The role of HMGB2 in the immune response of Nile tilapia (Oreochromis niloticus) to streptococcal infection.

High mobility group protein B2 (HMGB2) is an abundant chromatin-associated protein with pivotal roles in transcription, cell proliferation, differentiation, inflammation, and tumorigenesis. However, its immune function in Nile tilapia (Oreochromis niloticus) remains unclear. In this study, we identified a homologue of HMGB2 from Nile tilapia (On-HMGB2) and investigated its functions in the immune response against streptococcus infection. The open reading frame (ORF) of On-HMGB2 spans 642 bp, encoding 213 amino acids, and contains two conserved HMG domains. On-HMGB2 shares over 80 % homology with other fish species and 74%-76 % homology with mammals. On-HMGB2 was widely distributed in various tissues, with its highest transcript levels in the liver and the lowest in the intestine. Knockdown of On-HMGB2 promoted the inflammatory response in Nile tilapia, increased the bacterial load in the tissues, and led to elevated mortality in Nile tilapia following Streptococcus agalactiae infection. Taken together, On-HMGB2 significantly influences the immune system of Nile tilapia in response to streptococcus infection.

Duck HMGB2 Mediates Signaling Pathways in the Innate Immunity of Hosts Against Viral Infections.

High-mobility group box 2 (HMGB2) belongs to the HMG-box family that participates in a variety of biologic processes. Recent studies have suggested that HMGB2 plays an important role in the innate immunity of fish. Cherry Valley duck is the main duck bred for meat consumption in China, but there is limited research available on the impact of duck HMGB2 (duHMGB2) in antiviral innate immunity. Here, duHMGB2 genes were first cloned and analyzed from the spleen of Cherry Valley ducks. We show that duHMGB2 is widely distributed in most tissues of healthy ducks, and duHMGB2 was differentially expressed in three organs (the spleen, brain, and lung) of ducks during different viral infections. duHMGB2 is mainly expressed in the nucleus of duck embryo fibroblast (DEF) cells. However, duHMGB2 is released into the cytoplasm after viral infection. DuHMGB2 induced expression of several genes that regulate the immune response. Moreover, duHMGB2 activated and upregulatede transcription factor NF-κB promoter activity. We also used single gene manipulations (knockout or overexpression) to confirm that duHMGB2 can inhibit the replication of duck plague virus, duck Tembusu virus, and the novel duck reovirus in DEF cells. These data show that duHMGB2 can activate the antiviral innate immunity of the host. Thus, duHMGB2 may be considered an immune adjuvant against infectious diseases in duck.

Emergence of antibodies endowed with proteolytic activity against High-mobility group box 1 protein (HMGB1) in patients surviving septic shock.

High-mobility group box 1 (HMGB1) concentration in serum or plasma has been proposed as an important biological marker in various inflammation-related pathologies. We previously showed that low titer autoantibodies against HMGB1 could emerge during the course of sepsis. Importantly their presence was positively related with patients' survival. In this study, we focused on plasma samples from 2 patients who survived sepsis and exhibited high titer antibodies to HMGB1. These antibodies were proved to be specific for HMGB1 since they did not bind to HMGB2 or to human serum albumin. Following IgG purification, it has shown that both patients secreted HMGB1-hydrolyzing autoantibodies in vitro. These findings suggested that proteolytic antibodies directed against HMGB1 can be produced in patients surviving septic shock.

PfHMGB2 protects yellow catfish (Pelteobagrus fulvidraco) from bacterial infection by promoting phagocytosis and proliferation of PBL.

HMGB2, a member of the high mobility group box family, plays an important role in host immune responses. However, the mechanism of action of HMGB2 is not well understood. Herein, a homologue from yellow catfish (Pelteobagrus fulvidraco) was cloned and named PfHMGB2. The deduced amino acid sequence of PfHMGB2 possessed a typical tripartite structure (two DNA binding boxes and an acid tail) and shared 90% identity with the predicted HMGB2 from I. punctatus. The mRNA of PfHMGB2 was widely distributed in all 11 tested tissues in healthy fish bodies and was significantly induced in the liver and head kidney when yellow catfish were injected with inactivated Aeromonas hydrophila. Consistently, PfHMGB2 mRNA could also be induced in yellow catfish peripheral blood leucocytes (PBL) by lipopolysaccharide. The recombinant PfHMGB2 protein was purified from E. coli BL21 (DE3):pET-28a/PfHMGB2 and showed DNA-binding affinity. Moreover, rPfHMGB2 improved the phagocytosis and proliferation activity and upregulated the mRNA expression of the pro-inflammatory cytokine TNFα in yellow catfish PBL. These results indicated that PfHMGB2 could protect yellow catfish from pathogen infection by activating PBL.

High mobility group box 2 of black rockfish Sebastes schlegelii: Gene cloning, immunoregulatory properties and antibacterial effect.

High-mobility group box 2 (HMGB2) is a non-histone chromosomal protein that involved diverse functions such as transcriptional regulation and innate immune responses in mammalian. In teleost, very limited studies on HMGB2 proteins have been documented. Black rockfish (Sebastes schlegelii) is an economic fish species and cultured worldwide. However, the study of black rockfish about immunology is very scarce. In the present study, a HMGB2 homologue gene (SsHMGB2) was identified and characterized in black rockfish. The open reading frame of SsHMGB2 is 648 bp, and the deduced amino acid sequence of SsHMGB2 shares 74.4%-91.2% overall sequence identities with the HMGB2 proteins of several fish species. In silico analysis identified several conserved features, including two basic HMG boxes and an acidic C-terminal tail composed of 24 Asp/Glu residues. Expression of SsHMGB2 occurred in multiple tissues and was upregulated during pathogens infection. Recombinant SsHMGB2 (rSsHMGB2) exhibited apparent binding activities against DNA. In vivo studies showed that the expressions of multiple immune-related genes in head kidney were significantly enhanced when black rockfish were treated with rSsHMGB2. Furthermore, rSsHMGB2 reduced pathogen dissemination and replication in fish kidney and spleen. Taken together, these results suggest that SsHMGB2 possesses apparent immunoregulatory properties and played a role in fighting bacterial infection.

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.

Cross-reactivity of anti-HMGB1 antibodies for HMGB2.

HMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inflammation. Despite their relatively high homology, they may have distinct roles, making it essential to be able to differentiate between the two. Here we examine the specificity of five commercially-available anti-HMGB1 antibodies. By Western blotting of recombinant proteins and HMGB1-/- mouse embryonic fibroblasts, we identified only one HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting specific antibodies for HMGB1 and HMGB2 allowed identification of distinct HMGB1 and HMGB2 subcellular pools in primary neutrophils.

A small molecule binding HMGB1 and HMGB2 inhibits microglia-mediated neuroinflammation.

Because of the critical role of neuroinflammation in various neurological diseases, there are continuous efforts to identify new therapeutic targets as well as new therapeutic agents to treat neuroinflammatory diseases. Here we report the discovery of inflachromene (ICM), a microglial inhibitor with anti-inflammatory effects. Using the convergent strategy of phenotypic screening with early stage target identification, we show that the direct binding target of ICM is the high mobility group box (HMGB) proteins. Mode-of-action studies demonstrate that ICM blocks the sequential processes of cytoplasmic localization and extracellular release of HMGBs by perturbing its post-translational modification. In addition, ICM effectively downregulates proinflammatory functions of HMGB and reduces neuronal damage in vivo. Our study reveals that ICM suppresses microglia-mediated inflammation and exerts a neuroprotective effect, demonstrating the therapeutic potential of ICM in neuroinflammatory diseases.

Antibacterial and antiviral properties of tongue sole (Cynoglossus semilaevis) high mobility group B2 protein are largely independent on the acidic C-terminal domain.

High mobility group box (HMGB) proteins are known to be involved in diverse functions in mammalian cells. In teleost, very limited studies on HMGB proteins have been documented. In this study, we reported identification of a HMGB homologue (named CsHMGB2) from tongue sole (Cynoglossus semilaevis) and examined its biological property. CsHMGB2 is 245 residues in length and contains two basic HMG boxes and an acidic C-terminal tail composed of 23 Asp/Glu residues. Quantitative real time RT-PCR (qRT-PCR) analysis showed that CsHMGB2 expression occurred in multiple tissues and was upregulated by bacterial and viral infection in a time-dependent manner. In vitro studies showed that when tongue sole peripheral blood leukocytes were treated with recombinant CsHMGB2 (rCsHMGB2) and the mutant rCsHMGB2M, which bears a deletion of the C-terminal acidic region, significant and comparable increases in cellular resistance against bacterial infection were observed. qRT-PCR detected enhanced expression of proinflammatory cytokines and chemokines in rCsHMGB2-treated cells. In vivo studies showed that when tongues sole were administered with rCsHMGB2 or rCsHMGB2M before being subjected to bacterial and viral infection, the pathogen loads in the spleen and kidney of the fish were significantly reduced. Taken together, these results suggest that CsHMGB2 possesses immunoregulatory properties that promote resistance against bacterial and viral infection in a manner that is largely independent on the highly conserved C-terminal acidic domain.

Cloning and expression analysis of a novel high-mobility group box 2 homologue from Lampetra japonica.

High-mobility group box 2 (HMGB2) is a nonhistone architectural protein that plays important roles in many biological processes. In this study, we cloned a homologue of the HMGB2 from the lymphocyte-like cells of Lampetra japonica (L. japonica). Sequence analysis reveals that L. japonica HMGB2 contains two highly conserved motifs and shares more than 70 % identity with the homologues from other vertebrate species. Subsequently, Lj-HMGB2 was subcloned into the pET-28a(+) and pIRES2 AcGFP1-Nuc vector and expressed in Rosetta blue (DE3) and Hela cell lines, respectively. The recombinant L. japonica HMGB2 (rLj-HMGB2) with apparent molecular mass of 22 kDa was further purified by His-Bind affinity chromatography. Real-time quantitative PCR indicates that the expression level of Lj-HMGB2 was particularly up-regulated in intestines after challenged with lipopolysaccharide, while up-regulated in lymphocyte-like cells and heart after challenged with concanavalin A in vivo. In addition, rLj-HMGB2 could induce the generation of proinflammatory mediators in the activated human acute monocytic leukemia cell line (THP1), which suggested that Lj-HMGB2 may participate in the immune response of the lampreys.

Characterizations of two grass carp Ctenopharyngodon idella HMGB2 genes and potential roles in innate immunity.

High-mobility group box 2 (HMGB2) protein is a chromatin-associated nonhistone protein, involved in transcriptional regulation and nucleic-acid-mediated innate immune responses in mammalian. However, the function of piscine HMGB2 in innate immune responses is still unknown. In the present study, two HMGB2 homologue genes (CiHMGB2a, CiHMGB2b) were identified and characterized in grass carp (Ctenopharyngodon idella). Both CiHMGB2a and CiHMGB2b genes encode proteins with 213 amino acids, sharing 71.4% identities and containing two basic HMG boxes and an acidic tail. The deduced protein sequences showed the most identities to HMGB2a (93%) and HMGB2b (86.4%) of zebrafish (Danio rerio), respectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that CiHMGB2a and CiHMGB2b were constitutively expressed in all the 15 tested tissues. Post grass carp reovirus (GCRV) infection, mRNA levels of CiHMGB2a and CiHMGB2b were strongly up-regulated in spleen and head kidney and mildly modulated in C. idella kidney (CIK) cells. Meanwhile, mRNA expressions of CiHMGB2a and CiHMGB2b were significantly regulated by viral pathogen associated molecular patterns (PAMPs) polyinosinic-polycytidylic potassium salt (poly(I:C)) and bacterial PAMPs lipopolysaccharide (LPS), peptidoglycan (PGN) challenge in CIK cells. In CiHMGB2a and CiHMGB2b over-expression cells, expressions of CiHMGB2a and CiHMGB2b facilitated each other; transcription levels of CiTRIF, CiMyD88, CiIPS-1 and CiMx1 were remarkably enhanced, whereas CiIFN-I was inhibited, compared with those in cells transfected with pCMV (control plasmid); after GCRV challenge, all those tested genes were up-regulated with divergent expression profiles. Antiviral activities of CiHMGB2a and CiHMGB2b were manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield. All those results demonstrate that CiHMGB2a and CiHMGB2b not only mediate antiviral immune responses but also involve in responding to viral/bacterial PAMPs challenge, which provides novel insights into the essential role of HMGB2 in innate immunity.

Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach.

The innate immune system senses viral DNA that enters mammalian cells, or in aberrant situations self-DNA, and triggers type I interferon production. Here we present an integrative approach that combines quantitative proteomics, genomics and small molecule perturbations to identify genes involved in this pathway. We silenced 809 candidate genes, measured the response to dsDNA and connected resulting hits with the known signaling network. We identified ABCF1 as a critical protein that associates with dsDNA and the DNA-sensing components HMGB2 and IFI204. We also found that CDC37 regulates the stability of the signaling molecule TBK1 and that chemical inhibition of the CDC37-HSP90 interaction and several other pathway regulators potently modulates the innate immune response to DNA and retroviral infection.

Anti-high mobility group box 1 and box 2 non-histone chromosomal proteins (HMGB1/HMGB2) antibodies and anti-Saccharomyces cerevisiae antibodies (ASCA): accuracy in differentially diagnosing UC and CD and correlation with inflammatory bowel disease phenotype.

BACKGROUND: The development of a supportive diagnostic method has long been required to differentially diagnose ulcerative colitis (UC) and Crohn's disease (CD). Several antibodies circulate in the sera of patients with inflammatory bowel disease. We previously identified the high mobility group box 1 and box 2 non-histone chromosomal proteins (HMGB1 and HMGB2) as novel antigens of perinuclear type anti-neutrophil cytoplasmic antibodies (pANCA) and discovered anti-HMGB1/HMGB2 antibodies in sera from patients with UC. Here, we evaluated the ability of anti-HMGB1/HMGB2 antibodies combined with anti-Saccharomyces cerevisiae antibodies (ASCA) to differentially diagnose UC and CD. METHODS: We measured titers of anti-HMGB1/HMGB2 antibodies and ASCA in the sera of 213 patients with UC and 93 with CD, using enzyme-linked immunosorbent assays. RESULTS: Among the patients with UC, 26.8% were positive for anti-HMGB1/HMGB2 antibodies, with 85.0% specificity towards CD and a positive predictive value of 80.3%. Corticosteroids significantly suppressed the titer of anti-HMGB1/HMGB2 antibodies. Among the patients with CD, 24.7% were positive for ASCA, with 96.2% specificity towards UC and a positive predictive value of 74.2%. Interestingly, the positivity rate of anti-HMGB/HMGB2 antibodies was higher (35.7%) in patients with the ileitis type of CD than in patients with CD in the colon (6.2%; significant difference, P < 0.01). The specificity of anti-HMGB1/HMGB2 antibodies in UC for CD in the colon was 93.8%. CONCLUSIONS: CD in the colon and UC can be differentially diagnosed using anti-HMGB/HMGB2 antibodies combined with ASCA.

HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses.

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

High mobility group B2 is secreted by myeloid cells and has mitogenic and chemoattractant activities similar to high mobility group B1.

High mobility group B box (HMGB) proteins are a family of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. HMGB have a highly conserved sequence, but different expression pattern: HMGB1 is almost ubiquitous, whereas the others are highly expressed in only a few tissues in adults. We previously demonstrated that HMGB1 is released by necrotic cells and has chemoattractant activity for inflammatory and stem cells, via binding to receptor for advanced glycation endproducts (RAGE). HMGB1 can be actively secreted by inflammatory cells. Here, we report that also HMGB2 can be secreted by THP-1 cells, and promotes proliferation and migration of endothelial cells. These functions of HMGB2 are exerted via engagement of RAGE, whose blockade completely abrogates cell responses. Since extracellular HMGB2 has been detected in the blood and other biological fluids, it might be necessary to target HMGB2 at the same time as HMGB1 for therapeutical efficacy.

High mobility group box (HMGB) proteins of Plasmodium falciparum: DNA binding proteins with pro-inflammatory activity.

High mobility group box chromosomal protein 1 (HMGB1), known as an abundant, non-histone architectural chromosomal protein, is highly conserved across different species. Homologues of HMGB1 were identified and cloned from malaria parasite, Plasmodium falciparum. Sequence analyses showed that the P. falciparum HMGB1 (PfHMGB1) exhibits 45, 23 and 18%, while PfHMGB2 shares 42, 21 and 17% homology with Saccharomyces cerevisiae, human and mouse HMG box proteins respectively. Parasite PfHMGB1and PfHMGB2 proteins contain one HMG Box domain similar to B-Box of mammalian HMGB1. Electrophoretic Mobility Shift Assay (EMSA) showed that recombinant PfHMGB1 and PfHMGB2 bind to DNA. Immunofluorescence Assay using specific antibodies revealed that these proteins are expressed abundantly in the ring stage nuclei. Significant levels of PfHMGB1 and PfHMGB2 were also present in the parasite cytosol at trophozoite and schizont stages. Both, PfHMGB1 and PfHMGB2 were found to be potent inducers of pro-inflammatory cytokines such as TNFalpha from mouse peritoneal macrophages as analyzed by both reverse transcription PCR and by ELISA. These results suggest that secreted PfHMGB1 and PfHMGB2 may be responsible for eliciting/ triggering host inflammatory immune responses associated with malaria infection.

HMGB1, a novel inflammatory cytokine.

High mobility group box 1 (HMGB1) exhibits unique biochemical functions as a biologically intrinsic requisite factor and as a toxin. As such, it is imperative to understand the mechanism by which these seemingly and diametrically opposed functions are exerted. To effectively discriminate these actions is important to accurately and precisely determine the concentration of HMGB1 in biological samples. Research in this fascinating field, however, has been lacking due to the absence of a simple analytical system for HMGB1 that can be adapted for large sample numbers. In this report, we review the physiological and pathological significance of HMGB1 and describe the development of an assay method for this pleiotropic protein.

Autoantibodies to the transcriptional factor SOX13 in primary biliary cirrhosis compared with other diseases.

The molecule SOX13 was initially identified as an autoantigen (ICA12) in Type 1 diabetes. SOX13 is a member of the SOX family of transcriptional regulatory proteins that contain a high mobility group (HMG) motif with structural similarity to HMG proteins 1 and 2. Antibodies to HMG 1 and 2 occur in autoimmune diseases of the liver and in ulcerative colitis. We measured the occurrence and levels of anti-SOX13 by radioimmunoprecipitation in primary biliary cirrhosis (PBC) and other diseases, and compared frequencies with anti-HMG measured by ELISA. Anti-SOX13 was detected in 18% of patients with PBC, 13% with autoimmune hepatitis, 18% with Type 1 diabetes, at lower frequencies in other conditions including the multisystem autoimmune diseases, systemic lupus erythematosus and rheumatoid arthritis, and in 1% of normal sera. Anti-HMG1 and anti-HMG2 occurred at frequencies of 30% and 35% respectively in PBC. Serum levels of anti-SOX13 and anti-HMG correlated significantly for PBC although not for Type 1 diabetes. Anti-SOX13 in PBC may occur merely as an immune response to products of damage to parenchymal tissue, or may be illustrative of a general proclivity of transcriptional regulatory proteins to elicit autoimmune responses.