Mitochondrial calcification in juvenile dermatomyositis

幼年皮肌炎的线粒体钙化

基本信息

批准号：
10245228
负责人：
Jan Christian Lood
金额：
$ 18.67万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10245228
关键词：
Adolescent Adult Agonist Atherosclerosis Autoimmunity Calcinosis Calcium Cartoons Case Study Cell Death Cells Cellular Stress Chemicals Child Confocal Microscopy Crystallization Cutaneous DNA Data Deposition Dermatomyositis Disease Doctor of Philosophy Environment Experimental Models Flow Cytometry Generations Human Hydroxyapatites Hydroxychloroquine Hypoxia Immune Immunofluorescence Microscopy In Vitro Incubated Individual Inflammasome Inflammation Mass Spectrum Analysis Mediating Metabolism Mitochondria Mitochondrial DNA Mus Muscle Muscle Cells Muscle Fibers Organ Organelles Pathway interactions Patients Play Power Plants Process Retinoblastoma Role Signal Pathway Stress System Systemic Lupus Erythematosus Time Tissues base calcification calcium phosphate crystallinity cytokine extracellular in vivo inhibitor/antagonist innovation inorganic phosphate metabolomics mitochondrial dysfunction monocyte neutrophil novel phosphoproteomics sensor small molecule soft tissue uptake

项目摘要

Project Summary Mitochondria are intracellular organelles involved in metabolism, inflammation and cell death. Mitochondria also play an important role in regulating intracellular calcium levels, scavenging cytosolic calcium and inorganic phosphate, trapping it in its crystalline form (hydroxyapatite) within the mitochondria to rescue the cell from cytosolic Ca2+ overload. Intramitochondrial calcification has been observed in case reports of adult dermatomyositis (DM) as well as in experimental models of cutaneous calcinosis, a debilitating manifestation of juvenile DM (JDM). However, the underlying mechanisms and consequences of intramitochondrial calcification have not been investigated. Though mainly found intracellularly, mitochondria can be extruded upon ROS-mediated damage, partaking in induction of inflammation. The premise of this application is that JDM patients have mitochondrial calcification promoting mitochondrial extrusion, calcium crystal accumulation (calcinosis), and inflammation. To investigate this hypothesis we have two specific aims. The first aim will look into mechanisms contributing to mitochondrial calcification and extrusion in vitro. We hypothesize that mitochondrial ROS generation will support mitochondrial calcification and extrusion, contributing to inflammation and calcinosis. Experimentally, primary human skeletal muscle cells will be incubated in calcium rich medium in presence of stress agents, including hypoxia, TLR agonists, or JDM sera, and mitochondrial function assessed using flow cytometry, qPCR, immunofluorescence microscopy and metabolomics. Mass spectrometry-based phosphoproteomics will be used to establish which pathways are involved in mitochondrial calcification. Potential targets, including mitochondrial ROS, will be blocked by using small molecules, e.g. mitoTEMPO. The second aim will investigate mechanisms by which extruded mitochondria are cleared. We hypothesize that calcified mitochondria are phagocytosed, but not degraded, remaining in the cytosolic compartment triggering TLR9 and inflammasome activation. In brief, mitochondria will be incubated with primary human monocytes and neutrophils and assessed for uptake, intracellular localization, signaling pathways (phosphoproteomics) and cytokine induction. Key pathways involved in induction of mitochondrial- mediated inflammation, e.g. DNA sensors TLR9 and cGAS, as well as inflammasome activation, will be targeted using chemical inhibitors. The capacity of cells from JDM children and healthy individuals to support clearance of mitochondria will be compared in vitro. Finally, JDM immune cells will be analyzed for presence of hydroxyapatite-containing mitochondria in vivo using flow cytometry and confocal microscopy. We believe that our proposal is highly innovative and significant as i) we will define underlying mechanisms involved in mitochondrial calcification; and ii) we will for the first time explore if, and how, extruded mitochondria contribute to inflammation and calcinosis in JDM. These findings may also be applicable to other conditions in which the calcification process is prominent, including atherosclerosis.

项目概要线粒体是参与新陈代谢、炎症和细胞死亡的细胞内细胞器。线粒体在调节细胞内钙水平、清除细胞质钙和无机钙方面也发挥着重要作用。磷酸盐，以结晶形式（羟基磷灰石）将其捕获在线粒体内，以拯救细胞细胞质 Ca2+ 超载。在成人病例报告中观察到线粒体内钙化皮肌炎 (DM) 以及皮肤钙质沉着症（一种使人衰弱的表现）的实验模型青少年 DM (JDM)。然而，线粒体内的潜在机制和后果钙化尚未研究。虽然线粒体主要存在于细胞内，但可以被挤出在ROS介导的损伤后，参与炎症的诱导。这个应用的前提是 JDM患者存在线粒体钙化，促进线粒体挤出，钙晶体堆积（钙质沉着）和炎症。为了研究这个假设，我们有两个具体目标。第一个目标看起来研究有助于体外线粒体钙化和挤压的机制。我们假设线粒体 ROS 的产生将支持线粒体钙化和挤压，有助于炎症和钙质沉着。实验上，原代人类骨骼肌细胞将在钙中孵育存在应激剂（包括缺氧、TLR 激动剂或 JDM 血清和线粒体）的丰富培养基使用流式细胞术、qPCR、免疫荧光显微镜和代谢组学评估功能。大量的基于光谱测定的磷酸蛋白质组学将用于确定哪些途径参与线粒体钙化。潜在的目标，包括线粒体 ROS，将通过使用小分子来阻断，例如米托TEMPO。第二个目标是研究清除挤出的线粒体的机制。我们假设钙化的线粒体被吞噬，但没有降解，保留在细胞质中隔室触发 TLR9 和炎症小体激活。简而言之，线粒体将与原代人单核细胞和中性粒细胞，并评估摄取、细胞内定位、信号传导途径（磷酸蛋白质组学）和细胞因子诱导。参与线粒体诱导的关键途径介导的炎症，例如DNA 传感器 TLR9 和 cGAS 以及炎症小体激活将被使用化学抑制剂进行靶向治疗。 JDM 儿童和健康个体的细胞支持的能力将在体外比较线粒体的清除率。最后，将分析 JDM 免疫细胞是否存在使用流式细胞术和共聚焦显微镜在体内观察含有羟基磷灰石的线粒体。我们相信我们的提案具有高度创新性和重要意义，因为 i) 我们将定义涉及的基本机制线粒体钙化； ii）我们将首次探索挤出的线粒体是否以及如何贡献 JDM 中的炎症和钙质沉着。这些发现也可能适用于其他条件，其中钙化过程突出，包括动脉粥样硬化。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Juvenile Dermatomyositis: New Clues to Diagnosis and Therapy.

DOI：
10.1007/s40674-020-00168-5
发表时间：
2021-03
期刊：
Current treatment options in rheumatology
影响因子：
1.2
作者：
Pachman LM;Nolan BE;DeRanieri D;Khojah AM
通讯作者：
Khojah AM

Mitochondrial Calcification.

DOI：
10.20900/immunometab20210008
发表时间：
2021-01-01
期刊：
Immunometabolism
影响因子：
0
作者：
Duvvuri, Bhargavi;Lood, Christian
通讯作者：
Lood, Christian

Calcinosis in dermatomyositis: Origins and possible therapeutic avenues.

DOI：
10.1016/j.berh.2022.101768
发表时间：
2022-06
期刊：
BEST PRACTICE & RESEARCH IN CLINICAL RHEUMATOLOGY
影响因子：
5.2
作者：
Davuluri, Srijana;Duvvuri, Bhargavi;Lood, Christian;Faghihi-Kashani, Sara;Chung, Lorinda
通讯作者：
Chung, Lorinda

Changes in total body fat and body mass index among children with juvenile dermatomyositis treated with high-dose glucocorticoids.