Control of Cytokine Gene Expression in LymphoidMyeloid Cells

我们正在分析淋巴细胞中细胞因子和趋化因子基因表达的调节。我们选择干扰素-γ（IFN-γ）基因表达作为模型系统的控制基因表达的分析。我们正在利用慢性IFN-γ表达的小鼠模型来确定对宿主的生物学后果以及这种表型与人类疾病的相关性。我们已经使用生物信息学方法来鉴定IFN-γ mRNA的3'非翻译部分的保守区域。据信，这些保守区域代表基因结构中的重要调控元件，因为除非mRNA的非编码区提供一些进化优势，否则在进化中不存在用于保守的固有区域。基于该分析，我们靶向了鼠干扰素-γ 3'非翻译区的160-bp区域进行缺失，因为该区域富含AUUA序列，并且这些区域先前已被证明在细胞因子基因表达的调节中是重要的。敲除（KO）小鼠已经成功产生，并且我们的数据表明，该小鼠在用IL-12治疗后产生显著更多的干扰素-γ。此外，在基因敲除小鼠的血清中检测到低水平的干扰素-γ，但在野生型对照小鼠的血清中未检测到。此外，淋巴结、脾脏和胸腺的结构被破坏，肝脏表现出慢性炎症的迹象。随着CD 4+和CD 8 + T细胞的增加，T细胞稳态被破坏，并且小鼠中的T reg细胞具有更有效的抑制活性。对抗原刺激也有增加的TH 1应答和减少的TH 2应答。B细胞群也发生了改变，基线抗体产生也发生了偏移。 在胸腺中也观察到B细胞频率增加，因此表明IFN-γ可能改变B细胞运输。 除了表型结果外，B细胞对抗原的应答也被破坏，因为观察到IgM和Ig 2a ab应答增加，而IgG 1应答减少。 还观察到强的抗DNA和抗核抗原抗体应答，表明慢性IFN-γ表达可能在狼疮的发展中起作用。 奇怪的是，在Balb/c中没有看到狼疮样症状，其中也产生了160 bp的缺失。 这些小鼠的脾脏尺寸增大，淋巴细胞向淋巴结的运输存在明显缺陷。 这导致一个表型非常相似的再生障碍性贫血，我们正在阐明IFN-γ在这种疾病的发展中的作用。 此外，缺失的Balb/c小鼠似乎对肾癌细胞系RENCA的攻击更具抵抗力。总之，我们的方法对阐明干扰素-γ的调节中涉及的多种机制证明了免疫效应细胞中干扰素-γ基因表达调节的复杂性。 此外，我们现在已经开发了一种小鼠模型，用于理解和阐明长期慢性IFN-γ基因表达的系统生物学效应。

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 rthe 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 and our data indicates that this mouse produces significantly more interferon-gamma upon treatment with IL-12. In addition, low levels of interferon-gamma are detected in the serum of knockout mice but not wild type control mice. 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 where the 160 bp deletion has also been generated. 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. 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 approaches towards elucidating the multiple mechanisms involved in the regulation of interferon-gamma demonstrates the complexity by which interferon-gamma gene expression is regulated in immune effector cells. Furthermore we now have developed a mouse model for understanding and elucidating the systems biology effects of long term, chronic IFN-gamma gene expression.