Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells

122

Endotoxic shock is a life-threatening condition caused by exposure to bacterial LPS. LPS triggers the release of acute phase, proinflammatory, and Th1 cytokines that facilitate the development of endotoxic shock. Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited by a variety of immune stimuli. The current results demonstrate that suppressive ODN protect mice from LPS-induced endotoxic shock. Underlying this protective effect is the ability of suppressive ODN to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade mediated by LPS-induced IFN-β and IL-12. These findings suggest that suppressive ODN might be of use in the treatment of endotoxic shock.

Section: Discussionmentioning

confidence: 78%

Suppressive Oligodeoxynucleotides Protect Mice from Lethal Endotoxic Shock

Shirota

Gürsel

et al. 2005

122

“…It was also reported that sequences within the telomeres of the mammalian chromosome contain a motif that can down-regulate TLR-9 activation and that this could be an important mechanism to prevent autoimmunity (12). These observations have been strengthened by the identification of oligonucleotides that can effectively inhibit responses to CpG-containing sequences (7)(8)(9)(10)(11)(12). These ODNs are able to regulate TLR-9 activation; thus, we have named them IRS.…”

Section: Discussionmentioning

confidence: 99%

“…These sequences are derived from diverse sources, including viral sequences, mutated CpG sequences, and repeats of the TTAGGG motif present in mammalian telomeres (7)(8)(9)(10)(11)(12). We have termed these immunoregulatory DNA sequences (IRS), because they act to regulate immunostimulatory TLR-9 ligands.…”

Section: Inhibitors Of Tlr-9 Act On Multiple Cell Subsets In Mouse Anmentioning

confidence: 99%

Inhibitors of TLR-9 Act on Multiple Cell Subsets in Mouse and Man In Vitro and Prevent Death In Vivo from Systemic Inflammation

Duramad

Fearon

Chang

et al. 2005

111

125

In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-α and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.

“…Although a number of studies have shown an inhibitory action of viral (12) and vertebrate (13)(14)(15) DNA or specific ODN (12-14, 16 -20), there is no consensus in the literature about the nature of such motifs. The suggestions for inhibitory motifs include GC-rich sequences such as CCGG (12), GC-rich sequences when linked closely to CpG (21), methylated CpG motifs (15), certain G-rich sequences such as GGAGGGG (16,17), and deoxyguanosine (dG) homopolymer (13,14,22). In none of these cases is it known how an inhibitory effect is mediated, and competition for CpG DNA uptake has not always been eliminated as a mechanism.…”

mentioning

confidence: 99%

The Molecular Basis for the Lack of Immunostimulatory Activity of Vertebrate DNA

Stacey

Young

Clark

et al. 2003

162

193

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at ∼3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 μg/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.