Mammalian cells have evolved multiple sensing pathways to detect foreign invasion. Recent evidence? indicated that several proteins previously implicated in apoptosis also participate in innate immunity? through Toll-Like receptors (TLR)-dependent and independent pathways. These include TRAIL-R, an? apoptosis-inducing member of the tumor necrosis factor receptor family and FADD, a death-domain? containing adapter protein. TRAIL can be induced through TLR-> interferon pathway. Subsequent? activation of TRAIL-R by TRAIL results in negative feedback loop of NF-kappaB transcription factor. TRAIL-R-/-? dendritic cells/macrophages stimulated with TLR-3/4 ligands display enhanced cytokine levels and loss of? NF-kappaB homeostatic regulation. FADD, reminiscent of the Drosophila Imd-FADD innate immune response? system, was found to be crucial for intra-cellular dsRNA-activated gene expression in human/mouse. In? the presence of interferon, FADD-/- fibroblasts were not able to clear RNA viruses that include Influenza.? Thus, FADD is part of an alternative TLR-independent mammalian pathogen-sensing pathway. In this? application, we hypothesize that TRAIL-R and FADD play significant but distinct roles in the? innate immune responses against a variety of viruses and selected parasite.
In Aim 1, viruses from? various families, including several in the bio-defense category like Influenza, Vaccinia and LCMV (with? Project 3) and the intracellular parasite Toxoplasma gondii (with Project 1), will be used to determine the? role of FADD and TRAIL-R in regulating host responses. We will first examine FADD-/- and TRAIL-R-/-? fibroblasts for their ability to support viral replication. Microarray analysis will then be done to assess? altered global gene expression, if any, in these cells. Dendritic cells/macrophage-specific FADD-/- mice will? be generated. The host responses of these and TRAIL-R-/- mice against selected pathogens (Influenza,? Toxoplasma, Cytomegalovirus) will be examined. Two-photon imaging studies in fluorescent transgenic? mice in either TRAIL-R-/- or FADD tissue-specific deficient alleles will be used to assess host-pathogen? interaction.
In Aim 2, the signal transduction pathway leading to negative regulation of NF-KB by TRAIL? will be examined. Signaling proteins involved in FADD-mediated innate immunity will also be identified.? Microarray analysis will be performed to examine gene expression profile of TRAIL-R-/- macrophages.? Mass spectrometry will then be used to identify TRAIL-R- or FADD-associated proteins in experiments? involving Fas/TRAIL-R chimeric protein, tandem-affinity-protein technology and co-immunoprecipitation.? Finally, RNAi knockdown approach will be used to assess the functional significance of any newly? identified TRAIL-R or FADD associated proteins in innate immunity against selected bio-defense? organisms.
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