Remodeling of the electron transport chain in macrophages by a novel peptide-miRNA axis

通过新型肽-miRNA 轴重塑巨噬细胞中的电子传递链

• 批准号: 10312689
• 负责人: Megan Lynn Clark
• 金额: $ 3.35万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312689
• 关键词: Remodeling; electron; transport; chain; macrophages

Project Summary/Abstract: Macrophages are critical innate immune cells which are necessary for anti-pathogen immunity, cancer immunosurveillance and wound healing. Powering these diverse responses in macrophages is the dynamically regulated electron transport chain (ETC) within the mitochondria. An important regulator of ETC function is complex IV (CIV), which is critical for oxygen consumption, ATP generation, and cell survival. Humans with loss- of-function mutations in CIV subunits present with severe immunodeficiencies characterized by recurrent bacterial and viral infection, highlighting the importance of CIV in immune responses. Intriguingly, CIV is the only respiratory complex which remodels its protein subunit composition in response to environmental stimuli, such as hypoxia, to modulate its function. This is achieved through the upregulation of subunit isoforms which replace highly homologous core CIV subunits that function to improve cellular fitness. However, whether immune cells remodel CIV subunit composition during immune responses and whether this remodeling is important for myeloid cell function is unknown. Since inflammatory responses are powerful external stimuli that result in mitochondrial reprogramming, I hypothesized that CIV subunit composition is remodeled in macrophages and functions to regulate inflammatory responses. To this end, I identified a single transcript (AA467197) induced in pro- inflammatory macrophages which encodes a miRNA (miR-147) and a highly conserved peptide (NMES1) with striking homology to the CIV subunit NDUFA4. Intriguingly, my preliminary data and recent studies indicate that NMES1 and miR-147 work in concert to replace the core CIV subunit NDUFA4 with NMES1 in CIV. Strikingly, while remodeling of CIV subunit composition by NMES1 and miR-147 was dispensable for ATP production, I showed that this subunit switch played a critical role in regulating inflammatory cell death induced by NLRP3 inflammasome activation, termed pyroptosis. While mitochondrial function is modulated in macrophages during priming by pro-inflammatory signals such as LPS, it also can be critical in enhancing activation of the NLRP3 inflammasome through the production of ROS, release of mtDNA, externalization of cardiolipin, and regulation of ion flux in the mitochondria. Thus, our overall hypothesis is that NMES1 and miR-147 remodel CIV subunit composition in macrophages to increase NLRP3 inflammasome activation in vitro and in vivo. To explore this hypothesis, I will use macrophages sufficient or deficient in AA467197 to uncover how remodeling of CIV by NMES1 and miR-147 impacts CIV function to induce pyroptotic cell death. Additionally, I will conditionally delete AA467197 in relevant cell types and employ bacterial infection models to pinpoint how NMES1 and miR-147 regulate NLRP3 inflammasome activation and anti-bacterial immunity in vivo. Altogether, this proposal will elucidate the cellular and molecular mechanisms that underlie the impact of CIV remodeling in macrophages on shaping innate immune responses to bacterial infection.

项目概要/摘要： 巨噬细胞是重要的先天性免疫细胞，其对于抗病原体免疫、癌症免疫和免疫应答是必需的。 免疫监视和伤口愈合。在巨噬细胞中，这些不同的反应是动态的 调节线粒体内的电子传递链（ETC）。ETC功能的重要调节器是 复合体IV（CIV），对于氧气消耗、ATP产生和细胞存活至关重要。失去亲人的人- CIV亚单位的功能缺失突变表现为以复发性免疫缺陷为特征的严重免疫缺陷， 细菌和病毒感染，突出了CIV在免疫反应中的重要性。有趣的是，CIV是唯一 呼吸复合物，其响应于环境刺激而重塑其蛋白质亚基组成，例如 缺氧来调节它的功能。这是通过上调亚单位同种型来实现的， 高度同源的核心CIV亚基，其功能是改善细胞适应性。然而，免疫细胞是否 在免疫应答期间重塑CIV亚单位组成，以及这种重塑是否对骨髓细胞的免疫应答重要。 细胞功能未知。由于炎症反应是强大的外部刺激，导致线粒体 重编程，我假设CIV亚单位组成是在巨噬细胞和功能重塑， 来调节炎症反应为此，我鉴定了一个在pro-DNA中诱导的单一转录物（AA 467197）。 编码miRNA（miR-147）和高度保守肽（NMES 1）的炎性巨噬细胞， 与CIV亚基NDUFA 4具有惊人的同源性。有趣的是，我的初步数据和最近的研究表明， NMES 1和miR-147协同工作以在CIV中用NMES 1替换核心CIV亚基NDUFA 4。引人注目的是， 而NMES 1和miR-147对CIV亚基组成的重塑对ATP的产生是不利的， 表明这种亚基开关在调节NLRP 3诱导的炎性细胞死亡中起关键作用 炎性小体激活，称为焦亡。虽然线粒体功能在巨噬细胞中被调节， 通过促炎信号如LPS引发，它在增强NLRP 3的活化中也是关键的 炎症小体通过ROS的产生、mtDNA的释放、心磷脂的外化和调节 线粒体中离子流的变化。因此，我们的总体假设是NMES 1和miR-147重塑CIV亚基， 本发明提供了在巨噬细胞中使用组合物以增加体外和体内NLRP 3炎性体活化的方法。探讨 根据这一假设，我将使用AA 467197充足或缺乏的巨噬细胞来揭示CIV的重塑是如何通过 NMES 1和miR-147影响CIV功能以诱导热凋亡细胞死亡。另外，我将有条件地删除 AA 467197在相关细胞类型中的作用，并采用细菌感染模型来确定NMES 1和miR-147 在体内调节NLRP 3炎性体活化和抗细菌免疫。总的来说，这项建议将 阐明巨噬细胞中CIV重塑对巨噬细胞增殖的影响的细胞和分子机制。 形成对细菌感染的先天免疫反应。