Mechanism of action and therapeutic utility of immunosuppressive oligonucleotide

表达重复TTAGGG基序的合成寡核苷酸（ODN）在哺乳动物端粒中以高频率存在的六聚体序列之后图案化，下调由广泛的TLR配体引起的炎性免疫应答和由多克隆活化剂和抗原诱导的适应性免疫细胞应答。这些抑制性ODN可用于治疗以免疫反应过度旺盛为特征的疾病，包括感染性休克和自身免疫。 我们最近的研究表明，抑制性ODN也可用于预防/治疗二氧化硅吸入引起的危及生命的炎症（急性硅肺）。具体而言，抑制性ODN治疗显示在相关鼠模型中显著降低二氧化硅诱导的死亡率和发病率。我们正在研究是否二氧化硅诱导的肺肿瘤的易感性也可以通过早期Rx与抑制性ODN来降低。最近的研究结果表明，全身给予抑制性ODN可以改变宿主的免疫环境。我们正在利用这种效应来降低宿主对炎症诱导的癌症的易感性。正在评价抑制性ODN在化学诱导的皮肤癌的鼠模型中的作用。一系列的实验结果表明，ODN显着减少化学诱导的乳头状瘤的频率和大小。此外，在乳头状瘤形成后开始治疗可减小肿瘤大小。希望通过这些研究项目鉴定的抑制性ODN的治疗效用可以被利用来显著降低宿主对肿瘤发展和进展的易感性。尽管取得了这一进展，但对抑制性ODN的细胞靶点、负责其识别/摄取的受体或其作用机制知之甚少。我们正在使用微阵列技术来鉴定使抑制性ODN能够破坏正在进行的炎症反应并确定其体内免疫抑制活性的持续时间的基因和调控网络。通过这些研究获得的见解，我们计划鉴定负责识别这种抑制活性的关键TTAGGG基序的受体。微阵列研究表明，非常大量的基因被迅速下调后，抑制性ODN的管理。Sup ODN广泛降低基因表达的两种机制是正在进行的评估的焦点：i）Sup ODN靶向关键基因（如MAPK）调控区中存在的TTAGGG和/或CCCTAA基序，ii）Sup ODN抑制bZIP蛋白（共享碱性亮氨酸拉链结构域的调控DNA结合蛋白）的活性。 在MAPK调节的背景下，编码MAPK 1、MAPK 3和MAPK 14（分别刺激ERK-2、ERK-3和p38依赖性途径）的mRNA以及它们调节的转录因子（ATF 2、CREB 1、NFKB 1）在其调节区域中含有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MAPK对编码多种炎性基因的mRNA发挥稳定作用，因此下调其表达可能对先天免疫应答产生广泛影响。 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CREB 1、CEBPA和FOS）。事实上，由bZIP蛋白调节的TLR 9信号通路中79%的组分被Sup ODN显著下调（p小于0.0001）。我们研究了Sup ODN是否抑制bZIP蛋白与其靶DNA序列的结合，发现Sup ODN选择性地抑制三种不同类型的bZIP蛋白（CREB，C/EBPa和MAFg）与其靶DNA的结合。关于抑制性ODN作用的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. Our recent studies demonstrate that suppressive ODN are also useful in the prevention/treatment of the life-threatening inflammation caused by silica inhalation (acute silicosis). Specifically, suppressive ODN treatment was shown to significantly reduce silica-induced mortality and morbidity in a relevant murine model. We are in the process of examining whether susceptibility to silica-induced pulmonary tumors can also be reduced by early Rx with suppressive ODN. Recent results suggest that systemically administered suppressive ODN can alter the hosts immune milieu. We are harnessing this effect to reduce host susceptibility to inflammation-induced cancers. The effect of suppressive ODN in a murine model of chemically-induced skin cancer is being evaluated. Results from a series of experiments indicates that ODN significantly reduce the frequency and size of chemically induced papillomas. Moreover, initiation of therapy after papilloma formation reduces tumor size. It is hoped that the therapeutic utility of suppressive ODN identified through these research program can be harnessed to significantly reduce host susceptibility to tumor development and progression. Despite this progress, very little is known about the cellular targets of suppressive ODN, the receptors responsible for their recognition/uptake, or their mechanism of action. We are using microarray technology to identify the genes and regulatory networks that enable suppressive ODN to disrupt ongoing inflammatory responses and determine the duration of their immuno-inhibitory activity in vivo. With the insight gained from these studies, we plan to identify the receptor(s) responsible for the recognition of the TTAGGG motif key to this suppressive activity. Microarray studies indicate that very large numbers of genes are rapidly down-regulated following 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 less than .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.