Competition by inhibitory oligonucleotides prevents binding of CpG to C‐terminal TLR9

TLR9 recognizes unmethylated CpG‐containing DNA commonly found in bacteria. Synthetic oligonucleotides containing CpG‐motifs (CpG ODNs) recapitulate the activation of TLR9 by microbial DNA, whereas inversion of the CG dinucleotide within the CpG motif to GC (GpC ODNs) renders such ODNs inactive. This difference cannot be attributed to binding of ODNs to the full‐length TLR9 ectodomain, as both CpG and GpC ODNs bind comparably. Activation of murine TLR9 requires cleavage into an active C‐terminal fragment, which binds CpG robustly. We therefore compared the ability of CpG and GpC ODNs to bind to full‐length and C‐terminal TLR9, and their impact on the cleavage of TLR9. We found that CpG binds better to C‐terminal TLR9 when compared with GpC, despite comparably low binding of both ODNs to full‐length TLR9. Neither CpG nor GpC ODNs affected TLR9 cleavage in murine RAW 264.7 cells stably expressing TLR9‐Myc. Inhibitory ODNs (IN‐ODNs) block TLR9 signaling, but how they do so remains unclear. We show here that inhibitory ODNs do not impede TLR9 cleavage but bind to C‐terminal TLR9 preferentially, and thereby compete for CpG ODN binding both in RAW cells and in TLR9‐deficient cells transduced with TLR9‐Myc. Ligand binding to C‐terminal fragment thus determines the outcome of activation through TLR9.     

Immunotherapeutic potential of CpG oligodeoxynucleotides in veterinary species

Abstract

Innate immunity plays a critical role in host defense against infectious diseases by discriminating between self and infectious non-self. The recognition of infectious non-self involves germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). The PAMPs are the components of pathogenic microbes which include not only the cell wall constituents but also the unmethylated 2′-deoxy-ribo-cytosine-phosphate-guanosine (CpG) motifs. These CpG motifs present within bacterial and viral DNA are recognized by toll-like receptor 9 (TLR9), and signaling by this receptor triggers a proinflammatory cytokine response which, in turn, influences both innate and adaptive immune responses. The activation of TLR9 with synthetic CpG oligodeoxynucleotides (ODNs) induces powerful Th1-like immune responses. It has been shown to provide protection against infectious diseases, allergy and cancer in laboratory animal models and some domestic animal species. With better understanding of the basic biology and immune mechanisms, it would be possible to exploit the potential of CpG motifs for animal welfare. The research developments in the area of CpG and TLR9 and the potential applications in animal health have been reviewed in this article.     

Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG

Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference （RNAi） was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs transfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG. TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs may offer a potential tool to study the TLR9-CpG pathway.     

TLR9-/- and TLR9+/+ mice display similar immune responses to a DNA vaccine

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.     

Specific siRNA downregulated TLR9 and altered cytokine expression pattern in macrophage after CpG DNA stimulation

Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)candirectly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptorTLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial tothe subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are notfully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokineprofiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid uponCpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased whileTLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6,IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alterationof cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role ofCpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology.2005;2(2):130-135.     

Toll样受体-9的研究进展

Toll样受体-9(Toll-like receptor 9,TLR9)是哺乳动物TLRs家族中一员,作为细胞表面的天然模式识别受体,主要参与免疫刺激序列(CpG序列)激活免疫细胞的信号传导,从而在天然抗感染免疫及联系天然免疫和获得性免疫中发挥重要作用。通过对TLR9-CpG作用通路的研究,将促进天然免疫机制研究的进一步深入,有利于解决诸如:CpG佐剂、DNA疫苗、CpG抗感染、抑制肿瘤、预防过敏反应等实际应用过程中存在的问题。     

TLR9激活后影响人非小细胞肺癌细胞对多西他赛化疗敏感性的体外实验

目的:探讨含非甲基化Cp G基序的寡脱氧核苷酸(Cp G oligodeoxyribonucleotides 7909,Cp G ODN7909)与Toll样受体(Toll-like receptor,TLR)9结合后是否影响人肺癌A549和H520细胞对多西他赛(doctaxel,DOC)的化疗敏感性及相关机制。方法:设计并合成特异性干扰siRNA序列,转染细胞,Western blot法检测TLR9 siRNA的沉默效果,CCK-8法检测细胞活力。设空白对照组、阴性对照组和TLR9 siRNA干扰组,分别接受Cp G ODN7909和多西他赛单独或联合治疗,流式细胞术检测细胞周期及凋亡,Western blot法检测P38及Bax蛋白表达。结果:在2种细胞中,Cp G ODN7909单独处理后,细胞活力和凋亡率都无明显变化,G2/M期细胞比例P38和Bax蛋白的表达明显升高(P0.01);Cp G ODN7909处理TLR9 siRNA干扰细胞各指标均无明显变化。多西他赛单独处理后3组细胞的细胞活力明显下降,G2/M期细胞比例、凋亡率和Bax蛋白表达都明显升高(P0.01),P38表达无明显变化。与多西他赛单独处理比较,多西他赛与Cp G ODN7909联合治疗后细胞活力明显下降,G2/M期细胞比例、凋亡率和Bax蛋白表达都明显升高(P0.01),P38表达无明显变化;联合处理TLR9 siRNA干扰细胞各指标均无明显变化。结论:Cp G ODN7909与TLR9结合后可增强多西他赛抑制人肺癌细胞的细胞活力,诱导G2/M期阻滞,增强多西他赛对细胞的凋亡诱导作用进而提高其对多西他赛的化疗敏感性。     

胎盘组织TLR9分布与表达及与HBV宫内感染的关系

目的了解TLR9在外周血HBsAg阳性母亲胎盘组织中的表达及分布,探讨其与HBV宫内感染的关系。方法Abbott化学发光法检测新生儿外周血HBsAg;ELISA法检测母亲外周血HBV标志物;SABC免疫组织化学方法检测胎盘组织HBsAg和TLR9的表达和分布。结果胎盘HBV感染率77.27%(17/22),HBsAg分布于蜕膜细胞、滋养层细胞、间质细胞、绒毛毛细血管内皮细胞。TLR9在外周血HBsAg阴性母亲胎盘滋养层细胞表达,在外周血HBsAg阳性母亲胎盘滋养层细胞、间质细胞、绒毛毛细血管内皮细胞均有表达。外周血HBsAg阳性组比阴性组胎盘组织TLR9表达强,差异有统计学意义(P<0.05);而外周血HBsAg阳性母亲的胎盘,未感染HBV者比感染HBV者TLR9表达强,差异有统计学意义(P<0.05)。结论初步认为,胎盘组织TLR9表达与HBV宫内感染有一定关系,TLR9可能是HBV宫内感染的保护因素之一。     

Expression and function of Toll-like receptor 9 in severely injured patients prone to sepsis

The purpose of this prospective study was to enumerate Toll-like receptor 9 (TLR9)(+) cells and measure their function using synthetic oligonucleotides enriched in CG dinucleotide motifs (CpG)-induced proliferation within 48 h after trauma in severely injured patients prone to sepsis. Sixteen consecutive trauma patients with an injury severity score (ISS) > 21 and 16 blood donors (controls) were included in this study. Using two-colour flow cytometry, TLR9 expression was detectable intracellularly and also on the surface of B lymphocytes. The surface expression of TLR9 of B lymphocytes from whole blood and peripheral blood mononuclear cells (PBMC) stimulated with CpG was significantly increased in B cells of severely injured patients prone to sepsis compared to controls. No significant differences could be observed between CpG-induced proliferation of PBMC of severely injured patients prone to sepsis and controls. As a measure of immunosuppression, human leucocyte antigen (HLA)-DR expression of monocytes of the trauma patients was significantly diminished compared with controls in PBMC and in whole blood. Immunosuppression in the early phase after trauma seems not to be associated with a disturbed sensing of bacterial DNA.     

Identification and characteristic analysis of TLR28: A novel member of the TLR1 family in teleost

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish to recognize microorganisms. Fish TLRs have significant variety and distinct features. This study focuses on a novel TLR member that belongs to the TLR1 family and was first discovered in miiuy croaker (designated as TLR28, mmiTLR28). In phylogenetic analysis, the mmiTLR28 clustered in the TLR1 family. Further characteristic analysis showed a high homology with TLR2 despite some differences between them. The predicted tertiary structure of mmiTLR28 possesses a hydrophobic pocket in the ectodomain region. Expression analysis showed the high expression level in the liver of miiuy croaker. Further functional experiments on the liver after Vibrio anguillarum, Staphylococcus aureus, lipopolysaccharides (LPS), and poly (I:C) stimulation showed significant upregulation; these results indicate the potential role of mmiTLR28 in immune response. For LPS stimulation in miiuy croaker leukocytes, mmiTLR28 also displayed significant upregulation. The discovery of mmiTLR28 will enrich the information on TLR family; the functional experiments have shown the role of mmiTLR28 in immunity. The results of this study lay the foundation for future research on fish immune systems.     

Imbalance in the ratio of CpG and polyG contributes to impaired interferon-α expression

The secretion of interferon-α (IFN-α) is impaired during hepatitis B virus (HBV) infection. DNA sequences purified from distinct viruses, for example, HBV versus members of Herpesviridae, have been shown to differ in their IFN-α signaling properties. The present study found that DNA from HBV inhibited, while DNA from members of Herpesviridae induced, the expression of IFN-α. Furthermore, stimulatory cytosine-phosphate-guanosine (CpG) sequences derived from these DNA viruses could induce the secretion of IFN-α, while inhibitory guanosine-rich oligodeoxynucleoti (polyG) oligonucleotide sequences derived from these DNA viruses could inhibit CpG-induced IFN-α secretion. Using a computational analysis of genomic DNA sequences, the discrimination between the genomes of HBV and those of other DNA viruses that can also cause inflammation of the liver is based on different frequencies of the CpG and polyG motifs. The underrepresentation of stimulatory CpG motifs and overrepresentation of inhibitory polyG motifs were documented in HBV genomes, whereas the DNA from other viral genomes displayed the opposite trend. Moreover, it was demonstrated that HBV could suppress the activation of IFN-α via its own DNA through the high proportion of polyG motifs. To our knowledge, this is the first demonstration of a specific role for polyG motifs in the inhibition of the IFN-α response following DNA virus infection.     

Phosphorothioate‐modified CpG oligodeoxynucleotides mimic autoantigens and reveal a potential role for Toll‐like receptor 9 in receptor revision

Re‐expression of recombinase activating genes (RAG) in mature B cells may support autoreactivity by enabling revision of the B‐cell receptor (BCR). Recent reports suggest that administration of Toll‐like receptor 9 (TLR9) ‐stimulating CpG oligodeoxynucleotides (ODN) could trigger the manifestation of autoimmune disease and that TLR are involved in the selection processes eliminating autoreactive BCR. The mechanisms involved remain to be elucidated. This prompted us to ask, whether TLR9 could be involved in receptor revision. We found that phosphorothioate‐modified CpG ODN (CpG^PTO) induced expression of Ku70 and re‐expression of RAG‐1 in human peripheral blood B lymphocytes and Igλ expression in sorted Igκ⁺ B cells. Further results revealed unselective binding specificity of CpG^PTO‐induced immunoglobulin and suggested that CpG^PTO engage and/or mimic IgM receptor signalling, an important prerequisite for the initialization of receptor editing or revision. Altogether, our data describe a potential role for TLR9 in receptor revision and suggest that CpG^PTO could mimic chromatin‐bearing autoantigens by simultaneously engaging the BCR and TLR9 on IgM⁺ B cells.     

Toll样受体9与红斑狼疮易感性关系的研究进展

Toll样受体（Toll-like receptors, TLRs）作为连接人体天然免疫与获得性免 疫的桥梁,在自身免疫性疾病的发病中起着重要作用。其中TLR9与其特异性配体 CpGDNA结合,然后通过信号转导激活B细胞、树突状细胞,可产生各种细胞因子和自 身抗体。近年来研究发现,系统性红斑狼疮（Systemic lupus erythematosus ,SLE） 的易感性可能与TLR9基因变异有关,目前研究结论不一。本文将从鼠和人两个研究 层次来阐述TLR9与SLE易感性的关系,揭示TLR9在SLE发病中的作用机理。     

Brucella abortus induces Irgm3 and Irga6 expression via type-I IFN by a MyD88-dependent pathway,without the requirement of TLR2, TLR4, TLR5 and TLR9

The innate immune system senses bacterial pathogens by pattern recognition receptors, such as the well-characterised Toll-like Receptors (TLR). The activation of TLR signalling cascades depends on several adaptor proteins, among which MyD88 plays a key role in triggering innate immune responses. Here, we show in murine macrophages that Brucella abortus triggers expression of the interferon-inducible resistance proteins (IRGs, p47 GTPases) via type-I IFN secretion at late time points, when Brucella has reached its replication niche. This induction requires the adaptor molecule MyD88 but does not involve the TLRs normally implicated in sensing Gram-negative bacteria, namely TLR2, TLR4, TLR5 and TLR9. Brucella mutants lacking the functional VirB type-IV secretion system were not capable of inducing Irgm3 and Irga6 expression, suggesting that the type-IV secretion system is part of the triggering of the activation process. Our data suggest that Brucella is recognized intracellularly by an unknown receptor, different from the conventional ones used for Gram-negative sensing, but one that nevertheless signals through MyD88.     

Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trial

Background B‐type CpG oligodeoxynucleotides (ODN) is currently used in clinical trials because of its prolonged half–life, which is due to its phosphorothioate backbone. A‐type CpG ODN is a stronger inducer of IFN but has an unstable phosphodiester backbone that has so far prohibited its clinical use. However, upon association with virus‐like particles (VLP) consisting of the bacteriophage Qβ coat protein, A‐type CpG ODN can be stabilized and can become an efficient adjuvant in mice. Therefore, the phase I/IIa study presented represents the first test of A‐type CpGs in humans. Objective To test the safety, tolerability and clinical efficacy of QbG10 as an adjuvant for subcutaneous immunotherapy with a house dust mite (HDM) allergen extract in allergic patients. Methods A single centre, open‐label phase I/IIa study evaluated the safety, tolerability and clinical efficacy of QbG10 as an adjuvant to immunotherapy with a subcutaneous HMD allergen extract in 20 patients suffering from HDM allergy. Twenty‐one patients were enrolled between March and July 2005. Individual immunotherapy lasted 10 weeks. Clinical end‐points included questionnaires, conjunctival provocation, skin prick tests and the measurement of allergen‐specific IgG and IgE. Results QbG10 was well tolerated. Almost complete tolerance to the allergen was observed in conjunctival provocation testing after treatment with QbG10, and symptoms of rhinitis and allergic asthma were significantly reduced. Within 10 weeks of therapy, patients were nearly symptom‐free and this amelioration lasted for at least 38 weeks post‐treatment. Following injections of QbG10 and HDM allergen extract, allergen‐specific IgG increased, while there was a transient increase in allergen‐specific IgE titres. Skin reactivity to HDM was reduced. Conclusion The subcutaneous application of HDM allergen, together with A‐type CpG ODN packaged into VLP, was safe. All patients achieved practically complete alleviation of allergy symptoms after 10 weeks of immunotherapy. This promising clinical outcome calls for larger placebo‐controlled phase II studies.