PKR sensing of mitochondrial dsRNA in childhood Sjogrens disease

儿童干燥病线粒体 dsRNA 的 PKR 传感

基本信息

批准号：
10637496
负责人：
SEUNGHEE CHA
金额：
$ 64.13万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-08 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10637496
关键词：
Address Adhesions Adolescent Adult Affect Anti-Inflammatory Agents Area Autoimmune Autoimmune Diseases Autologous Award B-Cell NonHodgkins Lymphoma Bar Codes Binding Proteins Biological Biological Response Modifiers Biopsy CD14 gene CDW52 gene Cell Line Cells Child Childhood Clinical Co-Immunoprecipitations Collaborations Cytoplasm Data Data Set Deposition Development Diagnostic Disease Double-Stranded RNA Down-Regulation Early Intervention Elderly Endothelial Cells Enrollment Exocrine Glands Exploratory/Developmental Grant for Diagnostic Cancer Imaging Extravasation Female Foundations Funding Gene Amplification Gene Expression Gene Targeting Genes Genetic Goals Health Heterogeneity Homing Hospitals Immune Immunologics Impairment Incidence Inflammation Inflammatory Interferons Investigation Knowledge Laboratories Lip structure Mediating Medical Methylation Mitochondria Mitochondrial Proteins Mitochondrial RNA Molecular Molecular Analysis Molecular Disease National Institute of Arthritis, and Musculoskeletal, and Skin Diseases Natural History Nucleic Acids Organ Outcome Particulate Pathogenesis Pathway interactions Patients Peripheral Blood Mononuclear Cell Polyribonucleotide Nucleotidyltransferase Prevalence Prognostic Marker RNA RNA Binding RNA Degradation RNA Helicase RNA-Binding Proteins Rare Diseases Research STAT1 gene Sjogren&apos s Syndrome Stains Stratification Testing Tissues Transcript United States National Institutes of Health Up-Regulation Validation Xerostomia biomarker discovery cohort diagnostic biomarker diagnostic criteria disease diagnostic eye dryness genetic signature helicase immune activation innate immune mechanisms monocyte monomer multidisciplinary multiple omics novel overexpression personalized medicine precision medicine prevent protein kinase R sensor simulation single-cell RNA sequencing therapeutic RNA therapeutic target transcriptome sequencing transcriptomics

项目摘要

ABSTRACT Through our multidisciplinary clinical and research collaboration, we have established a rare cohort of children and adolescents who fulfilled the 2016 adult Sjögren’s disease (SjD) criteria (namely, cSjD for childhood Sjögren’s disease). Since autoimmune SjD typically affects elderly females, whether cSjD is a unique disease entity or an early manifestation of SjD is completely unknown. The overall goal is to characterize mechanisms by which mitochondrial dsRNAs (mtdsRNA) are released into the cytoplasm and activate the cytosolic dsRNA sensor, protein kinase R (PKR), for the proinflammatory signature found in cSjD CD14+ monocytes. Our preliminary data from scRNA-seq of cSjD monocytes revealed the aberrant expression of mtdsRNA processors, such as SUV3 (ATP-dependent RNA helicase, suppressor of variegation 3, SUPV3L1 gene) and PNPase (polyribonucleotide nucleotidyltransferase, PNPT1 gene), PKR, type I IFN signature (I-IFN), STAT1, and CD52. A higher percentage of cSjD monocytes were positive for particulate cytoplasmic dsRNA with downregulated SUV3 and upregulated PNPase when compared to healthy control monocytes. SUV3-deficient cell lines concurred consistently with cytoplasmic mtdsRNA accumulation, PKR activation, and ISG induction. In addition, we found mitochondrial RNA binding proteins (mtRBPs) crucial in maintaining mtdsRNA stability within the matrix and novel endogenous short duplex RNAs (e-sdRNA) bound to PKR, which may sequester PKR as a monomer to prevent its activation. Furthermore, our scRNA-seq data of PBMC clearly pinpoint CD52 downregulation detected only in cSjD monocytes compared to monocytes from other pediatric groups or adult SjD. Therefore, we propose our novel central hypothesis that defective mtdsRNA degradation by the aberrant SUV3 and PNPase expression in the mitochondria leads to cytoplasmic mtdsRNA accumulation, which activates the cytosolic dsRNA sensor, PKR, in cSjD monocytes. The three proposed aims included: Aim 1. Characterize PKR sensing of mtdsRNA in cSjD monocytes. We will determine if PKR is the key cytosolic sensor for autologous mtdsRNAs in I-IFN+CD14+cSjD Mo and if novel e-sdRNAs bound to PKR regulate its activation. Aim 2. Identify molecules involved in impaired mtdsRNA degradation and release into the cytoplasm. We will identify the impact of upregulated PNPase in cSjD Mo by hypothesizing that PNPase overexpression amplifies PKR activation through the degradation of e-sdRNAs bound to PKR. Additionally, novel mtRBPs will be tested to determine if they permit mtdsRNA to escape into the cytoplasm by altering the methylation status of mitochondrial transcripts. Aim 3. Stratify biological and clinical features of cSjD for precision medicine. We will test if the downregulation of anti- inflammatory, anti-adhesion CD52 in cSjD Mo is mediated by the mtdsRNA-PKR-STAT1 pathway to enhance extravasation through endothelial cells for tissue homing. We will also stratify transcriptomic, immunological, genetic, molecular, clinical, and laboratory features of cSjD by expanding our ongoing scRNA-seq of PBMC and paired biopsy tissues, and RNA-seq of Mo for validation. Our novel mechanistic studies on mtdsRNA, along with NGS, will lead to cSjD molecular barcodes and pertinent RNA therapy involving e-sdRNAs to dampen PKR activation in cSjD.

抽象的通过我们的多学科临床和研究合作，我们建立了一个罕见的儿童和符合 2016 年成人干燥病 (SjD) 标准（即儿童干燥病的 cSjD）的青少年由于自身免疫性 SjD 通常影响老年女性，因此无论 cSjD 是一种独特的疾病实体还是一种早期疾病。 SjD 的表现是完全未知的，总体目标是表征线粒体的机制。 dsRNA (mtdsRNA) 被释放到细胞质中并激活胞质 dsRNA 传感器、蛋白激酶 R (PKR)、我们在 cSjD CD14+ 单核细胞中发现的促炎症特征来自 cSjD 的 scRNA-seq。单核细胞揭示了 mtdsRNA 处理器的异常表达，例如 SUV3（ATP 依赖性 RNA 解旋酶，杂色抑制因子 3，SUPV3L1 基因）和 PNPase（多核糖核苷酸核苷酸转移酶，PNPT1 基因）， PKR、I 型 IFN 特征 (I-IFN)、STAT1 和 CD52 的 cSjD 单核细胞呈阳性的比例较高。与健康对照相比，颗粒细胞质 dsRNA SUV3 下调且 PNPase 上调 SUV3 缺陷的细胞系与细胞质 mtdsRNA 积累、PKR 激活一致，此外，我们发现线粒体 RNA 结合蛋白 (mtRBP) 对于维持功能至关重要。 mtdsRNA 在基质内的稳定性和与 PKR 结合的新型内源性短双链 RNA (e-sdRNA)，这可能此外，我们的 PBMC scRNA-seq 数据清楚地指出了这一点。与其他儿科群体或成人的单核细胞相比，仅在 cSjD 单核细胞中检测到 CD52 下调因此，我们提出了新的中心假设，即异常 SUV3 导致 mtdsRNA 降解缺陷。线粒体中的 PNPase 表达导致细胞质 mtdsRNA 积累，从而激活 cSjD 单核细胞中的胞质 dsRNA 传感器 PKR 提出的三个目标包括：目标 1. 表征 PKR。 cSjD 单核细胞中 mtdsRNA 的传感我们将确定 PKR 是否是自体细胞的关键胞质传感器。 I-IFN+CD14+cSjD Mo 中的 mtdsRNA 以及与 PKR 结合的新型 e-sdRNA 是否调节其激活。我们将鉴定参与受损 mtdsRNA 降解和释放到细胞质中的分子。通过假设 PNPase 过度表达放大 PKR 激活，观察 cSjD Mo 中 PNPase 上调的影响通过降解与 PKR 结合的 e-sdRNA 此外，还将测试新型 mtRBP 以确定它们是否有效。通过改变线粒体转录物的甲基化状态允许 mtdsRNA 逃逸到细胞质中。对 cSjD 的生物学和临床特征进行分层以进行精准医学我们将测试抗-SjD 是否下调。 cSjD Mo 中的炎症、抗粘连 CD52 由 mtdsRNA-PKR-STAT1 途径介导，以增强通过内皮细胞外渗进行组织归巢，我们还将对转录组、免疫学、遗传、通过扩展我们正在进行的 PBMC 的 scRNA-seq 和配对活检，了解 cSjD 的分子、临床和实验室特征我们对 mtdsRNA 的新颖机制研究以及 NGS 将导致 cSjD 分子条形码和相关 RNA 治疗 e-sdRNA 可抑制 cSjD 中的 PKR 激活。