T-cell production of IFN-gamma plays an essential role in disease initiation and severity of disease in animal models and Crohn's disease. Mucosal T-cells are different from peripheral T-cells in several ways. Mucosal T-cells are muted responders to activation via the T-cell receptor and are CD2 pathway dominant. During the previous award period the investigators used their ability to transfect primary T-cells to show unique mucosa-specific mechanisms by which T-cells produced Th1 cytokines. They demonstrated a role for AP1 in CD2 transactivation of the IL-2 gene. They showed that enhanced IL-2 production by CD2/CD28 co-activated peripheral T-cells occurs through mRNA stabilization and trans-activation in peripheral T-cells, however, in mucosal T-cells this effect can be solely the result of mRNA stabilization. This difference is due to the lack of activation of the AP1 and CD28 response elements in the IL-2 promotor in mucosal cells. They demonstrated several mucosal-specific mechanisms of IFN-gamma regulation and identified a unique role for TNF-alpha in mucosal T-cell IFN-gamma regulation. They defined the lamina propria mononuclear cell factor which can prime T-cells to respond to TNF-alpha. Furthermore, they demonstrated different cis elements in the IFN-gamma promotor that are used for transactivation of the IFN-gamma gene and mucosal T-cells as compared to peripheral T-cells. Finally, they demonstrated for the first time the enhancer function of the first intronic region of the IFN-gamma gene that involves the JAK/STAT pathway in mucosal T-cells but not in peripheral T-cells. Because of the vital importance of IFN-gamma in disease related mucosal inflammation yet its critical role for host defense, the knowledge of mucosa-specific targets, which would selectively attenuate mucosal IFN-gamma production without eliminating, would be a significant accomplishment. The overall objective of this continuation proposal is to more precisely define the mucosal specific regulatory pathways that are important for IFN-gamma regulation in T-cells and to use this knowledge to design approaches to modify IFN-gamma production in a mucosa-specific manner. This will be accomplished by the following specific aims: To identify through fine promotor analysis positive and negative cis-regulatory regions within the IFN-gamma promotor in which transcriptional regulation in lamina propria mononuclear cells differ from that in peripheral blood lymphocytes focusing on the previously identified regions of -204 to -108 (enhancer) and -528 to -204 base pair (repressor). To determine the mucosa-specific mechanisms of regulating the first intronic region of the IFN-gamma gene. From the results of Aim 1 and Aim 2 design molecular approaches including transfection of antisense oligonucleotides and or dominant negative transacting factors to attenuate in vitro IFN-gamma promotor expression in a mucosal-specific manner.
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