CpG Dinucleotide Motif

It is known from the 1980s that bacterial DNA stimulates the formation of cytotoxic IFN-a , jS, and IL-12 when the DNA is taken up by macrophages. It in turn leads to NK cell activation and production of pro-inflammatory cytokine IFN-y. This is accompanied by the proliferation of B-cells and therefore the reduction of apoptosis and release of IL-6 and IL-12 (246-249). These pro-inflammatory effects were found to be caused by some im-muno-stimulatory sequences in prokaryotic DNA that contained unmethylated CpG dinucleotide motif flanked by two 5 purines and two 3 pyrimidines. Plasmid DNA, which is derived from bacterial DNA, induces these immune responses. The unmethylated CpG motif-containing sequence occurs four times more frequently in prokaryote DNA than in eukaryotic DNA. Moreover, the CpG motifs are usually 75% more methylated in mammalian DNA than in prokaryotic DNA (250,251). On methylation of the cytosine bases in plasmids, the immuno-stimulatory effect is decreased considerably (252). Immature den-... 

Figure 22.1 Optimal CpG motifs. The stimulatory effects of a particular sequence of DNA are determined by the base context in which the CpG dinucleotides appear. The most immune stimulatory CpG motifs are preceded by any base except a C, and followed by any base except a G. The positions of two bases further from the CpG are less important, although a purine on the 5 side and pyrimidines on the 3 side generally lead to a more immune stimulatory CpG motif. Although there is some cross-stimula-tion, the preferred human and mouse motifs are slightly different, as shown above.

In parallel to these initial descriptions, observations in the antisense field of study revealed that some antisense ODNs displayed sequence-specific immunostimulatory properties (Branda et al., 1993 Krieg et al., 1989, 1993 McIntyre et al., 1993 Pisetsky and Reich 1994 Tanaka et al., 1992). Exhaustive study revealed that immunostimulation by these ODNs could be attributed to the presence of CpG dinucleotides, in particular to base contexts (most importantly a hexamer) that were not restricted to a palindrome (Krieg et al., 1995) (Figure 22.1). Further, methylation of the cytosine in the CpG dinucleotide completely abrogated any immunostimulatory properties, confirming that the immunostimulatory properties of these ODNs were dependent on the presence of the unmethylated CpG dinucleotides (Krieg et al., 1995). Such immunostimulatory sequences based on unmethylated CpG dinucleotides are now known as CpG motifs. 

On the other hand, extrinsic stimuli are often delivered by viruses and bacteria. Bacterial products like lipopolysac-charide (LPS) and DNA with motifs of unmethylated CpG dinucleotides are extremely potent inducers of neuroinflammation. Additionally, viruses themselves, or their products like retroviral coat protein gp41 and gpl20, double stranded RNA, and transcription factors like Tat, also induce inflammatory responses in brain cells. 

The dinudeotide CpG is significantly under-represented in vertebrate DNA and is usually methylated. In contrast, CpG dinucleotides are generally present at the expected firequency in bacterial DNA and are unmethyiated. These unmethylated CpG motifs induce B cells to secrete IL-6 and IgM and can induce NK and CD4 T cells to produce the immunoregulatory IFNy. 

The unmethylated CpG sequence, i.e., the CpG motif, is a danger signal for our immune system.20 A potent CpG motif consists of a central unmethylated CpG dinucleotide flanked by two 5 purines and two 3 pyrimidines. Compared with the DNA of eukaryotic cells (frequency of 1 64), bacterial genomic DNA contains a higher frequency of the dinucleotide sequence CpG (1 16). Prokaryotic DNA is relatively unmethylated compared with the eukaryotic form, in which approximately 80% of the cytosines are methylated, a modification known to eliminate immunostimulation. These differences allow the mammalian immune system to recognize and respond to foreign DNA of bacterial origin. In addition to the immu-... 

Sequence-dependent toxicity Palindromic sequences and dinucleotide motifs containing CpG sequences in particular, have been shown to posses potent immunostimulatory properties in rodents. Similar effects do not seem to occur in primates. 

As described above, certain chenucal modifications introduced within the CpG dinucleotide that alter stmcture and conformation lead to the loss of immune-stimulatory activity of agonists. One such modification is substitution of the methyl group at the 5-position of cytosine in the CpG motif of TLR9 agonists [66]. Vertebrates use this feature to distinguish self-DNA from that of bacterial DNA, which contains more tmmethylated CpG motifs. 

Kandimalla ER, Bhagat L, Zhu F-G et al (2003) A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species specific recognition observed with CpG motif. Proc Natl Acad Sci USA 100 14303-14308... 

CpG stands for cytosine phosphate guanine dinucleotide in a particular sequence context. CpG motifs are responsible for proliferative effects of antisense oligonucleotides, particularly with respect to B-lymphocytes. Die optimal immune-stimulatory consensus sequence surrounding CpG is R1R2CGY1Y2, where R1 is a purine (mild preference for G), R2 is a purine or T (preference for A), and Y1 and Y2 are pyrimidines (preference for T). 

DNA isolates from bacteria ehcit antitumor activity [33, 34], augment NK cell activity [35, 36] and induce interferon-a, interferon-p, and interferon-y production [36]. DNA from different bacterial species, but not mammalian DNA, stimulates prohferation of murine lymphoc3des [37]. Further studies showed that synthetic oligonucleotides containing certain self-complementary paUndromic sequences with CG dinucleotides, similar to those present in bacterial DNA, increase the cytolytic function of NK cells and induce secretion of IFN-y [38, 39]. Synthetic oligonucleotides containing CpG motifs were also shown to induce immune responses [40]. 

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