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Intracellular DNA sensors in immunity

Fumihiko Takeshita1 and Ken J Ishii2,3
Mammalian innate immunity possesses a distinct system to recognize aberrant DNA inside the cell. One class of DNA sensors is the Toll-like receptor 9, which is expressed in the specialized immune cells, binds to single-stranded DNA in the endosome to transmit cellular signaling through myeloid differentiation primary response protein 88 (MyD88). Another class of DNA sensors exists in the cytoplasm of most type of cells in the tissue, detecting double-stranded DNA to signal through TANK-binding kinase-1 (TBK1)-mediated type-I interferon production and apoptosis-associated speck-like protein containing a CARD (ASC)-mediated IL-1b secretion. Since DNA sensors have potential to recognize aberrant DNA of both self and nonself origin, their physiological roles in microbial infection, tissue damage, autoimmune diseases, and DNA-based therapeutic applications are being intensively investigated.
Addresses 1 Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama 236-0004, Japan 2 Department of Molecular Protozoology, Research Institute for Mirobial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Corresponding author: Takeshita, Fumihiko (takesita@yokohamacu.ac.jp) and Ishii, Ken J (kenishii@biken.osaka-u.ac.jp)

Current Opinion in Immunology 2008, 20:383388 This review comes from a themed issue on Host-Pathogen Interactions Edited by Tsuneyasu Kaisho and Hermann Wagner Available online 23rd June 2008 0952-7915/$ see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2008.05.009

Introduction
It has been more than four decades since Alick Isaacs who coined the term interferon, found that nucleic acids such as RNA and DNA are strong inducers of the interferon which is now designated as type I interferon (IFN) [1,2]. These seminal ndings, in particular that DNA is immunostimulatory, had been forgotten or largely ignored for the subsequent 20 years, until some microbial DNA or synthetic oligodeoxynucleotides (ODNs) encoding unmethylated CpG motifs (CpG DNA) was shown to stimulate immune cells to produce proinammatory
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cytokines, chemokines, and type-I IFNs [3,4]. At that time, most immunologists had believed that unmethylated CpG motif was the sole element required for robust innate immune activation by DNA. Furthermore, Tolllike receptor 9 (TLR9) was identied as a sole receptor for microbial genomic DNA and that any kind of CpG ODNs, [one of which is called A-type or D-type CpG DNA (CpG-A) and another was called B-type or K-type CpG DNA (CpG-B)], caused activation when puried as naked DNA and added into immune cell culture media [5,6]. The other line of evidence, however, suggested that double-stranded (ds), but not single-stranded (ss) DNA, possesses strong immunological activity without the need for specic sequences like CpG motifs, which can be thus derived from either host or pathogens [79]. This effect was observed when DNA was forced into cells by any means of transfection, and also when the DNA of righthanded B-form dsDNA (B-DNA) was aberrantly present inside the cell [8]. In fact, B-DNA induces production of type-I IFNs in various cell types including immune cells such as dendritic cells, macrophages and B cells, and nonimmune cells such as epithelial cells, broblasts, and thyroid cells through recognition and signaling pathways independently of TLR9 [8,9]. Such phenomena seem closer to what had been observed by Isaacs and physiologically relevant not only to host defense against microbial infection but also to autoimmune responses. For example, DNase-II knockout mice showed that an excess amount of undigested DNA results in hyperproduction of type-I IFNs, dysregulation of erythropoiesis, and rheumatoid arthritis (RA)-like symptoms in a TLR9independent manner, indicating that the self-genomic DNA can serve as an endogenous adjuvant to modulate immune responses under certain circumstances [10,11]. TANK-binding kinase-1 (TBK1), a pivotal kinase of interferon regulatory factors (IRFs), is crucial not only for B-DNA-mediated type-I IFN production and subsequent protection against DNA virus infection [8] but also for both innate and acquired immune responses to DNA vaccination without the need for TLR9 [12]. These reports, taken together, suggest that dsDNA serves as an exogenous as well as endogenous adjuvant and plays signicant roles in various immunological events, irrespective of TLR9-mediated DNA recognition. We will review recent progress on these TLR9dependent and TLR9-independent DNA recognition and signaling pathways, and their roles in protective and pathological immunity.

Mechanism of DNA sensing by TLR9

TLR9 is a type-I membrane protein shown to be recruited from the ER to the endosome. Its homodimerized form
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binds endocytosed CpG DNA through its ectodomain, changes its conformation, and allosterically transmits signaling through the cytoplasmic Toll/IL-1 receptor domain interaction with myeloid differentiation factor 88 (MyD88), TNF receptor-associated factor 6 (TRAF6), and IL-1 receptor-associated kinase 1 (IRAK1) and IRAK4 [13]. The maturation of the endosomes containing CpG DNA is essential for such signal activation, since treatment with chloroquine or balomycin A abrogates the signaling. In plasmacytoid dendritic cells (pDCs) (but not conventional DCs (cDCs) or macrophages), CpG-A activates kinases, such as IRAK1 and IkB kinase-a (IKK-a), and an E3 ligase, TRAF6, resulting in phosphorylation and ubiquitination of IRF-7, and robust production of IFN-a [14]. Such pDC-specic activation of IRF-7 takes place by the long-lasting retention of CpG DNA in the endosome of pDCs but not in those of cDCs or macrophages, though the precise molecular mechanism is as yet unknown [15]. CpGA-induces TRAF3 recruitment to the complex consisting of activated (=dimerized) MyD88, triggering type-I IFN production in pDCs. CpG-B elicits activation of mitogenactivated protein (MAP) kinases and NF-kB through TRAF6, which is pivotal for proinammatory cytokine and chemokine gene expression. CpG-B also induces activation of IRF-5 and production of IkBz to facilitate the transcriptional regulation of the proinammatory cytokine genes [16]. The level of IRF-1 expression is increased upon TLR ligand-stimulation or IFN-g-stimulation in cDCs. CpG-B induces IRF-1 interaction with MyD88, which crucially licenses IRF-1 transmigration into the nucleus and TLR9-mediated gene-induction programs such as IFN-b, inducible NO synthetase, and IL-12 p35 [17,18]. TLR9, on the contrary, can bind to a broad range of DNA with or without CpG motifs, suggesting that some additional mechanism(s) seem necessary for the TLR9mediated, CpG DNA-specic activation of innate immune responses. In fact, a conjugation of polyG at the 30 -end or transfection using N-[1-(2,3-dioleoyloxy)]N,N,N-trimethylammonium propan methylsulfate (DOTAP) enables non-CpG DNA to trigger TLR9 activation, while telomeric TTAGGG repeats conforming G-tetrads suppress TLR9 activation [15,19]. A recent study dissected and elucidated fundamental determinants for TLR9 activation by synthetic DNA. Homopolymeric, base-free natural (phosphodiester) 20 deoxyribose had stimulatory activity as a TLR9 agonist, which was further enhanced by addition of CpG motifs. By contrast, though phosphorothioate 20 -deoxyribose homopolymers acted as a TLR9 antagonist, CpG motifs transformed it from TLR9-inhibitory to stimulatory activity (=TLR9 agonist) [20]. TLR9 recognition of self-DNA may contribute to the pathogenesis of uncontrolled chronic inammation. For example, the chromatinIgG complex that is composed of
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self-DNA, nucleosome proteins, and the antinucleosome antibodies, which are often detected in sera of systemic lupus erythematosus (SLE) patients, are known to activate DCs and autoreactive B-lymphocytes mostly in a TLR9-depenedent manner. Such chromatinIgG complex is delivered to the TLR9-containing endosomes in a human FcgRIIa-dependent manner or mouse FcgRIIIdependent manner [21]. Recently, antimicrobial peptide LL37 (also known as cathelicidin antimicrobial peptide), overexpressed in psoriatic skin, was demonstrated to interact with self-DNA to form aggregated structures, which are also delivered to and retained in the early endosomes to trigger TLR9-mediated innate immune activation [22]. The subcellular localization of TLR9 is also crucial for DNA sensing. In intestinal epithelial cells, basolateral TLR9 signals through a canonical NFkB activation pathway by degrading IkB-a, whereas apical TLR9 elicits accumulation of ubiquitinated IkB in the cytoplasm, which prevents NF-kB activation [23]. The articial redistribution of TLR9 to the plasma membrane conferred cellular activation by self-DNA, suggesting that the TLR9 localization to the endosome but not to the plasma membrane is crucial for prohibition of self-DNA recognition and consequent innate immune activation [24]. Recently, a polytopic membrane protein, UNC93B1, was demonstrated to directly associate with TLR9 and crucially regulate its function by directing subcellular trafcking [25,26]. Several other factors have been shown to modulate TLR9-mediated signaling. High mobility group protein B1 (HMGB1) affects the chromatin structure by associating with the minor groove of the genomic DNA. This molecule is released from cells undergoing necrosis or cells stimulated with proinammatory cytokines or CpG DNA. Recombinant HMGB1 protein directly associates with CpG-A. Coadministration of HMGB1 with CpG-A synergistically activates pDCs to produce IFN-a through the association with cell surface receptor for advanced glycosylation end products (RAGEs) [27]. pDCs express a cell type-specic variant of triggering receptor expressed in myeloid cell (TREM) family members, PDC-TREM, which associates with cell surface semaphorin receptor plexin-A1 and adaptor protein DNAXactivation protein 12 (DAP12) [28]. Cell surface expression of PDC-TREM is upregulated upon CpG-A stimulation, whereas neutralization or knock down of this molecule result in a signicant reduction of CpG-Ainduced IFN-a production, suggesting that PDC-TREM is engaged in DAP12-mediated positive-feedback signal amplication to trigger robust production of IFN-a [28]. Cathepsin K is a lysosomal protease expressed not only in osteoclasts but also in cDCs. Deciency of cathepsin K resulted in a severe defect of CpG-B-induced TLR9 signaling in cDCs but not in macrophages, though the precise molecular mechanism remains to be elucidated [29].
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Mechanism of B-DNA sensing and subsequent signaling

Transfection of natural DNA or synthetic polynucleotides forming ds structure stimulates cells to produce type-I IFNs and induce cell-autonomous protection from viral replication. In contrast to CpG motifs for TLR9 activation, methylation of such dsDNA had no effect on the activity. Rather, poly(dAdT) poly(dT dA)-induced higher levels of type-I IFNs compared with poly(dGdC) poly(dCdG), suggesting that righthanded helical structure of B-form DNA (B-DNA) is essential for cellular activation for type-I IFN production [8]. The signaling pathway mediated by B-DNA is partly shared with that of the immunostimulatory RNA such as 50 -triphosphorylated RNA and dsRNA. In human, retinoic acid-inducible gene I (RIG-I), a sensor for viral-derived RNA, is also engaged in B-DNA-mediated type-I IFN production, though in vitro analysis demonstrated that RIG-I directly binds to 50 -triphosphorylated RNA but not to B-DNA [30]. RNA interference analysis clearly demonstrated that a signaling adaptor, IFN-b promoter stimulator-1 (IPS-1), is also involved in human B-DNA-mediated signaling [8,30,31]. However, RIG-I/ or IPS-1/ mouse embryonic broblasts (MEFs) did respond to B-DNA stimulation in a comparable fashion to WT mice. The response was completely abrogated in TBK1/ MEFs, suggesting that species-specic signaling pathways exist upstream of TBK1 in both human and mouse systems [8,32,33]. It is interesting to note that, in contrast to LPS, CpG DNA, or dsRNA, interferon stimulatory DNA (ISD), originally reported by Stetson and Medzhitov as having similar action to B-DNA, does not activate MAP kinases ERK, JNK and p38, which are required for consequent NF-kB activation [9]. Several candidate molecules in a panel of IFN-inducible genes were examined for their roles in B-DNA-sensing and subsequent signal transmission, since the expression levels of RNA sensors, such as RIG-I-like helicases, are upregulated by type-I IFN stimulation. Among these, overexpression of Z-DNA-binding protein-1 (ZBP1) enhanced levels of type-I IFN production in response to B-DNA in mouse connective tissue-derived cell line, L929 cells [34]. RNA interference of ZBP1 resulted in a partial but signicant suppression of B-DNA-mediated cellular activation, such as dimerization of IRF-3, DNA binding activity of NF-kB, and production of IFN-b and IL-6 in L929 cells [34]. Thus, because of such properties characterized by Takaoka et al., they proposed an alternative name of ZBP1 as DNA-dependent activator of IRFs (DAI, thereafter referred to as ZBP1/DAI) [34]. ZBP1/ DAI physically interacts with B-DNA and downstream signaling molecules TBK1 and IRF-3. Four domains of ZBP1/DAI are involved in its association with B-DNA and signal transmission. D3 domain located in the center
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is crucial for its interaction with B-DNA, whereas Za and Zb domains at the NH2-terminus and D3 domain almost equally contribute to its association with IRF-3 and signal transmission [35]. The function of TBK1/IRF-3 interacting domain at the COOH-terminus seems to be regulated by an as yet unknown serine/threonine kinase [35]. BDNA induces oligomerization of ZBP1/DAI that has been overexpressed in L929 cells, whereas articially oligomerized ZBP/DAI alone can activate signaling, suggesting that oligomerization of ZBP/DAI is a crucial step for BDNA-mediated signaling [35]. The scenario described above was solely observed in L929 cells but not in MEFs, suggesting a redundant role of ZBP1/DAI in B-DNAsensing system [12,34,35]. Alternatively, NACHT-leucine-rich repeat-PYD containing protein (NALP) family members, most of which are present in the inammasome, have been demonstrated to sense various stimuli inside the cell and trigger proinammatory responses [36]. NALP family proteins transmit cellular signaling via common signal adaptors, apoptosis-associated specklike protein containing a CARD (ASC) and caspase-1, by which secretory forms of IL-1b, IL-18, and IL-33 are processed from pre-existing precursors [36]. NALP3, also known as cryopyrin, regulates secretion of IL-1b protein in response to adenoviral infection [37]. Although NALP3/ macrophages are defective in IL-1b secretion after DNA virus infection, they respond to B-DNA stimulation comparably to WT macrophages [37]. By contrast, ASC/ macrophages are incapable of IL-1b secretion by either DNA virus infection or B-DNA stimulation [37]. Taking these observations together, it is suggested that ASC is a key to B-DNA-mediated production of proinammatory cytokines such as IL-1b, IL-18, and IL-33 whose secretion are regulated by caspase-1. However, it is still unclear whether the inammasome constitutes a molecular platform sensing B-DNA, since recent studies have shown that ASC plays an alternative role in pyroptosome by associating with a noninammasome protein, pyrin, for caspase-1 activation in a process leading to a distinct form of cell death, pyroptosis [38,39].

Roles of DNA-sensing system in autoimmunity and gene-mediated therapeutic applications

In terms of pathogenesis of autoimmune diseases triggered by host DNA, a couple of studies have demonstrated that deciency or mutations of each type of DNases, such as DNase I, II, or III, resulted in a systemic or local autoimmune disease with similarities to SLE, RA, or inammatory myocarditis, suggesting that accumulation of aberrant host DNA and exaggerated responses to DNA correlate with etiology of autoimmune diseases [10,11,4045]. Role of TLR9 in autoimmune diseases is somewhat contradictory. Although TLR9 triggers innate immune activation to induce antinucleosome antibody production, a defect of TLR9 in lupus-prone mice
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resulted in an increase of serum levels of IFN-a and an exacerbation of disease activity when compared with those of parental mice, suggesting that TLR9 regulates tolerance to autoantigens [4649]. As for gene-mediated therapeutic applications, more direct evidence indicated that TBK1 but not TLR9 is an essential element for innate and acquired immune responses raised by DNA vaccine [12]. Thus, DNA vaccine has a built-in adjuvant as B-DNA, by which the immunogenicity of the target antigen is upregulated through TBK1-mediated innate immune activation [12].
Figure 1

Concluding remarks
Evidence, as described above, has emerged that several distinct molecules and related regulatory elements are involved in immune recognition of aberrant DNA inside the cell. Elucidation of this innate immune recognition of DNA and its physiological role in DNA-immunology and DNA-biology will be further boosted by isolation of a sole cytoplasmic sensor(s) of ds B-DNA, clarication of intracellular and intercellular signaling pathways, including the adaptor(s) and association between TBK1-IRFsmediated type-I IFNs induction and ASC-dependent

Mechanism of DNA sensing in mammalian cells. Once TLR9 is delivered from the ER to the endosome by UNC93B1, it senses CpG DNA to trigger signaling via MyD88, IRAK1, and TRAF6. IRAK1 and IkB kinase-a (IKK-a) act as kinases, while TRAF6 acts as an E3 ligase for IRF-7 phosphorylation and ubiquitylation, which are required for hyperproduction of IFN-a in pDCs. In cDCs and macrophages, TRAF3 coordinates TANK-binding kinase-1 (TBK1)-mediated phosphorylation of IRF-7 for type-I IFN production, while TRAF6 mediates NF-kB activation to induce proinflammatory cytokine production. Human FcgRIIa or mouse FcgRIII is an essential player for TLR9 sensing of the self-DNAIg complex inside the cell. LL37 binds to and aggregates self-DNA to trigger TLR9 activation. HMGB1 association with CpG DNA facilitates receptor for advanced glycosylation end products (RAGEs)-mediated signal transduction, while PDC-TREM association with plexinA1 initiates DAP12-mediated positive-feedback signal activation for robust production of type-I IFNs. In human, RIG-I and IPS-1 are involved in B-DNA-mediated signaling for activation of IRF-3. ZBP-1/DAI directly interacts with B-DNA and transmits signaling via TBK1 and IRF-3 in L929 cells. ASC and caspase-1 are essential for B-DNA-induced IL-1b secretion. Current Opinion in Immunology 2008, 20:383388 www.sciencedirect.com

Intracellular DNA sensors Takeshita and Ishii 387

caspase-1-mediated, IL-1b secretion. There seem to have distinct host immune regulation of these DNA recognition among cell types, tissues, and species, and factors related to the DNA ligands. Thus, further elucidation of those complex mechanisms underlying recognition of DNA and consecutive signaling pathways may shed a light on etiology of autoimmune diseases and application of DNA-mediated immunostimulatory effects to therapeutic purposes (Figure 1).

innate immune response to dsDNA, and that was independent of DAI, recently identied as a cytosolic sensor for DNA. 13. Latz E, Verma A, Visintin A, Gong M, Sirois CM, Klein DC, Monks BG, McKnight CJ, Lamphier MS, Duprex WP et al.: Ligandinduced conformational changes allosterically activate Tolllike receptor 9. Nat Immunol 2007, 8:772-779. 14. Kumagai Y, Takeuchi O, Akira S: TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev 2008, 60:795-804. 15. Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A, Taya C, Taniguchi T: Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 2005, 434:1035-1040. 16. ONeill LA, Bowie AG: The family of ve: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007, 7:353-364. 17. Negishi H, Fujita Y, Yanai H, Sakaguchi S, Ouyang X, Shinohara M, Takayanagi H, Ohba Y, Taniguchi T, Honda K: Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program. Proc Natl Acad Sci U S A 2006, 103:15136-15141. 18. Schmitz F, Heit A, Guggemoos S, Krug A, Mages J, Schiemann M, Adler H, Drexler I, Haas T, Lang R et al.: Interferon-regulatoryfactor 1 controls Toll-like receptor 9-mediated IFN-beta production in myeloid dendritic cells. Eur J Immunol 2007, 37:315-327. 19. Ashman RF, Lenert P: Structural requirements and applications of inhibitory oligodeoxyribonucleotides. Immunol Res 2007, 39:4-14. 20. Haas T, Metzger J, Schmitz F, Heit A, Muller T, Latz E, Wagner H:  The DNA sugar backbone 20 deoxyribose determines toll-like receptor 9 activation. Immunity 2008, 28:315-323. This report demonstrated that the sugar-backbone, not the bases of DNA binds to TLR9 and is sufcient as a ligand to act as a mild agonist, and that this agonistic activity is further enhanced by the presence of a CpG motif. 21. Krug A: Nucleic acid recognition receptors in autoimmunity. Handb Exp Pharmacol 2008, 183:129-151. 22. Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH,  Homey B, Cao W, Wang YH, Su B, Nestle FO et al.: Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 2007, 449:564-569. This report together with Ref. [27] expands the list of factor(s) that can modify the selfnonself moiety of DNA, thereby inuencing TLR9mediated descrimination between microbial (nonself) DNA and host (self) DNA. 23. Lee J, Mo JH, Shen C, Rucker AN, Raz E: Toll-like receptor signaling in intestinal epithelial cells contributes to colonic homoeostasis. Curr Opin Gastroenterol 2007, 23:27-31. 24. Barton GM, Kagan JC, Medzhitov R: Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat Immunol 2006, 7:49-56. 25. Brinkmann MM, Spooner E, Hoebe K, Beutler B, Ploegh HL, Kim YM: The interaction between the ER membrane protein UNC93B and TLR3, 7 and 9 is crucial for TLR signaling. J Cell Biol 2007, 177:265-275. 26. Kim YM, Brinkmann MM, Paquet ME, Ploegh HL: UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 2008, 452:234-238. 27. Tian J, Avalos AM, Mao SY, Chen B, Senthil K, Wu H, Parroche P,  Drabic S, Golenbock D, Sirois C et al.: Toll-like receptor 9dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 2007, 8:487-496. See Annotation to Ref. [22]. 28. Watarai H, Sekine E, Inoue S, Nakagawa R, Kaisho T, Taniguchi M: PDC-TREM, a plasmacytoid dendritic cell-specic receptor, is responsible for augmented production of type I interferon. Proc Natl Acad Sci U S A 2008, 105:2993-2998. Current Opinion in Immunology 2008, 20:383388

Acknowledgements
We sincerely thank Dr Shizuo Akira, Cevayir Coban, Shohei Koyama, Satoshi Uematsu, Taro Kawai, Osamu Takeuchi, and Toshihiro Horii for helpful discussions. FT and KJI are supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.

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