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Cytoplasmic mitochondrial dsRNA in pediatric Sjogren's syndrome

小儿干燥综合征中的细胞质线粒体 dsRNA

基本信息

批准号：
10287866
负责人：
SEUNGHEE CHA
金额：
$ 36.91万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-22 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10287866
关键词：
Address Adolescent Adult Affect Applications Grants Autoimmune Autoimmunity B-Cell Lymphomas Bacterial Infections Biological Biological Assay Biopsy Specimen CD14 gene Cell Differentiation process Cell Line Cell physiology Cells Child Childhood Clinical Research Coculture Techniques Collaborations Communication Complement Control Groups Cytoplasm Data Diagnosis Diagnostic Disease Double-Stranded RNA Elderly Female Gene Expression Gene Targeting Genes Gland Goals High Prevalence Immune Impairment Inflammation Institutes Interdisciplinary Study Interferons Interleukin-17 Investigation Knowledge Lacrimal gland structure Lead Lymphoma Mitochondria Mitochondrial RNA Mus Nature Nucleic Acids Pain Parotitis Particulate Pathogenesis Pathology Pathway interactions Patients Pediatric cohort Peripheral Peripheral Blood Mononuclear Cell Polyribonucleotide Nucleotidyltransferase Production Prognosis Property RNA Degradation RNA Helicase Recurrence Regulation Regulatory T-Lymphocyte Reporting Role Salivary Glands Signal Transduction Sjogren&apos s Syndrome Stains Swelling Tissues Transfection Transfer RNA United States National Institutes of Health Viral Virus Diseases autoimmune inflammation cohort effector T cell evidence base experimental study genetic signature helicase high risk in vitro Assay indexing innovation intercellular communication knock-down macrophage mitochondrial genome monocyte multidisciplinary novel novel therapeutics sensor single-cell RNA sequencing small hairpin RNA therapeutic target transcriptome sequencing transcriptomics

项目摘要

ABSTRACT Through our multidisciplinary clinical and research collaboration, we have recently discovered a rare cohort of children and adolescents who fulfill the Sjögren’s syndrome (SS) diagnostic criteria (pediatric SS, PSS). As autoimmune SS typically affects elderly females, whether PSS is a unique disease entity or a mere reflection of an early stage of SS is completely unknown. PSS is commonly misdiagnosed as infectious recurrent parotitis (RP), since glandular swelling and pain without a viral/bacterial infection occurs more frequently in PSS than in SS. The overall goal of this application is to characterize the pathology of robust target tissue inflammation in PSS with RP. Our recent, preliminary discovery by RNA-sequencing of SS monocytes revealed aberrant expression of mitochondrial-dsRNA (mtdsRNA) regulators such as SUV3 (ATP-dependent RNA helicase, suppressor of variegation 3), along with the type I IFN signature and a tendency of M1 polarization-associated genes. Consistently, SUV3 gene expression was downregulated in PSS monocytes, detected by RT-qPCR, and knockdown of SUV3 by shRNA transfection led to massive accumulation of mtdsRNA in a cell line. More interestingly, cytoplasmic accumulation of particulate dsRNA was detected in PSS and SS monocytes, which prompted us to ask fundamental questions regarding the nature of the detected dsRNA and its potential impact on monocytes and subsequent immune dysregulation in PSS. To address these questions, we propose our central hypothesis that aberrant mtdsRNA degradation by altered RNA helicase expression and activity elicits amplified priming sensitivity of PSS monocytes by mimicking a viral infection. Our two Specific Aims are: Aim 1. To characterize the nature of cytoplasmic dsRNA involving SUV3 in monocytes of PSS. We hypothesize that cytoplasmic accumulation of mtdsRNA by aberrant mtdsRNA degradation triggers type I IFN production in monocytes of PSS. Cytoplasmic dsRNA transfer and mitochondrial genome depletion will be applied to identify potential mitochondriopathies involving SUV3, which will be complemented by RNA-sequencing of purified monocytes. Aim2. To determine the impact of dsRNA- positive monocytes on macrophage polarization and regulatory T cell (Treg) conversion. We hypothesize that dsRNA and type I IFN signature in PSS monocytes confer propensity toward intensified M1polarization impacting T reg plasticity. Various in vitro assays, such as co-culture experiments and functional assays, will be carried out in parallel with scRNA-seq of PMBC and paired biopsy specimens to profile cellular communications between macrophage and Tregs in the target tissue, which we hypothesize to be influenced by cytoplasmic dsRNA in PSS monocytes. To our knowledge, this is the first study to delineate the properties and impact of cytoplasmic mtdsRNA on monocyte dysregulation and robust inflammation in RP, as well as the first investigation of PSS pathogenesis in the field. Our novel hypothesis involving mtRNA helicases will provide a new opportunity to fill the current knowledge gap.

摘要通过我们的多学科临床和研究合作，我们最近发现了一个罕见的队列，符合干燥综合征（SS）诊断标准（儿科SS，PSS）的儿童和青少年。作为自身免疫性SS通常影响老年女性，无论PSS是一种独特的疾病实体还是仅仅反映了一种疾病， SS的早期阶段完全未知。PSS常被误诊为感染性复发性腮腺炎（RP），因为没有病毒/细菌感染的腺体肿胀和疼痛在PSS中比在SS中更频繁地发生。的本申请的总体目标是表征具有RP的PSS中的强烈靶组织炎症的病理学。我们最近通过对SS单核细胞进行RNA测序的初步发现揭示了双链dsRNA（mtdsRNA）调节剂，如SUV 3（ATP依赖性RNA解旋酶，杂色抑制因子 3），沿着I型IFN特征和M1极化相关基因的趋势。一致性，SUV 3基因通过RT-qPCR检测到PSS单核细胞中SUV 3的表达下调，并通过shRNA敲低SUV 3 转染导致mtdsRNA在细胞系中大量积累。更有趣的是，细胞质积累的在PSS和SS单核细胞中检测到颗粒状dsRNA，这促使我们提出基本问题关于检测到的dsRNA的性质及其对单核细胞和随后的免疫系统的潜在影响， PSS失调。为了解决这些问题，我们提出了我们的中心假设，即异常的mtdsRNA 通过改变RNA解旋酶表达和活性的降解增强PSS单核细胞的引发敏感性通过模仿病毒感染我们的两个具体目标是：目标1。为了表征细胞质的性质， PSS单核细胞中涉及SUV 3的dsRNA。我们假设线粒体双链RNA的细胞质积累是由异常的mtdsRNA降解触发PSS单核细胞中I型IFN的产生。细胞质dsRNA转移和线粒体基因组缺失将被应用于鉴定涉及SUV 3的潜在的线粒体病，将通过纯化的单核细胞的RNA测序来补充。目标2。为了确定dsRNA的影响- 阳性单核细胞对巨噬细胞极化和调节性T细胞（Treg）转化的影响。我们假设 PSS单核细胞中的dsRNA和I型IFN特征赋予增强的M1极化的倾向，其影响T reg 可塑性各种体外试验，如共培养实验和功能试验，将平行进行使用PMBC和配对活检标本的scRNA-seq来分析巨噬细胞和巨噬细胞之间的细胞通讯，我们假设这受到PSS单核细胞中胞质dsRNA的影响。据我们所知，这是第一个研究描绘的性质和影响的细胞质mtdsRNA对单核细胞 RP中的失调和强烈炎症，以及该领域对PSS发病机制的首次调查。我们一个新的假说涉及的mtDNA解旋酶将提供一个新的机会，以填补目前的知识空白。