Mechanism of action and therapeutic utility of immunosuppressive oligonucleotide

表达多个TTAGGG基序的合成寡核苷酸（ODN）可以下调多种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。为了证实和扩展这一发现，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。这使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（香烟烟雾中存在的主要致癌物）会增加小鼠患肺癌的风险。我们现在已经证明，Sup ODN可以用来显著减少矽肺炎症，这将使nnk诱导的肺癌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，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。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，炎症增加了严重程度，延长了病理持续时间。我们的研究结果表明，Sup ODN治疗有效地减少了炎症，并显着降低了这两种疾病状态的严重程度。我们假设需要用Sup ODN进行慢性治疗来降低宿主对癌症的易感性。不幸的是，ODN治疗通常是肠外给药，因此很难设想长期的人体研究/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。进行了局部（GI）和全身免疫的多项研究。结果表明口服给药ODN主要作用于肠道免疫细胞，而对全身的影响很小。此外，包封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反复给药Sup ODN来改变宿主的免疫环境是否会改变对炎症性疾病（包括癌症）的易感性。我们最近的合作工作记录了cGAS在介导对该结果至关重要的免疫抑制中所起的作用。我们还扩大了这些研究的范围，以检查用TLR7/8激动剂R848治疗小鼠的效果。结果表明，R848无论是经肠外还是口服，都能提高小鼠的免疫反应，从而显著降低小鼠对感染的易感性。

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.