Intracellular immunity, cytosolic DNA sensing by cyclic GAMP synthase, and macrophages in ischemic injury and cardiac remodeling

项目概要/摘要 最近的CANTOS试验证明，以白细胞介素-1 β为靶点的抗炎症治疗可以降低 心血管事件的发生率。然而，我们缺乏可以限制炎症损伤触发的治疗方法， 急性缺血，即使它显然与更糟糕的临床结果有关。知识的一个关键差距， 理解危险识别，特别是细胞内危险识别，起着重要的作用，因为 危险的检测决定了炎症的范围。我们的长期目标是开发免疫调节剂， 修改危险识别以包含炎症介导的损伤。本建议的总体目标是 确定DNA及其胞质受体环GAMP合酶（cGAS）如何传播由 缺血受损的心肌富含线粒体（每个细胞有数千个拷贝）和线粒体（每个细胞有数千个拷贝）。 心肌细胞）和核DNA。大量的DNA对心肌修复构成严重威胁， 巨噬细胞，专业吞噬细胞，检测到它，并作出反应，与强大的炎症反应 从进化论的角度来看是为了消灭病原体。这个项目的核心假设是， cGAS对DNA的识别通过激活I型干扰素（IFN）维持炎性巨噬细胞 促进AIM 2（在黑色素瘤2中不存在）炎性因子的途径;因此，cGAS在缺血诱导的炎症反应中至关重要。 重塑这一假设是根据初步数据和最近发表的工作， 申请人的实验室这项研究的基本原理是，了解细胞内免疫， 缺血触发的炎症具有发现限制炎症相关损伤的有效方法的潜力。 在强有力的初步数据的指导下，这一假设将通过追求以下具体目标进行检验： 确定巨噬细胞中的cGAS活化驱动病理性重塑和HF; 2）确定 cGAS介导的信号激活AIM 2炎性体通路。cGAS激活1型干扰素- 调节鸟苷酸结合蛋白（GBP）表达的介导的信号传导。GBP使 吞噬体并导致DNA释放到胞质溶胶中并触发AIM 2炎性小体激活; 3） 确定抑制cGAS通路以减少缺血后重构和HF的有效方法 损伤目的1将使用cGASf/f小鼠系来确定巨噬细胞作为负责细胞类型。 在第二个目标下，cGAS依赖性AIM 2炎性因子激活和GBP的重要作用是研究炎症因子的表达。 将使用体外和体内模型的功能丧失或获得实验进行检查。目标3将测试 对抑制cGAS介导的信号传导的免疫调节剂的潜在保护，包括两种临床上 可用的代理人。该研究在概念上是新颖的，通过靶向DNA及其胞质传感系统， 在心肌缺血的情况下，传统上被视为病毒反应途径。获得的知识将 垂直推进我们对细胞内免疫在缺血性损伤中的关键作用的理解。缺血性心脏 疾病是一个巨大的负担，往往是一个毁灭性的条件，我们的退伍军人，拟议的研究移动领域 通过寻找新的战略来减轻负担和改善护理。

PROJECT SUMMARY/ABSTRACT The recent CANTOS Trial has proved that anti-infammation therapy targeting the interleukin-1β lowers the incidence of cardiovascular events. However, we lack therapies that can limit the inflammatory injury triggered by acute ischemia, even though it clearly links to worse clinical outcomes. A critical gap of knowledge in understanding danger recognition, especially intracellular danger recognition, plays a significant role, because detecting danger dictates the scope of inflammation. Our long-term goal is to develop immune modulators that modify danger recognition to contain inflammation-mediated injury. The overall objective of this proposal is to determine how DNA and its cytosolic receptor the cyclic GAMP synthase (cGAS) propagate injury triggered by ischemia. The damaged myocardium is enriched with both mitochondrial (thousands of copies per cardiomyocyte) and nuclear DNA. The large amount of DNA poses a serious threat to myocardial repair when macrophages, the professional phagocytes, detect it and respond with the robust inflammatory responses intended to get rid of pathogens from the evolutionary standpoint. The central hypothesis of this project is that recognition of DNA by cGAS sustains the inflammatory macrophages via activation of the type I interferon (IFN) pathway that promotes AIM2 (absent in melanoma 2) inflammasone; as a result, cGAS is crucial in ischemia-induced remodeling. This hypothesis has been formulated on the preliminary data and the recently published work from the applicants’ laboratory. The rationale for the proposed research is that understanding the intracellular immunity in ischemic-triggered inflammation has the potential to discover effective ways of limiting inflammation-related injury. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following specific aims: 1) Determine that cGAS activation in macrophages drives pathological remodeling and HF; 2) Determine that cGAS-mediated signaling activates the AIM2 inflammasome pathway. cGAS activates type 1 interferon- mediated signaling that governs the expression of the guanylate binding proteins (GBPs). GBPs destabilize the phagosome and cause the release of DNA into the cytosol and triggers AIM2 inflammasome activation; 3) Identify effective approaches for inhibition of the cGAS pathway to reduce remodeling and HF after ischemic injury. Aim 1 will be addressed using a cGASf/f mouse line to determine macrophage as the responsible cell type. Under the second aim, the cGAS-dependent AIM2 inflammasone activation and the essential roles of GBPs will be examined using loss or gain of function experiments with in vitro and in vivo models. Aim 3 will test potential protection from immune modulators that inhibit the cGAS-mediated signaling, including two clinically available agents. The study is conceptually novel by targeting DNA and its cytosolic sensing system, traditionally viewed as a viral response pathway, in the setting of myocardial ischemia. Knowledge acquired will vertically advance our understanding of the critical role of intracellular immunity in ischemic injury. As ischemic heart disease is an enormous burden and often a devastating condition to our veterans, the proposed study moves the field forward by finding novel strategies alleviating the burden and improve care.