Reduced nitric oxide production and iNOS mRNA expression in IFN-gamma-stimulated chicken macrophages transfected with iNOS siRNAs.
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-gamma-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-gamma, however, when transfected with iNOS siRNAs, the production of NO is significantly decreased. We observed a 14-28% reduction in NO production by IFN-gamma-stimulated HD11 cells at 48h after initial siRNA transfection compared to non-transfected IFN-gamma-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-gamma-stimulated macrophages and to those treated with a negative control siRNA. The IFN-gamma-stimulated chicken macrophages transfected with iNOS siRNAs did not show altered levels of mRNA expression for genes involved in IFN-gamma signaling and iNOS pathways (IL-1beta, IL-6, IFN-gamma, TGF-beta4, 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-gamma-induced changes in the other genes tested.
Cheeseman
Jennifer H
JH
Department of Animal Science, Iowa State University, Ames, IA 50011, USA.
Lillehoj
Hyun S
HS
Lamont
Susan J
SJ
D000818:Animals; D002460:Cell Line; D002645:Chickens; D016207:Cytokines / Q000737:chemistry / Q000235:genetics; D015536:Down-Regulation; D007371:Interferon-gamma / Q000494:pharmacology*; D008262:Macrophage Activation / Q000276:immunology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D014162:Transfection / Q000662:veterinary
D016207:Cytokines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II