Mechanism of action and therapeutic utility of immunosuppressive oligonucleotide

根据哺乳动物端粒中存在的高频重复序列，合成表达多个TTAGGG基序的寡核苷酸(ODN)，下调广泛TLR配体诱导的炎性免疫反应以及多克隆激活剂和抗原诱导的获得性免疫细胞反应。这些抑制性ODN在治疗以过度活跃的免疫反应为特征的疾病方面很有用，包括感染性休克和自身免疫。来自我的团队的结果表明，系统地给药的Sup ODN改变了宿主的免疫环境，可以被利用来降低对炎症诱导的癌症的易感性。这项研究最初的重点是考察Sup ODN在DMBA/TPA皮肤癌模型中的作用。我们发现，在这种炎症相关肿瘤形成的小鼠模型中，Sup ODN显著减少了发生DMBA/TPA依赖的乳头状瘤的小鼠数量和乳头状瘤/动物的数量。为了证实和扩展这一发现，我们启动了一个完全不同的炎症促进肿瘤发生的模型系统的研究。首先，我们证明了Sup ODN在预防/治疗由矽肺(一种影响美国和国外许多矿工的疾病)引起的危及生命的肺部炎症方面是有效的。对这类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因暴露在香烟烟雾中而导致肺癌的易感性。这导致我们开发了一种新的模型，在该模型中，暴露于二氧化硅粉尘和NNK(香烟烟雾中存在的一种主要致癌物质)会增加小鼠患肺癌的风险。我们现在已经证明，Sup ODN可以用于显著减少矽肺炎症，并且这将使对NNK诱导的肺癌的易感性恢复到背景水平。为了阐明Sup ODN抑制肿瘤发展的机制，我们检测了各种炎症指标。在所研究的两种模型中，白细胞的渗透以及促炎细胞因子和趋化因子的产生显著减少，而对照ODN没有明显的影响。我们正在扩大这些研究的范围，以包括其他导致肺部炎症和疾病的因素。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，非常大量的基因迅速下调。最近，我们将这项研究扩展到评估Sup ODN在慢性阻塞性肺疾病(COPD)和缺血性中风小鼠模型中的作用。在这两种疾病中，炎症都会增加病情严重程度，延长病程。我们的研究结果表明，Sup ODN治疗有效地减少了炎症，并显著降低了这两种疾病的严重程度。我们假设，需要使用Sup ODN进行慢性治疗，以降低宿主对癌症的易感性。不幸的是，ODN治疗通常是非肠道给药，这使得很难想象长期的人体研究/治疗。因此，我们启动了一项研究，以确定ODN是否可以口服并保持其活性。进行了多项局部(GI)和全身免疫研究。结果表明，口服ODN主要作用于肠道内的免疫细胞，而全身作用很小。此外，包裹ODN以防止胃内降解对其摄取或活性没有显著影响。该项目的长期目标是确定通过重复注射Sup ODN来改变宿主的免疫环境是否会改变包括癌症在内的炎症性疾病的易感性。我们最近的合作工作记录了cGAS在调节对这一结果至关重要的免疫抑制中所发挥的作用。我们还扩大了这些研究的范围，以检查TLR7/8激动剂R848对小鼠的治疗效果。结果表明，无论是非肠道给药还是口服给药，R848都能改善小鼠的免疫反应，从而显著降低它们对感染的敏感性。

Synthetic oligonucleotides (ODN) expressing multiple TTAGGG motifs patterned after the repetitive 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 reduced 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 and extend that 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 in which exposure to silica dust plus NNK (a major carcinogen present in cigarette smoke) increases the risk of lung cancer in mice. We've now shown that Sup ODN can be used to significantly reduce silicotic inflammation, and that this returns susceptibility to NNK-induced lung cancer to background levels. 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. Most recently we extended this research to evaluating the effect of Sup ODN in murine models of chronic obstructive pulmonary disease (COPD) and ischemic stroke. In both of those diseases, inflammation increases the severity and extends the duration of pathology. Our findings demonstrate that treatment with Sup ODN effectively reduced inflammation and significantly reduce the severity of both these disease states. We hypothesize that chronic treatment with Sup ODN will be needed to reduce host susceptibility to cancer. Unfortunately, ODN therapy is typically administered parenterally making it difficult to envision long-term human studies/therapy. We've therefore initiated a study to determine if ODN could be delivered orally and retain their activity. Multiple studies of local (GI) and systemic immunity were performed. Results indicate that orally delivered ODN have their primary effect on immune cells in the gut and very little effect systemically. Moreover, encapsulating the ODN to prevent degradation in the stomach has no significant effect on their uptake or activity. The long term goal of this project is to determine whether changing the immune milieu of the host by repetitive administration of Sup ODN alter susceptibility to inflammatory diseases, including cancer. Our recent collaborative work documented the role played by cGAS in mediating the immune suppression critical to that outcome. We also expanded the scope ofthese studies to examine the effect of treating mice with the TLR7/8 agonists R848. Results show that R848, administered either parenterally or orally, improves the immune response of mice such that their susceptibility to infection is significantly reduced.