Control of Cytokine Gene Expression in LymphoidMyeloid Cells

淋巴髓样细胞中细胞因子基因表达的控制

• 批准号: 8157227
• 负责人: Howard Young
• 金额: $ 79.01万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157227
• 关键词: Cells; Cytokine Gene; Gene Expression

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.

我们正在分析淋巴样细胞中细胞因子和趋化因子基因表达的调节。我们选择了干扰素 - γ（IFN-GAMMA）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。我们正在继续剖析人干扰素 - γ基因的区域，以确定哪些区域对细胞外信号的响应增强/抑制基因转录。 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.我们正在表征参与对佛波尔12-麦糖酸酯13-乙酸盐（PMA）或Bryostatin + bryostatin + IL-12的协同诱导的生化途径。我们的最初结果表明，在临床试验中测试了两种药物，将双肌抑素和IL-12结合起来，导致在体外和体内小鼠模型系统中对干扰素 - 伽马的协同诱导，因此表明这些药物的组合可以代表一种强大的新方法对癌症免疫疗法。当前的研究表明，当IL-12与乳杆菌素结合时，通过蛋白激酶C的信号传导可能会扩展。我们已经使用了一种生物信息学方法来识别干扰素γmRNA的3'未翻译部分的保守区域。人们认为，这些保守的区域代表基因结构中的重要调节元素，因为除非mRNA的非编码区域提供了一定的进化优势，否则不会通过进化来保护固有的区域。基于此分析，我们针对鼠干扰素3'未翻译区域的160 bp区域进行缺失，因为该区域富含AUUA序列，并且此类区域先前已被证明对细胞因子基因表达的调节很重要。敲除（KO）小鼠已成功创建，我们的数据表明，用IL-12处理后，该小鼠会产生更多的干扰素 - γ。另外，在敲除小鼠的血清中检测到低水平的干扰素γ，但未检测到野生型对照小鼠。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CD4+和CD8+ T细胞增加，并且小鼠中的T Reg细胞具有更有效的抑制活性，因此T细胞稳态受到了破坏。 TH1反应也增加，TH2对抗原刺激的反应减少。 B细胞群也会改变，基线抗体的产生偏斜。除表型后果外，由于IgM增加和Ig2a AB反应的增加，IgG1响应的降低也会破坏B细胞对抗原的反应。还观察到了强抗DNA和抗核抗原抗体反应，这表明慢性IFN-GAMMA表达可能在狼疮的发育中起作用。代谢组分析表明，血清代谢产物的变化与患者患者中看到的变化相似。总而言之，我们阐明调节干扰素调节的多种机制的方法证明了在免疫效应细胞中调节干扰素 - γ基因表达的复杂性。此外，我们现在开发了一种小鼠模型，以理解和阐明长期，慢性IFN-gamma基因表达的系统生物学影响。