Distinguishing cytosolic sensing of DNA and cyclic dinucleotides in vivo

描述(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。我们特别感兴趣的是对细菌产生的独特核酸的先天免疫反应，这种核酸被称为环二核苷酸。这些核苷酸包括c-di-GMP和c-di-AMP，最为人所知的是细菌生理的关键调节因子。然而，由于它们不是由哺乳动物细胞产生的，环二核苷酸是天然免疫识别的理想靶标。事实上，我们发现，宿主细胞胞浆中存在环二核苷酸可诱导一种先天免疫反应，其特征是产生称为I型干扰素(IFN)的重要细胞因子。我们最近发现，宿主蛋白刺是环二核苷酸的直接先天免疫感受器。STING与胞浆中的环二核苷酸结合，对于随后诱导宿主I型干扰素反应是必不可少的。然而，有趣的是，它 研究还表明，刺痛对于诱导宿主干扰素对胞浆DNA的应答是必不可少的。由于干扰素对DNA和环二核苷酸的反应都需要STING，因此该领域现在面临着一个重大问题，即无法在体内确定任何给定的细菌病原体诱导干扰素是由于细胞内对DNA的识别还是由于环二核苷酸的识别。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，我们在STING(R231A)中发现了一个特定的点突变，它选择性地取消了对环二核苷酸的干扰素反应，而不影响对DNA的胞浆反应。这个突变体提供了一个独特的工具来区分对DNA和环二核苷酸的反应。在这项应用中，我们建议利用我们的发现来追求两个特定的目标：(1)培育“敲入”小鼠，其中编码野生型刺痛的基因被编码刺痛R231a突变体的基因取代；(2)使用各种诱导剂和感染模型来验证和测试刺痛R231a敲入小鼠。我们计划将STINR231A型敲入鼠不受限制地分发给提出要求的调查人员。我们希望这些小鼠将成为该领域一种独特而强大的试剂，并允许研究人员确定任何感兴趣的病原体是通过DNA还是通过环二核苷酸来诱导宿主干扰素反应。

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.