Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro.

This research was conducted to explore the effect of different nucleic acid preparations on the production of cytokines, specifically type I interferons and interleukin-6, in horse cells. It was observed that both bacterial plasmid DNA and short synthetic oligodeoxyribonucleotides containing unmethylated CpG-motifs induced the production of interferons in the presence of lipofectin, but not without. Additionally, the presence of poly-guanine sequences was associated with increased interferon output.

Study Method

• To stimulate cytokine production, researchers added bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides to cultures of peripheral blood mononuclear cells from horses, a type of white blood cell that plays a key role in immune response.
• The study assessed the effects of these DNA preparations both with and without the transfection reagent lipofectin, a compound that transports nucleic acids into cells.
• They measured cytokine activity through bioassays and detected cytokine mRNA expression using RT-PCR, a method used to amplify and quantify DNA.

Key Findings

• The researchers found that both the bacterial plasmid DNA and the synthetive oligodeoxyribonucleotides that contained an unmethylated CpG-motif induced the production of interferons only in the presence of lipofectin. Interferon production did not occur without lipofectin.
• Neither the bacterial plasmid DNA nor the synthetic oligodeoxyribonucleotides followed a consistent pattern in their ability to stimulate Interferon production, as the levels varied from case to case and were often quite low. If the CpG sequence was methylated or removed entirely, no Interferon was produced.
• The synthetic oligodeoxyribonucleotides that included poly-guanine sequences induced higher levels of cytokine, especially those linked with a phosphorothioate backbone.
• Conversely, the cytokine interleukin-6 was produced in baseline levels in control cultures, and was also increased threefold following the introduction of the nucleic acid preparations.
• Additionally, there was evidence to suggest that some of the oligodeoxyribonucleotides increased the expression of IL-12p40 mRNA, a subunit of the cytokine interleukin-12.

Conclusion

• The findings concluded that horse peripheral blood mononuclear cells responded to CpG-DNA with the production of both type I interferons and/or Interleukin-6. Various factors such as CpG-motifs, the presence of poly-G sequences, the oligodeoxyribonucleotide’s backbone, and the presence of lipofectin influenced the quantity of cytokine production and the type of cytokine produced.