PMC:3537549 / 29782-32449 JSONTXT

{"project":"2_test","denotations":[{"id":"22906110-11449369-143530980","span":{"begin":235,"end":237},"obj":"11449369"}],"text":"ODN 2216 influence type I IFN and proinflammatory gene expression in primary feline immune cells\nThrough interaction with the TLR9, CpG-A typically induce expression of both type I IFN and proinflammatory cytokines in stimulated cells[12]. In order to understand whether ODN 2216 exert similar effects in the cat, treated cultures of feline PBMCs, FEA, CrFK and fcwf-4 cells were systematically screened for increased mRNA expression of IFNα and IL-6 following stimulation. Although all tested immortalized feline cell lines expressed TLR9 mRNA (data not shown), they failed to respond to stimulation with ODN 2216 (Figure3A and B and data not shown). However, this molecule exhibited potent immunomodulatory properties in feline PBMCs: when measured 24 h post stimulation, a concentration of only 1 μg/mL ODN 2216 was sufficient to enhance transcription of IFNα by 40-fold, and a maximum induction of this gene was observed when 4 μg/mL ODN were used (Figure3C). Although affected in a similar pattern, the mRNA expression of the proinflammatory cytokine IL-6 remained comparatively low at all concentrations tested (Figure3D). In experiments foreseen to determine gene expression kinetics in feline cells after stimulation with the ODN 2216 molecule, an influence on IFNα transcription could be measured as early as 3 h after treatment of PBMCs, whereas increased levels of IL-6 mRNA were observed only as of 6 h post stimulation (Figure3E and F). Notably, the highest induction of both genes was measured 24 h after addition of ODN 2216 to the cultures, with transcription increasing by 9000-fold and 39-fold at this time point for IFNα and IL-6 respectively (Figure3E and F). The observed effects were specifically conferred by the CpG motifs comprised in the 2216 ODN, since specific control ODN 2243 only induced slight elevations in the expression of the genes tested (Figure3C-F). Importantly, trypan blue exclusion experiments indicated no evidence of cellular toxicity after treatment neither with ODN 2216 nor with ODN 2243 at all concentrations tested (data not shown).\nFigure 3 ODN 2216 influence IFNα and IL-6 expression in primary feline immune cells. mRNA expression factors of IFNα and IL-6 were measured in (A, B) fcwf-4 cells stimulated with ODN 2216 and (C-F) feline PBMCs from one cat belonging to group 2 stimulated with either ODN 2216 or control ODN 2243. The transcription of both genes was assessed either 24 h after treatment of the cells with increasing concentrations of ODN (A, C, D), or over time after a single stimulation with 4 μg/mL ODN (B, E, F). Experiments C-F were repeated for 4 adult cats and representative results are shown."}