The arthritogenic and immunostimulatory properties of phosphorothioate oligodeoxynucleotides rely on synergy between the activities of the nuclease-resistant backbone and CpG motifs.

Experiments with immunostimulatory unmethylated CpG-containing DNA are usually conducted with nuclease-protected phosphorothioate oligodeoxynucleotides (S-ODNs), rather than phosphodiester oligodeoxynucleotides (O-ODNs). We compared the murine immune responses to S-ODNs and O-ODNs that either contained or lacked CpG motifs. Both CpG and non-CpG S-ODNs induced synovitis, as did sequence-matched CpG O-ODN, but not GpC O-ODN. There was a minimum length requirement for arthritogenic S-ODNs since a CpC dinucleotide S-ODN did not induce arthritis. There were both sequence- (CpG > non-CpG) and backbone-dependent (S-ODN > O-ODN) differences in the levels of DNA-induced arthritis upon intra-articular injection with the ODNs. However, CpG O-ODN being an exception, induced more severe arthritis than the GpC S-ODN. The levels of in vitro proliferation and production of IL-6, TNF-alpha, IL-12, and RANTES by splenocytes following exposure to CpG S-ODN were significantly higher than those induced by CpG O-ODN. In addition, both proliferative responses and cytokine production induced by S-ODN-stimulated splenocytes increased significantly when the S-ODN contained a CpG motif. Transcription factor NFkappaB was activated by both CpG S-ODN and CpG O-ODN but interestingly not by GpC S-ODN. This indicates that the NFkappaB signal pathway modulates CpG-mediated immunostimulation, while sequence-independent immune activation by the phosphorothioate backbone is probably signalled via a different pathway.