EBI3 Research Articles

Human interleukin-27: wide individual variation in plasma levels and complex inter-relationships with interleukin-17A

Although it is widely believed that interleukin (IL)-27 is anti-inflammatory, its role in controlling human immune responses is not fully established. In particular, its interactions with T helper type 17 (Th)17 cytokines are unclear. Our aims were to establish the relationships between IL-27 and proinflammatory cytokines, including IL-17A, in human sera and cultures of peripheral blood mononuclear cells. Plasma IL-27 levels in 879 healthy humans from 163 families varied widely, but with relatively low heritability (19%). Despite IL-27 including a subunit encoded by Epstein-Barr virus-induced gene 3 (EBI3), there was no correlation of levels with serological evidence of infection with the virus. Although IL-27 has been reported to inhibit IL-17A production, we demonstrated a strong positive correlation in sera, but lower correlations of IL-27 with other proinflammatory cytokines. We verified that IL-27 inhibited IL-17A production by human peripheral blood T cells in vitro, but not that it stimulated IL-10 secretion. Importantly, addition of IL-17A decreased IL-27 production by stimulated T cells but had the opposite effect on resting T cells. Together, these data suggest a model whereby IL-27 and IL-17A exerts complex reciprocal effects to boost inflammatory responses, but restrain resting cells to prevent inappropriate activation.

Expression of the G-protein coupled receptor EBI2 in T cells is highly regulated and confers pathogenicity to myelin specific Th17 cells

Epstein–Barr virus-induced gene 2 (EBI2) is a recently de-orphanized G-protein coupled receptor. Expression of EBI2, as well as generation of its most potent ligand, 7alpha,25-dihydroxycholesterol (7alpha-OHC) play important roles in positioning and function of B cells and dendritic cells (DCs). However, up to now only little is known about the expression and function of EBI2 in T cells. Using a novel reporter-knockin/knockout (KO) mouse model, we found that EBI2 expression is highly regulated in T cells.

Decreased IL-35 levels in patients with immune thrombocytopenia

IL-35 is a novel heterodimeric anti-inflammatory cytokine consisting of Epstein–Barr virus-induced gene 3 (EBI3) and the p35 subunit of IL-12. IL-35 has been shown to possess the potency of inhibiting the CD4+ effector T cells and alleviating autoimmune diseases. In the study we investigated the levels of IL-35 as well as its prospective role in immune thrombocytopenia (ITP).ELISA was adopted to measure plasma IL-35, TGF-β and IL-10 levels. The mRNA expression levels of P35 and EBI3 in peripheral blood mononuclear cells (PBMCs) were studied based on real-time quantitative PCR. The correlation between plasma cytokine levels and clinical parameters was analyzed. Significantly lower plasma IL-35 levels were found in active ITP patients compared with those in remission (p=0.017) and the healthy controls (p<0.001). In active ITP patients, the plasma IL-35 levels displayed a significantly positive correlation with platelet counts (r=0.5335, p<0.0008). Further, P35 mRNA expression levels were lower in patients with active ITP than patients in remission (p=0.033) and normal controls (p=0.016).Thus, for the first time, this research reported a dramatically decreased IL-35 levels in ITP patients, suggesting that IL-35 may be involved in the pathogenesis of ITP.

EBI3 is pivotal for the initiation of experimental autoimmune uveitis

Murine experimental autoimmune uveitis (EAU) is a model for human autoimmune uveitis, whose pathogenesis is caused by both Th1 and Th17 cell responses. Epstein–Barr virus-induced gene 3 (EBI3) is a component of the heterodimeric cytokines: interleukin (IL)-27 and IL-35. Although IL-27 was shown to initiate Th1 cell development, it is also recognized as a negative regulator of fully activated CD4+ T cells, including Th17 cells. Recently, IL-35 also has also been reported to play immunosuppressive roles in autoimmunity. To investigate the roles of EBI3 in EAU, EBI3−/− mice were immunized with human interphotoreceptor retinoid binding protein peptide 1–20 (IRBP) to induce EAU. We observed that the clinical score in EBI3−/− mice was diminished compared with that in EBI3+/+ mice up to day 22 after immunization, whereas the score in EBI3−/− mice reached the same levels as that of EBI3+/+ mice after day 28. Histological analysis revealed a significant reduction of cellular infiltration into the retina in EBI3−/− mice on day 16. Although Th1 cell responses and IRBP-specific IL-10 production were reduced in EBI3−/− mice, the development of Th17 cell responses was unaffected on day 9. On day 21, Th1 cell responses and IRBP-specific IL-10 production was restored to the same levels as that in EBI3+/+ mice, and Th17 cell responses significantly increased in EBI3−/− mice. Furthermore, Foxp3 expression in CD4+ T cells was comparable between EBI3+/+ and EBI3−/− mice on days 9 and 21. Therefore, these results indicate that EBI3 may be important in EAU initiation by Th1 cell responses and may suppress EAU by inhibition of both Th1 and Th17 cell responses in the late/maintenance phase.

Identification and Characterization of Small Molecule Modulators of the Epstein–Barr Virus-Induced Gene 2 (EBI2) Receptor

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) ( Nature 2011 , 475 , 524 ; 519 ). EBI2 is highly expressed in immune cells ( J. Biol. Chem. 2006 , 281 , 13199 ), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes ( Nature 2010 , 467 , 460 ). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (1), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (2) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (4m). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells.

IL-35: a potential target for the treatment of atherosclerosis.

The imbalance of anti- inflammatory/pro-inflammatory cytokines plays an important role in the process of atherosclerosis. IL-35 is an anti-inflammatory cytokine comprising the p35 subunit of IL-12 and the subunit Epstein-Barr virus (EBV) -induced gene 3(EBI3). Accumulating evidence showed that IL-35 up-regulates the expression of anti-inflammatory cytokines, induces the generation of CD4 + regulatory T cells, inhibits CD4 + effector T cells response and other target cells activity, and reduces the progression of inflammatory and autoimmune diseases. In addition, it has been found that Ebi3 and p35 strongly coexpressed in human advanced lesions. Therefore, we hypothesize that IL-35 may become a novel target for the treatment of atherosclerosis. Further studies are required to investigate the precise effect and the signaling pathway of IL-35 in atherosclerosis process.

Gastric Epithelial Expression of IL-12 Cytokine Family in Helicobacter pylori Infection in Human: Is it Head or Tail of the Coin?

Recently, there has been a growing interest in an expanding group of cytokines known as “IL-12 family”. The so far gained knowledge about these cytokines, as crucial playmakers in mucosal immunity, has not yet been sufficiently investigated in the context of Helicobacter pylori infection. All genes encoding the monomeric components of these cytokines and their corresponding receptors were examined in gastric epithelial cell lines (AGS and MKN-28) after being infected with 4 H. pylori strains: BCM-300, P1 wild-type, and P1-derived isogenic mutants lacking cytotoxin-associated gene A (cagA) or virulence gene virB7 (multiplicity of infection=50). Both infected and uninfected samples were analyzed after 24h and 48h using real-time quantitative polymerase chain reaction (RT-qPCR). Gene expression analysis demonstrated a strong upregulation of IL23A (encodes p19) by infection, whereas IL23R, Epstein–Barr virus-induced gene 3 (EBI3), IL6ST, IL12A, and IL27RA were found to be expressed, but not regulated, or to a lesser extent. Transcripts of IL12RB2, IL12B, IL12RB1, and IL27A were not detected. Interestingly, P1 resulted in stronger alterations of expression than CagA mutant and BCM-300, particularly for IL23A (59.7-fold versus 32.4- and 6.7-fold, respectively in AGS after 48h, P<.05), whereas no changes were seen with VirB7 mutant. In a proof-of-principle experiment, we demonstrated epithelial-derived expression of IL-12, p19, and Ebi3 in gastric mucosa of gastritis patients using immunohistochemistry (IHC). Unlike IL-12 and Ebi3, increased immunostaining of p19 was observed in H. pylori gastritis. Herein, we highlight the potential role of gastric epithelial cells in mucosal immunity, not only because they are predominant cell type in mucosa and initial site of host-bacterial interaction, but also as a major contributor to molecules that are thought to be primarily expressed by immune cells so far. Of these molecules, p19 was the most relevant one to H. pylori infection in terms of expression and localization.

First characterization of a teleost Epstein-Barr virus-induced gene 3 (EBI3) reveals a regulatory effect of EBI3 on the innate immune response of peripheral blood leukocytes

Epstein-Barr virus-induced gene 3 (EBI3) encodes a protein that in mammals is known to be a subunit of interleukin (IL)-27 and IL-35, both which regulate cytokine production and inflammatory response. To date, no studies on fish EBI3 have been documented. In this work, we report the identification of an EBI3 homologue, CsEBI3, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsEBI3 is composed of 245 amino acid residues and possesses a Fibronectin type 3 (FN3) domain that is preserved in lower and higher vertebrates. Expression of CsEBI3 was detected in a wide range of tissues, in particular those of immune relevant organs, and upregulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) enhanced CsEBI3 expression and caused extracellular secretion of CsEBI3. Purified recombinant CsEBI3 (rCsEBI3) stimulated the respiratory burst activity of PBL and upregulated the expression of IL-1β, IL-8, Myd88, interferon-induced gene 15, CD28, and chemokines. In contrast, rCsEBI3M, a mutant CsEBI3 that lacks the FN3 domain failed to activate PBL and induced much weaker expression of the immune genes. Treatment of PBL with rCsEBI3, but not with the mutant rCsEBI3M, enhanced cellular resistance against bacterial invasion, whereas antibody blocking of CsEBI3 on PBL significantly reduced cellular resistance against bacterial infection. Taken together, these results indicate for the first time that a teleost EBI3 possesses immunoregulatory property in a manner that is dependent on the conserved FN3 domain, and that CsEBI3 is involved in the innate immune defense of PBL against microbial pathogens.

Overexpression of Epstein–Barr virus-induced gene 3 protein (EBI3) in MRL/lpr mice suppresses their lupus nephritis by activating regulatory T cells

To identify the effect of an imbalance of Th1/Th2 cytokines on the development of autoimmune glomerulonephritis (lupus nephritis), we studied the modification of pathological changes in diffuse proliferative glomerulonephritis (DPGN) and membranous glomerulonephritis (MGN) in MRL/lpr mice, which are animal models of systemic lupus erythematosus (SLE). Transgenic MRL/lpr mice (Tg) that overexpressed Epstein--Barr virus-induced gene 3 (EBI3) showed almost normal renal function, which was demonstrated by healing of glomerulonephritis upon renal histology, as compared to the wild-type MRL/lpr (Wt) mice. The levels of anti-dsDNA antibodies and IgE decreased in the Tg mice compared to Wt mice. Quantitative real-time PCR indicated an increase in the mRNA levels of FoxP3, and a decrease in that of IFNγ in the splenocytes of Tg mice as compared to Wt mice. In addition, flow cytometric analysis showed an increase in CD4+CD25+FoxP3+-T cells in the former, as compared to the latter. Our findings suggest that EBI3-overexpression in MRL/lpr mice induces generation of regulatory T cells, which causes suppression of autoimmune and inflammatory reactions by affecting the Th1/Th2 cytokine balance.

High Beta-Palmitate Fat Controls the Intestinal Inflammatory Response and Limits Intestinal Damage in Mucin Muc2 Deficient Mice

BackgroundPalmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha’-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha’-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer.MethodsMuc2−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed.ResultsBoth HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis.ConclusionsThis study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2−/− mice by inducing an immunosuppressive Treg cell response.

Interleukin 27 inhibits atherosclerosis via immunoregulation of macrophages in mice

Chronic inflammation in arterial wall that is driven by immune cells and cytokines plays pivotal roles in the development of atherosclerosis. Interleukin 27 (IL-27) is a member of the IL-12 family of cytokines that consists of IL-27p28 and Epstein-Barr virus induced gene 3 (EBI3) and has anti-inflammatory properties that regulate T cell polarization and cytokine production. IL-27-deficient (Ldlr-/-Ebi3-/-) and IL-27 receptor-deficient (Ldlr-/-WSX-1-/-) Ldlr-/- mice were generated and fed with a high-cholesterol diet to induce atherosclerosis. Roles of bone marrow-derived cells in vivo and macrophages in vitro were studied using bone marrow reconstitution by transplantation and cultured peritoneal macrophages, respectively. We demonstrate that mice lacking IL-27 or IL-27 receptor are more susceptible to atherosclerosis compared with wild type due to enhanced accumulation and activation of macrophages in arterial walls. The number of circulating proinflammatory Ly6C(hi) monocytes showed no significant difference between wild-type mice and mice lacking IL-27 or IL-27 receptor. Administration of IL-27 suppressed the development of atherosclerosis in vivo and macrophage activation in vitro that was indicated by increased uptake of modified low-density lipoprotein and augmented production of proinflammatory cytokines. These findings define a novel inhibitory role for IL-27 in atherosclerosis that regulates macrophage activation in mice.

Lipopolysaccharide-Induced M2 to M1 Macrophage Transformation for IL-12p70 Production Is Blocked by Candida albicans Mediated Up-Regulation of EBI3 Expression

Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rβ2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host’s health.

Stimulated human peripheral T cells produce high amounts of IL-35 protein in a proliferation-dependent manner

The p35 subunit of IL-12 and the Epstein–Barr virus-induced gene 3 (EBI3) have been shown to form a heterodimeric cytokine, named interleukin-35 (IL-35). Recently, mRNA expression of both IL-12p35 and EBI3 was clearly shown in stimulated human T effector cells.Here, we investigated the production of IL-35 protein in human anti-CD3/CD28-stimulated pan T cells as well as T cell subpopulations using a specific human IL-35 ELISA system. We measured high concentrations of IL-35 (up to 3ng/ml) in cell culture supernatants of stimulated pan T cells as well as CD4+, CD8+ and CD4+CD25− T cell subpopulations at 72h after stimulation. Very low amounts of IL-35, in the range of 100pg/ml, were detectable in supernatants of resting T cells. These observations could be confirmed using a dot-blot assay for IL-12p35 and EBI3. High concentrations of IL-35 could be also measured in cell culture supernatants of both, resting and stimulated CD4+CD25+ T cells.In order to learn more about the regulation of IL-35 production, we studied the effect of dexamethasone, cyclosporine A and rapamycin on IL-35 production of anti-CD3/CD28-stimulated human pan T cells as well as CD4+ and CD8+ T cell subpopulations. All three drugs significantly suppressed IL-35 production of these cells in a proliferation-dependent manner.In summary, we could show that stimulated human peripheral blood T cells of healthy donors produce high amounts of IL-35 protein. However, the biological function of this cytokine remains to be elucidated.

Abstract 2370: Epstein-Barr Virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer.

Abstract Lung cancer is the leading cause of cancer deaths in the world. The number of patients responding well to current standard therapies is still small, and no tumor marker has been entirely useful for screening curative stage cancers. To identify molecules which could be useful for the development of diagnostic biomarkers and novel drugs, we have established an effective screening strategy as follows; i) To identify up-regulated genes in 120 lung cancers by genome-wide screening using the cDNA microarray representing 27,648 genes and pure populations of tumor cells taken from cancer tissues by laser microdissection, ii) To verify the candidate genes for their very low level of expression in normal tissues by northern-blot analyses, iii) To validate the clinicopathological significance of its over-expression with tissue microarray containing hundreds of archived lung-cancers, iv) To verify whether the target gene is essential for the growth of cancer cells by RNAi and cell growth assays, v) To evaluate it for usefulness as a serum diagnostic biomarker by ELISA, if they are tumor-specific transmembrane or secretory proteins. As a result of this process, we identified 35 druggable oncoproteins. We further identified the Epstein-Barr virus-induced gene 3 (EBI3), which encodes a secretory glycoprotein. Immunohistochemical staining of EBI3 using tissue microarrays revealed that strong EBI3 expression was associated with a poor prognosis (P = 0.0014), and multivariate analysis confirmed it to be an independent prognostic factor (P = 0.0439; N=414). In addition, we established an ELISA to measure serum EBI3 and found that the proportion of serum EBI3-positive cases was 47.1% (73 of 155) for lung adenocarcinoma, 54.5% (24 of 44) for lung squamous cell carcinoma, and 41.3% (31 of 75) for small cell lung cancer, while 3 (2.4%) of 126 healthy volunteers were falsely diagnosed. The sensitivity of serum EBI3 for cancer detection was 44.4% for stage I-II lung adenocarcinoma, 50.0% for stage I-II lung squamous cell carcinoma, and 35.2% for small cell lung cancer with limited disease (LD) stage, thereby proving it was superior to conventional tumor markers of CEA and CYFRA21-1 in terms of both sensitivity and specificity. Validation analysis using another independent set of serum samples (132 lung cancers and 41 healthy volunteers) confirmed significant elevation of EBI3 in the sera of lung cancer patients. Furthermore, reduction in EBI3 expression by siRNA suppressed cancer cell proliferation, while induction of EBI3 conferred growth-promoting activity. EBI3 should be a biomarker for early cancer detection and for the prediction of prognosis, as well as being a suitable therapeutic target. Citation Format: Yataro Daigo, Atsushi Takano, Yusuke Nakamura. Epstein-Barr Virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2370. doi:10.1158/1538-7445.AM2013-2370 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

A protective role of IL-30 via STAT and ERK signaling pathways in macrophage-mediated inflammation

IL-30, the p28 subunit of IL-27, interacts with the Epstein–Barr virus-induced gene 3 (EBI3) to form IL-27, which modulates the inflammatory responses in autoimmune and infectious diseases. Several previous studies have provided evidence for the role of IL-30 in the anti-inflammatory process. However, the effect of IL-30 in macrophage-mediated immune responses is not well understood. With the recent observation in our experiment, we found that IL-30 exerted potent anti-inflammatory effects in the RAW 264.7 macrophages and in a lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced mouse model. IL-30 decreased the production of tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. In the macrophage-mediated GalN and LPS model of acute liver injury, IL-30 prevented liver injury by suppressing serum enzyme activity and down-regulating the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and interferon (IFN)-γ. IL-30 treatment decreased apoptosis in liver tissue and increased glutathione (GSH) levels. We postulated that IL-30 might function through gp130-mediated signaling pathways and then demonstrated that IL-30 affects LPS-induced inflammation through the STAT1, STAT3, and ERK signaling pathways. These data indicate that IL-30 can provide critical protection against macrophage-mediated liver inflammation through anti-apoptotic, anti-oxidant, and anti-inflammatory activities.

Interleukin-35: The Future of Hyperimmune-Related Diseases?

Interleukin (IL-35) is a newly identified heterodimeric cytokine belonging to the IL-12 family. It contains Epstein-Barr virus-induced gene 3 subunit and IL-27 p35 subunit. Although its receptor and signaling pathway are not clear, we presumed that its receptor is composed by two chains that might be similar to those receptors of IL-12, IL-23, and IL-27. We also believe that the signal transducer activator of transcription family members is involved in its signaling pathway. It was reported that IL-35 could suppress Teff cell proliferation and Th17 development. It was considered to have a potential therapeutic effect against immune diseases. In our perspective, the finding of IL-35 is of great significance, since it can regulate T cells, which is an important therapeutic target of immunological disorders. IL-35 would promote the development of different kinds of vaccines, even vaccine for special cancer, and be promising to cure autoimmune and inflammatory diseases.

Some Polymorphisms in Epstein-Barr Virus–induced Gene 3 Modify the Risk for Chronic Rhinosinusitis

Forkhead box protein 3 (FOXP3), a transcription factor required for development and function of regulatory T cell, and Epstein-Barr virus-induced gene 3 (EBI3), a downstream target of FOXP3, may be important in the development of chronic rhinosinusitis (CRS) with or without sinonasal polyposis (CRSwNP and CRSsNP, respectively). Our objective was therefore to examine the presence of any associations between specific single nucleotide polymorphisms (SNPs) in/around the FOXP3 and EBI3 genes and risk for CRS in Chinese subjects. A population-based case-control association analysis was performed. DNA extracted from peripheral blood leukocytes from 667 subjects with CRS (360 CRSwNP and 306 CRSsNP) and 330 healthy controls was assessed for SNPs selected and complemented with tagging SNPs. A total of seven SNPs (4 in FOXP3 and 3 in EBI3 genes) were genotyped and genetic association tests were performed. In the single-locus analyses of CRS risk, the allele frequencies of rs428253 (p = 0.0191) in EBI3 gene and rs2232365 (p = 0.0307) and rs3761548 (p = 0.0221) in FOXP3 gene were significantly different between the CRS cases and the controls. Furthermore, genotype association analysis showed a significant protective effect only between EBI3_rs428253 (CG/CC; p = 0.029) and CRS after adjustment. Similarly, stratified analysis of CRS risk on category showed rs428253 of the EBI3 gene to play a protective role among both CRSwNP (CG/CC; p = 0.002) and CRSsNP (GG/CC; p = 0.023) subjects. Haplotype analysis of the FOXP3 gene region further indicated that CRS risk was higher in individuals carrying the haplotype GG in rs2294018-rs2232365 block, compared with wild-type AG haplotype, and AG in rs3761548-4824747 block, compared with wild-type CG haplotype. The findings of this study suggest that SNPs in FOXP3 and EBI3 genes modify the risk for development of CRS.

Epstein-Barr virus-Induced gene-3(EBI3)knockoutマウスにおける 精巣内免疫微小環境の変化

Epstein-Barr virus-Induced gene-3(EBI3)knockoutマウスにおける 精巣内免疫微小環境の変化

IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

Epstein–Barr virus-induced gene 3 negatively regulates neuroinflammation and T cell activation following coronavirus-induced encephalomyelitis

Epstein–Barr virus-induced gene 3 (EBI3) associates with p28 and p35 to form the immunomodulatory cytokines IL-27 and IL-35, respectively. Infection of EBI3−/− mice with the neuroadapted JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality that was not associated with impaired ability to control viral replication but enhanced T cell and macrophage infiltration into the CNS. IFN-γ secretion from virus-specific CD4+ and CD8+ T cells isolated from infected EBI3−/− mice was augmented while IL-10 expression muted in comparison to infected WT mice. These data demonstrate a regulatory role for EBI3-associated cytokines in controlling host responses following CNS viral infection.