TLR9 as a key receptor for the recognition of DNA

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Abstract

Unmethylated DNA with CpG-motifs is recognized by Toll-like receptor 9 (TLR9) and pleiotropic immune responses are elicited. Macrophages and conventional dendritic cells (cDCs) produce proinflammatory cytokines to B/K-type CpG-DNA, whereas plasmacytoid DCs induce type I interferons to A/D-type CpG-DNA and DNA viruses. The TLR9 mediated signaling pathway is not only responsible for activation of innate immune cells, but also for mounting acquired responses. Thus, it has been attempted to exploit TLR9 ligands as a vaccine adjuvant for anti-cancer immunotherapy. Further, TLR9 mediated signaling is implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. Nevertheless, recent studies revealed that double-stranded DNA can be recognized by intracellular receptor(s) in a TLR9-independent manner. This review will focus on the roles of TLR9 in immune responses, and its signaling pathways.

Introduction

Upon microbial infection, the host evokes various responses to eliminate the invading pathogens. Host immune cells elicit an innate immune response by detecting pathogen specific molecular patterns (PAMPs) and mount a strong acquired immune response as well [1]. Bacterial DNA has long been known as one of the key immunostimulatory PAMPs. The DNA fraction of Mycobacterium bovis BCG has been shown to be capable of activating human and mouse non-B/T cells [2]. Bacterial DNA also causes septic shock [3]. On the other hand, mammalian DNA does not induce such responses. Immunostimulatory DNA has a specific pattern of unmethylated CpG motifs (reviewed in [4]). Unmethylated CpG-DNA activates not only innate immunity but also acquired immunity. These motifs are not detected in mammalian DNA because most CpG sequences are methylated in mammals.

During the 1990s, several lines of accumulated evidence indicated that Toll-like receptors (TLRs) can act as pattern-recognition receptors (PRRs) that detect PAMPs. The Toll receptor was first identified in the fruit fly Drosophila melanogaster where it was found to be involved in an anti-fungal response [5]. Subsequently, mammalian TLRs were cloned, and their roles in the recognition of microbial components were elucidated by a mouse model with both forward and reverse genetic approaches (reviewed in [6]). TLR2 (together with TLR1 or TLR6) and TLR4 are the receptors mainly involved in the recognition of bacteria-derived ligands, such as microbial lipoproteins and LPS, respectively [7], [8], [9]. TLR3 and 7 (and possibly 8) are involved in the recognition of double-stranded (ds) and single-stranded (ss) RNA, respectively [10], [11], [12], [13].

TLR9 was first cloned and identified as a receptor for unmethylated CpG-DNA as well as for bacterial DNA in 2000 [14]. Analysis of TLR9-deficient mice revealed that TLR9 is essential not only for pro-inflammatory cytokine production and other inflammatory responses, but it also plays a role in the induction of T helper 1 (Th1) acquired immune response and in the proliferation of B cells.

After the identification of TLR9, the mechanisms of various immune responses elicited by DNA were clarified. It has been shown that several DNA viruses are recognized and are able to elicit antiviral responses through TLR9. The TLR9-mediated recognition of DNA viruses and CpG-containing oligodeoxynucleotides (CpG-ODNs) induces type I interferon (IFN) production in plasmacytoid dendritic cells (pDCs) [15], [16], [17]. TLR7 is also highly expressed on pDCs and stimulation with ssRNA as well as RNA viruses produced vast amount of type I IFNs via TLR7.

Nevertheless, recent studies revealed that cytosolic PRRs called Retinoic acid-inducible gene (RIG-I)-like helicases (RLHs) can recognize viral RNA in the cytosol [18]. RIG-I and Melanoma differentiation-associated gene 5 (MDA5) consist of two caspase recruitment domains (CARDs) and a RNA helicase moiety. The recognition of viral RNAs by these helicases induces production of type I IFNs and an associated antiviral response. Double-stranded DNA without the CpG motif may also be recognized by cytoplasmic PRR(s) [19]. In this review, we will discuss the current understanding of the role of TLR9 in immunity from several points of view, and outline what is still not yet known.

Section snippets

The TLR9 ligands

The TLR9 ligands, CpG-ODNs, are classified into two different subtypes, B/K-type and A/D-type [20]. The B/K-type CpG-ODNs are phosphorothioate-modified throughout the sequence and are known to induce DC maturation and B cell proliferation. On the other hand, A/D-type CpG-ODNs, which are characterized by a phosphodiester backbone CpG motif and phosphorothioate-modified poly G stretches at the 5′ and 3′ ends, induce type I IFNs in pDCs [21].

TLR9 also recognizes bacterial and viral DNA. Upon

TLR9 signaling

TLR consists of leucine-rich repeats (LRRs), a transmembrane domain and a cytoplasmic Toll/interleukin-1 receptor homology (TIR) domain. TLR9 is localized at the intracellular membrane compartment, such as the endoplasmic reticulum (ER), the endosome, and the lysosome [33]. The LRR of TLR9 is on the inside of the membrane compartment, while the TIR domain is located on the cytosolic side. Stimulation of TLR9 with its ligands leads to activation of various transcription factors, including

Cell types utilizing TLR9

TLR9 is expressed on various cell types (Fig. 2). Upon B/K-type CpG-DNA stimulation, cDCs and macrophages produce proinflammatory cytokines, such as TNF-α, IL-6 and IL-12, and upregulate surface expression of MHC class II and co-stimulatory molecules [21]. DCs maturated via TLR9 act on T cells to mount an acquired immune response [14]. Indeed, CpG-ODNs are known as very strong adjuvants that polarize helper T cell responses to Th1 [20].

TLR9 is highly expressed on human and mouse pDC, a cell

TLR9-induced type I IFN production in pDCs

Type I IFNs, comprised of multiple IFN-αs and single IFN-β, are a critical part of cytokine antiviral immunity [67]. They act on cells infected with viruses to induce apoptotic cell death, they potentiate antiviral activity to surrounding cells, and they also play an important role in the development of adaptive immune responses.

pDCs are known to highly express TLR9 and TLR7, and are capable of producing large amounts of type I IFNs upon stimulation with A/D-type CpG-DNA and ssRNA [11], [12],

TLR9 in autoimmunity

Type I IFN is known to be important for the pathogenesis of certain autoimmune diseases like systemic lupus erythematosus (SLE) (reviewed in [82]). IFN-α levels in sera from SLE patients are known to be correlated with the severity of the disease. Moreover, SLE was found to be induced during the course of type I IFN therapy. From these observations, some researchers hypothesize that excess levels of type I IFN breaks peripheral tolerance and consequently leads to autoimmunity.

In SLE patients,

Concluding remarks

A vast array of data indicates that TLR9 plays a key role in DNA-induced innate immunity, and links it with a role in acquired immunity through the activation of various cell types, such as pDC, cDC, and B cells. Its intracellular signaling pathway has been elucidated at the molecular level. The ligands for TLR9 have also been extensively studied. Several biological studies have concluded that spatial and temporal regulation is critical for proper and optimal TLR9 signaling. As well, the

Acknowledgements

The authors appreciate all the members of Akira's lab for their helpful discussions. This work is supported in part by NIH (PO1 AI070167).

References (105)

  • Y. Kumagai et al.

    Alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with RNA viruses

    Immunity

    (2007)
  • M. Lucas et al.

    Dendritic cells prime natural killer cells by trans-presenting interleukin 15

    Immunity

    (2007)
  • D.B. Stetson et al.

    Type I interferons in host defense

    Immunity

    (2006)
  • A. Prakash et al.

    Tissue-specific positive feedback requirements for production of type I interferon following virus infection

    J. Biol. Chem.

    (2005)
  • J. Banchereau et al.

    Type I interferon in systemic lupus erythematosus and other autoimmune diseases

    Immunity

    (2006)
  • S.R. Christensen et al.

    Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus

    Immunity

    (2006)
  • D.B. Stetson et al.

    Recognition of cytosolic DNA activates an IRF3-dependent innate immune response

    Immunity

    (2006)
  • T. Tokunaga et al.

    Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity

    J. Natl. Cancer Inst.

    (1984)
  • T. Sparwasser et al.

    Bacterial DNA causes septic shock

    Nature

    (1997)
  • B. Beutler et al.

    Genetic analysis of resistance to viral infection

    Nat. Rev., Immunol.

    (2007)
  • A. Poltorak et al.

    Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene

    Science

    (1998)
  • K. Hoshino et al.

    Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product

    J Immunol

    (1999)
  • L. Alexopoulou et al.

    Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3

    Nature

    (2001)
  • S.S. Diebold et al.

    Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA

    Science

    (2004)
  • F. Heil et al.

    Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8

    Science

    (2004)
  • H. Hemmi et al.

    Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway

    Nat. Immunol.

    (2002)
  • H. Hemmi et al.

    A Toll-like receptor recognizes bacterial DNA

    Nature

    (2000)
  • J. Lund et al.

    Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells

    J. Exp. Med.

    (2003)
  • T. Kawai et al.

    Innate immune recognition of viral infection

    Nat. Immunol.

    (2006)
  • K.J. Ishii et al.

    A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA

    Nat. Immunol.

    (2006)
  • D.M. Klinman

    Immunotherapeutic uses of CpG oligodeoxynucleotides

    Nat. Rev. Immunol.

    (2004)
  • H. Hemmi et al.

    The roles of Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets

    J. Immunol.

    (2003)
  • A. Bafica et al.

    TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

    J. Exp. Med.

    (2005)
  • R. Copin et al.

    MyD88-dependent activation of B220-CD11b+LY-6C+dendritic cells during Brucella melitensis infection

    J. Immunol.

    (2007)
  • A.E. Anderson et al.

    TLR9 polymorphisms determine murine lymphocyte responses to Helicobacter: results from a genome-wide scan

    Eur. J. Immunol.

    (2007)
  • J. Zhu et al.

    Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and-independent pathways

    J. Virol.

    (2007)
  • C. Coban et al.

    Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin

    J. Exp. Med.

    (2005)
  • P. Parroche et al.

    Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9

    Proc. Natl. Acad. Sci. U. S. A.

    (2007)
  • C. Coban et al.

    Pathological role of Toll-like receptor signaling in cerebral malaria

    Int. Immunol.

    (2007)
  • J. Vollmer et al.

    Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation

    Immunology

    (2004)
  • T.L. Roberts et al.

    Cutting edge: species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified oligonucleotides

    J. Immunol.

    (2005)
  • E.R. Kandimalla et al.

    Immunomodulatory oligonucleotides containing a cytosine-phosphate-2′-deoxy-7-deazaguanosine motif as potent Toll-like receptor 9 agonists

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • E. Latz et al.

    TLR9 signals after translocating from the ER to CpG DNA in the lysosome

    Nat. Immunol.

    (2004)
  • H. Hacker et al.

    Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6

    J. Exp. Med.

    (2000)
  • L.A. O'Neill et al.

    The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling

    Nat. Rev. Immunol.

    (2007)
  • T. Kawagoe et al.

    Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling

    J. Exp. Med.

    (2007)
  • C. Wang et al.

    TAK1 is a ubiquitin-dependent kinase of MKK and IKK

    Nature

    (2001)
  • M. Yamamoto et al.

    Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling

    Nat. Immunol.

    (2006)
  • S. Sato et al.

    Essential function for the kinase TAK1 in innate and adaptive immune responses

    Nat. Immunol.

    (2005)
  • A. Takaoka et al.

    Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors

    Nature

    (2005)
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