Impact of Airway Inflammation on Mitochondria

气道炎症对线粒体的影响

• 批准号: 10225165
• 负责人: Y. S. Prakash
• 金额: $ 68.37万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10225165
• 关键词: Acute; Affect; Airway Disease; Asthma; Awareness; Biogenesis; COVID-19 pandemic; Chemicals; Consumption; Contractile Proteins; Endoplasmic Reticulum; Enzymes; Experimental Designs; Failure; Generations; Goals; Human; Individual; Inflammation; Inflammatory; Inositol; Lung diseases; Mediating; Messenger RNA; Mitochondria; Molecular Chaperones; Muscle Mitochondria; Oxidative Stress; PINK1 gene; Pathway interactions; Phosphorylation; Plasmids; Proteins; RNA Splicing; Reactive Oxygen Species; Research; Signal Transduction; Site; Small Interfering RNA; Smooth Muscle Myocytes; Stress; Structure; TNF gene; Testing; Transfection; Ubiquitination; airway inflammation; cytokine; density; endoplasmic reticulum stress; knock-down; loss of function; meetings; mutant; novel; overexpression; pandemic disease; parkin gene/protein; protein expression; respiratory; respiratory smooth muscle; response

The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNFα), and underlies a number of respiratory diseases. A fundamental question is why are some individuals more susceptible than others to the negative impact of airway inflammation. We will explore a novel homeostatic mechanism, which protects airway smooth muscle (hASM) cells from the negative impact of inflammation-induced reactive oxygen species (ROS) formation and protein unfolding (endoplasmic reticulum (ER) stress). We believe that a failure in this homeostatic mechanism leads to increased ROS formation thereby exacerbating oxidative and ER stress. Overall Hypothesis: TNFα-induced ROS formation and protein unfolding activates the pIRE1α/XBP1s ER stress pathway in hASM, which initiates a homeostatic response directed towards increasing mitochondrial biogenesis and mitochondrial volume density to reduce O2 consumption and ROS formation by individual mitochondrion, while still meeting the increase in ATP demand – sharing the energetic load across mitochondria. Furthermore, reduced Mfn2 disrupts mitochondrial tethering to the ER, thereby decreasing mitochondrial Ca2+ influx and maximum respiratory capacity of mitochondria. Aim 1: TNFα-induced activation of pIRE1α/XBP1s ER stress pathway increases mitochondrial volume density and reduces O2 consumption and ROS formation per mitochondrion. In hASM cells, the downstream impact of TNFα-induced activation of the pIRE1α/XBP1s ER stress pathway will be explored using transfection of a non-phosphorylatable IRE1α mutant plasmid (DP-IRE1α) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the effects of siRNA knockdown of PGC1α and Mfn2 overexpression on TNFα-induced changes in mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation. Aim 2: TNFα-induced reduction in Mfn2 disrupts mitochondrial tethering to ER, decreases mitochondrial Ca2+ influx and reduces maximum respiratory capacity of mitochondria. In hASM cells, we will examine the impact of DP-IRE1α or uXBP1 mRNA transfection and siRNA Mfn2 knockdown on TNFα-induced disruption of mitochondrial/ ER tethering, decreased mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria. Aim 3: The impact of TNFα on activation of the pIRE1α/XBP1s ER stress pathway and downstream effects are mitigated by ROS scavenging and chemical chaperone treatment. In hASM cells, the mitigating effects of ROS scavenging and chemical chaperone treatment on TNFα-induced activation of the pIRE1α/XBP1s ER stress pathway will be examined.

急性气道注射的影响是由促炎性细胞因子（例如TNFα）， 并构成了许多呼吸道疾病。一个基本的问题是为什么有些 比其他人更容易受到气道注射的负面影响。我们将 探索一种新型的稳态机制，该机制可保护气道平滑肌（HASM）细胞免受 炎症诱导的活性氧（ROS）形成的负面影响和 蛋白质展开（内质网（ER）应力）。我们相信这种体内平衡的失败 机制导致ROS的形成增加，从而加剧氧化和ER应激。 总体假设：TNFα诱导的ROS形成和蛋白质展开激活 HASM中的Pire1α/XBP1S ER应力途径，该途径启动了定向的稳态反应 旨在增加线粒体生物发生和线粒体体积密度以降低O2 单个线粒体的消费和ROS形成，同时仍达到增加 ATP需求 - 共享线粒体的能量负载。此外，MFN2降低 破坏线粒体绑扎到ER，从而降低线粒体Ca2+影响和 线粒体的最大呼吸能力。 AIM 1：TNFα诱导的PIRE1α/XBP1S ER应力途径的激活增加了线粒体 量密度并降低每线粒体的O2消耗和ROS形成。 在HASM细胞中，TNFα诱导的PIRE1α/XBP1S ER激活的下游影响 将使用非磷酸化IRE1α突变体的转化来探索应力途径 质粒（DP-ire1α）或不可裂解的XBP1（UXBP1）mRNA。此外，我们将研究 PGC1α和MFN2过表达的siRNA敲低对TNFα诱导的变化的影响 线粒体生物发生，线粒体体积密度，O2消耗和ROS形成。 AIM 2：TNFα诱导的MFN2降低会破坏线粒体束缚至ER，下降 线粒体Ca2+影响并降低线粒体的最大呼吸能力。 在HASM细胞中，我们将检查DP-IRE1α或UXBP1 mRNA转染和 TNFα诱导的线粒体/ ER链接的破坏，siRNA MFN2敲低降低 线粒体CA2+影响线粒体的最大呼吸能力。 AIM 3：TNFα对PIRE1α/XBP1S ER应力途径激活的影响 通过ROS清除和化学伴侣治疗可以减轻下游效应。 在HASM细胞中，ROS清除和化学链酮处理的缓解作用 将检查TNFα诱导的PIRE1α/XBP1S ER应力途径的激活。