Distinguishing cytosolic sensing of DNA and cyclic dinucleotides in vivo

描述（由申请人提供）：先天免疫系统通过种系编码受体识别特定的微生物配体来检测感染。我们特别感兴趣的是描述先天免疫对细菌产生的称为环二核苷酸的独特核酸的反应。这些核苷酸，包括c-di- gmp和c-di- AMP，被认为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，环二核苷酸是先天免疫识别的理想目标。事实上，我们发现宿主细胞质中存在环二核苷酸可诱导先天免疫反应，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STING是环二核苷酸的直接先天免疫传感器。STING与细胞质中的环二核苷酸结合，对随后诱导宿主I型干扰素应答至关重要。然而，有趣的是，它

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.