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

项目总结 我们长期致力于了解IKK依赖的NF-κB信号是如何 控制炎症和免疫集中在对NLRP3炎症小体的正向和负向调节上 通过核因子-κB和线粒体(MT)代谢。我们将致力于彻底阐明一部小说。 我们实验室发现的信号机制，通过它参与Toll样受体(TLR)的传递 巨噬细胞(M)对应激、损伤信号和触发NLRP3炎症体的微粒的反应 激活并诱导IL-1β和IL-18的产生。持续的NLRP3炎症小体激活参与了 几种神经退行性、代谢性和炎症性疾病，例如阿尔茨海默病、II型糖尿病和 骨关节炎(OA)，但对其缺乏了解阻碍了新型NLRP3特异性抗肿瘤药物的开发 发炎药。此外，以前通过靶向IKK依赖的NF-1来缓解炎症的尝试 由于κ炎症小体激活增强，NLRP3B信号转导失败。通过研究NF-κB如何负性地 调节NLRP3炎症体，我们确定了线粒体在控制炎症体中的关键作用 活动。然而，依赖于NLRP3B和P62的丝裂原吞噬作用终止了κ炎症体的激活。 受刺激的M、TLR4或TLR3参与通过一种基于激活的新途径触发线粒体DNA复制 IRF-1的表达和核苷酸激酶CMPK2的诱导。这一途径对于氧化(Ox)的产生是必不可少的。 TLR激活的M-中线粒体DNA暴露于不同的NLRP3炎性小体激活剂，如三磷酸腺苷，黑素， 明矾和DOTAP脂质体。我们的结果表明，Ox-mtDNA是最终的NLRP3配体，负责 炎性小体的组装和激活。我们将继续研究这一途径，并调查是否适合 它的目标是治疗目前无法治愈的炎症性疾病，如骨性关节炎。因此，我们将 确定CMPK2基因敲除和敲除小鼠是否表现出缺陷的NLRP3炎性小体激活和 因此能抵抗羟基磷灰石引起的关节炎症。我们还将确定CMPK2如何- 依赖的mtDNA复制支持Ox-mtDNA的生产，并检查后者是否与NLRP3结合 直接绘制结合部位图，并进行生化和结构研究，以确定Ox-mtDNA如何 结合诱导NLRP3与炎症体支架蛋白ASC结合。我们将调查如何 其他线粒体信号和代谢物，活性氧(ROS)和衣康酸(IA)，调节合成 前IL-1β、前IL-18等细胞因子。这些研究将集中在氧化剂反应的作用上。 转录因子NRF2在细胞因子基因表达中的作用，并将探讨mtROS和IA如何影响NF-κB 活动及其与NRF2的串扰。这些研究将扩大我们对根本的理解 控制炎症的机制，将为开发新型抗炎药物奠定基础 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.