Cytosine-phosphate-guanine (CpG) motifs are sensitizing agents for lipopolysaccharide in toxic shock model

Objective: Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides are highly frequent motifs in bacterial DNA and rare in the mammalian genome. They are potent inducers of inflammatory cytokines and act synergistically with lipopolysaccharide (LPS) for the induction of tumor necrosis factor alpha (TNF-alpha) production in vivo. It has therefore been suggested that innate immune reaction to bacterial unmethylated CpG motifs might contribute to the development of septic shock. We designed this study to assess the sensitization role of CpG motifs in LPS-induced shock using the D-galactosamine (D-GalN)-sensitized mouse model. Design: A prospective, randomized in vivo animal laboratory study. Setting: Experimental research laboratory. Intervention: We performed experiments in which CpG, LPS and D-GalN were administrated sequentially in various orders or simultaneously in 8week-old BALB/c mice. Measurements and results: Cytosine-phosphate-guanine treatment potentiated LPS action only if injected prior to LPS. A combination of predefined sublethal doses of CpG (1 nmol/mouse) and LPS (1 ng/mouse) not only had a synergetic effect on TNF-alpha production (20.3 +/- 9.2 IU/ml versus 2.5 +/- 1.4 IU/ml and 5.6 +/- 3.4 IU/ml for CpG and LPS C, groups, respectively, p < 0.05), but also led to animal death (5/5). An CpG effect requires de novo mRNA synthesis, since the sensitizing effect was inhibited by co-administration of mRNA transcription inhibitors such as D-GalN and pentoxifylline, which is a specific TNF-alpha transcription inhibitor. Furthermore, CpG treatment provoked a strong TNF-alpha mRNA production in the liver that was dramatically reduced by pre-treatment with D-GalN. Conclusion: Our findings indicate that CpG motifs act synergistically with LPS by initializing the synthesis of TNF-alpha and/or TNF-alpha regulating factors, thereby acting as a sensitizing agent.