NF-kappaB and Mitochondrial Signals as Positive and Negative Regulators of Inflammation

项目摘要 我们长期致力于了解IKK依赖的NF-κB信号通路是如何被激活的， 控制炎症和免疫的重点是NLRP 3炎性体的正向和负向调节 通过NF-κB和线粒体（mt）代谢。我们将致力于对一部小说的完整阐释 信号传导机制，在我们的实验室中确定，通过Toll样受体（TLR）的参与， 巨噬细胞（M β）对应激、损伤信号和触发NLRP 3炎性体的微粒有反应 活化并诱导IL-1β和IL-18产生。持续的NLRP 3炎性小体激活参与了 几种神经变性、代谢和炎性疾病，例如阿尔茨海默病、II型糖尿病和 骨关节炎（OA），但其认识不足，阻碍了新的NLRP 3特异性抗- 消炎药此外，以前通过靶向IKK依赖性NF-κ B来缓解炎症的尝试也失败了。 κB信号传导由于增强的NLRP 3炎性体激活而失败。通过研究NF-κB B如何负性地 调节NLRP 3炎性体，我们确定了线粒体在控制炎性体中的关键作用， 活动然而，NF-κB-和p62-依赖的线粒体自噬终止NLRP 3炎性小体激活， 受刺激的M β、TLR 4或TLR 3参与通过基于激活的新途径触发mtDNA复制 IRF-1和核苷酸激酶CMPK 2的诱导。该途径对于氧化（Ox）的产生至关重要。 暴露于不同NLRP 3炎性体激活剂，例如ATP，尼日利亚菌素， 明矾和DOTAP脂质体。我们的研究结果表明，Ox-mtDNA是最终的NLRP 3配体，负责 炎性小体组装和活化。我们将继续研究这一途径，并调查 其靶向治疗目前无法治愈的炎症性疾病，如OA。因此，我们将 确定CMPK 2敲除和敲入小鼠是否表现出缺陷性NLRP 3炎性小体活化， 因此对羟基磷灰石诱导的关节炎症具有抗性。我们还将确定CMPK 2- 依赖性mtDNA复制支持Ox-mtDNA的产生，并检查后者是否与NLRP 3结合 直接绘制结合位点，并进行生化和结构研究，以确定Ox-mtDNA 结合诱导NLRP 3与炎性体支架蛋白ASC的结合。我们将调查如何 其他mt信号和代谢物，活性氧（ROS）和衣草酸（IA），调节合成的 pro-IL-1β、pro-IL-18和其他细胞因子。这些研究将集中在氧化反应的作用， 转录因子NRF 2在细胞因子基因表达中的作用，并探讨线粒体ROS和IA如何影响NF-κB 活性及其与NRF 2的串扰。这些研究将扩大我们对基本的 控制炎症的机制，并为开发新型抗炎药物奠定基础 其抑制IL-1β和IL-18的产生而不干扰抗微生物免疫。

PROJECT SUMMARY This renewal application of our long-term effort to understand how IKK-dependent NF-κB signaling controls inflammation and immunity is focused on positive and negative regulation of the NLRP3 inflammasome by NF-κB and mitochondrial (mt) metabolism. Our effort will be placed on complete elucidation of a novel signaling mechanism, identified in our laboratory, through which engagement of Toll-like receptors (TLR) renders macrophages (M) responsive to stress, damage signals and microparticles that trigger NLRP3 inflammasome activation and induce IL-1β and IL-18 production. Persistent NLRP3 inflammasome activation is involved in several neurodegenerative, metabolic and inflammatory diseases, e.g. Alzheimer’s disease, type II diabetes and osteoarthritis (OA), but its poor understanding has prevented development of novel NLRP3-specific anti- inflammatory drugs. Furthermore, previous attempts to alleviate inflammation by targeting IKK-dependent NF- κB signaling have failed due to enhanced NLRP3 inflammasome activation. By studying how NF-κB negatively regulates the NLRP3 inflammasome, we identified a critical role for mitochondria in control of inflammasome activity. Whereas, NF-κB- and p62-dependent mitophagy terminates NLRP3 inflammasome activation in stimulated M, TLR4 or TLR3 engagement triggers mtDNA replication via a novel, pathway based on activation of IRF-1 and induction of the nucleotide kinase CMPK2. This pathway is essential for production of oxidized (Ox) mtDNA in TLR-activated M that were exposed to diverse NLRP3 inflammasome activators, e.g. ATP, nigericin, alum and DOTAP liposomes. Our results suggest that Ox-mtDNA is the ultimate NLRP3 ligand responsible for inflammasome assembly and activation. We will continue to study this pathway and investigate the suitability of its targeting for treatment of currently incurable inflammatory diseases, such as OA. Accordingly, we will determine whether CMPK2 knockout and knockin mice exhibit defective NLRP3 inflammasome activation and are therefore resistant to hydroxyapatite-induced joint inflammation. We will also determine how CMPK2- dependent mtDNA replication supports Ox-mtDNA production and examine whether the latter binds NLRP3 directly, map the binding site and conduct biochemical and structural studies to determine how Ox-mtDNA binding induces the association of NLRP3 with the inflammasome scaffold protein ASC. We will investigate how other mt signals and metabolites, reactive oxygen species (ROS) and itaconic acid (IA), modulate synthesis of pro-IL-1β, pro-IL-18 and other cytokines. These studies will focus on the role of the oxidant-responsive transcription factor NRF2 in cytokine gene expression and will also explore how mtROS and IA affect NF-κB activity and its crosstalk with NRF2. These studies will expand our basic understanding of the fundamental mechanisms that control inflammation and will lay the foundation for developing novel anti-inflammatory drugs that inhibit IL-1β and IL-18 production without interfering with antimicrobial immunity.