Project 1: Initiation of immune responses to Toxoplasma gondii. Shastri/Robey PIs
Project 2: The role of “apoptotic proteins” in regulation of innate immunity. Winoto PI
Project 3: Induction of NK cell ligands by viruses. Coscoy/Raulet PIs
Project 1: Initiation of immune responses to Toxoplasma gondii Shastri/Robey PIs
The intracellular parasite Toxoplasma gondii is a major food borne pathogen of humans and livestock, but very little is known about how the parasite is recognized and controlled by the immune system. In Aim 1 we will examine the impact of innate immune signaling pathways on T. gondii infection using fibroblasts, dendritic cells, macrophage and mice containing defined genetic lesions. Particular emphasis will be placed on TRAIL-R and FADD (in collaboration with Project 2) and NK cells and the NK ligand NKG2D (in collaboration with Project 3). In Aim 2 we will identify the major natural antigens recognized by CD8 T cells during Toxoplasma infection. We hypothesize that the parasite antigens recognized by CD8 T cells will vary depending on the stage of infection and the type of antigen presenting cells involved. In Aim 3, we will examine the impact of innate immune responses and CD4 T cells on the response of CD8 T cells. We will quantitate CD8 T cell responses to particular antigens in mice lacking CD4 T cells or the innate immune pathways identified in Aim 1. We will also use 2-photon imaging to examine the dynamic aspects of immune responses in Aims 1 and 3.
Project 2: The role of “apoptotic proteins” in regulation of innate immunity. Winoto PI
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 ®interferon pathway. Subsequent activation of TRAIL-R by TRAIL results in negative feedback loop of NF-kB transcription factor. TRAIL-R-/- dendritic cells/macrophages stimulated with TLR-3/4 ligands display enhanced cytokine levels and loss of NF-kB 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.
Project 3: : Induction of NK cell ligands by viruses Coscoy/Raulet PIs
Natural killer cells provide protection from numerous viruses in the early stages of the infection. NK cell activity is controlled by cytokines and cell surface stimulatory and inhibitory receptors. The NKG2D stimulatory receptor has been implicated in the recognition of cells infected with viruses. NKG2D recognizes the RAET1 (including several subfamilies) and MIC families of cell surface ligands, which are poorly expressed on the surface of most normal cells. MIC and RAET1 family members are transcriptionally upregulated in cells infected with herpesviruses and most likely other viruses. Our expertise on immune recognition by NK cells (Raulet) and viral immune evasion strategies (Coscoy) will be combined to investigate our hypothesis that viral infection leads to generic signals that upregulate transcription of NKG2D ligands, but that some viruses evade recognition by preventing ligand expression at the cell surface. The long term aim of these studies is to provide routes to develop therapeutic agents that enhance NK cell responses to viruses, especially in the context of biodefense. Our aims are to 1) focus on two herpesviruses to elucidate mechanisms that upregulate expression of NKG2D ligands in virus-infected cells, including the role of Toll like receptors, cytokines, interferons and the DNA damage pathway; 2) Examine representatives of 8 virus families, including several relevant to biofense, for their capacity to upregulate NKG2D ligand mRNAs and downregulate ligand proteins; by examining a panel of mutant fibroblasts defective in a broad array of protective functions, we expect to learn the factors responsible for ligand upregulation in different viral systems; 3) Determine how MHV68, a mouse gamma herpesvirus, evades NKG2D ligand upregulation, and, using two-photon microscopy, determine how NKG2D-ligand interactions impact NK cell-target cell interactions in vivo; 4) Determine whether the pathways uncovered in the mouse system in aims 1-3 regulate human NKG2D ligands in human cells infected with two herpesviruses, HCMV and KSHV. Importantly, aspects of this project will be part of a collaborative effort between the different groups participating in this PO1 application.
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