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，用于分析自然杀伤细胞（NK细胞）和T细胞中基因表达的控制。我们正在继续解剖人类干扰素-γ基因的区域，以确定哪些区域响应细胞外信号增强/抑制基因转录。特别是，我们正在利用自然杀伤细胞系来阐明白细胞介素-2（IL-2）、白细胞介素-12（IL-12）、白细胞介素-15（IL-15）、白细胞介素-18（IL-18）或干扰素-α（IFN-α）诱导或抑制干扰素-γ基因表达的转录和转录后机制。我们正在表征参与干扰素-γ基因表达响应佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）或苔藓抑素+ IL-12的协同诱导的生化途径。我们的初步结果表明，结合苔藓抑素和IL-12，目前正在临床试验中测试的两种药物，结果在体外和体内小鼠模型系统中的干扰素-γ的协同诱导，从而表明这些药物的组合可以代表一个强大的新方法对癌症免疫治疗。目前的研究表明，当IL-12与苔藓抑素组合时，通过蛋白激酶C的信号传导可能会延长。我们已经使用生物信息学方法来确定干扰素-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。由于存在增加的CD 4+和CD 8 + T细胞，并且小鼠中的T reg细胞具有更有效的抑制活性，T细胞稳态已被破坏。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。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应答降低。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。代谢物组学分析显示，血清代谢物的变化与SLE患者中观察到的变化相似。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 in natural killer cells (NK cells) and T cells. We are continuing to dissect the regions of the human interferon-gamma gene to determine which regions enhance/repress gene transcription in response to extracellular signals. In particular, we are utilizing natural killer cell lines to elucidate the mechanisms, both transcriptional and post-transcriptional, by which interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) or interferon-alpha (IFN-alpha) induce or inhibit interferon-gamma gene expression. We are characterizing the biochemical pathways involved in the synergistic induction of interferon-gamma gene expression in response to phorbol 12-myristate 13-acetate (PMA) or bryostatin + IL-12. Our initial results indicate that combining bryostatin and IL-12, two drugs currently being tested in clinical trials, results in synergistic induction of interferon-gamma both in vitro and in in vivo mouse model systems, thus suggesting that the combination of these drugs could represent a powerful new approach towards cancer immunotherapy. Current studies have revealed that the signaling through Protein Kinase C may be extended when IL-12 is combined with bryostatin. 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. 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. Metabolome analysis has revealed that alterations in serum metabolites parallel those seen in patients suffering from SLE. 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.