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Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity
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Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity Hermann Wagner The astounding adjuvanticity and Th1-polarizing immunobiology of bacterial CpG-DNA and mimicking CpG-oligonucleotides continue to mirror promising therapeutic potential. The past year has witnessed some particularly impressive progress in knowledge of its molecular mode of action. Accordingly, CpG-DNA acts as a ‘pathogen-associated’ molecular pattern that is recognized by TLR9 expressed, in particular, by dendritic cells. Interactions between CpG-DNA and TLR9 rapidly activate antigen-presenting dendritic cells through the ancient Toll/IL-1-receptor signaling pathway to upregulate co-stimulatory molecules and to produce Th1-polarizing cytokines, such as interleukin-12 and interleukin-18. Thus, interactions between CpG-DNA and TLR9 effectively bridge innate and acquired immunity. Addresses Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstraße 9, 81675 Munich, Germany; e-mail: [email protected] Current Opinion in Microbiology 2002, 5:62–69 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations APC antigen-processing cell DC dendritic cell GFP green fluorescent protein h human HSP heat shock protein IFN-γγ interferon-γ IL interleukin LPS lipopolysaccharide m murine MDDC monocyte-derived dendritic cell MHC major histocompatibility complex NK natural killer ODN oligonucleotide p precursor PAMP pathogen-associated molecular pattern PRR pattern recognition receptor TIR Toll/interleukin 1R domain Th T helper TLR Toll-like receptor α TNF-α tumor necrosis factor-α TRAF TNF-receptor-associated factor
Introduction The functioning of the immune system in higher organisms is based on two distinct recognition systems: innate (that is, non-clonal) and adaptive (that is, clonal) host defenses. The former builds on the function of innate immune cells, such as macrophages, dendritic cells (DCs) and natural killer (NK) cells, and traditionally has been viewed as non-specific, whereas the latter is founded on clonally distributed B and T cells and is typified by specificity and memory [1].
In this review, I discuss the recent advances made in the understanding of the interactions between DNA containing unmethylated CpG motifs from pathogenic microorganisms and the Toll-like receptor (TLR) 9 of DCs, and how these interactions stimulate the innate and adaptive immune responses. First, I provide a brief overview of the innate and adaptive immune responses.
The immune response When higher organisms are exposed to pathogenic microorganisms, innate immune responses occur immediately, both in terms of cell activation and inflammation. On one hand, they are characterized by uptake (that is, phagocytosis or endocytosis) and subsequent destruction or degradation of pathogens. On the other hand, the innate immune cells — macrophages and DCs — are acutely activated, resulting in the following examples: first, the upregulation of co-stimulatory cell surface molecules and major histocompatibility complex (MHC) class I and II molecules [2]; second, the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, and effector cytokines, such as IL-12 and type I interferon [3]; and third, the enhanced presentation of the products of pathogen degradation (antigenic peptides) via the MHC class I or II presentation pathway to antigenreactive T cells [2] and the production of bactericidal effector substances such as nitric oxide. Thus, innate immune cells represent not only a first line of defense towards infections but play a decisive and instructive role in emanating the adaptive immune responses [3]. A major challenge to innate immune cells is the discrimination of foreign pathogens from self. As originally perceived by Janeway [4,5], innate immune cells such as antigen-processing cells (APCs) possess germline-encoded pattern recognition receptors (PRRs) that recognize and are triggered by evolutionary conserved molecules essential to pathogen function but absent in the host. These pathogen-associated molecular patterns (PAMPs) are widespread and include cell wall components such as mannans in the yeast cell wall, lipopolysaccharide (LPS) in Gram-negative bacteria, lipoproteins, peptidoglycans and DNA containing unmethylated CpG motifs. There are at least two distinct classes of PRRs: those that mediate acute phagocytosis (internalization) and those that cause immediate cell activation. For example, scavenger receptors such as the mannose receptor (or related molecules such as langerin, DEC 205 and other lectins) mediate internalization of microorganisms primarily to direct them into detoxification pathways, yet they appear not to activate innate immune cells. On the other hand, upon cellular pathogen uptake, members of the TLR family become recruited to early phagosomes to screen their
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content for ligands from foreign pathogens and subsequently to trigger cell activation upon ligand recognition [6]. This apparent ‘division of labor’ (internalization versus cell activation) between scavenger receptors and TLRs bears a caveat, given that crosslinking of the scavenger receptor CD36 profoundly modulates LPS-driven DC maturation [7]. Adaptive immunity is controlled by the generation of MHC-restricted effector T cells and production of cytokines [8]. When stimulated by professional APCs such as DCs, naïve T helper (Th) cells may differentiate into Th1- versus Th2-polarized subsets; Th1 cells secrete primarily interferon IFN-γ, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13. Cytokines such as IL-12 (and, in man, type I interferons [9]) trigger via STAT4 IFN-γ the production of CD4+ T cells (Th1 polarization), whereas IL-4 induces Th2 differentiation. Upon activation, APCs (signal 1) upregulate the expression of co-stimulatory molecules, such as CD80 and CD86 (signal 2), thereby increasing immunogenicity of peptide antigens presented. Finally, APC activation triggers production of ‘instructive’ cytokines, such as IL-12, IL-18, IL-4 or IL-10, that are able to polarize emerging T cell responses (signal 3). Adding to the complexity, it is to date not clear whether all forms of activation of APCs necessarily result in increased immunogenicity. For example, certain forms of activation could selectively increase delivery of signal 1 without signal 2, giving rise to tolerogenic rather than immunogenic APCs. Furthermore, APCs can produce different cytokines (signal 3) in response to different activating stimuli [10]. A case in point is the observation that murine DCs phagocytosing either yeast or hyphae of Candida albicans produce either IL-12 or IL-4, and in vivo drive either Th1 or Th2 differentiation, respectively [11•]. It follows that APCs such as DCs and macrophages serve as a decisive interface bridging the innate and adaptive immune system [12], given that induction of productive T cell responses depends upon activation of APCs. Activation is brought about by PAMPs that are recognized by cell-activating PRRs. Furthermore, it is almost certain that the quality (intensity or avidity) of the individual ligand (PAMPs)–receptor (PRRs) interaction determines the functional state of APCs [13].
The Toll-like receptors and their ligands TLRs are transmembrane proteins and represent a newly recognized family of vertebrate PRRs. Today, we have come to realize that, for millions of years, recognition of pathogens by plants, insects and vertebrates has depended upon a system of receptors that share a characteristic domain now termed TIR (Toll/interleukin 1R domain) [14]. Surprisingly, the cytoplasmic TIR domain has remained conserved and functions in anti-pathogen responses in plants, insects and mammals alike [15]. The extracellular portion of the IL-1R family members contains, however, three immunoglobulin-like domains, whereas the extracellular portion of TLR family members contains leucine-rich repeats. To date, ten TLRs (TLRs 1–10) have been reported [16,17].
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Building on the role of Toll receptors in innate immunity of Drosophila [18], Janeway and colleagues identified in a landmark report [19] the first human homolog of Drosophila Toll. Equally important, subsequent positional cloning in C3H/HeJ mice revealed that this Toll homolog encodes the long-hunted signal transduction and receptor component for LPS, now termed TLR4 [20]. Indeed TLR4–/– mice turned out to be hyporesponsive to LPS [21] and, in man, TLR4 mutations are associated with LPS hyporesponsiveness [22•]. Notably, for TLR4-mediated signaling, CD14 must ‘individualize’ LPS (from micelles) and the secreted molecule MD2 [23] must associate with the extracellular position of TLR4. As it turns out, TLR4–MD2 not only recognizes LPS [24] but, in mice, also recognizes the structurally unrelated antitumor agent taxol complexed to heat shock proteins (HSPs) [25], as well as glucuronoxylmannan, a capsule component of the fungus Crytococcus neoformans [26], and even the outer envelope protein of respiratory syncytial virus [27•]. Furthermore, ‘endogeneous’ ligands potentially released from stressed or necrotic cells such as HSPs-60 [28•,29•] or HSP gp96 (RM Vabulas, S Braedel, N Hilf et al., unpublished data) engage TLR4 (as well as TLR2), presumably to activate DCs in the absence of infection. Finally, even cellular fibronectin fragments reportedly engage TLR4 [30]. Obviously TLR4 binds both ‘exogeneous’ ligands (that is, PAMPs) and ‘endogeneous’ ligands (such as HSPs). TLR2 recognizes lipoproteins (proteins triacylated at the amino-terminal cysteine residue) derived from Grampositive and Gram-negative bacteria including Borrelia, Treponema, Mycoplasma and Mycobacterium tuberculosis [31–34], as well as peptidoglycans [35], glycolipids and lipoteichoic acid [36], and atypical leptospiral LPS [37] and HSPs [29•]. It is difficult to reconcile the increasing list of putative TLR4 and TLR2 ligands with the concept of ligandspecific PAMP–TLR interactions. There is still the caveat of potential contaminations, given that, in equivalent assays, far higher concentrations (for example, of peptidoglycan) are required when compared with the concentration of bacterial lipopeptides. More interestingly, the ligand specificities of TLR2 may reflect a combinatorial repertoire generated by TLR2 heterodimerization, for example, with TLR1 or TLR6 [38•,39•]. Flagellin from Escherichia coli or Salmonella effects IL-8 release from intestinal epithelial cells [40] and causes NFκB activation in murine macrophages [41]. Very recently, flagellin-mediated cell activation has been shown to occur via TLR5 [42••].
TLR9 recognizes bacterial CpG-DNA Bacterial DNA, invertebrate DNA and DNA from some viruses such as herpes simplex virus type 1 (HSV-1) differ structurally from vertebrate DNA. First, bacterial DNA displays the expected frequency of CpG dinucleotides predicted by random usage (1:16) [43], whereas this frequency is suppressed fourfold in vertebrate DNA.
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Second, eukaryotic 5′-CpG-3′ motifs are preferentially (80%) methylated at a given time. Building on the pioneering work of Yamamoto and co-workers [44] and Krieg et al. [45], it is now accepted that, owing to these structural differences, unmethylated CpG motifs within bacterial DNA or within mimicking synthetic oligonucleotides (ODNs) directly activate murine B cells [45], macrophages [46,47] and DCs [48,49], but not T cells and probably not NK cells [50]. In the quest of the postulated CpG-DNA receptor, it turned out that cells of both TLR2- and TLR4-deficient mice responded to CpG-DNA, yet TLR9-deficient cells did not [51••]. This observation was consistent with the view that TLR9 represents an essential component of the postulated CpG-DNA receptor. However, mice lacking the DNA-dependent protein kinase (DNA-PK) also lacked responsiveness to CpG-DNA [52•]. This was unexpected, as SCID mice are known to harbor an inactivating mutation in the DNA-PK and yet can still respond to CpG-DNA [50]. Subsequently, it was certified that TLR9 expression in immune cells correlated with responsiveness to bacterial CpG-DNA and that responsiveness to CpG-DNA is brought about in human TLR9-deficient cells by genetic complementation with either human (h) TLR9 or murine (m) TLR9. Notably CpG-DNA responsiveness turned out to be independent of CD14 and MD2 (presumably because of the hydrophilic nature of CpG-DNA), yet required species-specific CpG-DNA motifs (that is, CpG-DNA motifs able to activate either human- or mouse-derived TLR9 in genetic complementation tests). For example, h-TLR9-transfected cells respond poorly to murine CpG motifs, and vice versa [53•]. Overall, these data imply that TLR9 conveys CpG-DNA responsiveness by directly recognizing CpG-DNA.
TLR9-mediated signaling There are remarkable structural and functional similarities between signaling pathways mediated by mammalian IL-1R [54] and those initiated by TLRs [55]. Binding of its respective ligands presumably causes TLR dimerization, which in turn triggers recruitment of the adaptor protein MyD88 via the cytoplasmatic TIRs of the TLR. This facilitates association with the IL-1R-associated kinase (IRAK) via the death domains [56]. Upon autophosphorylation, IRAK dissociates from the receptor complex and interacts with TRAF6, a member of the TNF-receptor-associated factor (TRAF) family of adaptor proteins [57]. TRAF6 participates in signaling initiated by TLRs and CD40. Unusual ubiquitination [58] allows TRAF6 to activate TAK-1 (TGF-β activated kinase), which in turn stimulates the MAP kinases, leading to JNK activation [59], or via NIK (NFκB-inducing kinase), which activates the IκB kinase complex to allow liberation of NFκB [60]. Furthermore, TRAF6 reportedly recruits evolutionary conserved signaling intermediate in Toll pathways (ECSIT), which takes place in macrophages/dendritic cells and activates via the MAP-3 kinase MEKK1 the transcription factors AP-1 and NFκB [61].
In order to unravel the molecular mechanisms of CpGDNA-driven cell activation, the cellular uptake of CpG-ODNs was studied, as well as the need for endosomal maturation and the role of the stress kinase pathway both in macrophages and DCs [62]. CpG-DNA induced activation of JNK1/2 and p38, leading to AP-1 activity. Furthermore, cellular uptake via receptor-mediated endocytosis and subsequent endosomal maturation was essential for signaling, given that competition by non-stimulating DNA as well as compounds blocking endosomal maturation (chloroquine or bafulomycin A) prevented all aspects of cellular activation [62]. Finally, CpG-DNA activated cells through the TLR/IL-1R signal pathway via MyD88 and TRAF6, and both macrophages and DCs of MyD88-deficient mice failed to show any sign of cellular activation upon exposure to CpG-DNA [63•]. This is in contrast to LPS-mediated activation of MyD88-deficient macrophages: LPS triggers TLR4 independently of MyD88, resulting in the partial activation of NFκB, JNK and p38 [64], and the MyD88independent LPS activation is able to drive IP-10 secretion and the expression of activation markers such as CD86, but not proinflammatory cytokines [32,64]. Obviously, MyD88-dependent and -independent signaling pathways exist and become activated upon LPS recognition by TLR4 [65•], whereas CpG-ODNs absolutely rely on signaling via MyD88 [63•,65•].
Bottlenecks in TLR signaling To understand the role of TLRs in mammalian immunity, it is essential to define their expression patterns in normal cells. First, distinct TLRs are differentially expressed among innate immune cells [66]. Second, TLR2 and TLR4, but not TLR9, are expressed at the cell surface and modulated upon maturation of human DCs from macrophages; for example, TLR3 expression increases and that of TLR5 is lost [67]. This raises the possibility that TLR9 becomes primarily recruited to intracellular cytoplasmatic compartments where they function by interacting with internalized PAMPs. If so, effective internalization would be a prerequisite for TLR-initiated signaling. A case in point represents CpG-DNA→TLR9 signaling in murine Raw macrophages. Raw macrophages expressing MyD88 tagged with green fluorescent protein (GFP) recruit this fusion protein to cytoplasmatic ‘endosome-like’ structures upon internalization of CpG-DNA, where these components co-localize with TLR9 (P Ahmad-Nejad, H Häcker, M Rutz et al., unpublished data). In contrast, without the need of endocytosis, ‘soluble’ LPS recruits GFP-tagged MyD88 to the cell membrane to initiate signaling. These data imply that ‘soluble’ LPS induced signaling at the cell membrane, whereas receptor-mediated endocytosis of CpGDNA is a condition for CpG-DNA/TLR9 initiated signaling. It is worth noting that the structure of the cell surface receptor that binds and translocates single-stranded DNA to endosomes in a sequence non-specific manner has not yet been characterized. It has been noted, however, that CpG-DNA covalently tagged to proteinaceous antigen
Interactions between bacterial CpG-DNA and TLR9 Wagner
effectively improves antigen uptake by DCs, whereas the cell-activating capacities of CpG-DNA are maintained [68,69•,70•]. Perhaps the ‘translocating’ DNA-binding receptor improves antigen uptake by DCs via receptormediated endocytosis.
Dendritic cells as sensors of infection Activation of Drosophila (d) Toll induces expression of an antifungal peptide, drosomycin, whereas ligation of its homologue, 18-wheeler, induces an antibacterial peptide, attacin [15]. Obviously, d-TLRs are capable of discriminating between fungi and bacteria. Mammalian DCs express TLR1–9, even though individual DC-subtypes differ with regard to TLR expression. For example, h-CD11c− CD123+ plasmacytoid precursor (p) DCs express TLR9 but low levels of TLR4, and are responsive to CpG-DNA but not to LPS. In contrast, monocyte-derived DCs (MDDCs) lack TLR9 but abundantly express TLR4 (and respond to LPS but not to CpG-DNA) [53•]. Furthermore, h-CD11c– CD123+ pDCs produce, in response to certain CpG-DNA motifs, large amounts of type I IFN but not IL-12 [71•–73•], whereas LPS causes MDDCs to produce IL-12 or TNF-α but not type I IFNs. It follows that, because of the different expression patterns of individual TLRs, DCs may not only sense the type of infection by recognizing pathogen-typifying PAMPs, but also respond in a cell-type-restricted manner. Perhaps type I IFN-producing plasmacytoid pDCs have evolved to control virus infections by members of the Herpes simplex virus family that are rich in immunostimulating CpG-motifs. DCs may not only sense the type of infecting pathogen via specific recognition of pathogen-typifying PAMPs, but also deliver critical information to antigen-reactive T cells about the context in which the ligand was encountered. Thus, DCs may convey information about the nature of the infectious agent and be able to influence the type of ensuing T cell responses. It is possible that the biological outcome of ligand-driven homo-(hetero)dimerization of TLR2, TLR4, TLR5 or TLR9 in terms of DC-derived cytokine production (type I IFN versus IL-12, IL-18) or chemokine production will turn out to be distinct. Furthermore, the magnitude of signal 1, of signal 2 and of signal 3 (see earlier) conveyed by DCs to antigen-reactive T cells is likely to influence the outcome of the adaptive T cell responses (that is, Th1 versus Th2). For example, CpG-DNA profoundly enhances the ability of DCs to support Th1 cell differentiation by inducing the release of IL-12 (signal 3), upregulation of CD40, CD80 and CD86 (signal 2), as well as the efficiency of antigen processing or presentation (signal 1).
The immunobiology of CpG-DNA As far as it is known, TLR9, which recognizes CpG-DNA, is not expressed at the cell surface of innate immune cells; upon CpG-DNA driven activation, both TLR9 and MyD88 co-localize at ‘endosome-like’ cytoplasmatic structures. CpG-ODNs ‘cross’ the cell membrane via
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sequence-non-specific receptor-mediated endocytosis. Initiation of signaling via the Toll/IL-1R-signal pathway requires endosomal maturation [74]. Given the wealth of information that has accumulated recently on the role of TLR9 as the recognition structure for CpG-DNA able to activate DCs via the Toll/IL-1R signal pathway, the immunobiology of CpG-DNA is less astounding, at least retrospectively. Originally, however, it came as a surprise to observe that challenge of mice with a mixture of CpG-DNA (used as adjuvants) and proteinaceous antigens triggered strong peptide-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes (LNs) [75,76]. Furthermore, specific antibody responses were augmented and the CpG-ODNs switched the isotype to a Th1 profile (that is, antigen-specific Ig2a evolved as dominant) [75,77]. It also became evident that CpG motifs in naked plasmid DNA derived from bacteria used for subcutaneous or intramuscular gene vaccination promoted the Th1-polarizing adjuvanticity of plasmid DNA [78], as if the CpG motifs in the plasmid DNA backbone directly induced maturation and activation of DCs. In contrast, genegun administration of DNA vaccines preferentially induced Th2 responses. Obviously, the amount of plasmid DNA used correlates with Th1 polarization. Finally, insect DNA and CpG-ODNs surpassed the activity of complete Freund’s adjuvant, as measured by the parameters ‘proliferative T responses’, IFN-γ responses and specific IgG2a antibody responses [79]. A consensus, thus, emerged that immunostimulatory CpG-DNA can be viewed almost as an ideal adjuvant with the propensity to polarize the adaptive immune system towards a Th1 response. Compared with humans (see below), mice may be classified as ‘high’ CpG-DNA responders. In fact, systemic challenge of mice with CpG-ODNs causes a transient (1–6 hour) ‘cytokine storm’ in the blood; substantial concentrations of proinflammatory cytokines, including TNF-α, IL-6, IL-1β, IL-12 and GM-CSF, are found. As a consequence, D-galactosamine (D-Gal)-sensitized mice challenged with CpG-DNA succumb within 24 hours to TNF-α-mediated acute liver-cell apoptosis [80]. Presumably, owing to liberation in vivo of hematopoietic active factors such as GM-CSF, CpG-ODNs also cause transient extramedullary hematopoiesis [81] with up to a threefold increase in spleen weight. Splenic GM-CFUs as well as BFU-E and CFU-S become dramatically enhanced. It is worth noting that the enlarged spleen (day six) represents a convenient experimental source of DC progenitors. Epidemiological studies indicate an inverse relationship between Th1-promoting infections and the propensity of individuals to develop atopical disorders [82]. This raised the question of whether or not bacterial CpG-DNA would mimic the Th1-promoting effect of bacterial infection. Challenge of mice with CpG-ODN mixed with Schistosoma eggs [58] or Amb1 (ragweed allergen) [82] led to an amazing attenuation of the Th2 bias of the subsequent
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immune responses. Covalent conjugation of CpG-DNA to proteinaceous allergen was up to 100-fold more efficient [68]. Infection of BALB/c mice with Leishmania major represents another model of Th2-driven disease. CpG-DNA converted BALB/c mice from a L. major-susceptible Th2 phenotype to a Th1-resistant phenotype associated with IL-12 production and sustained expression of the IL-12 receptor β chain (IL-12 Rβ) in draining LN cells. Strikingly, CpG-ODN could cure Leishmania-infected mice when administered as late as 15–20 days after infection [83]. Aseptic immunostimulation of mice with CpG-DNA alone induced local and transient lymphadenopathy associated with sustained IL-12 production by DCs and γ-IFN production by IL12Rβ2 positive T cells for up to 7–14 days. During this time period, mice were predisposed for Th1-biased subsequent adaptive immune responses [84]. Challenge up to two weeks earlier with CpG-DNA protected mice from lethal challenges with Listeria monocytogenes or Francisella tularensis [85,86], presumably because activated innate immune cells efficiently controlled replication of intracellular bacteria. Overall, these data suggest that TLR9-mediated CpG-DNA recognition by innate immune cells predisposes local lymphoid organs to organize an IL-12 and γ-IFN driven, long-lasting, elevated and locally stored potential for enhanced adaptive and Th1-biased immune responses [84]. An interesting feature of CpG-DNA as an adjuvant refers to the finding that it drives in mice robust maturation of IL-12-producing CD11c DCs (APCs) in the regional LN that allow T-helper-cell-independent CTL responses toward soluble proteins, as well as MHC class I restricted T cell epitopes [69•,87,88•]. Again CpG covalently bound to proteinaceous antigen strongly enhanced Th-independent CD8+ CTL responses, presumably by promoting antigen uptake via DNA-binding receptors on DCs.
Conclusions and future directions In mice and humans, innate immune cells appear to recognize CpG-DNA via TLR9. If one uses a TLR9 complementation system [53•] and NFκB-driven luciferase as ‘read out’ to compare species-specific CpG responsiveness, one is left with the conclusion that mouse cells express a high-responder phenotype, and human cells a low-responder phenotype [53•]. Altogether, this may turn out not to be a disadvantage for ongoing attempts to unravel the full therapeutic benefit of CpG-DNA in humans; the systemic side effects of CpG challenge observed in mice are likely to be nonexistent in humans. Independently, use of CpGDNA covalently conjugated to proteinaceous antigen may be, if necessary, the key to avoiding systemic side effects, as observed in mice. Another level of complexity may represent CpG-sequencedependent production, by DCs, of potentially harmful or useful cytokines [89]. In human, for example, CD11c– CD123+ plasmacytoid pDCs respond to optimal CpG motifs with rapid activation and maturation, yet this
response is associated with poor type I IFN production. On the other hand, stimulation with sequence-modified CpG motifs triggers high type I IFN production [71•,72•] but poor DC maturation. If it turns out that, in human DC subsets, the ‘quality’ (avidity) of CpG-DNA/TLR9 interactions controls the type of cytokine produced, tailoring of cytokine production may be possible. Exploiting the unique and potent immunobiology of CpG-DNA for therapeutic application in humans may be one of the challenges in biomedically oriented science.
Update Since submission of this review, evidence has been provided by Alexopoulou et al. that TLR3 recognizes double-stranded RNA [90]. This study describes a ligand for the orphan receptor TLR3.
Acknowledgements I thank the lab members for useful discussions and Georg Häcker for critical reading of the manuscript. This work is supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung and Coley Pharmaceutical GmbH, Hilden.
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48. Sparwasser T, Koch ES, Vabulas RM, Heeg K, Lipford GB, Ellwart JW, Wagner H: Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol 1998, 28:2045-2054. 49. Jakob T, Walker PS, Krieg AM, Udey MC, Vogel JC: Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J Immunol 1998, 161:3042-3049.
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50. Wagner H: Bacterial CpG DNA activates immune cells to signal infectious danger. Adv Immunol 1999, 73:329-368. 51. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, •• Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S: A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408:740-745. This work is the first to demonstrate that TLR9 represents an essential receptor component for bacterial CpG-DNA. 52. Chu W, Gong X, Li Z, Takabayashi K, Ouyang H, Chen Y, Lois A, • Chen DJ, Li GC, Karin M, Raz E: DNA-PKcs is required for activation of innate immunity by immunostimulatory DNA. Cell 2000, 103:909-918. Although the mechanisms are not defined, these findings implicate DNAdependent protein kinases in the signal pathways triggered by bacterial CpG-DNA. 53. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, • Wagner H, Lipford GB: Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001, 98:9237-9242. This study is the first to show gain-of-function to bacterial CpG-DNA by genetic complementation with TLR9, and defines species specificity of ligand–receptor interaction. 54. O’Neill LA, Greene C: Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J Leukocyte Biol 1998, 63:650-657. 55. Medzhitov R, Janeway CA Jr: Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997, 91:295-298. 56. Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z: MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 1997, 7:837-847. 57.
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58. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ: TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 2001, 412:346-351. 59. Shirakabe K, Yamaguchi K, Shibuya H, Irie K, Matsuda S, Moriguchi T, Gotoh Y, Matsumoto K, Nishida E: TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase. J Biol Chem 1997, 272:8141-8144. 60. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M: A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature 1997, 388:548-554. 61. Kopp E, Medzhitov R, Carothers J, Xiao C, Douglas I, Janeway CA, Ghosh S: ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes Dev 1999, 13:2059-2071. 62. Hacker H, Mischak H, Miethke T, Liptay S, Schmid R, Sparwasser T, Heeg K, Lipford GB, Wagner H: CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J 1998, 17:6230-6240. 63. Hacker H, Vabulas RM, Takeuchi O, Hoshino K, Akira S, Wagner H: • Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptorassociated factor (TRAF)6. J Exp Med 2000, 192:595-600. The finding that MyD88 controls signaling triggered by CpG-DNA was instrumental for the identification of the receptor TLR9 (see annotation to [51••]). 64. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S: Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 1999, 11:115-122. 65. Kaisho T, Takeuchi O, Kawai T, Hoshino K, Akira S: Endotoxin • induced maturation of MyD88-deficient dendritic cells. J Immunol 2001, 166:5688-5694. Activation of TLR4, but not of TLR9 or TLR2, drives APC maturation via a MyD88-independent pathway. 66. Muzio M, Bosisio D, Polentarutti N, D’amico G, Stoppacciaro A, Mancinelli R, van’t Veer C, Penton-Rol G, Ruco LP, Allavena P, Mantovani A: Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 2000, 164:5998-6004. 67.
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68. Tighe H, Takabayashi K, Schwartz D, Marsden R, Beck L, Corbeil J, Richman DD, Eiden JJ Jr, Spiegelberg HL, Raz E: Conjugation of protein to immunostimulatory DNA results in a rapid, long-lasting and potent induction of cell-mediated and humoral immunity. Eur J Immunol 2000, 30:1939-1947. 69. Cho HJ, Takabayashi K, Cheng PM, Nguyen MD, Corr M, Tuck S, • Raz E: Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism. Nat Biotechnol 2000, 18:509-514. Papers [69•,87,88•] demonstrate that in vitro and in vivo CpG-DNA activates (licenses) professional APCs to allow induction of T-helper-cell-independent cytotoxic CD8+ T cell responses. 70. Shirota H, Sano K, Hirasawa N, Terui T, Ohuchi K, Hattori T, Shirato K, • Tamura G: Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells. J Immunol 2001, 167:66-74. This study demonstrates a role for enhanced antigen uptake by DCs of CpG-DNA covalently coupled to proteinaceous antigen. 71. Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, • Ballas ZK, Endres S, Krieg AM, Hartmann G: Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol 2001, 31:2154-2163. See annotation to [73•]. 72. Kadowaki N, Antonenko S, Liu YJ: Distinct CpG DNA and • polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c– type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN. J Immunol 2001, 166:2291-2295. See annotation to [73•]. 73. Bauer M, Redecke V, Ellwart JW, Scherer B, Kremer JP, Wagner H, • Lipford GB: Bacterial CpG-DNA triggers activation and maturation of human CD11c–, CD123+ dendritic cells. J Immunol 2001, 166:5000-5007. Papers [71•–73•] demonstrate that human CD11c− CD123+ dendritic cells (DCs) are CpG-DNA responsive, whereas CD11c+ DCs are not. 74. Krieg AM, Wagner H: Causing a commotion in the blood: immunotherapy progresses from bacteria to bacterial DNA. Immunol Today 2000, 21:521-526. 75. Lipford GB, Bauer M, Blank C, Reiter R, Wagner H, Heeg K: CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur J Immunol 1997, 27:2340-2344. 76. Sparwasser T, Koch ES, Vabulas RM, Heeg K, Lipford GB, Ellwart JW, Wagner H: Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol 1998, 28:2045-2054. 77.
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78. Sato Y, Roman M, Tighe H, Lee D, Corr M, Nguyen MD, Silverman GJ, Lotz M, Carson DA, Raz E: Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 1996, 273:352-354. 79. Sun S, Kishimoto H, Sprent J: DNA as an adjuvant: capacity of insect DNA and synthetic oligodeoxynucleotides to augment T cell responses to specific antigen. J Exp Med 1998, 187:1145-1150. 80. Sparwasser T, Miethke T, Lipford G, Erdmann A, Hacker H, Heeg K, Wagner H: Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor-alpha-mediated shock. Eur J Immunol 1997, 27:1671-1679. 81. Sparwasser T, Hultner L, Koch ES, Luz A, Lipford GB, Wagner H: Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis. J Immunol 1999, 162:2368-2374. 82. Shirakawa T, Enomoto T, Shimazu S, Hopkin JM: The inverse association between tuberculin responses and atopic disorder. Science 1997, 275:77-79. 83. Zimmermann S, Egeter O, Hausmann S, Lipford GB, Rocken M, Wagner H, Heeg K: CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J Immunol 1998, 160:3627-3630. 84. Lipford GB, Sparwasser T, Zimmermann S, Heeg K, Wagner H: CpG-DNA-mediated transient lymphadenopathy is associated with a state of Th1 predisposition to antigen-driven responses. J Immunol 2000, 165:1228-1235.
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85. Krieg AM, Love-Homan L, Yi AK, Harty JT: CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J Immunol 1998, 161:2428-2434. 86. Elkins KL, Rhinehart-Jones TR, Stibitz S, Conover JS, Klinman DM: Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria. J Immunol 1999, 162:2291-2298. 87.
Vabulas RM, Pircher H, Lipford GB, Hacker H, Wagner H: CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses. J Immunol 2000, 164:2372-2378.
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88. Sparwasser T, Vabulas RM, Villmow B, Lipford GB, Wagner H: • Bacterial CpG-DNA activates dendritic cells in vivo: T helper cellindependent cytotoxic T cell responses to soluble proteins. Eur J Immunol 2000, 30:3591-3597. See annotation to [69•]. 89. Lipford GB, Sparwasser T, Bauer M, Zimmermann S, Koch ES, Heeg K, Wagner H: Immunostimulatory DNA: sequencedependent production of potentially harmful or useful cytokines. Eur J Immunol 1997, 27:3420-3426. 90. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA: Recognition of κB by Toll-like double-stranded RNA and activation of NF-κ receptor 3. Nature 2001, 413:732-738.
Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity Hermann Wagner The astounding adjuvanticity and Th1-polarizing immunobiology of bacterial CpG-DNA and mimicking CpG-oligonucleotides continue to mirror promising therapeutic potential. The past year has witnessed some particularly impressive progress in knowledge of its molecular mode of action. Accordingly, CpG-DNA acts as a ‘pathogen-associated’ molecular pattern that is recognized by TLR9 expressed, in particular, by dendritic cells. Interactions between CpG-DNA and TLR9 rapidly activate antigen-presenting dendritic cells through the ancient Toll/IL-1-receptor signaling pathway to upregulate co-stimulatory molecules and to produce Th1-polarizing cytokines, such as interleukin-12 and interleukin-18. Thus, interactions between CpG-DNA and TLR9 effectively bridge innate and acquired immunity. Addresses Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstraße 9, 81675 Munich, Germany; e-mail: [email protected] Current Opinion in Microbiology 2002, 5:62–69 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations APC antigen-processing cell DC dendritic cell GFP green fluorescent protein h human HSP heat shock protein IFN-γγ interferon-γ IL interleukin LPS lipopolysaccharide m murine MDDC monocyte-derived dendritic cell MHC major histocompatibility complex NK natural killer ODN oligonucleotide p precursor PAMP pathogen-associated molecular pattern PRR pattern recognition receptor TIR Toll/interleukin 1R domain Th T helper TLR Toll-like receptor α TNF-α tumor necrosis factor-α TRAF TNF-receptor-associated factor
Introduction The functioning of the immune system in higher organisms is based on two distinct recognition systems: innate (that is, non-clonal) and adaptive (that is, clonal) host defenses. The former builds on the function of innate immune cells, such as macrophages, dendritic cells (DCs) and natural killer (NK) cells, and traditionally has been viewed as non-specific, whereas the latter is founded on clonally distributed B and T cells and is typified by specificity and memory [1].
In this review, I discuss the recent advances made in the understanding of the interactions between DNA containing unmethylated CpG motifs from pathogenic microorganisms and the Toll-like receptor (TLR) 9 of DCs, and how these interactions stimulate the innate and adaptive immune responses. First, I provide a brief overview of the innate and adaptive immune responses.
The immune response When higher organisms are exposed to pathogenic microorganisms, innate immune responses occur immediately, both in terms of cell activation and inflammation. On one hand, they are characterized by uptake (that is, phagocytosis or endocytosis) and subsequent destruction or degradation of pathogens. On the other hand, the innate immune cells — macrophages and DCs — are acutely activated, resulting in the following examples: first, the upregulation of co-stimulatory cell surface molecules and major histocompatibility complex (MHC) class I and II molecules [2]; second, the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, and effector cytokines, such as IL-12 and type I interferon [3]; and third, the enhanced presentation of the products of pathogen degradation (antigenic peptides) via the MHC class I or II presentation pathway to antigenreactive T cells [2] and the production of bactericidal effector substances such as nitric oxide. Thus, innate immune cells represent not only a first line of defense towards infections but play a decisive and instructive role in emanating the adaptive immune responses [3]. A major challenge to innate immune cells is the discrimination of foreign pathogens from self. As originally perceived by Janeway [4,5], innate immune cells such as antigen-processing cells (APCs) possess germline-encoded pattern recognition receptors (PRRs) that recognize and are triggered by evolutionary conserved molecules essential to pathogen function but absent in the host. These pathogen-associated molecular patterns (PAMPs) are widespread and include cell wall components such as mannans in the yeast cell wall, lipopolysaccharide (LPS) in Gram-negative bacteria, lipoproteins, peptidoglycans and DNA containing unmethylated CpG motifs. There are at least two distinct classes of PRRs: those that mediate acute phagocytosis (internalization) and those that cause immediate cell activation. For example, scavenger receptors such as the mannose receptor (or related molecules such as langerin, DEC 205 and other lectins) mediate internalization of microorganisms primarily to direct them into detoxification pathways, yet they appear not to activate innate immune cells. On the other hand, upon cellular pathogen uptake, members of the TLR family become recruited to early phagosomes to screen their
Interactions between bacterial CpG-DNA and TLR9 Wagner
content for ligands from foreign pathogens and subsequently to trigger cell activation upon ligand recognition [6]. This apparent ‘division of labor’ (internalization versus cell activation) between scavenger receptors and TLRs bears a caveat, given that crosslinking of the scavenger receptor CD36 profoundly modulates LPS-driven DC maturation [7]. Adaptive immunity is controlled by the generation of MHC-restricted effector T cells and production of cytokines [8]. When stimulated by professional APCs such as DCs, naïve T helper (Th) cells may differentiate into Th1- versus Th2-polarized subsets; Th1 cells secrete primarily interferon IFN-γ, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13. Cytokines such as IL-12 (and, in man, type I interferons [9]) trigger via STAT4 IFN-γ the production of CD4+ T cells (Th1 polarization), whereas IL-4 induces Th2 differentiation. Upon activation, APCs (signal 1) upregulate the expression of co-stimulatory molecules, such as CD80 and CD86 (signal 2), thereby increasing immunogenicity of peptide antigens presented. Finally, APC activation triggers production of ‘instructive’ cytokines, such as IL-12, IL-18, IL-4 or IL-10, that are able to polarize emerging T cell responses (signal 3). Adding to the complexity, it is to date not clear whether all forms of activation of APCs necessarily result in increased immunogenicity. For example, certain forms of activation could selectively increase delivery of signal 1 without signal 2, giving rise to tolerogenic rather than immunogenic APCs. Furthermore, APCs can produce different cytokines (signal 3) in response to different activating stimuli [10]. A case in point is the observation that murine DCs phagocytosing either yeast or hyphae of Candida albicans produce either IL-12 or IL-4, and in vivo drive either Th1 or Th2 differentiation, respectively [11•]. It follows that APCs such as DCs and macrophages serve as a decisive interface bridging the innate and adaptive immune system [12], given that induction of productive T cell responses depends upon activation of APCs. Activation is brought about by PAMPs that are recognized by cell-activating PRRs. Furthermore, it is almost certain that the quality (intensity or avidity) of the individual ligand (PAMPs)–receptor (PRRs) interaction determines the functional state of APCs [13].
The Toll-like receptors and their ligands TLRs are transmembrane proteins and represent a newly recognized family of vertebrate PRRs. Today, we have come to realize that, for millions of years, recognition of pathogens by plants, insects and vertebrates has depended upon a system of receptors that share a characteristic domain now termed TIR (Toll/interleukin 1R domain) [14]. Surprisingly, the cytoplasmic TIR domain has remained conserved and functions in anti-pathogen responses in plants, insects and mammals alike [15]. The extracellular portion of the IL-1R family members contains, however, three immunoglobulin-like domains, whereas the extracellular portion of TLR family members contains leucine-rich repeats. To date, ten TLRs (TLRs 1–10) have been reported [16,17].
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Building on the role of Toll receptors in innate immunity of Drosophila [18], Janeway and colleagues identified in a landmark report [19] the first human homolog of Drosophila Toll. Equally important, subsequent positional cloning in C3H/HeJ mice revealed that this Toll homolog encodes the long-hunted signal transduction and receptor component for LPS, now termed TLR4 [20]. Indeed TLR4–/– mice turned out to be hyporesponsive to LPS [21] and, in man, TLR4 mutations are associated with LPS hyporesponsiveness [22•]. Notably, for TLR4-mediated signaling, CD14 must ‘individualize’ LPS (from micelles) and the secreted molecule MD2 [23] must associate with the extracellular position of TLR4. As it turns out, TLR4–MD2 not only recognizes LPS [24] but, in mice, also recognizes the structurally unrelated antitumor agent taxol complexed to heat shock proteins (HSPs) [25], as well as glucuronoxylmannan, a capsule component of the fungus Crytococcus neoformans [26], and even the outer envelope protein of respiratory syncytial virus [27•]. Furthermore, ‘endogeneous’ ligands potentially released from stressed or necrotic cells such as HSPs-60 [28•,29•] or HSP gp96 (RM Vabulas, S Braedel, N Hilf et al., unpublished data) engage TLR4 (as well as TLR2), presumably to activate DCs in the absence of infection. Finally, even cellular fibronectin fragments reportedly engage TLR4 [30]. Obviously TLR4 binds both ‘exogeneous’ ligands (that is, PAMPs) and ‘endogeneous’ ligands (such as HSPs). TLR2 recognizes lipoproteins (proteins triacylated at the amino-terminal cysteine residue) derived from Grampositive and Gram-negative bacteria including Borrelia, Treponema, Mycoplasma and Mycobacterium tuberculosis [31–34], as well as peptidoglycans [35], glycolipids and lipoteichoic acid [36], and atypical leptospiral LPS [37] and HSPs [29•]. It is difficult to reconcile the increasing list of putative TLR4 and TLR2 ligands with the concept of ligandspecific PAMP–TLR interactions. There is still the caveat of potential contaminations, given that, in equivalent assays, far higher concentrations (for example, of peptidoglycan) are required when compared with the concentration of bacterial lipopeptides. More interestingly, the ligand specificities of TLR2 may reflect a combinatorial repertoire generated by TLR2 heterodimerization, for example, with TLR1 or TLR6 [38•,39•]. Flagellin from Escherichia coli or Salmonella effects IL-8 release from intestinal epithelial cells [40] and causes NFκB activation in murine macrophages [41]. Very recently, flagellin-mediated cell activation has been shown to occur via TLR5 [42••].
TLR9 recognizes bacterial CpG-DNA Bacterial DNA, invertebrate DNA and DNA from some viruses such as herpes simplex virus type 1 (HSV-1) differ structurally from vertebrate DNA. First, bacterial DNA displays the expected frequency of CpG dinucleotides predicted by random usage (1:16) [43], whereas this frequency is suppressed fourfold in vertebrate DNA.
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Second, eukaryotic 5′-CpG-3′ motifs are preferentially (80%) methylated at a given time. Building on the pioneering work of Yamamoto and co-workers [44] and Krieg et al. [45], it is now accepted that, owing to these structural differences, unmethylated CpG motifs within bacterial DNA or within mimicking synthetic oligonucleotides (ODNs) directly activate murine B cells [45], macrophages [46,47] and DCs [48,49], but not T cells and probably not NK cells [50]. In the quest of the postulated CpG-DNA receptor, it turned out that cells of both TLR2- and TLR4-deficient mice responded to CpG-DNA, yet TLR9-deficient cells did not [51••]. This observation was consistent with the view that TLR9 represents an essential component of the postulated CpG-DNA receptor. However, mice lacking the DNA-dependent protein kinase (DNA-PK) also lacked responsiveness to CpG-DNA [52•]. This was unexpected, as SCID mice are known to harbor an inactivating mutation in the DNA-PK and yet can still respond to CpG-DNA [50]. Subsequently, it was certified that TLR9 expression in immune cells correlated with responsiveness to bacterial CpG-DNA and that responsiveness to CpG-DNA is brought about in human TLR9-deficient cells by genetic complementation with either human (h) TLR9 or murine (m) TLR9. Notably CpG-DNA responsiveness turned out to be independent of CD14 and MD2 (presumably because of the hydrophilic nature of CpG-DNA), yet required species-specific CpG-DNA motifs (that is, CpG-DNA motifs able to activate either human- or mouse-derived TLR9 in genetic complementation tests). For example, h-TLR9-transfected cells respond poorly to murine CpG motifs, and vice versa [53•]. Overall, these data imply that TLR9 conveys CpG-DNA responsiveness by directly recognizing CpG-DNA.
TLR9-mediated signaling There are remarkable structural and functional similarities between signaling pathways mediated by mammalian IL-1R [54] and those initiated by TLRs [55]. Binding of its respective ligands presumably causes TLR dimerization, which in turn triggers recruitment of the adaptor protein MyD88 via the cytoplasmatic TIRs of the TLR. This facilitates association with the IL-1R-associated kinase (IRAK) via the death domains [56]. Upon autophosphorylation, IRAK dissociates from the receptor complex and interacts with TRAF6, a member of the TNF-receptor-associated factor (TRAF) family of adaptor proteins [57]. TRAF6 participates in signaling initiated by TLRs and CD40. Unusual ubiquitination [58] allows TRAF6 to activate TAK-1 (TGF-β activated kinase), which in turn stimulates the MAP kinases, leading to JNK activation [59], or via NIK (NFκB-inducing kinase), which activates the IκB kinase complex to allow liberation of NFκB [60]. Furthermore, TRAF6 reportedly recruits evolutionary conserved signaling intermediate in Toll pathways (ECSIT), which takes place in macrophages/dendritic cells and activates via the MAP-3 kinase MEKK1 the transcription factors AP-1 and NFκB [61].
In order to unravel the molecular mechanisms of CpGDNA-driven cell activation, the cellular uptake of CpG-ODNs was studied, as well as the need for endosomal maturation and the role of the stress kinase pathway both in macrophages and DCs [62]. CpG-DNA induced activation of JNK1/2 and p38, leading to AP-1 activity. Furthermore, cellular uptake via receptor-mediated endocytosis and subsequent endosomal maturation was essential for signaling, given that competition by non-stimulating DNA as well as compounds blocking endosomal maturation (chloroquine or bafulomycin A) prevented all aspects of cellular activation [62]. Finally, CpG-DNA activated cells through the TLR/IL-1R signal pathway via MyD88 and TRAF6, and both macrophages and DCs of MyD88-deficient mice failed to show any sign of cellular activation upon exposure to CpG-DNA [63•]. This is in contrast to LPS-mediated activation of MyD88-deficient macrophages: LPS triggers TLR4 independently of MyD88, resulting in the partial activation of NFκB, JNK and p38 [64], and the MyD88independent LPS activation is able to drive IP-10 secretion and the expression of activation markers such as CD86, but not proinflammatory cytokines [32,64]. Obviously, MyD88-dependent and -independent signaling pathways exist and become activated upon LPS recognition by TLR4 [65•], whereas CpG-ODNs absolutely rely on signaling via MyD88 [63•,65•].
Bottlenecks in TLR signaling To understand the role of TLRs in mammalian immunity, it is essential to define their expression patterns in normal cells. First, distinct TLRs are differentially expressed among innate immune cells [66]. Second, TLR2 and TLR4, but not TLR9, are expressed at the cell surface and modulated upon maturation of human DCs from macrophages; for example, TLR3 expression increases and that of TLR5 is lost [67]. This raises the possibility that TLR9 becomes primarily recruited to intracellular cytoplasmatic compartments where they function by interacting with internalized PAMPs. If so, effective internalization would be a prerequisite for TLR-initiated signaling. A case in point represents CpG-DNA→TLR9 signaling in murine Raw macrophages. Raw macrophages expressing MyD88 tagged with green fluorescent protein (GFP) recruit this fusion protein to cytoplasmatic ‘endosome-like’ structures upon internalization of CpG-DNA, where these components co-localize with TLR9 (P Ahmad-Nejad, H Häcker, M Rutz et al., unpublished data). In contrast, without the need of endocytosis, ‘soluble’ LPS recruits GFP-tagged MyD88 to the cell membrane to initiate signaling. These data imply that ‘soluble’ LPS induced signaling at the cell membrane, whereas receptor-mediated endocytosis of CpGDNA is a condition for CpG-DNA/TLR9 initiated signaling. It is worth noting that the structure of the cell surface receptor that binds and translocates single-stranded DNA to endosomes in a sequence non-specific manner has not yet been characterized. It has been noted, however, that CpG-DNA covalently tagged to proteinaceous antigen
Interactions between bacterial CpG-DNA and TLR9 Wagner
effectively improves antigen uptake by DCs, whereas the cell-activating capacities of CpG-DNA are maintained [68,69•,70•]. Perhaps the ‘translocating’ DNA-binding receptor improves antigen uptake by DCs via receptormediated endocytosis.
Dendritic cells as sensors of infection Activation of Drosophila (d) Toll induces expression of an antifungal peptide, drosomycin, whereas ligation of its homologue, 18-wheeler, induces an antibacterial peptide, attacin [15]. Obviously, d-TLRs are capable of discriminating between fungi and bacteria. Mammalian DCs express TLR1–9, even though individual DC-subtypes differ with regard to TLR expression. For example, h-CD11c− CD123+ plasmacytoid precursor (p) DCs express TLR9 but low levels of TLR4, and are responsive to CpG-DNA but not to LPS. In contrast, monocyte-derived DCs (MDDCs) lack TLR9 but abundantly express TLR4 (and respond to LPS but not to CpG-DNA) [53•]. Furthermore, h-CD11c– CD123+ pDCs produce, in response to certain CpG-DNA motifs, large amounts of type I IFN but not IL-12 [71•–73•], whereas LPS causes MDDCs to produce IL-12 or TNF-α but not type I IFNs. It follows that, because of the different expression patterns of individual TLRs, DCs may not only sense the type of infection by recognizing pathogen-typifying PAMPs, but also respond in a cell-type-restricted manner. Perhaps type I IFN-producing plasmacytoid pDCs have evolved to control virus infections by members of the Herpes simplex virus family that are rich in immunostimulating CpG-motifs. DCs may not only sense the type of infecting pathogen via specific recognition of pathogen-typifying PAMPs, but also deliver critical information to antigen-reactive T cells about the context in which the ligand was encountered. Thus, DCs may convey information about the nature of the infectious agent and be able to influence the type of ensuing T cell responses. It is possible that the biological outcome of ligand-driven homo-(hetero)dimerization of TLR2, TLR4, TLR5 or TLR9 in terms of DC-derived cytokine production (type I IFN versus IL-12, IL-18) or chemokine production will turn out to be distinct. Furthermore, the magnitude of signal 1, of signal 2 and of signal 3 (see earlier) conveyed by DCs to antigen-reactive T cells is likely to influence the outcome of the adaptive T cell responses (that is, Th1 versus Th2). For example, CpG-DNA profoundly enhances the ability of DCs to support Th1 cell differentiation by inducing the release of IL-12 (signal 3), upregulation of CD40, CD80 and CD86 (signal 2), as well as the efficiency of antigen processing or presentation (signal 1).
The immunobiology of CpG-DNA As far as it is known, TLR9, which recognizes CpG-DNA, is not expressed at the cell surface of innate immune cells; upon CpG-DNA driven activation, both TLR9 and MyD88 co-localize at ‘endosome-like’ cytoplasmatic structures. CpG-ODNs ‘cross’ the cell membrane via
65
sequence-non-specific receptor-mediated endocytosis. Initiation of signaling via the Toll/IL-1R-signal pathway requires endosomal maturation [74]. Given the wealth of information that has accumulated recently on the role of TLR9 as the recognition structure for CpG-DNA able to activate DCs via the Toll/IL-1R signal pathway, the immunobiology of CpG-DNA is less astounding, at least retrospectively. Originally, however, it came as a surprise to observe that challenge of mice with a mixture of CpG-DNA (used as adjuvants) and proteinaceous antigens triggered strong peptide-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes (LNs) [75,76]. Furthermore, specific antibody responses were augmented and the CpG-ODNs switched the isotype to a Th1 profile (that is, antigen-specific Ig2a evolved as dominant) [75,77]. It also became evident that CpG motifs in naked plasmid DNA derived from bacteria used for subcutaneous or intramuscular gene vaccination promoted the Th1-polarizing adjuvanticity of plasmid DNA [78], as if the CpG motifs in the plasmid DNA backbone directly induced maturation and activation of DCs. In contrast, genegun administration of DNA vaccines preferentially induced Th2 responses. Obviously, the amount of plasmid DNA used correlates with Th1 polarization. Finally, insect DNA and CpG-ODNs surpassed the activity of complete Freund’s adjuvant, as measured by the parameters ‘proliferative T responses’, IFN-γ responses and specific IgG2a antibody responses [79]. A consensus, thus, emerged that immunostimulatory CpG-DNA can be viewed almost as an ideal adjuvant with the propensity to polarize the adaptive immune system towards a Th1 response. Compared with humans (see below), mice may be classified as ‘high’ CpG-DNA responders. In fact, systemic challenge of mice with CpG-ODNs causes a transient (1–6 hour) ‘cytokine storm’ in the blood; substantial concentrations of proinflammatory cytokines, including TNF-α, IL-6, IL-1β, IL-12 and GM-CSF, are found. As a consequence, D-galactosamine (D-Gal)-sensitized mice challenged with CpG-DNA succumb within 24 hours to TNF-α-mediated acute liver-cell apoptosis [80]. Presumably, owing to liberation in vivo of hematopoietic active factors such as GM-CSF, CpG-ODNs also cause transient extramedullary hematopoiesis [81] with up to a threefold increase in spleen weight. Splenic GM-CFUs as well as BFU-E and CFU-S become dramatically enhanced. It is worth noting that the enlarged spleen (day six) represents a convenient experimental source of DC progenitors. Epidemiological studies indicate an inverse relationship between Th1-promoting infections and the propensity of individuals to develop atopical disorders [82]. This raised the question of whether or not bacterial CpG-DNA would mimic the Th1-promoting effect of bacterial infection. Challenge of mice with CpG-ODN mixed with Schistosoma eggs [58] or Amb1 (ragweed allergen) [82] led to an amazing attenuation of the Th2 bias of the subsequent
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immune responses. Covalent conjugation of CpG-DNA to proteinaceous allergen was up to 100-fold more efficient [68]. Infection of BALB/c mice with Leishmania major represents another model of Th2-driven disease. CpG-DNA converted BALB/c mice from a L. major-susceptible Th2 phenotype to a Th1-resistant phenotype associated with IL-12 production and sustained expression of the IL-12 receptor β chain (IL-12 Rβ) in draining LN cells. Strikingly, CpG-ODN could cure Leishmania-infected mice when administered as late as 15–20 days after infection [83]. Aseptic immunostimulation of mice with CpG-DNA alone induced local and transient lymphadenopathy associated with sustained IL-12 production by DCs and γ-IFN production by IL12Rβ2 positive T cells for up to 7–14 days. During this time period, mice were predisposed for Th1-biased subsequent adaptive immune responses [84]. Challenge up to two weeks earlier with CpG-DNA protected mice from lethal challenges with Listeria monocytogenes or Francisella tularensis [85,86], presumably because activated innate immune cells efficiently controlled replication of intracellular bacteria. Overall, these data suggest that TLR9-mediated CpG-DNA recognition by innate immune cells predisposes local lymphoid organs to organize an IL-12 and γ-IFN driven, long-lasting, elevated and locally stored potential for enhanced adaptive and Th1-biased immune responses [84]. An interesting feature of CpG-DNA as an adjuvant refers to the finding that it drives in mice robust maturation of IL-12-producing CD11c DCs (APCs) in the regional LN that allow T-helper-cell-independent CTL responses toward soluble proteins, as well as MHC class I restricted T cell epitopes [69•,87,88•]. Again CpG covalently bound to proteinaceous antigen strongly enhanced Th-independent CD8+ CTL responses, presumably by promoting antigen uptake via DNA-binding receptors on DCs.
Conclusions and future directions In mice and humans, innate immune cells appear to recognize CpG-DNA via TLR9. If one uses a TLR9 complementation system [53•] and NFκB-driven luciferase as ‘read out’ to compare species-specific CpG responsiveness, one is left with the conclusion that mouse cells express a high-responder phenotype, and human cells a low-responder phenotype [53•]. Altogether, this may turn out not to be a disadvantage for ongoing attempts to unravel the full therapeutic benefit of CpG-DNA in humans; the systemic side effects of CpG challenge observed in mice are likely to be nonexistent in humans. Independently, use of CpGDNA covalently conjugated to proteinaceous antigen may be, if necessary, the key to avoiding systemic side effects, as observed in mice. Another level of complexity may represent CpG-sequencedependent production, by DCs, of potentially harmful or useful cytokines [89]. In human, for example, CD11c– CD123+ plasmacytoid pDCs respond to optimal CpG motifs with rapid activation and maturation, yet this
response is associated with poor type I IFN production. On the other hand, stimulation with sequence-modified CpG motifs triggers high type I IFN production [71•,72•] but poor DC maturation. If it turns out that, in human DC subsets, the ‘quality’ (avidity) of CpG-DNA/TLR9 interactions controls the type of cytokine produced, tailoring of cytokine production may be possible. Exploiting the unique and potent immunobiology of CpG-DNA for therapeutic application in humans may be one of the challenges in biomedically oriented science.
Update Since submission of this review, evidence has been provided by Alexopoulou et al. that TLR3 recognizes double-stranded RNA [90]. This study describes a ligand for the orphan receptor TLR3.
Acknowledgements I thank the lab members for useful discussions and Georg Häcker for critical reading of the manuscript. This work is supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung and Coley Pharmaceutical GmbH, Hilden.
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