Identification of TLR signaling network

描述（由申请人提供）：toll样受体在先天和适应性免疫反应的启动中起关键作用。TLR家族成员识别保守的微生物结构并激活导致针对微生物感染的免疫应答的信号通路。所有的tlr都激活了共同的通路来诱导一系列核心的刻板反应，比如炎症。然而，单个tlr也可以诱导针对特定微生物感染的免疫反应。人们对这些不同反应的机制和组成部分知之甚少。鉴于tlr在宿主防御中的重要性，解剖这些通路是合理设计免疫调节剂和佐剂的关键。为了解决TLR信号网络的复杂性，必须采用能够系统检查信号转导的技术。ATTAGENE公司开发了一种可逆的遗传方法，可以筛选成千上万的cdna表达文库，以识别信号中间体。我们使用这种方法确定了介导白细胞介素-1诱导的转录因子NF-kB激活的途径的一些新成分。我们的研究表明，可逆遗传方法为系统的全基因组信号转导鉴定提供了一种高度通用的工具。这种全面的办法不依赖于先入为主的观念，它有消除假阳性背景的固有程序。在这项研究中，我们将采用可逆遗传方法来系统地识别TLR信号网络的组成部分。我们的目标是：(1)确定将TLR家族的个体成员与转录因子NF-kB的激活联系起来的信号转导成分；(2)将这些成分标注为正/负中间体和微分/共同中间体；(3)体外检测鉴定出的介质在先天免疫应答中的生物学功能；(4)建立敲除动物模型，以评估鉴定的介质作为潜在靶点来调节对不同病原体的先天和适应性免疫反应，包括单核细胞增生李斯特菌和鼠伤寒沙门菌。该计划的成功实施将提供控制病原体免疫反应的分子机制的全面知识，从而极大地促进高度特异性免疫调节剂和佐剂的开发。

DESCRIPTION (provided by applicant): Toll-like receptors play a critical role in the initiation of the innate and adaptive immune responses. Members of the TLR family recognize conserved microbial structures and activate signaling pathways that result in immune responses against microbial infections. All TLRs activate common pathways to induce a core set of stereotyped responses, such as inflammation. However, individual TLRs can also induce immune responses that are tailored to a given microbial infection. The mechanisms and components of these varied responses are poorly understood. Given the importance of TLRs in host defense, dissection of these pathways is key to the rational design of immunomodulators and adjuvants. To address the complexity of the TLR signaling network, it is imperative to put in place technologies enabling systematic examination of the signal transduction. ATTAGENE Inc. has developed a reversible genetic approach that affords screening expression libraries of tens of thousands of cDNAs to identify signaling intermediates. We used this methodology to identify a number of novel components of the pathways that mediate interleukin-1-inducible activation of the transcription factor NF-kB. Our studies indicate that the reversible genetic approach offers a highly versatile tool for a systematic, genome-wide identification of signal transduction. This comprehensive approach does not rely on preconceived notions and it has built-in procedures that eliminate false-positive background. In this study, we will adapt the reversible genetic approach to systematic identification of components of the TLR signaling network. Our objectives are (1) to identify the components of signal transduction that link individual members of the TLR family with activation of the transcription factor NF-kB; (2) to annotate those components as positive/negative and differential/common intermediates; (3) to examine biological functions of the identified mediators in innate immune responses in vitro; and (4) to create knock-out animal models in order to assess the identified mediators as potential targets to modulate the innate and adaptive immune responses to different pathogens, including Listeria monocytogenes and Salmonella typhimurium. Successful implementation of the proposed plan should provide comprehensive knowledge of molecular mechanisms controlling immune responses to pathogens, thus greatly facilitating the development of highly specific immunomodulators and adjuvants.