The Ets transcription factor Fli1 clearly has effects on the immune system and the pathogenesis of lupus as evidenced by the immune phenotype of Fli1 transgenic mice and the profound effect of Fli1 heterozygosity on a mouse model of lupus. Based on these studies and our preliminary data, we hypothesize that Fli1 fails to be down-regulated following activation of T cells from lupus prone mice, due mechanistically to the loss of binding of negative regulators to the Fli1 promoter, contributing to the over-expression of Fli1 in lupus T cells with specific impact on cell number and regulatory function. We propose to study cell proliferation, apoptosis and suppressor and effector functions of T cells in mice expressing reduced Fli1 activity and to identify the mechanisms involved in the down-regulation of Fli1 expression upon T cell activation that are dysfunctional in lupus. Specific Aims include: 1) Demonstrate that reduced Fli1 activity increases CD8+ T cell proliferation and regulatory function and 2) Identify the mechanisms controlling down-regulation of Fli1 in activated T cells and identify in MRL/lpr lupus T cells which of these mechanisms is dysfunctional. The effects of reduced Fli1 expression on T cell number and function will be determined before and after exposure to inflammatory stimuli in lymphocyte co-cultures from lupus prone and control mice expressing a Fli1 protein that exhibits reduced Fli1 activity compared to their wild-type counterparts. Cell proliferation and apoptosis will be analyzed by standard flow cytometry assays. T suppressor and cytotoxic T lymphocyte (CTL) function will be analyzed by standard suppression and CTL assays, respectively. Mechanisms involved in the down-regulation of Fli1 in activated T cells in lupus prone and control mice will be analyzed by identifying proteins binding the endogenous Fli1 promoter using chromatin immunoprecipitation (IP) and analyses of expression levels, post-translational modifications and interactions with co-factors of the proteins that control the Fli1 promoter by western immunoblots, real-time RTPCR, co-IP and DNA affinity assays in T cells. The proposed studies will directly test our hypotheses and add valuable knowledge in the pathogenesis of lupus and immune regulation in general. Most importantly, it will provide the necessary groundwork for our long-term goal of identifying targets in the Fli1 regulatory pathway and/or methods of down-regulating Fli1 transcription for translation into human lupus studies leading to possible treatments for lupus patients. Lupus predominantly afflicts women and African Americans/Hispanics. As the population of females and minorities in the military continues to rise, the number of individuals with lupus in the VA system will significantly increase. The proposed studies will eventually lead to identifying earlier screening methods and interventions for the treatment of this disease;thus relieving the burden of increased health care costs for these patients and improving their quality of life. PUBLIC HEALTH RELEVANCE: The Ets factor Fli1 has been implicated as a key modulator of lupus disease pathogenesis. Increasing the levels of Fli1 in normal mice results in the development of a lupus-like disease. Fli1 is over- expressed in two lupus prone mouse strains and in lupus patients. The expression of Fli1 in lupus patients correlated with disease activity. Importantly, genetically lowering the levels of Fli1 in two lupus prone mouse strains by 50% significantly improved disease and prolonged survival. Preliminary data generated in our laboratory indicates that Fli1 transcription fails to be turned off when T cells are activated due to dysregulation of transcription in a lupus prone mouse model. We hypothesize that Fli1 fails to be down- regulated upon activation of T cells from lupus prone mice, due mechanistically to the loss of binding of negative regulators to the Fli1 promoter, contributing to the over-expression of Fli1 in lupus T cells with specific impact on T cell number and regulatory function. We propose to study the function of T cells in mice expressing reduced Fli1 activity and to identify the mechanisms involved in the down-regulation of Fli1 expression upon T cell activation that are dysfunctional in lupus. Specific Aims include: 1) Demonstrate that reduced Fli1 activity increases T cell proliferation and regulatory function and 2) Identify the mechanisms controlling down-regulation of Fli1 in activated T cells and identify in lupus prone T cells which of these mechanisms is dysfunctional. Fli1 expression clearly influences the pathogenesis of lupus and warrants further study. Little is known about the role of Fli1 in the function of T cells or how its expression is regulated in the immune system. These studies will provide the necessary groundwork for our long-term goal of identifying targets in the Fli1 regulatory pathway for translation into human lupus studies leading to possible treatments for lupus patients. Lupus afflicts women 10 times more frequently than men. It also is 4-5 times more common in African Americans and Hispanics than Caucasians, and morbidity and mortality are greater in African Americans/Hispanics with lupus. As the population of females and minorities in the military continues to rise, the number of individuals with lupus in the VA system will significantly increase. There are already thousands of veterans with lupus being cared for in the VA system. Due to its severity, patients with lupus use a disproportionate amount of health care dollars including VA resources. Our proposed studies on lupus will lead to a better understanding of the pathogenesis of lupus and the factors involved and could lead to the development of better treatments/therapies as well as potential screening tools for individuals at risk for developing lupus. Additionally, insight will be gained into the regulation of T cell function in both health and disease with relevance to an array of immune and neoplastic diseases.
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