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 4: Host Pathogen Interactions between Salmonella and Toll-like receptors  Barton PI
Project 1: Initiation of immune responses to Toxoplasma gondii.   
Shastri/Robey PIs

We are using mouse infection by the protozoan parasite Toxoplasma gondii as an experimental system for understanding the mammalian immune response to an intracellular pathogen, with particular relevance for the design of vaccines for CD8 mediated protection and oral pathogens.   We have identified the natural T cells epitopes recognized in both resistant (BALB/c; H-2d) and sensitive (C57Bl/6 ; H-2b) strains, and have begun to characterize the interactions between parasites, T cells, and potential antigen presenting cells in lymph nodes.  We are currently investigating the mechanisms that make certain T cell responses effective.  We are also examining the impact of innate immune responses on CD8 T cell responses, and are using 2-photon imaging to examine the dynamic aspects of immune responses and the fate of parasites.

Project 2: The role of “apoptotic proteins” in regulation of innate immunity. 
Winoto PI

Cell death is an integral part of the immune responses to pathogenic infection, and different forms of cell death can have different immunological consequences. For example, apoptosis is thought to clear viral infected cells and to control run-away immune responses whereas pyroptosis of macrophages can result in the release of cytokines. The Tumor Necrosis Factor (TNF) Receptor family of death receptors can trigger apoptosis but also an alternative form of death called programmed necrosis or necroptosis. Necroptosis can occur when apoptosis is blocked and is dependent on the death-domain containing kinase RIP1.  However, little is known on the biological role of necroptosis during infection or its signal transduction pathway.  One hypothesis stipulates that necroptosis of infected cells may represent a “danger” signal for the immune system to stimulate inflammation to control infection. To test this, we plan to utilize mice deficient in FADD at different cell types, challenging them with pathogens like Toxoplasma gondii, Influenza, Salmonella and Sendai Virus.  FADD or Fas-associated death domain protein is a known adapter for all the TNF-R death receptors and thus FADD-deficient cells are resistant to death receptor-mediated apoptosis. However, stimulation of FADD-deficient cells leads to increased necroptosis. This happens in TNF-induced FADD-deficient fibroblasts and in T-cell receptor stimulated FADD-deficient T cells. Using this approach, we plan to examine how lack of apoptosis but increased necroptosis of different innate immune cells might affect the mouse immune system against infection.
We are also interested in the biochemical transduction pathway of necroptosis and innate immunity involving FADD.  We have shown recently that TRIM21, an E3 ubiquitin ligase, associates constitutively with FADD and RIP1.  TRIM21 and FADD together can negatively regulate the interferon alpha pathway during Sendai viral infection.  How this process relates to RIP1-dependent necroptosis is an open question that we plan to pursue.

 Project 3: Induction of NK cell ligands by viruses.
Coscoy/Raulet PIs

Natural killer (NK) cells are key effectors of the innate immune system. Recognition of target cells by NK cells is regulated by the balance of signaling by inhibitory and stimulatory receptors specific for cell surface ligands. NKG2D is a stimulatory receptor that recognizes members of the RAET1 and MIC families of proteins (altogether known as the NKG2D ligands). NKG2D ligands are poorly expressed on the surface of most normal cells but are induced upon various stress conditions including transformation, DNA damage and viral infection. Engagement of the NKG2D ligands by NKG2D drives NK cells activation and subsequent lysis of infected cells or tumor cells. Aberrant expression of the NKG2D ligands has been associated with severe forms of autoimmunity. The mechanism of NKG2D ligand induction is not well understood, yet is the key step in determining whether this arm of NK recognition is effective in limiting disease.
In this proposal, we use the mouse cytomegalovirus (MCMV) model to study the regulation of NKG2D ligand expression in the context of a viral infection. In particular, we will i) identify the mechanisms that regulate expression of the Rae-1 family of mouse NKG2D ligands in infected cells and ii) study the consequences of Rae-1 induction with respect to NK cell localization, NK cell migration within lymphoid tissue, destruction of infected cells and virus elimination.
Our first specific aim is to determine the molecular mechanisms responsible for regulation of NKG2D ligands by two pathways that we have implicated in this process, one dependent on IRF3 and the other dependent on PI3-kinase. The second specific aim will address the role of IRF3- and PI3K-dependent induction of NKG2D ligands in regulating NK cell activation, migration and function in vivo in infected mice. The third specific aim is to identify the mediator of a third pathway necessary for induction of NKG2D ligands, based on preliminary evidence that a specific virus deletion mutant lacks the capacity to induce NKG2D ligands in infected cells. The outcome of our study will help elucidate the principles and molecules that regulate NK cell function in the context of viral infections and surveillance of cancer.

Project 4: Host Pathogen Interactions between Salmonella and Toll-like receptors.  Barton PI
Interactions between bacterial pathogens and host innate immunity often determine the outcome of an infection.  Toll-like receptors (TLRs) induce antimicrobial mechanisms, but many bacteria have evolved virulence strategies that allow them to evade aspects of this host response.  The importance of TLRs for immunity to Salmonella typhimurium has been demonstrated using cells or mice deficient in TLRs or TLR signaling components.  A caveat of these studies, however, is that they are typically performed with highly susceptible inbred mouse strains due to a non-functional allele of the Nramp-1 gene (Nramp-1S/S).  We hypothesize that the extreme susceptibility of Nramp-1S/S mice may mask host-pathogen interactions between S. typhimurium and the host innate immune system.  Accordingly, we have generated and analyzed TLR-deficient mice with functional Nramp-1.  These analyses have resulted in a surprising finding: TLR function is required for S. typhimurium survival and replication in macrophages and for virulence in mice.  In macrophages lacking TLR function, S. typhimurium fails to induce virulence genes required for intracellular survival and replication. We are now working to identify the TLR-dependent signal used by S. typhimurium to coordinate virulence gene induction as well as to define in which cell types TLR signaling is required for S. typhimurium virulence in vivo.  In addition, we are studying the host side of this host-pathogen interaction and are examining why TLR4 plays such a dominant role in the host response to S. typhimurium infection. 

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