Derstine, Randall2014-06-102014-06-102013http://udspace.udel.edu/handle/19716/13035For animals, the innate immune system is the first line of defense against foreign pathogens. It is a non-specific response to infectious challenge that is independent of antigen recognition, utilizing a combination of anatomical, physiological, and chemical factors to eliminate a pathogen before it can colonize its target host. The toll-like receptor (TLR) family of proteins are a critical group of pattern recognition receptors (PRRs) which are present on multiple cell types, such as neutrophils, natural killer (NK) cells, dendritic cells, and macrophages. In mammals, one of these PRRs, toll-like receptor 7 (TLR7), has been demonstrated to bind single-stranded RNA (ssRNA) of viral and synthetic origin. The toll-like receptor pathway consists of multiple types of transmembrane receptor proteins that recognize unique microbial structures, also known as pathogen associated molecular patterns (PAMPs). The binding of a PAMP to its respective TLR leads to signal transduction to the cell nucleus by the use of multiple adaptor proteins and signal mediators, resulting in the transcription of pro-inflammatory cytokines and chemokines. In mammals, a transcription factor, interferon regulatory factor 7 (IRF7), is known to mediate the transcription of type I interferons and helps to induce an anti-viral response. Interferons play a critical role in the anti-viral response by inhibiting protein translation in virally infected cells, as well as by enhancing antigen presentation to cytotoxic T lymphocytes. The goal of this study was to clarify the functional homology of avian TLR7 and IRF7 to mammalian TLR7 and IRF7, and to determine if their respective roles in mediating the pro-inflammatory response to ssRNA are conserved. It was hypothesized that avian TLR7 is functionally homologous to mammalian TLR7 in binding ssRNA to mediate a pro-inflammatory response. We further hypothesized that avian IRF7 functions in a manner similar to mammalian IRF7 in regulating the transcription of type I interferons. To test these hypotheses, a siRNA-mediated RNAi method for transient gene knockdown in the immortalized macrophage cell line HD11 of Gallus gallus was developed. With this method, TLR7 and IRF7 were silenced in HD11s by an average of 60% at 24 hours post-transfection with TLR7 and IRF7 specific siRNAs. These cultures were subsequently exposed to the ssRNA chemical analog R848. The roles of avian TLR7 and IRF7 in mediating the pro-inflammatory response to ssRNA were elucidated by measuring the transcription levels of two pro-inflammatory genes, iNOS and IFN-α. The iNOS and IFN-α genes were selected based on their ability to be induced by 25.3 fold at 2 hours and 15.6 fold at 8 hours, respectively, in response to a 10 ug/mL R848 treatment. With TLR7 silenced, HD11 cultures displayed a 23.7% reduction in iNOS expression and a 23.7% reduction in IFN-α expression when treated with R848 (p = ˂ .0001). With IRF7 silenced, HD11 cultures displayed a 14.3% reduction in iNOS expression (p = ˂ .0022) and a 34% reduction in IFN-α expression (p = ˂ .0001). The combined knockdown of TLR7 and IRF7 resulted in an additive reduction in iNOS and IFN-α expression in response to R848, with a 42.3% reduction in iNOS (p = ˂ .0001) and a 46.7% reduction in IFN-α (p = ˂ .0001). The results of this study provide strong evidence for the roles of avian TLR7 and IRF7 in mediating the pro-inflammatory response to ssRNA.RNA.Chickens.Inflammation.Proteins.Thesis