Cytoplasmic mitochondrial dsRNA in pediatric Sjogren's syndrome

摘要 通过我们的多学科临床和研究合作，我们最近发现了一组罕见的 符合干燥综合征(SS)诊断标准(儿童SS，PSS)的儿童和青少年。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反映了 SS的早期阶段是完全未知的。PSS常被误诊为传染性复发性腮腺炎(RP)， 由于无病毒/细菌感染的腺体肿胀和疼痛在PSS中比SS更常见。这个 这项应用的总体目标是用RP描述PSS中强健的靶组织炎症的病理特征。 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SS单核细胞异常表达 线粒体-dsRNA(MtdsRNA)调节因子，如SUV3(ATP依赖的RNA解旋酶，杂色抑制因子 3)，伴随I型干扰素特征和M1极化相关基因的趋势。一直以来，SUV3基因 RT-qPCR检测到PSS单核细胞表达下调，shRNA抑制SUV3的表达 转染导致mtdsRNA在细胞系中大量积累。更有趣的是，细胞质中积累的 在PSS和SS单核细胞中检测到颗粒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免疫的潜在影响 PSS中的调节失调。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 RNA解旋酶表达和活性变化对PSS单核细胞启动敏感性的影响 通过模仿病毒感染。我们的两个具体目标是：目标1.描述细胞质的性质 PSS患者单核细胞中含有SUV3的dsRNA。我们假设mtdsRNA的细胞质积累通过 MtdsRNA降解异常可触发PSS单核细胞产生I型干扰素。胞质dsRNA转移和 线粒体基因组耗竭将被用于识别涉及SUV3的潜在线粒体疾病，该疾病 将通过对纯化的单核细胞进行RNA测序来补充。AIM2.为了确定dsRNA的影响- 单核细胞对巨噬细胞极化和调节性T细胞(Treg)转化的影响。我们假设 PSS单核细胞中的dsRNA和I型干扰素信号具有增强M1极化影响T细胞的倾向 可塑性。各种体外测试，如共培养实验和功能测试，将并行进行。 用PMBC的scRNA-seq和配对的活检标本来描述巨噬细胞和 靶组织中的Tregs，我们假设它受到PSS单核细胞胞质dsRNA的影响。 据我们所知，这是第一次描述细胞质mtdsrna的性质及其对单核细胞的影响。 RP中的调节失调和强烈的炎症，以及PSS发病机制的第一次现场研究。我们的 涉及mtRNA解旋酶的新假说将为填补目前的知识空白提供新的机会。

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.