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Reduced nitric oxide production and iNOS mRNA expression in IFN-γ-stimulated chicken macrophages transfected with iNOS siRNAs

Cheeseman, Jennifer H., Lillehoj, Hyun S., Lamont, Susan J.
Veterinary immunology and immunopathology 2008 v.125 no.3-4 pp. 375
chickens, immune system, immune response, biochemical pathways, genes, gene expression, messenger RNA, nitric oxide, enzymes, nitric oxide synthase, interferons, signal transduction, in vitro studies, cell lines, macrophages, transfection, RNA interference, gene expression regulation, inhibitors, enzyme inhibition, interferon-gamma
Utilizing RNA interference technology with siRNA in the HD11 macrophage cell line, we determined how the inhibition or knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-γ-induced macrophage production of nitric oxide (NO) and mRNA expression of genes involved in this biological pathway in the chicken. Chicken macrophages produce NO when stimulated with recombinant chicken IFN-γ, however, when transfected with iNOS siRNAs, the production of NO is significantly decreased. We observed a 14-28% reduction in NO production by IFN-γ-stimulated HD11 cells at 48h after initial siRNA transfection compared to non-transfected IFN-γ-stimulated macrophages. Significant knock-down of iNOS mRNA expression (15 to 50-fold lower) was observed for each of four iNOS siRNAs, when compared to non-transfected IFN-γ-stimulated macrophages and to those treated with a negative control siRNA. The IFN-γ-stimulated chicken macrophages transfected with iNOS siRNAs did not show altered levels of mRNA expression for genes involved in IFN-γ signaling and iNOS pathways (IL-1β, IL-6, IFN-γ, TGF-β4, or SOCS-3) suggesting that the observed decrease in NO production is a direct result of siRNA mediated knock-down of iNOS, rather than IFN-γ-induced changes in the other genes tested.