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.

抽象的 通过我们的多学科临床和研究合作，我们建立了罕见的儿童队列 满足2016年成人Sjögren病（SJD）标准的青少年（即CSJD儿童时期Sjögren's 疾病）。由于自身免疫性SJD通常会影响老年女性，无论CSJD是独特的疾病实体还是早期 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）， 对于CSJD CD14+单核细胞中发现的促炎性签名。我们来自CSJD的Scrna-Seq的初步数据 单核细胞揭示了mTDSRNA处理器的异常表达，例如suv3（依赖ATP的RNA解旋酶， Variegation 3，Supv3L1基因）和PNPase（多迪比替迪核苷酸核苷酸转移酶，PNPT1基因）的抑制剂，PNPT1基因）， PKR，I型IFN签名（I-IFN），STAT1和CD52。 CSJD单核细胞的较高百分比为阳性 与健康对照相比 单核细胞。 SUV3缺乏细胞系与细胞质MTDSRNA积累，PKR激活，一致同意 和ISG诱导。此外，我们发现线粒体RNA结合蛋白（MTRBP）在维持 矩阵中的mtDSRNA稳定性和与PKR结合的新型内源性短双链RNA（E-SDRNA），可能 隔离PKR作为单体以防止其激活。此外，我们的PBMC的SCRNA-seq数据明确查明了 与其他儿科或成人的单核细胞相比，CD52仅在CSJD单核细胞中检测到的下调 SJD。因此，我们提出了我们的新型中心假设，即异常SUV3有缺陷的mtDSRNA降解 线粒体中的PNPase表达会导致细胞质mTDSRNA积累，这激活了 CSJD单核细胞中的胞质DSRNA传感器，PKR。提出的三个目的包括：目标1。表征PKR 在CSJD单核细胞中传感mtDSRNA。我们将确定PKR是否是自体的关键胞质传感器 I-IFN+CD14+CSJD MO中的mtDSRNA，如果与PKR结合的新型E-SDRNA会调节其激活。目标2。确定 MTDSRNA降解并释放到细胞质中的分子。我们将确定 通过假设PNPase过表达放大器PKR激活，上调PNPase在CSJD MO中的影响 通过与PKR结合的E-SDRNA的降解。此外，将测试新的MTRBP，以​​确定它们是否 通过改变线粒体转录本的甲基化状态，允许mtDSRNA逃脱到细胞质中。目标3。 将CSJD的生物学和临床特征分层用于精确医学。我们将测试是否下调抗 MTDSRNA-PKR-STAT1途径介导了CSJD MO中的炎症性抗粘附CD52以增强 通过内皮细胞进行渗出以进行组织寄养。我们还将对转录组，免疫学，遗传学， CSJD的分子，临床和实验室特征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的新机械研究以及NGS将导致 CSJD分子条形码和相关的RNA疗法涉及CSJD中Damen PKR激活的E-SDRNA。