Mechanism of Action of Inhibitory CpG Oligonucleotides

描述（由申请人提供）：细菌DMA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。短合成的寡脱氧核糖核苷酸（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，从而共同触发体液和TH1免疫，这使得它们作为疫苗佐剂以及癌症和过敏的治疗具有宝贵的潜在作用。只要改变2个碱基，强ST-ODN就会转化为阻断TLR9而不阻断其他tlr的抑制剂（IN-ODN）。在当前基金的支持下，已经确定了小鼠细胞中in - odn作用的序列要求。15对位置中只有3对决定IN-ODN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。我们请求继续支持，以解决以下问题：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)是ST-ODN还是IN-ODN将TLR9分子连接在一起？ODN与自己或彼此聚合的能力是否允许它们这样做？4)细胞中ST-ODN和in - odn的竞争发生在什么地方？5) in - odn能否阻断体内ST-ODN活性？同样微妙的结构要求也适用吗？将使用的例子是odn诱导的“感染性休克”模型和小鼠炎症性结肠炎。6)人类细胞的IN-ODN结构要求是什么？它们在不同的ODN类型之间有区别吗？这些问题的答案与理解ODN反应的机制和进一步发展在人类疾病中的应用有关，特别是用IN-ODN治疗狼疮和类风湿性关节炎的前景。

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.