Now I Know My CpGs

[编者的话]

The vertebrate innate immune system detects the presence of infection by the recognition of molecular patterns that are present in many pathogens but absent from host tissues. For example, lipoproteins, peptidoglycans and lipopolysaccharides are all detected by one or more of the Toll-like receptors (TLRs), which have emerged as key receptors in this innate immune function. A recent study reports that a CpG motif, an immune stimulatory sequence characteristic of bacterial but not vertebrate DNA, is also detected by the TLR system.

The vertebrate immune system is frequently faced with new antigens. Some antigens are associated with pathogens and necessitate a rapid response if the organism is to survive. Other antigens might initially be expressed as the result of physiological changes (e.g. puberty) or can be ingested in food, in which case immune responses should be avoided. One of the central problems for the immune system is in deciding which antigens to respond to - how does it tell when it is really being infected? The answer to this question has much greater implications than merely satisfying scientific curiosity. If it were possible to 'trick' the immune system at will into reacting as if a particular antigen was from an infectious pathogen, then it should be possible to design safer and more effective vaccines and cancer immunotherapies.

Activation of Innate Immunity

The proinflammatory effects of bacterial extracts have been recognized for centuries. In fact, crude extracts known as 'Coley's toxins' were used in treating >1000 patients with advanced malignancy, with some remarkable successes (reviewed in reference 1). Mycobacterium bovis bacillus Calmette-Guérin (BCG) is highly active in many mouse tumor models and continues to be useful in the immunotherapy of bladder cell cancer in humans. Studies to determine the active component within BCG revealed the surprising finding that the DNA itself was the component capable of inducing anti-tumor immune responses in mice [2,3]. Subsequently, we demonstrated that bacterial DNA activates several cell types (figure 1) and that the immune stimulatory effects resulted from the presence of CpG dinucleotides in particular base contexts.

These CpG motifs distinguish bacterial DNA from vertebrate DNA in three ways. First, vertebrate genomes uniformly show an unexplained feature termed CpG suppression - the frequency of CpG dinucleotides is only ~25% of the frequency that would be expected if base usage was random. Second, vertebrate genomes show CpG skewing, that is, the CpG dinucleotides that are present more commonly occur in base contexts that are not immune stimulatory than in base contexts that are [4]. Third, the CpG dinucleotides in vertebrate genomes are usually methylated, which abolishes their immune stimulatory effect [5].

That the unmethylated CpG motifs in bacterial DNA are responsible for its immune stimulatory activity is demonstrated by the fact that this activity is lost when these motifs are methylated5. However, demethylation of the CpG motifs in vertebrate DNA does not restore immune stimulatory activity [6], probably owing to the skewing in the CpG motifs and the presence of inhibitory sequences [4].

Toll-like Receptors

In recent years, a new paradigm has arisen to explain the mechanisms used by the immune system to detect infections rapidly. This function is fulfilled by the innate immune system, through cells such as dendritic cells (DCs), macrophages, monocytes and neutrophils. These innate immune cells lack the sophisticated and exquisitely antigen-specific receptors of T and B cells, but have instead evolved a set of pattern recognition receptors (PRRs) that enable them partially to sense the world around them. Different classes of pathogens, be they viruses, Gram-negative or Gram-positive bacteria, protozoans or multicellular parasites, possess certain molecular signatures that are shared across many genera and families of microorganisms, but are absent from vertebrate cells. Termed pathogen-associated molecular patterns (PAMPs) by Janeway and colleagues [7], these motifs bind to the PRRs on innate immune cells. Some PRRs appear to be expressed only on particular cell subsets, whereas others are expressed more widely. Depending on the cell types that are activated and the molecular pathways that are triggered by particular PRRs or PRR combinations, the immune system as a whole appears to integrate the resulting signals, which presumably allows it to then trigger the type of immune response that has been evolutionarily selected for this pattern or combination of molecular patterns [8].

A major recent discovery in immunology is the identity of a family of key PRRs, the toll-like receptors (TLRs) [9,10]. Of the ten reported mammalian TLRs, until recently only two had identified ligands (Table 1). Immune activation by bacterial endotoxins was known to require TLR-4 [11], whereas recognition of bacterial lipoproteins and peptidoglycans required TLR-2 [12,13]. In a recent issue of Nature, Hemmi et al. reported that TLR-9 is required for immune activation by a CpG motif in a synthetic oligodeoxynucleotide (CpG ODN) [14]. This exciting finding reinforces the role of the TLRs in innate immune recognition of PAMPs and provides novel insights into the molecular mechanism of action of CpG DNA. To interpret this finding fully, it is necessary to review the prior state of knowledge concerning the mechanism of action of CpG DNA.

Cell Uptake and Endosomal Acidification and/or Maturation

The previously identified TLR ligands are thought to induce innate immune cell signaling by interacting with their microbial ligands (and specific host-produced cofactors) at the cell membrane. By contrast, CpG DNA requires cell uptake for activation to occur, suggesting that if it binds the CpG motifs directly, TLR-9 might not work at the cell membrane [5]. Unidentified cell surface proteins bind DNA in a sequence-independent manner, internalizing it into acidified endosomal vesicles [15]. In contrast to cellular activation by lipopolysaccharide (LPS), endosomal acidification and/or maturation is required for CpG-induced activation [16-18]. This internalization is accompanied by the rapid generation of reactive oxygen species and the induction of mitogen-activated protein kinase signaling pathways, culminating in the activation of transcription factors such as nuclear factor (NF)-kB and activator protein 1 (AP-1) [15]. TLR-9 has a transmembrane domain; however, these findings suggest that its role in mediating CpG-induced signaling could be fulfilled through expression in an intracellular compartment such as the endosome, or perhaps through recruitment of additional cofactors, which might only occur within the endosomes or other intracellular sites. It is worth noting that direct associations have generally not been reported between other TLRs and their ligands. Instead, cofactors might be required, such as CD14 and LPS-binding protein in the case of TLR-4. Potential cofactors for TLR-9 have not yet been identified although, like most other TLRs, signaling requires the presence of the adapter protein MyD88 [19,20].

Other Mechanisms Involved in Recognition of CpG Motifs

It is worth pointing out that the requirement for TLR-9 in CpG-induced signaling has only been demonstrated using a single type of CpG motif in a synthetic ODN [14]. Because bacterial DNA has a wide range of CpG motifs, and these are in a native phosphodiester backbone instead of the phosphorothioate backbone that is commonly used with CpG ODN, it remains possible that TLR-9 could be only part of the story. It is therefore intriguing that an independent group of researchers has recently suggested a role for the DNA-dependent protein kinase, DNA-PK, in mediating immune activation by both bacterial DNA and CpG ODN [21]. In this study, Chu et al. reported that CpG DNA sequences induce the catalytic activity of DNA-PK in vitro and that mice genetically lacking the catalytic subunit of DNA-PK fail to respond to CpG stimulation. Although the activity of DNA-PK had previously been shown to be induced in a sequence-independent fashion by the ends of double-stranded DNA (dsDNA) and by other DNA structures, these investigators reported that the optimal activation of DNA-PK activity required the presence of unmethylated CpG motifs, and was reduced by DNA methylation. DNA-PK activation had been previously associated with the cell activation response to damaged DNA, but is now suggested by these investigators to be more generally linked to the activation of host defenses in response to foreign DNA.

These results do not clarify whether the sequence-specific immune activation observed is caused by preferential binding of DNA-PK to CpG DNA, or whether a multi-protein complex, possibly including TLR-9 or other factors, is involved. Although the findings of Chu et al. are provocative and stimulating, the hypothesis that CpG-induced DNA-PK catalytic activity is required for the resulting immune stimulation is inconsistent with previous reports that CpG DNA is a highly effective immune stimulator in severe combined immunodeficient (SCID) mice, as these mice have no DNA-PK catalytic function [22]. Previous studies have reported that dsDNA sequences without CpG motifs can trigger cellular activation through pathways that appear to be independent of those activated by CpG motifs [23]. Given the known ability of dsDNA sequences to activate DNA-PK, this latter observation can be more easily reconciled with a role for DNA-PK than can CpG-specific regulation. Resolution of the questions raised by these disparate studies will require substantial additional investigations.

Different CpG DNA Molecules Trigger Distinct Immune Effects

Multiple mechanisms are likely to be involved in the immune stimulatory effects of CpG ODN, as ODN with different backbones and different sequence motifs can induce dramatically different profiles and kinetics of immune activation [24-27]. For example, ODN containing phosphodiester backbones are particularly effective at activating NK cells, whereas CpG motifs in nuclease-resistant phosphorothioate backbones have dramatically enhanced B-cell stimulatory properties but reduced NK-cell stimulation [5,24]. Based on these properties, we have suggested that several distinct families of CpG ODN can be recognized (Table 2) [28]. These observations suggest that distinct CpG ODN activate multiple independent as well as shared signaling pathways in parallel. A possible explanation for this is that the ODN might serve as a kind of scaffold upon which various CpG-specific or non-specific DNA-binding proteins can form different complexes, depending on the DNA backbone, the CpG motifs and the other DNA motifs present. In considering these various models, it must be kept in mind that long DNA sequences are not required for immune activation; sequences as short as six bases show some activity, although perhaps not the full range of effects that can be observed with longer ODN [29].

Conclusion

The ability of the innate immune system to distinguish microbial from self DNA based on differences in the content of unmethylated CpG sequences is an elegant demonstration of the PRR principle. To the reductionist, it is pleasing that the immune system should have solved the problem of self-non-self discrimination by using a single family of proteins, the TLRs, to detect such a diverse group of microbial molecules (Table 1). At this time, the identity of the protein(s) that specifically bind to CpG motifs remains unclear, as well as whether there are different CpG-binding proteins for different motifs or different cofactors that are recruited into a multi-protein complex. Regardless of the ultimate resolution of these outstanding questions, the recent reports of proteins required for CpG-induced immune activation provide important new directions for research in this exciting field.