cells and focus on the clinical relevance of using these danger signals in chemoimmunotherapeutic strategies against cancer.

High mobility group box 1 enables bacterial lipids to trigger receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis and apoptosis in mice.

Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of programmed cell death and inflammation during viral infection or sterile tissue injury. Whether and how bacterial infection also activates RIPK3-dependent immune responses remains poorly understood. Here we show that bacterial lipids (lipid IVa or lipid A) form a complex with high mobility group box 1 (HMGB1), released by activated immune cells or damaged tissue during bacterial infection, and that this complex triggers RIPK3- and TIR domain-containing adapter-inducing IFN-ß (TRIF)-dependent immune responses. We found that these responses lead to macrophage death, interleukin (IL)-1α release, and IL-1ß maturation. In an air-pouch inflammatory infiltration model, genetic deletion of Ripk3, Trif, or IL-1 receptor (Il-1R), or monoclonal antibody-mediated HMGB1 neutralization uniformly attenuated inflammatory responses induced by Gram-negative bacteria that release lipid IVa and lipid A. These findings uncover a previously unrecognized mechanism by which host factors and bacterial components work in concert to orchestrate immune responses.