Mechanism of action and therapeutic utility of immunosuppressive oligonucleotide

表达重复TTAGGG基序的合成寡核苷酸（ODN）在哺乳动物端粒中高频出现六聚体序列后，可下调多种TLR配体引发的炎症免疫反应以及多克隆激活剂和抗原诱导的适应性免疫细胞反应。这些抑制性ODN可用于治疗以过度旺盛的免疫反应为特征的疾病，包括感染性休克和自身免疫。我的研究小组的结果表明，全身给药的Sup ODN改变了宿主的免疫环境，可以用来减少对炎症诱导的癌症的易感性。本研究的最初重点是研究Sup ODN在DMBA/TPA皮肤癌变模型中的作用。我们发现Sup ODN显著减少了发生DMBA/TPA依赖性乳头状瘤的小鼠数量和炎症相关肿瘤发生的小鼠模型中乳头状瘤/动物的数量。为了证实这一发现，我们在一个完全不同的炎症促进肿瘤发生的模型系统中开展了研究。首先，我们证明了Sup ODN在预防/治疗由矽肺引起的危及生命的肺部炎症（一种影响美国和国外许多矿工的疾病）方面是有效的。对这些矿工的流行病学研究表明，二氧化硅颗粒（以及石棉和煤尘）引起的炎症增加了他们对暴露于香烟烟雾引起的肺癌的易感性。这促使我们开发了一种新的模型来研究暴露于二氧化硅粉尘和NNK（香烟烟雾中存在的主要致癌物）是否会增加小鼠患肺癌的风险。我们的研究结果证明了这一点（提供了第一个二氧化硅诱导的肺癌敏感性小鼠模型）。更重要的是，我们发现用Sup ODN治疗矽肺炎症不仅可以减少肺部炎症，还可以使癌症易感性恢复到背景。为了阐明Sup ODN抑制肿瘤发展的机制，研究了各种炎症措施。在研究的两种模型中，白细胞浸润和促炎细胞因子和趋化因子的产生都显著减少，而对照ODN没有显著影响。我们正在将这些研究扩展到其他引起肺部炎症和疾病的药物。我们还使用微阵列技术来识别由抑制性ODN触发的基因和调控网络。这些微阵列研究表明，在给予抑制性ODN后，大量基因迅速下调。Sup ODN广泛降低基因表达的两种机制是正在进行的评估的重点：1)Sup ODN靶向关键基因（如MAPKs）调控区域中存在的TTAGGG和/或CCCTAA基序；2)Sup ODN抑制bZIP蛋白（共享基本亮氨酸拉链结构域的调节DNA结合蛋白）的活性。在MAPK调控的背景下，编码MAPK1、MAPK3和MAPK14（分别刺激ERK-2、ERK-3和p38依赖通路）的mRNA及其调控的转录因子（ATF2、CREB1、NFKB1）在其调控区域含有TTAGGG和/或CCCTAA基序，这些基序可以通过反义机制被Sup ODN靶向。由于MAPKs对编码多种炎症基因的mrna具有稳定作用，因此下调其表达可能对先天免疫反应产生广泛影响。bZIP蛋白是广泛影响基因表达的转录因子。转录结合位点分析表明，许多被Sup ODN下调的基因含有bZIP蛋白识别的调控结构域（如CREB1、CEBPA和FOS）。事实上，在bZIP蛋白调控的TLR9信号通路中，79%的组分被Sup ODN显著下调（p < .0001）。我们检测了Sup ODN是否抑制了bZIP蛋白与其靶DNA序列的结合，发现Sup ODN选择性地抑制了三种不同类型的bZIP蛋白（CREB、C/EBPa和MAFg）与靶DNA的结合。收集到的关于抑制性ODN的作用靶点和机制的信息将支持旨在探索其治疗效用的研究。

Synthetic oligonucleotides (ODN) expressing repetitive TTAGGG motifs patterned after hexameric sequences present at high frequency in mammalian teleomeres down-regulate the inflammatory immune responses elicited by a broad range of TLR ligands and the adaptive immune cell responses induced by polyclonal activators and antigens. These suppressive ODN are useful in the treatment of diseases characterized by over-exuberant immune responses, including septic shock and autoimmunity. Results from my group show that systemically administered Sup ODN alter the host immune milieu and can be harnessed to reduce susceptibility to inflammation-induced cancer. The initial focus of this research examined the effect of Sup ODN in the DMBA/TPA model of skin carcinogenesis. We found that Sup ODN significantly reduce both the number of mice that develop DMBA/TPA dependent papillomas and the number of papillomas/animal in this murine model of inflammation-associated tumorigenesis. To confirm this finding, we initiated studies in a completely different model system of inflammation-promoted tumorigenesis. First, we demonstrated that Sup ODN were effective in preventing/treating the life-threatening pulmonary inflammation caused by silicosis (a disease that affects many miners in the US and abroad). Epidemiologic studies of such miners suggest that the inflammation induced by silica particles (as well as asbestos and coal dust) increases their susceptibility to lung cancer induced by exposure to cigarette smoke. This led us to develop a novel model to examine whether exposure to silica dust plus NNK (the major carcinogen present in cigarette smoke) increase the risk of lung cancer in mice. Our findings demonstrate this to be the case (providing the first murine model of silica-induced sensitivity to lung cancer). More importantly, we show that treating silicotic inflammation with Sup ODN not only reduces pulmonary inflammation but returns cancer susceptibility to background. To clarify the mechanism by which Sup ODN inhibit tumor development, various measures of inflammation were examined. In both models studied, leukocyte infiltration and the production of pro-inflammatory cytokines and chemokines were significantly reduced whereas control ODN had no significant effect. We are extending these studies to include other agents that cause pulmonary inflammation and disease. We are also using microarray technology to identify the genes and regulatory networks triggered by suppressive ODN. These microarray studies indicate that very large numbers of genes are rapidly down-regulated following the administration of suppressive ODN. Two mechanisms by which Sup ODN broadly reduce gene expression are the focus of ongoing evaluation: i) that Sup ODN target TTAGGG and/or CCCTAA motifs present in the regulatory regions of critical genes (such as MAPKs) and ii) that Sup ODN inhibit the activity of bZIP proteins (regulatory DNA binding proteins that share a basic leucine zipper domain). In the context of MAPK regulation, mRNA encoding MAPK1, MAPK3 and MAPK14 (which stimulate the ERK-2, ERK-3 and p38 dependent pathways, respectively), as well as the transcription factors they regulate (ATF2, CREB1, NFKB1), contain TTAGGG and/or CCCTAA motifs in their regulatory regions that could be targeted by Sup ODN via an anti-sense mechanism . Since MAPKs exert a stabilizing effect on mRNAs encoding multiple inflammatory genes, down-regulating their expression could have a broad effect on the innate immune response. bZIP proteins are transcription factors that broadly influence gene expression. Transcription binding site analysis shows that many genes down-regulated by Sup ODN contain regulatory domains recognized by bZIP proteins (e.g. CREB1, CEBPA, and FOS). Indeed, 79% of the components in the TLR9 signaling pathway regulated by bZIP proteins are significantly down-regulated by Sup ODN (p < .0001). We examined whether Sup ODN inhibited the binding of bZIP proteins to their target DNA sequences and found that Sup ODN selectively inhibited the binding of three different classes of bZIP protein (CREB, C/EBPa and MAFg) to their target DNAs. Information gathered on the targets and mechanism(s) of action of suppressive ODN will support studies designed to explore their therapeutic utility.