PKR sensing of mitochondrial dsRNA in childhood Sjogrens disease

儿童干燥病线粒体 dsRNA 的 PKR 传感

• 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
• 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' 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

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.

摘要 通过我们的多学科临床和研究合作，我们已经建立了一个罕见的儿童队列， 符合2016年成人干燥病（SjD）标准（即儿童干燥病的cSjD）的青少年 疾病）。由于自身免疫性SjD通常影响老年女性，因此cSjD是否是一种独特的疾病实体或早期疾病， SjD的表现完全未知。总体目标是描述线粒体 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）， 在cSjD CD 14+单核细胞中发现的促炎信号。我们从cSjD的scRNA-seq获得的初步数据 单核细胞显示出mtdsRNA加工器的异常表达，如SUV 3（ATP依赖性RNA解旋酶， 杂色抑制基因3，SUPV 3L 1基因）和PNPase（多核糖核苷酸核苷酸转移酶，PNPT 1基因）， PKR、I型IFN特征（I-IFN）、STAT 1和CD 52。更高百分比的cSjD单核细胞对 当与健康对照相比时，具有下调的SUV 3和上调的PNR的颗粒细胞质dsRNA 单核细胞SUV 3缺陷细胞系与细胞质mtDNA积累、PKR激活一致， 和ISG诱导。此外，我们发现线粒体RNA结合蛋白（mtRBP）在维持细胞内的 mtdsRNA在基质内的稳定性和与PKR结合的新的内源性短双链RNA（e-sdRNA）， 螯合PKR作为单体以防止其活化。此外，我们的PBMC的scRNA-seq数据清楚地指出， 与来自其他儿科组或成人的单核细胞相比，仅在cSjD单核细胞中检测到CD 52下调 SjD。因此，我们提出了一个新的中心假设，即异常SUV 3 线粒体中的PNTR表达导致细胞质中mtdsRNA的积累，这激活了线粒体中的线粒体。 cSjD单核细胞中的胞质dsRNA传感器PKR。这三个目标包括：目标1。表征PKR cSjD单核细胞中的mtdsRNA的传感。我们将确定PKR是否是自体免疫的关键胞质传感器。 I-IFN+ CD 14 +cSjD Mo中的mtdsRNA和新的e-sdRNA结合PKR调节其活化。目标二。识别 参与受损的mtdsRNA降解和释放到细胞质中的分子。我们将确定 通过假设PNTR过表达放大PKR活化对cSjD Mo中PNTR上调的影响 通过降解与PKR结合的e-sdRNA。此外，将测试新的mtRBP，以确定它们是否 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。目标3. 对cSjD的生物学和临床特征进行分层，用于精准医学。我们将测试抗- cSjD Mo中的炎性、抗粘附CD 52由mtdsRNA-PKR-STAT 1通路介导，以增强 通过内皮细胞外渗以用于组织归巢。我们还将对转录组学，免疫学，遗传学， 通过扩展我们正在进行的PBMC和配对活检的scRNA-seq， 组织和Mo的RNA-seq进行验证。我们对mtdsRNA和NGS的新机制研究沿着，将导致 cSjD分子条形码和涉及e-sdRNA以抑制cSjD中PKR活化的相关RNA疗法。