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Title: REDUCED NITRIC OXIDE PRODUCTION AND INOS MRNA EXPRESSION IN IFN-G STIMULATED CHICKEN MACROPHAGES TRANSFECTED WITH INOS SIRNAS

Author
item CHEESEMAN, JENNIFER - DEPT ANIMAL SCI. IA SU
item Lillehoj, Hyun
item LAMONT, SUSAN - DEPT ANIMAL SCI. IA SU

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2008
Publication Date: 9/1/2008
Citation: Cheeseman, J.H., Lillehoj, H.S., Lamont, S.J. 2008. Reduced nitric oxide production and INOS mrna expression in ifn-g stimulated chicken macrophages transfected with inos sirnas. Veterinary Immunology and Immunopathology. 125:375-80.

Interpretive Summary: Recent progress in poultry genomics technology is being used to address fundamental questions concerning host immunity to pathogens and the role different immune response gene plays. In this paper, scientists at the Iowa State University collaborated with an ARS scientist to apply the RNA interference (RNAi) methodology to develop gene knock-down model in chickens. RNAi is a powerful tool to examine the function of specific genes and their potential role(s) in biological pathways and the resulting loss of function can illuminate intricate gene interactions involved in fundamental biological processes such as growth and development, reproduction, cellular homeostasis, and immune responses. The genes targeted for knock down was chicken inducible nitric oxide synthase (iNOS) gene which control nitric oxide production in the chicken macrophages. Chicken iNOS, also known as NOS-2, is an enzyme that produces nitric oxide (NO) which has antimicrobial and anti-tumor functions. Increased NO production by chicken macrophages infected with various Salmonella and Eimeria species indicates a role of NO and, therefore, likely iNOS activity, in avian immunity to disease. In this study, we demonstrate how knock-down of the iNOS gene would alter nitric oxide production in the chicken macrophages. This method will help poultry scientists to investigate host gene function in disease process in poultry disease research.

Technical Abstract: Utilizing RNA interference technology with siRNA in the HD-11 macrophage cell line, we determined how the inhibition or knock-down of the iNOS (inducible nitric oxide synthase) gene affected IFN-y' 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-y, however, when transfected with iNOS siRNAs, the production of NO is significantly decreased. We observed a 14-28% reduction in NO production by IFN-y stimulated HD-11 cells at 48 hours after initial siRNA transfection compared to non-transfected IFN-y-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-y stimulated macrophages and to those treated with a negative control siRNA. The IFN-y-stimulated chicken macrophages transfected with iNOS siRNAs did not show altered levels of mRNA expression for genes involved in IFN-y signaling and iNOS pathways (IL-1ß, IL-6, IFN-y, TGF-ß4, 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-y-induced changes in the other genes tested.