title: Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro.
abstract: This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, containing an unmethylated CpG-motif, were able to induce IFN production in the presence of lipofectin but not without. The levels of IFN varied with individuals and were often quite low. Moreover, methylation or removal of the CpG sequence completely abolished IFN induction. CpG-containing ODN with poly-guanine (G) sequences in the 5' and 3' ends induced considerably higher levels of IFN, especially when the poly-G sequences had a phosphorothioate backbone. ODN with poly-G sequences also had the ability to induce IFN in the absence of lipofectin but the levels of IFN induced were radically reduced compared to those induced with lipofectin. In contrast to IFN, which was only detected upon induction, low spontaneous IL-6 production was observed in unstimulated control cultures. Nevertheless, plasmid DNA and CpG-containing ODN were able to increase the IL-6 production threefold. All the IFN inducing ODN also induced IL-6 production and the levels of IL-6 induced seemed influenced by addition of lipofectin and presence of poly-G sequences in the same way as was observed for the IFN-production. However, a complete phosphorothioate ODN with a central CpG-motif and poly-C sequences, that did not induce IFN, readily induced IL-6 both in the presence and absence of lipofectin. In addition, there was also evidence that some ODN induced increased expression of IL-12p40 mRNA. To conclude, equine PBMC were able to recognize CpG-DNA and respond with both IFN-alpha/beta and/or IL-6 production. The levels of cytokine induced, and sometimes which cytokine induced, varied with, e.g., CpG-motifs used, the presence of poly-G sequences, ODN backbone chemistry and presence of lipofectin.
authors.affiliation: Section of Veterinary Immunology and Virology, Department of Molecular Biosciences, Swedish University of Agricultural Sciences, SE-75123 Uppsala, Sweden. Eva.Wattrang@adm.slu.se
authors.forename: Eva
authors.initials: E
authors.lastname: Wattrang
authors.forename: Mikael
authors.initials: M
authors.lastname: Berg
authors.forename: Mattias
authors.initials: M
authors.lastname: Magnusson
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006736:Horses / Q000276:immunology*; D066298:In Vitro Techniques; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology*; D008297:Male; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D016207:Cytokines; D017931:DNA Primers; D007370:Interferon Type I; D015850:Interleukin-6; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004247:DNA