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.

抽象的通过我们的多学科临床和研究合作，我们最近发现了一组罕见的符合干燥综合征 (SS) 诊断标准的儿童和青少年（儿童 SS、PSS As）。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是某种疾病的反映 SS 的早期阶段完全未知，PSS 通常被误诊为传染性复发性腮腺炎 (RP)，因为 PSS 中不伴有病毒/细菌感染的腺体肿胀和疼痛比 SS 更常见。该应用的总体目标是表征 PSS 和 RP 中强烈靶组织炎症的病理学特征。我们最近通过 SS 单核细胞的 RNA 测序初步发现，线粒体-dsRNA (mtdsRNA) 调节因子，例如 SUV3（ATP 依赖性 RNA 解旋酶、杂色抑制因子 3），以及I型IFN特征和M1极化相关基因的趋势一致，SUV3基因。通过 RT-qPCR 检测到 PSS 单核细胞中的表达下调，并通过 shRNA 敲低 SUV3 转染导致细胞系中 mtdsRNA 大量积累，更有趣的是，细胞质中积累了 mtdsRNA。在 PSS 中检测到颗粒 dsRNA 并引发 SS 单核细胞，我们对此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免疫的潜在影响为了解决这些问题，我们提出了我们的中心假设：mtdsRNA 异常。 RNA 解旋酶表达和活性改变引起的降解可增强 PSS 单核细胞的引发敏感性我们的两个具体目标是：目标 1. 表征细胞质的性质。 PSS 单核细胞中涉及 SUV3 的 dsRNA 我们通过以下方法捕获了 mtdsRNA 的细胞质积累。异常的 mtdsRNA 降解会触发 PSS 的单核细胞产生 I 型 IFN。线粒体基因组耗竭将用于识别涉及 SUV3 的潜在线粒体病理，这将通过纯化单核细胞的 RNA 测序来补充，以确定 dsRNA- 的影响。单核细胞对巨噬细胞极化和调节性 T 细胞 (Treg) 转化呈阳性。 PSS 单核细胞中的 dsRNA 和 I 型 IFN 特征赋予增强 M1 极化影响 T reg 的倾向可塑性的各种体外测定，例如共培养实验和功能测定，将同时进行。使用 PMBC 的 scRNA-seq 和配对活检标本来分析巨噬细胞和靶组织中的 Tregs，我们探索其受到 PSS 单核细胞中细胞质 dsRNA 的影响。据我们所知，这是第一项描述细胞质 mtdsRNA 对单核细胞的特性和影响的研究 RP 中的失调和强烈炎症，以及该领域对 PSS 发病机制的首次研究。涉及 mRNA 解旋酶的新假设将为填补当前的知识空白提供新的机会。