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来分析自然杀伤细胞 (NK 细胞) 和 T 细胞中基因表达的控制。我们正在继续剖析人类干扰素-γ基因的区域，以确定哪些区域响应细胞外信号而增强/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) 的转录和转录后机制。 、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。我们正在表征响应佛波醇 12-肉豆蔻酸酯 13-乙酸酯 (PMA) 或苔藓抑素 + IL-12 协同诱导干扰素-γ 基因表达的生化途径。我们的初步结果表明，目前正在临床试验中测试的两种药物苔藓抑素和 IL-12 的组合，可在体外和体内小鼠模型系统中协同诱导干扰素-γ，因此表明这些药物的组合可以代表一种强大的癌症免疫治疗新方法。目前的研究表明，当 IL-12 与苔藓抑素结合时，通过蛋白激酶 C 的信号传导可能会延长。我们使用生物信息学方法来鉴定干扰素-γ mRNA 3'非翻译部分的保守区域。据信，这些保守区域代表了基因结构中的重要调控元件，因为除非mRNA的非编码区域提供了一些进化优势，否则在进化过程中不会存在用于保守的固有区域。基于此分析，我们针对鼠干扰素-γ 3'非翻译区的160 bp区域进行了删除，因为该区域富含AUUA序列，并且该区域先前已被证明在细胞因子基因的调节中很重要表达。敲除 (KO) 小鼠已成功创建，我们的数据表明该小鼠在使用 IL-12 治疗后产生显着更多的干扰素-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 CD4+ 和 CD8+ T 细胞的增加，T 细胞稳态已被破坏，并且小鼠中的 T reg 细胞具有更有效的抑制活性。对抗原刺激的 TH1 反应增加，TH2 反应减少。 B 细胞群也发生改变，基线抗体产生发生偏差。除了表型后果外，B 细胞对抗原的反应也被破坏，因为 IgM 和 Ig2a ab 反应增加，而 IgG1 反应减少。还观察到强烈的抗 DNA 和抗核抗原抗体反应，表明慢性 IFN-γ 表达可能在狼疮的发展中发挥作用。代谢组分析显示，血清代谢物的变化与 SLE 患者的变化相似。总之，我们阐明干扰素γ调节涉及的多种机制的方法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。