Mechanism of Action of Inhibitory CpG Oligonucleotides

描述(申请人提供)：细菌DNA的CpG基序是被我们的先天免疫系统识别为“危险信号”的微生物分子模式之一，需要Toll样受体(TLR)9。短的合成寡核苷酸(ODN)促进了结构-功能关系的研究。刺激性(ST-)ODN能够直接激活B细胞、巨噬细胞和树突状细胞，从而共同激发体液免疫和TH1免疫，使它们成为疫苗佐剂以及癌症和过敏的治疗手段。只要改变2个碱基，就可以将一个强大的ST-ODN转化为一种抑制TLR9而不是其他TLR的抑制剂(IN-ODN)。在当前资助的支持下，已经确定了小鼠细胞中IN-ODN作用的序列要求。在15个位置中只有3对决定了IN-ODN的活性。同一组IN-ODN可以阻断B细胞(B型)和非B细胞(A型)首选的ST-ODN。为了解决这些问题，我们请求继续提供支持：1)IN-ODN是否与ST-ODN竞争TLR9结合？2)ST-ODN和IN-ODN与TLR9的结合强度是否决定了它们的生物活性？3)ST-ODN或IN-ODN是否将TLR9分子结合在一起？ODN自身或彼此聚集的能力是否允许它们这样做？4)ST-ODN和IN-ODN之间的竞争发生在细胞的什么地方？5)IN-ODN能否在体内阻断ST-ODN的活性？同样微妙的结构要求也适用吗？使用的例子是ODN诱导的小鼠“感染性休克”和炎症性结肠炎的模型。6)IN-ODN对人类细胞的结构要求是什么？它们在不同的ODN类型之间有区别吗？这些问题的答案对于理解ODN反应的机制和开发人类疾病的进一步应用，特别是利用IN-ODN治疗狼疮和类风湿性关节炎的前景都是相关的。

DESCRIPTION (provided by applicant): The CpG motifs of bacterial DMA are among the microbial molecular patterns recognized as "danger signals" by our innate immune system, requiring Toll-Like Receptor (TLR) 9. Short synthetic oligodeoxyribonucleotides (ODN) have facilitated studies of structure-function relationships. The ability of stimulatory (ST-) ODN to activate B cells, macrophages, and dendritic cells directly, thus triggering humoral and TH1 immunity together, gives them valuable potential roles as vaccine adjuvants and as therapy in cancer and allergy. Changing as few as 2 bases converts a strong ST-ODN into an inhibitor (IN-ODN) that blocks TLR9 but not the other TLRs. With support from the current grant the sequence requirements for IN-ODN action in mouse cells have been defined. Only 3 pairs of positions out of 15 determine IN-ODN activity. The same panel of IN-ODN can block the ST-ODN preferred by B cells (Type B) and non-B cells (Type A). We request continued support in order to address these questions: 1) Do IN-ODN compete with ST-ODN for TLR9 binding? 2) Does the strength of binding of ST-ODN and IN-ODN for TLR9 determine their biologic activity? 3) Do ST-ODN or IN-ODN tie TLR9 molecules together? Does the ability of ODN to aggregate with themselves or each other allow them to do this? 4) Where in the cell does the competition between ST-ODN and IN-ODN take place? 5) Can IN-ODN block ST-ODN activity in vivo? Do the same subtle structural requirements apply? Examples to be used are a model of ODN-induced "septic shock" and an inflammatory colitis in mice. 6) What are the IN-ODN structural requirements for human cells? Do they differ between ODN types? The answers to these questions are pertinent both to understanding the mechanism of ODN responses and to developing further applications to human disease, especially the prospect of treating lupus and rheumatoid arthritis with IN-ODN.