Accepted Article
Received Date : 25-Mar-2014 Revised Date
: 03-Jun-2014
Accepted Date : 09-Jun-2014 Article type
: Review-Symposium
Nucleic acid sensing and beyond: virtues and vices of HMGB1
Hideyuki Yanai1,2 & Tadatsugu Taniguchi1,2
From the 1Department of Molecular Immunology, Institute of Industrial Science, Max Planck-The University of Tokyo Center for Integrative Inflammology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8505, Japan, and 2Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan
Correspondence: Tadatsugu Taniguchi (e-mail: [email protected]); .Komaba 4-6-1, Meguro-ku, Tokyo 153-8505, Japan
Abstract High-mobility group box 1 (HMGB1) was first described as an architectural chromatin-binding protein. Today, a wealth of evidence indicates that this protein is very versatile and serves an amazing assortment of roles in the nucleus, cytoplasm and extracellular milieu. As a result,
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HMGB1 is fast becoming recognized as a key regulator of protective and pathological immune responses. While acknowledging the many functions of HMGB1 and its family members, we focus this review on their role as broad effectors of immune responses mediated by nucleic acids. In addition, we touch upon the recent progress in determining the in vivo role of HMGB1 as revealed by the study of mice conditionally null for the Hmgb1 gene.
Keywords: nucleic acids, HMGB1, infection, inflammation, autophagy, sepsis.
Introduction
HMGB1 is an evolutionarily conserved protein that is abundantly present in the nucleus of almost all eukaryotic cells. It was long thought that this protein to simply functions as a nonhistone chromatin-binding protein, stabilizing chromatin structure and modulating gene transcription along with other HMGB family members such as HMGB2, 3 and 4, whose expression is more restricted to particular cell types [1, 2]. A more versatile and perhaps more interesting role of HMGB1 stems from its function as a factor promoting regeneration of neurite-type cytoplasmic processes in brain neurons [3]. Further, pioneering work characterizing HMGB1 as a critical mediator of endotoxin lethality [4] has opened a new avenue of research on the remarkable functional versatility of the protein in evoking inflammatory responses [5-10]. Extracellular HMGB1 has proinflammatory cytokine-like and chemo-attractant functions, which are mediated by its interaction with several receptors and partner proteins, such as Toll-like receptors (TLRs), RAGE (receptor for advanced glycation end product), and the chemokine CXCL12 that, paired with HMGB1, binds CXCR4 chemokine receptor [5-8, 10-12]
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(Fig. 1). An intracellular function of HMGB1 has also been reported; specifically, the protein appears to have a role in autophagy, a cellular response closely linked with immunity and cancer [13, 14]. Collectively, these studies place HMGB1 at the centre of research on immunity and inflammation [5]. Of particular note, the versatility of HMGB1 is controlled by its modifications, such as acetylation [15] and redox states [16]. Indeed, the biological and clinical importance of HMGB1 is underscored by this protein being involved in numerous pathological conditions, including sepsis, ischemia–reperfusion (I/R) injury, arthritis, and cancer [5-10]. Furthermore, HMGB research has advanced in yet another direction: HMGB1 and its family members are sentinels for immunogenic nucleic acids that work in conjunction with signal-transducing, nucleic acid–sensing innate receptors [17-20]. Many excellent reviews already exist on HMGB1 [5-10], so we shall refrain from tallying the multiple functions of this protein, but rather focus on its role in nucleic acid sensing and recent data generated from the study of conditional knockout mice.
Sensing of nucleic acids by innate receptors Nucleic acids derived from infected pathogens or dead cells can activate immune responses, and indeed, multiple innate receptors have evolved to recognize RNA and DNA in mammals. These include TLRs, retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and several cytosolic DNA sensors. In particular, TLR3, TLR7 and TLR9 are present in endosomes where they sense double-stranded RNA (dsRNA), single-stranded RNA (ssRNA)
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and short dsRNA and hypomethylated CpG-motif–containing oligodeoxynucleotides (CpG ODNs), respectively [21, 22]. RLRs, namely RIG-I, melanoma differentiation associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), are best known as RNA-sensing receptors in the cytosol [23]. Numerous cytosolic DNA-sensing receptors also trigger the innate immune system, and these include RLRs, stimulator of interferon genes (STING), DNA-dependent activator of interferon (IFN) regulatory factors (IRFs), cyclic CMP-AMP (cGAMP) synthase (cGAS), absent in melanoma 2 (AIM2), IFN-inducible protein 16 (IFI16)/IFN activated gene 204 (IFI204), and (Asp-Glu-Ala-Asp) (DEAD) box polypeptide 41 (DDX41), when DNA is exposed to the cytosol [21, 24-30]. More recently, it has been shown that cyclic dinucleotides activate DNA-sensing receptors. Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) produced by bacteria are sensed by DDX41 and STING [31-33]. Furthermore, cGAMP, synthesized by the DNA sensor cGAS in response to cytosolically exposed DNA, activates the STING signalling pathway [26, 29]. A hallmark of the innate immune response activated by these nucleic acid–sensing pattern recognition receptors (PRRs) is the induction of type I IFN [21, 34], proinflammatory cytokines and chemokines, with the exception of AIM2, which instead activates the DNA-mediated inflammasome pathway [25]. Several diseases are linked to the aberrant activation of the nucleic acid–sensing system, most notably autoimmunity [35-40].
Binding of HMGB1 and its family members to nucleic acids and their analogues HMGB1 and its family members were identified as being involved in cytosolic DNA recognition because these proteins could bind to a synthetic DNA, poly(dA-dT)·poly(dT-dA) (B-DNA hereafter), that potently activates immune responses when delivered to the cytosol [41]. Recombinant HMGBs bind to various types of nucleic acids to activate innate responses in vitro, and immune activation by these nucleic acids correlates with their binding affinity to
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HMGBs. Indeed, both HMGB1 and HMGB2 bind strongly to the highly immunogenic B-DNA, but weakly to DNA derived from calf thymus or bacteria, which only weakly activates an immune response. HMGB1 also binds with high affinities to immunogenic double-stranded RNA [polyinosinic-polycytidylic acid; poly(I:C)) or single-stranded RNA [poly(U)), but HMGB2 does not [19]. HMGB3 binds both DNA and RNA [19]. HMGB1 participates in TLR9 signalling in response to CpG-B ODN, as activation of TLR9 is substantially decreased in macrophages and dendritic cells (DCs) deficient in HMGB1 [42]. Of note, HMGB1 binds to CpG-B ODN in vitro even more strongly than it does to DNAs and RNAs that activate cytosolic innate receptors. Strong binding of HMGB1 was also observed with poly(U), a well-known TLR7 agonist. On the other hand, HMGB1 does not bind to R837, another TLR7 agonist but not a nucleic acid. As such, a good correlation is seen between the affinity of a type of nucleic acid to HMGB and its immunogenicity [19, 20].
Contribution of HMGBs to the innate receptor pathways by nucleic acids Several in vitro and in vivo experiments demonstrated the role of HMGB proteins in cytosolic nucleic acid–mediated activation of innate immune responses. The mRNA induction for type I IFN, IL-6 and RANTES was significantly reduced when cells were cytosolically stimulated by B-DNA or poly(I:C) in mouse embryonic fibroblasts (MEFs) from HMGB1-deficient mice (Hmgb1−/− MEFs) [18, 19]. Whilst the induction of these mRNAs was also reduced in Hmgb2−/− MEFs upon stimulation by B-DNA, such reduction was not seen upon poly(I:C) stimulation, an observation consistent with HMGB2 binding DNA, but not RNA [19]. Further, HMGB2 is required for the activation of innate responses by IFN-stimulatory DNA–derived [43] or HSV-derived dsDNA [17]. Finally, in MEFs expressing an siRNA designed to interfere with the expression of HMGB1, 2 and 3 (referred to as pan-HMGB siRNA), the induction of
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these cytokine mRNAs upon cytosolic stimulation by B-DNA or poly(I:C) was more severely impaired as compared to their induction in MEFs lacking either HMGB1 or 2 [19]. Since the siRNA does not affect the induction of proinflammatory cytokine genes by lipopolysaccharide (LPS) stimulation in these cells, gene transcription is not generally affected. Further, activation of IRF3, NF-κB and extracellular signal-regulated kinase (ERK) are all suppressed upon B-DNA or poly(I:C) stimulation in these cells, collectively supporting the notion that HMGB proteins function upstream of nucleic acid–sensing cytosolic receptor signalling pathways, but not on gene transcription [19]. In macrophages, cytosolic DNA stimulation also induces the formation of the inflammasome through activation of AIM2, the DNA sensor that triggers the secretion of proinflammatory cytokines such as IL-1β [25]. Interestingly, the secretion of IL-1β in response to cytosolic B-DNA is also impaired in Hmgb1−/− macrophages or RAW264.7 cells expressing pan-HMGB siRNA [19]. Taken together, HMGBs are required for the full-blown activation of innate immune responses by these classes of cytosolic nucleic acid–sensing receptors.
As expected from the preceding observations, infection of MEFs expressing the pan-HMGB siRNA with RNA or DNA virus results in reduced type I IFN gene induction, causing higher levels of viral replication than in control cells [19]. These findings confirm that sensing of these nucleic acids by HMGBs is critical for mounting effective antiviral responses. Where and how HMGBs bind to virus-derived nucleic acids remain to be clarified. On the other hand, evidence exists that nuclear HMGBs assist with the replication of some viruses [44-46]. The requirement of HMGB1 for the activation of nucleic acid–sensing TLRs has also been demonstrated by mRNA induction of proinflammatory cytokines upon stimulation of TLR3 by poly(I:C) or TLR9 by CpG-B ODN being impaired in conventional DCs from
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Hmgb1−/− mice [19]. The induction of proinflammatory cytokine genes by these ligands is more severely undermined in RAW 264.7 cells expressing the pan-HMGB siRNA, which suggests that other HMGBs also participate in these TLR responses. In plasmacytoid DCs, a small subset of DCs that produce type I IFN at high levels via TLR7 or TLR9 signalling [47-49], type I IFN mRNA induction in response to TLR7 agonist poly(U) or TLR9 agonist CpG-A ODN is impaired in Hmgb1−/− plasmacytoid DCs, whereas the TLR7 response induced by the R837 agonist, which does not bind to HMGB proteins, remains normal [19]. Overall, HMGB1 and its members serve as universal sentinels via their promiscuous sensing for all immunogenic nucleic acids examined to achieve the full-blown nucleic acid–induced activation of innate immune responses mediated by the more discriminative innate receptors. In this context, it is interesting that T-cell immunoglobulin mucin-3 (TIM-3) interacts with HMGB1, interfering with the recruitment of nucleic acids into endosomes and subsequent nucleic acid–mediated immune responses, be they mediated by cytosolic receptors or TLRs [50].
Nucleic acid–bound HMGBs may collectively serve as essential partners or co-ligands for the entire nucleic acid–sensing innate receptors; the recognition by and activation of discriminative nucleic acid–sensing receptors are contingent on nucleic acids binding to HMGBs. Along this line, a physical association between HMGB1 and TLR9 has been demonstrated [42], and interaction of HMGB1 with RIG-I was observed by co-immunoprecipitation assay using cells that overexpressed RIG-I and HMGB1 upon cytosolic stimulation of the cells with nucleic acids (H.Y.; unpublished observation). In addition, HMGB2 has also been reported to interact with IFI204/IFI16 at the endoplasmic reticulum [17]. It remains to be further clarified where HMGBs interact with immunogenic nucleic acids and derivatives in the cell and how these proteins function in activating innate receptors.
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Suppression and implications of HMGB-mediated immune responses by decoy nucleotides Since nucleic acids are common to both the host and infectious microbes, the recognition of self versus nonself nucleic acids by innate receptors must maintain a delicate balance [36, 38]. Indeed, ample evidence exists for nucleic acid–mediated immune responses being involved in the animal models of autoimmune diseases and human diseases such as systemic lupus erythematosus [35-37, 39, 40, 51-53]. In view of the function of HMGB proteins described above, designing HMGB inhibitors for nucleic acid–mediated immune responses might be a useful therapeutic strategy to suppress these immunological diseases.
To explore this issue, an array of HMGB-binding non-immunogenic ODNs were examined. Binding affinities were found to be rather independent of nucleotide sequences and instead dependent on the length and structure of the deoxyribose backbone (phosphorothioate backbone instead of the usual phosphodiester backbone) [20]. One ODN, termed ISM ODN, was chosen for more detailed examination because it had high binding affinity to HMGB proteins but did not evoke innate immune responses [20]. ISM ODN pretreatment reduces the activation of innate immune responses by cytosolic stimulation with B-DNA or poly(I:C) based on type I IFN and proinflammatory cytokine gene induction. Further, a profoundly reduced TLR7- or TLR9-mediated innate response is also observed upon pretreatment with ISM ODN. As such, ISM ODN functions as a ‘decoy’ for the HMGB-mediated activation of nucleic acid–sensing innate receptors. STING acts as a receptor for the bacterial second messenger molecules c-di-AMP and c-di-GMP [31], which are potent inducers of type I IFNs and may function as major triggers of type I IFN production in macrophages [33]. STING directly binds to these molecules and to cGAMP, an endogenous second messenger generated in response to DNA, to trigger the
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IRF3-mediated induction of IFN-β in response to transfected DNA or DNA viruses [26, 29]. DDX41 also serves as a PRR involved in detecting c-di-GMP and c-di-AMP to trigger TBK1-IRF3 signalling via STING [32]. In this context, it is interesting that ISM ODN also interferes with type I IFN gene induction by these second messengers (Yanai, H.; unpublished data). These observations suggest but do not prove that HMGB1 and family members also bind to these second messengers.
The in vivo immunosuppressive role of ISM ODN has also been investigated. ISM ODN treatment of mice suppressed the induction of adaptive immune responses instigated by B-DNA or CpG-A. It also suppressed the development of experimental autoimmune encephalomyelitis [20], an animal autoimmune demyelinating disease model of human multiple sclerosis exacerbated by nucleic acid–sensing TLRs [54, 55]. Interestingly, LPS-mediated death by endotoxin shock is also suppressed by ISM ODN, accompanied with a reduction of proinflammatory cytokines such as TNF-α and IL-6 in the serum as compared to control mice [20]. One may speculate that ISM ODN treatment affects LPS-TLR4 interaction by inhibiting the cytokine action of secreted HMGB1 [4] through binding to its B box DNA binding domain, which is critical for its inflammatory function [56]. Alternatively, ISM ODN may inhibit the association between HMGB1 and LPS, which strongly activates TLR4 [6, 20, 57]. Lastly, since HMGB1 plays a critical role in dead cells activating the immune system [5, 6, 58], an intriguing possibility is that HMGB1 released by dead cells upon LPS stimulation may be bound by nucleic acids and may enervate some aspect or aspects of the LPS-mediated inflammatory reaction in vivo. Obviously, further study is required to clarify how ISM ODN functions in the in vivo LPS response.
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HMGB and nucleic acids in immunity: some speculations Studies on the role of HMGB1 (and its family) in nucleic acid sensing suggest that under normal physiological conditions chronic cell death in vivo results in self nucleic acid–bound HMGB1 being released, albeit at levels much lower than those that occur during infection or inflammation, and this complex may keep the immune system in a weakly activated state. Although seemingly futile, this weak, steady-state activation may serve as a “ready-to-go” state that is necessary for mounting rapid and robust immune responses upon encounter with pathogens [59, 60]. In this regard, it is interesting that type I IFN genes are constitutively expressed in normal individuals [61] and that weak production of type I IFN is critical for type I IFN and other cytokines to mount robust response [60]. We can speculate that these type I IFNs are, at least in part, produced in response to self nucleic acid–bound HMGB1.
When enhanced cell death occurs locally (e.g. by injury), levels of HMGB1 bound with self nucleic acid, in addition to free HMGB1, may increase and function as danger-associated molecular patterns that enhance the state of immune cell activation [5]. Although this immune system priming could be critical for resolving a local challenge to the host, sustained and/or uncontrolled cell death accompanied by defects in nucleic acid metabolism may render HMGB1/nucleic acid complex–mediated immune activation pathogenic, likely by triggering a type I IFN response, causing autoimmunity [52, 62]. In this speculative scenario, the discrimination of self and nonself may be a matter of quantity; that is, ‘increased self’ may be the foundation of pathogenesis. Upon infections occurring, however, pathogen-derived, nonself nucleic acids bind HMGB proteins with greater affinity compared to self nucleic acids and thereby evoke a more robust immune response to control the infection. This type of response is, however, transient and returns to steady-state when the immune system resolves the infection.
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In this sense, HMGBs may serve as a master switch from self to nonself recognition of nucleic acids. Obviously, these speculations require further elaboration and consolidation.
Progress on in vivo roles of HMGB1 by conditional gene ablation in mice Although numerous studies have revealed many of HMGB1’s biological functions, study of this protein in vivo has been hampered by the prematureearly death of conventional Hmgb1-deficient mice due to hypoglycaemia [63]. Recently, several groups, including ours, have generated HMGB1 conditional knockout mice, enabling investigators to ablate HMGB1 expression in a manner specific to the cell type or tissue [9, 64-67] (see also Table 1). Although the lethality of conventional Hmgb1-deficient mice has been suggested to result from defects in hepatocytes, hepatic HMGB1–deleted mice grow and develop normally [64, 65, 67], and dexamethasone-induced hepatic gene expression profiles appear to remain unaffected [65]. Therefore, the lethality of conventional Hmgb1-deficient mice may arise from other defects such as reduced autophagy [9], a critical cellular response for survival during ontogeny [68]. Indeed, several reports have shown that HMGB1 regulates autophagy via a direct interaction with autophagy-related protein Beclin-1, a Bcl-2–homology (BH)-3 domain-only protein that is essential for this process [14, 69]. On the other hand, HMGB1 may not be required for autophagy in the liver and heart [65], and the cause of death for conventional Hmgb1-deficient mice needs further clarification. Studies on the conditional Hmgb1 knockout mice have revealed an unexpected protective role of cytosolic HMGB1 in the host defence against inflammation and bacterial infection [64, 66, 67]. Mice with HMGB1 ablation in myeloid cells grow normally and show no abnormalities in myeloid cell populations; however, they are vulnerable to LPS-induced endotoxin shock and Listeria monocytogenes infection [67, 70]. Although myeloid cells are
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known to release HMGB1 en masse upon LPS stimulation [4, 71], plasma HMGB1 levels are only marginally affected in myeloid cells after LPS stimulation [67]. Thus, the increased vulnerability to LPS shock reflects a function of intracellular HMGB1 rather than extracellular HMGB1. In mutant mice with targeted HMGB1 ablation in myeloid cells, IL-1β and IL-18 production in plasma was increased whilst TNF-α, IL-6 and IL-12p40 production remained unaffected. Intracellular HMGB1 in myeloid cells might act as a negative regulator for inflammasome and caspase-1 activation resulting in the maturation of IL-1β and IL-18 [72, 73]. In this context, LPS-induced autophagosome formation was significantly decreased in Hmgb1-deficient macrophages [67]. Since previous reports indicate that autophagy negatively regulates LPS-induced inflammasome activation [74-76], HMGB1-promoted autophagosome formation might be involved in the regulatory pathway of inflammasome and partly suppress excessive inflammation (Fig. 2). Interestingly, decreased autophagosome formation was also observed in Hmgb1-deficient macrophages infected with L. monocytogenes, whilst mutant mice succumbed to normally sublethal doses of L. monocytogenes [67]. Since autophagy is required for the effective clearance of intracellular bacteria [77], cytosolic HMGB1-mediated activation of autophagy might be a critical component for bacterial killing (Fig. 2). Although the precise mechanism for how HMGB1 regulates autophagy in response to LPS stimulation or bacterial infection remains to be clarified further, these observations offer in vivo evidence for the protective aspect of cytosolic HMGB1 against endotoxaemia and bacterial infection. A protective function for intracellular HMGB1 has also been reported in acute pancreatitis (AP) and liver I/R models [64, 66]. AP is an inflammatory disease that causes human morbidity and mortality [78]. In patients with AP, serum levels of HMGB1 are significantly increased and correlate with disease severity [79, 80]. Mice with pancreas-specific ablation of Hmgb1 showed severe tissue injury with high levels of serum amylase, acinar cell death, leukocyte infiltration, interstitial oedema and high lethality to experimental AP induced
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by L-arginine or cerulein injection [66]. The enhanced severity was associated with increased nuclear catastrophe represented by phosphorylation of γ-H2AX, a specific marker for DNA damage, and massive cell death. Cell death releases nucleosomal proteins, such as histones H3 and H4, along with DNA, which leads to subsequent activation of innate immune effecter cells, and neutralizing extracellular histones have been shown to confer protection against AP [66]. As such, intracellular HMGB1 appears to serve as a negative regulator of inflammation-limiting nuclear catastrophe after AP injury with resultant release of inflammatory nucleosomes.
HMGB1 may also function in the inflammation of hepatic I/R. HMGB1 is rapidly mobilized from the nucleus to the cytosol and is released into the extracellular space in response to hepatic I/R [81, 82]. HMGB1 released from hepatocytes is thought to act as a danger-associated molecular pattern [81, 82]. In this context, mice with hepatocyte-specific ablation of Hmgb1 were subjected to a nonlethal warm I/R challenge [64]. Interestingly, deletion of HMGB1 in hepatocytes greatly exacerbated hepatic I/R injury. Mutant mice showed high serum titres of sALT, TNF-α and IL-6; massive activation of MAPKs and NF-κB; and increased recruitment of inflammatory cells. The lack of HMGB1 leads to several pathological responses upon I/R or oxidative stress, including 1) increased DNA damage and decreased chromatin accessibility to repair; 2) excessive nuclear instability and release of nucleosome components leading to activation of innate immune responses through TLR9; and 3) increase and activation of the nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1), leading to decreased energy stores [83] and subsequent mitochondrial injury, reactive oxygen species (ROS) production, and cell death [84]. These responses accelerate the massive inflammation of I/R injury. Since HMGB1 is essential for mitochondrial quality control and loss of HMGB1 leads to excessive ROS production [85], which in turn induces DNA damage [86], HMGB1 may suppress I/R-induced inflammation at the level of mitochondrial quality control processes.
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Collectively, the preceding findings obtained through the study of conditional HMGB1 knockout mice reveal several hitherto unidentified protective roles of HMGB1 in inflammation and infection. The conditional knockout mice will continue to be a valuable tool in continued study of the in vivo functions of HMGB1. Nevertheless, a potential caveat is the difficulty in distinguishing where HMGB1 functions (intracellular versus extracellular) in these mice. For example, the LPS experiments show that myeloid cell–derived HMGB1 is crucial for the survival of the mice [67], but in terms of the phenotype, this seems to conflict with the observations that anti-HMGB1 antibody protects against LPS-induced shock [4, 5, 87]. The function of intracellular HMGB1 likely dominates over that of extracellular HMGB1 for certain processes, thereby masking the role of this protein in the extracellular milieu during endotoxin shock, for example. Thus, although these mice will be useful, a careful approach and interpretation are required in future studies on this multifunctional protein.
CONFLICT OF INTEREST STATEMENT None of the authors has any conflicts of interest to declare.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant-In-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST). The Department of Molecular Immunology is supported by BONAC Corporation and Kyowa Hakko Kirin Co., Ltd.
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Figure 1. Extracellular interaction of HMGB1 on a variety of cell-surface receptors. HMGB1 is released thorough multiple pathways. The translocation can
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represent an active process in immune cells such as macrophages upon pathogen infections or by a variety of stimuli, such as LPS. HMGB1 is also passively released during primary or secondary necrosis. Extracellular HMGB1 activates multiple signalling pathways via TLR2, TLR4, RAGE and CD24. Association of HMGB1 with TLRs evokes MyD88-dependent NF-κB activation and induces maturation of DCs and production of proinflammatory cytokines and chemokines such as TNF-α, IL-6, IL-1 and MIP-1. HMGB1 is also recognized by RAGE, and this binding has been implicated in cell growth, autophagy, differentiation and migration of inflammatory cells such as neutrophils and monocytes. On the other hand, HMGB1 bound to CD24 and Siglec-10 impedes proinflammatory cytokine release induced by HMGB1-TLR4 signalling. Thrombomodulin (TM) sequesters HMGB1 from TLRs and RAGE and promotes proteolytic cleavage of HMGB1 by thrombin (upper panel). HMGB1 also makes highly immunostimulatory complexes with cytokines and other molecules, such as LPS, Pam3CSK4, IL-1β, nucleosomes, CXCL12 and nucleic acids [5, 6, 8, 10] (lower panel). These complexes stimulate immune receptors such as TLRs, RAGE, CXCR4 and others and evoke robust immune responses [5, 6, 8, 10].
Figure 2. Schematic illustration of HMGB1-mediated activation of autophagy. Activation of TLR signalling and infection by pathogens cause posttranslational modification of HMGB1 such as acetylation, methylation, phosphorylation and oxidation [10, 14]. This modification enables HMGB1 translocation from the nucleus into the cytoplasm. Then, cytoplasmic HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2 and promotes autophagy [9, 13, 14]. Activation of autophagy eliminates intracellular pathogens [77], including L. monocytogenes, suppresses inflammasome activation [74-76, 88] and exhibits other functions.
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Received Date : 25-Mar-2014 Revised Date
: 03-Jun-2014
Accepted Date : 09-Jun-2014 Article type
: Review-Symposium
Nucleic acid sensing and beyond: virtues and vices of HMGB1
Hideyuki Yanai1,2 & Tadatsugu Taniguchi1,2
From the 1Department of Molecular Immunology, Institute of Industrial Science, Max Planck-The University of Tokyo Center for Integrative Inflammology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8505, Japan, and 2Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan
Correspondence: Tadatsugu Taniguchi (e-mail: [email protected]); .Komaba 4-6-1, Meguro-ku, Tokyo 153-8505, Japan
Abstract High-mobility group box 1 (HMGB1) was first described as an architectural chromatin-binding protein. Today, a wealth of evidence indicates that this protein is very versatile and serves an amazing assortment of roles in the nucleus, cytoplasm and extracellular milieu. As a result,
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/joim.12285 This article is protected by copyright. All rights reserved.
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HMGB1 is fast becoming recognized as a key regulator of protective and pathological immune responses. While acknowledging the many functions of HMGB1 and its family members, we focus this review on their role as broad effectors of immune responses mediated by nucleic acids. In addition, we touch upon the recent progress in determining the in vivo role of HMGB1 as revealed by the study of mice conditionally null for the Hmgb1 gene.
Keywords: nucleic acids, HMGB1, infection, inflammation, autophagy, sepsis.
Introduction
HMGB1 is an evolutionarily conserved protein that is abundantly present in the nucleus of almost all eukaryotic cells. It was long thought that this protein to simply functions as a nonhistone chromatin-binding protein, stabilizing chromatin structure and modulating gene transcription along with other HMGB family members such as HMGB2, 3 and 4, whose expression is more restricted to particular cell types [1, 2]. A more versatile and perhaps more interesting role of HMGB1 stems from its function as a factor promoting regeneration of neurite-type cytoplasmic processes in brain neurons [3]. Further, pioneering work characterizing HMGB1 as a critical mediator of endotoxin lethality [4] has opened a new avenue of research on the remarkable functional versatility of the protein in evoking inflammatory responses [5-10]. Extracellular HMGB1 has proinflammatory cytokine-like and chemo-attractant functions, which are mediated by its interaction with several receptors and partner proteins, such as Toll-like receptors (TLRs), RAGE (receptor for advanced glycation end product), and the chemokine CXCL12 that, paired with HMGB1, binds CXCR4 chemokine receptor [5-8, 10-12]
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(Fig. 1). An intracellular function of HMGB1 has also been reported; specifically, the protein appears to have a role in autophagy, a cellular response closely linked with immunity and cancer [13, 14]. Collectively, these studies place HMGB1 at the centre of research on immunity and inflammation [5]. Of particular note, the versatility of HMGB1 is controlled by its modifications, such as acetylation [15] and redox states [16]. Indeed, the biological and clinical importance of HMGB1 is underscored by this protein being involved in numerous pathological conditions, including sepsis, ischemia–reperfusion (I/R) injury, arthritis, and cancer [5-10]. Furthermore, HMGB research has advanced in yet another direction: HMGB1 and its family members are sentinels for immunogenic nucleic acids that work in conjunction with signal-transducing, nucleic acid–sensing innate receptors [17-20]. Many excellent reviews already exist on HMGB1 [5-10], so we shall refrain from tallying the multiple functions of this protein, but rather focus on its role in nucleic acid sensing and recent data generated from the study of conditional knockout mice.
Sensing of nucleic acids by innate receptors Nucleic acids derived from infected pathogens or dead cells can activate immune responses, and indeed, multiple innate receptors have evolved to recognize RNA and DNA in mammals. These include TLRs, retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and several cytosolic DNA sensors. In particular, TLR3, TLR7 and TLR9 are present in endosomes where they sense double-stranded RNA (dsRNA), single-stranded RNA (ssRNA)
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and short dsRNA and hypomethylated CpG-motif–containing oligodeoxynucleotides (CpG ODNs), respectively [21, 22]. RLRs, namely RIG-I, melanoma differentiation associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), are best known as RNA-sensing receptors in the cytosol [23]. Numerous cytosolic DNA-sensing receptors also trigger the innate immune system, and these include RLRs, stimulator of interferon genes (STING), DNA-dependent activator of interferon (IFN) regulatory factors (IRFs), cyclic CMP-AMP (cGAMP) synthase (cGAS), absent in melanoma 2 (AIM2), IFN-inducible protein 16 (IFI16)/IFN activated gene 204 (IFI204), and (Asp-Glu-Ala-Asp) (DEAD) box polypeptide 41 (DDX41), when DNA is exposed to the cytosol [21, 24-30]. More recently, it has been shown that cyclic dinucleotides activate DNA-sensing receptors. Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) produced by bacteria are sensed by DDX41 and STING [31-33]. Furthermore, cGAMP, synthesized by the DNA sensor cGAS in response to cytosolically exposed DNA, activates the STING signalling pathway [26, 29]. A hallmark of the innate immune response activated by these nucleic acid–sensing pattern recognition receptors (PRRs) is the induction of type I IFN [21, 34], proinflammatory cytokines and chemokines, with the exception of AIM2, which instead activates the DNA-mediated inflammasome pathway [25]. Several diseases are linked to the aberrant activation of the nucleic acid–sensing system, most notably autoimmunity [35-40].
Binding of HMGB1 and its family members to nucleic acids and their analogues HMGB1 and its family members were identified as being involved in cytosolic DNA recognition because these proteins could bind to a synthetic DNA, poly(dA-dT)·poly(dT-dA) (B-DNA hereafter), that potently activates immune responses when delivered to the cytosol [41]. Recombinant HMGBs bind to various types of nucleic acids to activate innate responses in vitro, and immune activation by these nucleic acids correlates with their binding affinity to
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HMGBs. Indeed, both HMGB1 and HMGB2 bind strongly to the highly immunogenic B-DNA, but weakly to DNA derived from calf thymus or bacteria, which only weakly activates an immune response. HMGB1 also binds with high affinities to immunogenic double-stranded RNA [polyinosinic-polycytidylic acid; poly(I:C)) or single-stranded RNA [poly(U)), but HMGB2 does not [19]. HMGB3 binds both DNA and RNA [19]. HMGB1 participates in TLR9 signalling in response to CpG-B ODN, as activation of TLR9 is substantially decreased in macrophages and dendritic cells (DCs) deficient in HMGB1 [42]. Of note, HMGB1 binds to CpG-B ODN in vitro even more strongly than it does to DNAs and RNAs that activate cytosolic innate receptors. Strong binding of HMGB1 was also observed with poly(U), a well-known TLR7 agonist. On the other hand, HMGB1 does not bind to R837, another TLR7 agonist but not a nucleic acid. As such, a good correlation is seen between the affinity of a type of nucleic acid to HMGB and its immunogenicity [19, 20].
Contribution of HMGBs to the innate receptor pathways by nucleic acids Several in vitro and in vivo experiments demonstrated the role of HMGB proteins in cytosolic nucleic acid–mediated activation of innate immune responses. The mRNA induction for type I IFN, IL-6 and RANTES was significantly reduced when cells were cytosolically stimulated by B-DNA or poly(I:C) in mouse embryonic fibroblasts (MEFs) from HMGB1-deficient mice (Hmgb1−/− MEFs) [18, 19]. Whilst the induction of these mRNAs was also reduced in Hmgb2−/− MEFs upon stimulation by B-DNA, such reduction was not seen upon poly(I:C) stimulation, an observation consistent with HMGB2 binding DNA, but not RNA [19]. Further, HMGB2 is required for the activation of innate responses by IFN-stimulatory DNA–derived [43] or HSV-derived dsDNA [17]. Finally, in MEFs expressing an siRNA designed to interfere with the expression of HMGB1, 2 and 3 (referred to as pan-HMGB siRNA), the induction of
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these cytokine mRNAs upon cytosolic stimulation by B-DNA or poly(I:C) was more severely impaired as compared to their induction in MEFs lacking either HMGB1 or 2 [19]. Since the siRNA does not affect the induction of proinflammatory cytokine genes by lipopolysaccharide (LPS) stimulation in these cells, gene transcription is not generally affected. Further, activation of IRF3, NF-κB and extracellular signal-regulated kinase (ERK) are all suppressed upon B-DNA or poly(I:C) stimulation in these cells, collectively supporting the notion that HMGB proteins function upstream of nucleic acid–sensing cytosolic receptor signalling pathways, but not on gene transcription [19]. In macrophages, cytosolic DNA stimulation also induces the formation of the inflammasome through activation of AIM2, the DNA sensor that triggers the secretion of proinflammatory cytokines such as IL-1β [25]. Interestingly, the secretion of IL-1β in response to cytosolic B-DNA is also impaired in Hmgb1−/− macrophages or RAW264.7 cells expressing pan-HMGB siRNA [19]. Taken together, HMGBs are required for the full-blown activation of innate immune responses by these classes of cytosolic nucleic acid–sensing receptors.
As expected from the preceding observations, infection of MEFs expressing the pan-HMGB siRNA with RNA or DNA virus results in reduced type I IFN gene induction, causing higher levels of viral replication than in control cells [19]. These findings confirm that sensing of these nucleic acids by HMGBs is critical for mounting effective antiviral responses. Where and how HMGBs bind to virus-derived nucleic acids remain to be clarified. On the other hand, evidence exists that nuclear HMGBs assist with the replication of some viruses [44-46]. The requirement of HMGB1 for the activation of nucleic acid–sensing TLRs has also been demonstrated by mRNA induction of proinflammatory cytokines upon stimulation of TLR3 by poly(I:C) or TLR9 by CpG-B ODN being impaired in conventional DCs from
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Hmgb1−/− mice [19]. The induction of proinflammatory cytokine genes by these ligands is more severely undermined in RAW 264.7 cells expressing the pan-HMGB siRNA, which suggests that other HMGBs also participate in these TLR responses. In plasmacytoid DCs, a small subset of DCs that produce type I IFN at high levels via TLR7 or TLR9 signalling [47-49], type I IFN mRNA induction in response to TLR7 agonist poly(U) or TLR9 agonist CpG-A ODN is impaired in Hmgb1−/− plasmacytoid DCs, whereas the TLR7 response induced by the R837 agonist, which does not bind to HMGB proteins, remains normal [19]. Overall, HMGB1 and its members serve as universal sentinels via their promiscuous sensing for all immunogenic nucleic acids examined to achieve the full-blown nucleic acid–induced activation of innate immune responses mediated by the more discriminative innate receptors. In this context, it is interesting that T-cell immunoglobulin mucin-3 (TIM-3) interacts with HMGB1, interfering with the recruitment of nucleic acids into endosomes and subsequent nucleic acid–mediated immune responses, be they mediated by cytosolic receptors or TLRs [50].
Nucleic acid–bound HMGBs may collectively serve as essential partners or co-ligands for the entire nucleic acid–sensing innate receptors; the recognition by and activation of discriminative nucleic acid–sensing receptors are contingent on nucleic acids binding to HMGBs. Along this line, a physical association between HMGB1 and TLR9 has been demonstrated [42], and interaction of HMGB1 with RIG-I was observed by co-immunoprecipitation assay using cells that overexpressed RIG-I and HMGB1 upon cytosolic stimulation of the cells with nucleic acids (H.Y.; unpublished observation). In addition, HMGB2 has also been reported to interact with IFI204/IFI16 at the endoplasmic reticulum [17]. It remains to be further clarified where HMGBs interact with immunogenic nucleic acids and derivatives in the cell and how these proteins function in activating innate receptors.
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Suppression and implications of HMGB-mediated immune responses by decoy nucleotides Since nucleic acids are common to both the host and infectious microbes, the recognition of self versus nonself nucleic acids by innate receptors must maintain a delicate balance [36, 38]. Indeed, ample evidence exists for nucleic acid–mediated immune responses being involved in the animal models of autoimmune diseases and human diseases such as systemic lupus erythematosus [35-37, 39, 40, 51-53]. In view of the function of HMGB proteins described above, designing HMGB inhibitors for nucleic acid–mediated immune responses might be a useful therapeutic strategy to suppress these immunological diseases.
To explore this issue, an array of HMGB-binding non-immunogenic ODNs were examined. Binding affinities were found to be rather independent of nucleotide sequences and instead dependent on the length and structure of the deoxyribose backbone (phosphorothioate backbone instead of the usual phosphodiester backbone) [20]. One ODN, termed ISM ODN, was chosen for more detailed examination because it had high binding affinity to HMGB proteins but did not evoke innate immune responses [20]. ISM ODN pretreatment reduces the activation of innate immune responses by cytosolic stimulation with B-DNA or poly(I:C) based on type I IFN and proinflammatory cytokine gene induction. Further, a profoundly reduced TLR7- or TLR9-mediated innate response is also observed upon pretreatment with ISM ODN. As such, ISM ODN functions as a ‘decoy’ for the HMGB-mediated activation of nucleic acid–sensing innate receptors. STING acts as a receptor for the bacterial second messenger molecules c-di-AMP and c-di-GMP [31], which are potent inducers of type I IFNs and may function as major triggers of type I IFN production in macrophages [33]. STING directly binds to these molecules and to cGAMP, an endogenous second messenger generated in response to DNA, to trigger the
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IRF3-mediated induction of IFN-β in response to transfected DNA or DNA viruses [26, 29]. DDX41 also serves as a PRR involved in detecting c-di-GMP and c-di-AMP to trigger TBK1-IRF3 signalling via STING [32]. In this context, it is interesting that ISM ODN also interferes with type I IFN gene induction by these second messengers (Yanai, H.; unpublished data). These observations suggest but do not prove that HMGB1 and family members also bind to these second messengers.
The in vivo immunosuppressive role of ISM ODN has also been investigated. ISM ODN treatment of mice suppressed the induction of adaptive immune responses instigated by B-DNA or CpG-A. It also suppressed the development of experimental autoimmune encephalomyelitis [20], an animal autoimmune demyelinating disease model of human multiple sclerosis exacerbated by nucleic acid–sensing TLRs [54, 55]. Interestingly, LPS-mediated death by endotoxin shock is also suppressed by ISM ODN, accompanied with a reduction of proinflammatory cytokines such as TNF-α and IL-6 in the serum as compared to control mice [20]. One may speculate that ISM ODN treatment affects LPS-TLR4 interaction by inhibiting the cytokine action of secreted HMGB1 [4] through binding to its B box DNA binding domain, which is critical for its inflammatory function [56]. Alternatively, ISM ODN may inhibit the association between HMGB1 and LPS, which strongly activates TLR4 [6, 20, 57]. Lastly, since HMGB1 plays a critical role in dead cells activating the immune system [5, 6, 58], an intriguing possibility is that HMGB1 released by dead cells upon LPS stimulation may be bound by nucleic acids and may enervate some aspect or aspects of the LPS-mediated inflammatory reaction in vivo. Obviously, further study is required to clarify how ISM ODN functions in the in vivo LPS response.
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HMGB and nucleic acids in immunity: some speculations Studies on the role of HMGB1 (and its family) in nucleic acid sensing suggest that under normal physiological conditions chronic cell death in vivo results in self nucleic acid–bound HMGB1 being released, albeit at levels much lower than those that occur during infection or inflammation, and this complex may keep the immune system in a weakly activated state. Although seemingly futile, this weak, steady-state activation may serve as a “ready-to-go” state that is necessary for mounting rapid and robust immune responses upon encounter with pathogens [59, 60]. In this regard, it is interesting that type I IFN genes are constitutively expressed in normal individuals [61] and that weak production of type I IFN is critical for type I IFN and other cytokines to mount robust response [60]. We can speculate that these type I IFNs are, at least in part, produced in response to self nucleic acid–bound HMGB1.
When enhanced cell death occurs locally (e.g. by injury), levels of HMGB1 bound with self nucleic acid, in addition to free HMGB1, may increase and function as danger-associated molecular patterns that enhance the state of immune cell activation [5]. Although this immune system priming could be critical for resolving a local challenge to the host, sustained and/or uncontrolled cell death accompanied by defects in nucleic acid metabolism may render HMGB1/nucleic acid complex–mediated immune activation pathogenic, likely by triggering a type I IFN response, causing autoimmunity [52, 62]. In this speculative scenario, the discrimination of self and nonself may be a matter of quantity; that is, ‘increased self’ may be the foundation of pathogenesis. Upon infections occurring, however, pathogen-derived, nonself nucleic acids bind HMGB proteins with greater affinity compared to self nucleic acids and thereby evoke a more robust immune response to control the infection. This type of response is, however, transient and returns to steady-state when the immune system resolves the infection.
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In this sense, HMGBs may serve as a master switch from self to nonself recognition of nucleic acids. Obviously, these speculations require further elaboration and consolidation.
Progress on in vivo roles of HMGB1 by conditional gene ablation in mice Although numerous studies have revealed many of HMGB1’s biological functions, study of this protein in vivo has been hampered by the prematureearly death of conventional Hmgb1-deficient mice due to hypoglycaemia [63]. Recently, several groups, including ours, have generated HMGB1 conditional knockout mice, enabling investigators to ablate HMGB1 expression in a manner specific to the cell type or tissue [9, 64-67] (see also Table 1). Although the lethality of conventional Hmgb1-deficient mice has been suggested to result from defects in hepatocytes, hepatic HMGB1–deleted mice grow and develop normally [64, 65, 67], and dexamethasone-induced hepatic gene expression profiles appear to remain unaffected [65]. Therefore, the lethality of conventional Hmgb1-deficient mice may arise from other defects such as reduced autophagy [9], a critical cellular response for survival during ontogeny [68]. Indeed, several reports have shown that HMGB1 regulates autophagy via a direct interaction with autophagy-related protein Beclin-1, a Bcl-2–homology (BH)-3 domain-only protein that is essential for this process [14, 69]. On the other hand, HMGB1 may not be required for autophagy in the liver and heart [65], and the cause of death for conventional Hmgb1-deficient mice needs further clarification. Studies on the conditional Hmgb1 knockout mice have revealed an unexpected protective role of cytosolic HMGB1 in the host defence against inflammation and bacterial infection [64, 66, 67]. Mice with HMGB1 ablation in myeloid cells grow normally and show no abnormalities in myeloid cell populations; however, they are vulnerable to LPS-induced endotoxin shock and Listeria monocytogenes infection [67, 70]. Although myeloid cells are
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known to release HMGB1 en masse upon LPS stimulation [4, 71], plasma HMGB1 levels are only marginally affected in myeloid cells after LPS stimulation [67]. Thus, the increased vulnerability to LPS shock reflects a function of intracellular HMGB1 rather than extracellular HMGB1. In mutant mice with targeted HMGB1 ablation in myeloid cells, IL-1β and IL-18 production in plasma was increased whilst TNF-α, IL-6 and IL-12p40 production remained unaffected. Intracellular HMGB1 in myeloid cells might act as a negative regulator for inflammasome and caspase-1 activation resulting in the maturation of IL-1β and IL-18 [72, 73]. In this context, LPS-induced autophagosome formation was significantly decreased in Hmgb1-deficient macrophages [67]. Since previous reports indicate that autophagy negatively regulates LPS-induced inflammasome activation [74-76], HMGB1-promoted autophagosome formation might be involved in the regulatory pathway of inflammasome and partly suppress excessive inflammation (Fig. 2). Interestingly, decreased autophagosome formation was also observed in Hmgb1-deficient macrophages infected with L. monocytogenes, whilst mutant mice succumbed to normally sublethal doses of L. monocytogenes [67]. Since autophagy is required for the effective clearance of intracellular bacteria [77], cytosolic HMGB1-mediated activation of autophagy might be a critical component for bacterial killing (Fig. 2). Although the precise mechanism for how HMGB1 regulates autophagy in response to LPS stimulation or bacterial infection remains to be clarified further, these observations offer in vivo evidence for the protective aspect of cytosolic HMGB1 against endotoxaemia and bacterial infection. A protective function for intracellular HMGB1 has also been reported in acute pancreatitis (AP) and liver I/R models [64, 66]. AP is an inflammatory disease that causes human morbidity and mortality [78]. In patients with AP, serum levels of HMGB1 are significantly increased and correlate with disease severity [79, 80]. Mice with pancreas-specific ablation of Hmgb1 showed severe tissue injury with high levels of serum amylase, acinar cell death, leukocyte infiltration, interstitial oedema and high lethality to experimental AP induced
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by L-arginine or cerulein injection [66]. The enhanced severity was associated with increased nuclear catastrophe represented by phosphorylation of γ-H2AX, a specific marker for DNA damage, and massive cell death. Cell death releases nucleosomal proteins, such as histones H3 and H4, along with DNA, which leads to subsequent activation of innate immune effecter cells, and neutralizing extracellular histones have been shown to confer protection against AP [66]. As such, intracellular HMGB1 appears to serve as a negative regulator of inflammation-limiting nuclear catastrophe after AP injury with resultant release of inflammatory nucleosomes.
HMGB1 may also function in the inflammation of hepatic I/R. HMGB1 is rapidly mobilized from the nucleus to the cytosol and is released into the extracellular space in response to hepatic I/R [81, 82]. HMGB1 released from hepatocytes is thought to act as a danger-associated molecular pattern [81, 82]. In this context, mice with hepatocyte-specific ablation of Hmgb1 were subjected to a nonlethal warm I/R challenge [64]. Interestingly, deletion of HMGB1 in hepatocytes greatly exacerbated hepatic I/R injury. Mutant mice showed high serum titres of sALT, TNF-α and IL-6; massive activation of MAPKs and NF-κB; and increased recruitment of inflammatory cells. The lack of HMGB1 leads to several pathological responses upon I/R or oxidative stress, including 1) increased DNA damage and decreased chromatin accessibility to repair; 2) excessive nuclear instability and release of nucleosome components leading to activation of innate immune responses through TLR9; and 3) increase and activation of the nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1), leading to decreased energy stores [83] and subsequent mitochondrial injury, reactive oxygen species (ROS) production, and cell death [84]. These responses accelerate the massive inflammation of I/R injury. Since HMGB1 is essential for mitochondrial quality control and loss of HMGB1 leads to excessive ROS production [85], which in turn induces DNA damage [86], HMGB1 may suppress I/R-induced inflammation at the level of mitochondrial quality control processes.
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Collectively, the preceding findings obtained through the study of conditional HMGB1 knockout mice reveal several hitherto unidentified protective roles of HMGB1 in inflammation and infection. The conditional knockout mice will continue to be a valuable tool in continued study of the in vivo functions of HMGB1. Nevertheless, a potential caveat is the difficulty in distinguishing where HMGB1 functions (intracellular versus extracellular) in these mice. For example, the LPS experiments show that myeloid cell–derived HMGB1 is crucial for the survival of the mice [67], but in terms of the phenotype, this seems to conflict with the observations that anti-HMGB1 antibody protects against LPS-induced shock [4, 5, 87]. The function of intracellular HMGB1 likely dominates over that of extracellular HMGB1 for certain processes, thereby masking the role of this protein in the extracellular milieu during endotoxin shock, for example. Thus, although these mice will be useful, a careful approach and interpretation are required in future studies on this multifunctional protein.
CONFLICT OF INTEREST STATEMENT None of the authors has any conflicts of interest to declare.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant-In-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST). The Department of Molecular Immunology is supported by BONAC Corporation and Kyowa Hakko Kirin Co., Ltd.
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Figure 1. Extracellular interaction of HMGB1 on a variety of cell-surface receptors. HMGB1 is released thorough multiple pathways. The translocation can
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represent an active process in immune cells such as macrophages upon pathogen infections or by a variety of stimuli, such as LPS. HMGB1 is also passively released during primary or secondary necrosis. Extracellular HMGB1 activates multiple signalling pathways via TLR2, TLR4, RAGE and CD24. Association of HMGB1 with TLRs evokes MyD88-dependent NF-κB activation and induces maturation of DCs and production of proinflammatory cytokines and chemokines such as TNF-α, IL-6, IL-1 and MIP-1. HMGB1 is also recognized by RAGE, and this binding has been implicated in cell growth, autophagy, differentiation and migration of inflammatory cells such as neutrophils and monocytes. On the other hand, HMGB1 bound to CD24 and Siglec-10 impedes proinflammatory cytokine release induced by HMGB1-TLR4 signalling. Thrombomodulin (TM) sequesters HMGB1 from TLRs and RAGE and promotes proteolytic cleavage of HMGB1 by thrombin (upper panel). HMGB1 also makes highly immunostimulatory complexes with cytokines and other molecules, such as LPS, Pam3CSK4, IL-1β, nucleosomes, CXCL12 and nucleic acids [5, 6, 8, 10] (lower panel). These complexes stimulate immune receptors such as TLRs, RAGE, CXCR4 and others and evoke robust immune responses [5, 6, 8, 10].
Figure 2. Schematic illustration of HMGB1-mediated activation of autophagy. Activation of TLR signalling and infection by pathogens cause posttranslational modification of HMGB1 such as acetylation, methylation, phosphorylation and oxidation [10, 14]. This modification enables HMGB1 translocation from the nucleus into the cytoplasm. Then, cytoplasmic HMGB1 directly interacts with the autophagy protein Beclin1 displacing Bcl-2 and promotes autophagy [9, 13, 14]. Activation of autophagy eliminates intracellular pathogens [77], including L. monocytogenes, suppresses inflammasome activation [74-76, 88] and exhibits other functions.
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