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。自从加入NCI以来，我们证明了抑制性ODN在预防/治疗由矽肺引起的危及生命的炎症方面也很有用。具体来说，抑制性ODN显著降低了小鼠矽肺模型的死亡率和发病率。我们正在研究是否早期服用抑制ODN的Rx也能降低慢性二氧化硅诱导炎症和肺部肿瘤的易感性。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引起的癌症的易感性。我们正在进行的研究的主要重点是检查Sup ODN在DMBA/TPA皮肤癌变模型中的作用。我们发现Sup ODN显著减少了发生乳头瘤的小鼠数量和炎症相关肿瘤发生的小鼠模型中乳头瘤/动物的数量。剂量反应研究证实，50ug的Sup ODN可使乳头瘤的发生率和每只动物乳头瘤的数量降低90 - 95% (p = 0.027)，且剂量越低，效果越差。无论是局部还是全身，Sup ODN都能减少乳头状瘤的形成，而对照ODN对乳头状瘤的形成没有影响。为了阐明Sup ODN抑制乳头状瘤形成的机制，我们检测了TPA诱导炎症的各项参数。TPA引起皮肤水肿、白细胞浸润，并引发编码趋化因子和其他炎症介质（包括CXCL2、CCL2、ODC和COX-2）的mRNA增加3 - 18倍。给予Sup ODN可显著降低所有炎症指标，而对照ODN则无显著影响。即使在TPA治疗停止后，炎症环境仍能维持已形成的乳头状瘤的增殖，导致乳头状瘤大小在3个月内增加3倍。DMBA/TPA引起的乳头状瘤在TPA停止后4周（第20周）开始用Sup ODN治疗。晚期给予Sup ODN可阻止已形成的乳头状瘤的进一步增殖，而对照ODN则没有效果（p. < 0.01）。这些结果表明，Sup ODN可能抑制炎症相关肿瘤的持续生长和初始发展。我们正在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. Since joining the NCI we demonstrated that suppressive ODN are also useful in the prevention/treatment of the life-threatening inflammation caused by silicosis. Specifically, suppressive ODN significantly reduced mortality and morbidity in a murine silicosis model. We are in the process of examining whether susceptibility to chronic silica-induced inflammation and pulmonary tumors can also be reduced by early Rx with suppressive ODN. Recent results suggest that systemically administered suppressive ODN can alter the host immune milieu. We are harnessing this effect to reduce host susceptibility to inflammation-induced cancer. The major focus of our ongoing research involves examining the effect of Sup ODN in the DMBA/TPA model of skin carcinogenesis. We find that Sup ODN significantly reduce both the number of mice that develop papillomas and the number of papillomas/animal in this murine model of inflammation-associated tumorigenesis. Dose-response studies established that 50 ug of Sup ODN reduced the incidence of papillomas and the number of papillomas/animal by 90 - 95% (p = 0.027), with lower doses being progressively less effective. Sup ODN reduced papilloma formation when delivered either locally or systemically, whereas control ODN had no effect on papilloma formation. To clarify the mechanism by which Sup ODN inhibit papilloma formation, various parameters of TPA induced inflammation were examined. TPA causes cutaneous edema, leukocyte infiltration and triggers a 3 - 18 fold increase in mRNA encoding chemokines and other mediators of inflammation (including CXCL2, CCL2, ODC and COX-2). The administration of Sup ODN significantly reduced all measures of inflammation, whereas control ODN had no significant effect. The proliferation of established papillomas is maintained by the inflammatory milieu that persists even after TPA treatment ceases, leading to a >3-fold increase in papilloma size over 3 months. Papillomas elicited by DMBA/TPA were treated with Sup ODN starting 4 wk after the cessation of the TPA (at week 20). This late administration of Sup ODN prevented the further proliferation of established papillomas, whereas control ODN had no effect (p. <.01). These results demonstrate that the continued growth as well as the initial development of inflammation-associated tumors may be inhibited by Sup ODN. We are 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.