We are analyzing the regulation of cytokine and chemokine gene expression in lymphoid cells. We have chosen interferon-gamma (IFN-gamma) gene expression, as a model system for analysis of the control of gene expression. We are utilizing mouse models of chronic IFN-gamma expression to determine the biological consequences to the host and the relevance of this phenotype to human disease. We have used a bioinformatics approach to identify conserved regions of the 3'untranslated portion of the interferon-gamma mRNA. It is believed that these conserved regions represent important regulatory elements in the gene structure, as there would be no inherent region for conservation through evolution unless the non-coding regions of the mRNA provided some evolutionary advantage. Based on this analysis, we have targeted a 160-bp region of the murine interferon-gamma 3'untranslated region for deletion as this region is rich in AUUA sequences, and such regions have been previously shown to be important in the regulation of cytokine gene expression. The knockout (KO) mouse has been successfully created on the C57 BL/6 genetic background and our data indicates that this mouse produces significantly more interferon-gamma at a basal level and upon treatment with IL-12 as compared to the wild-type mouse. Furthermore, the architecture of lymph nodes, spleen, and thymus is disrupted and the liver exhibits signs of chronic inflammation. T cell homeostasis has been disrupted as increased CD4+ and CD8+ T cells are present and the T reg cells in the mouse have more potent suppressor activity. There is also an increased TH1 response and a decreased TH2 response to antigenic stimulation. The B cell population is also altered and baseline antibody production is skewed. B cells are also observed in the thymus at increased frequency, thus indicating that IFN-gamma may alter B cell trafficking. In addition to the phenotypic consequences, the B cell response to antigen is also disrupted as increased IgM and Ig2a ab responses are seen with a decrease in the IgG1 response. Strong anti-DNA and anti-nuclear antigen antibody responses are also observed suggesting that chronic IFN-gamma expression may play a role in the development of lupus. Curiously, lupus-like symptoms are not seen in the Balb/c mouse, where the 160-bp deletion has also been crossed onto that genetic background. These mice have increased spleen sizes and an apparent defect in lymphocyte trafficking to the lymph nodes. This results in a phenotype very similar to aplastic anemia and we are elucidating the role of IFN-gamma in the development of this disease. These effects appear to be a direct consequence of interferon-gamma inhibition of hematopoietic progenitors in this mouse strain. In addition, the Balb/c mice with the deletion appear to be more resistant to challenge with the renal carcinoma line RENCA. In summary, our approach towards elucidating the multiple mechanisms involved in the biology of interferon-gamma demonstrates the complexity by which interferon-gamma gene expression alters host homeostasis. Furthermore, we now have developed a mouse model for understanding and elucidating the systems biology effects of long term chronic IFN-gamma gene expression.
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