Distinguishing cytosolic sensing of DNA and cyclic dinucleotides in vivo

描述（由申请人提供）：先天免疫系统通过识别特定微生物配体的生殖系编码受体检测感染。我们一直特别感兴趣的特征先天免疫反应的独特的核酸产生的细菌称为环状二核苷酸。这些核苷酸，包括c-di-GMP和c-di- AMP，是细菌生理学的关键调节剂。然而，由于它们不是由哺乳动物细胞产生的，因此环状二核苷酸是先天免疫识别的理想靶标。事实上，我们发现，在宿主细胞胞质溶胶中的环状二核苷酸的存在下诱导先天性免疫反应，其特征在于生产的重要的细胞因子称为I型干扰素（IFN）。我们最近发现，宿主蛋白STING是环二核苷酸的直接先天免疫传感器。STING与胞质溶胶中的环状二核苷酸结合，并且对于随后诱导宿主I型干扰素应答是必需的。有趣的是，它 还显示STING对于诱导宿主对胞质DNA的干扰素应答是必需的。由于STING是针对DNA和环状二核苷酸的IFN应答所必需的，因此该领域现在面临着一个重要的问题，即，没有办法在体内确定任何给定细菌病原体的干扰素诱导是否是由于DNA或环状二核苷酸的胞质识别。这是一个关键问题，因为几乎所有的细菌病原体都能够产生两种刺激配体。为了解决这个问题，我们确定了一个特定的点突变STING（R231 A），选择性地取消了IFN响应环状二核苷酸，而不影响细胞溶质响应DNA。这种突变体提供了一种独特的工具，用于区分DNA与环状二核苷酸的反应。在本申请中，我们提出利用我们的发现来追求两个具体目标：（1）产生“敲入”小鼠，其中编码野生型STING的基因被编码STING R231 A突变体的基因替换;（2）使用各种诱导剂和感染模型来鉴定和测试STING R231 A敲入小鼠。我们计划将STING R231 A基因敲入小鼠不受限制地分发给要求它们的研究者。我们的希望是，这些小鼠将代表该领域的一个独特的和强大的试剂，并允许研究人员确定任何感兴趣的病原体是否诱导宿主IFN应答是通过DNA或环状二核苷酸。

DESCRIPTION (provided by applicant): The innate immune system detects infection via germ-line encoded receptors that recognize specific microbial ligands. We have been particularly interested in characterizing the innate immune response to unique nucleic acids produced by bacteria called cyclic dinucleotides. These nucleotides, which include c-di-GMP and c-di- AMP, are best known as key regulators of bacterial physiology. However, since they are not produced by mammalian cells, cyclic dinucleotides are ideal targets for innate immune recognition. Indeed, we found that the presence of cyclic dinucleotides in the host cell cytosol induces an innate immune response characterized by the production of important cytokines called type I interferons (IFNs). We recently found that the host protein STING is a direct innate immune sensor of cyclic dinucleotides. STING binds to cyclic dinucleotides in the cytosol and is essential for subsequent induction of a host type I interferon response. Interestingly, however, it has also been shown that STING is essential for induction of the host interferon response to cytosolic DNA. Since STING is required for the IFN response to both DNA and cyclic dinucleotides, the field now faces a significant problem, namely, there is no way to determine in vivo whether interferon induction by any given bacterial pathogen is due to cytosolic recognition of DNA or cyclic dinucleotides. This is a critical issue since virtually all bacterial pathogens ar capable of producing both stimulatory ligands. To address this problem we identified a specific point mutation in STING (R231A) that selectively abolishes the IFN response to cyclic dinucleotides without affecting the cytosolic response to DNA. This mutant provides a unique tool for distinguishing the responses to DNA versus cyclic dinucleotides. In this application we propose to leverage our findings to pursue two specific aims: (1) Generate "knock-in" mice in which the gene encoding wild-type STING is replaced by a gene encoding a STING R231A mutant (2) Validate and test STING R231A knock-in mice using various inducers and infection models. We plan to distribute the STING R231A knock-in mice without restriction to investigators that request them. Our hope is that these mice will represent a unique and powerful reagent for the field and allow investigators to determine for any pathogen of interest whether induction of the host IFN response is via DNA or cyclic dinucleotides.