Cellular Sources of Self Antigen in SLE

摘要 系统性红斑狼疮(SLE)是一种典型的自身免疫性疾病，其特征是产生 抗核抗体(ANA)与严重的多系统炎症性疾病表现相关。 ANA靶向广泛的核大分子，可以通过形成免疫来介导疾病 沉积在组织中以促进炎症和损害的复合体；这些复合体还可以刺激 包括1型干扰素在内的细胞因子的产生，以驱动全身免疫系统紊乱。 在形成免疫复合体的抗体中，抗DNA抗体(抗DNA)是SLE的血清学标志 以及诊断和预后的标志物。这些抗体与单链和双链DNA结合，并 形成免疫复合体，沉积在组织中，特别是肾脏，以促进炎症；此外， 这些免疫复合体可以刺激浆细胞样树突状细胞产生细胞因子，触发 DNA的内部传感器。虽然抗DNA抗体的性质已经被广泛研究，但许多 关于抗原性DNA的起源、它进入免疫系统的途径以及它独特的分子，人们知之甚少 属性。一般而言，这种DNA的来源被认为是起源于核的，其产物是 死亡和濒临死亡的细胞。我们的研究为这个问题提供了一个新的视角，证明了两个 胞外DNA的重要特征：1)它以微粒的形式存在；2)它的存在 这些线粒体颗粒(Mt)以及核DNA(NDNA)。线粒体DNA的这种存在是很重要的 由于线粒体DNA具有与nDNA不同的结构特征，因此在本质上具有免疫刺激作用。 这些涉及线粒体DNA中的CpG基序，这是细菌线粒体起源的结果，以及氧化 鸟苷残留量。此外，我们还表明，从细胞中释放的线粒体具有 与微粒相似，提示线粒体在狼疮中可能作为自身抗原， 有助于免疫活动归因于微粒并提供免疫复合体的核心 队形。在前期工作的基础上，拟议的研究将集中在三个主要假设上：1)DNA 自身抗原可以从经历各种死亡形式的细胞中释放出来，并以游离态和颗粒态存在。 2)细胞死亡过程中DNA的新陈代谢影响血液中的数量、大小和 以颗粒或可溶性形式表达；以及3)抗DNA抗体可与多种抗原形式结合 DNA，无论是游离的还是微粒的，对这些形式的抗体的检测提供了更多的信息生物标志物。 我们将追求三个具体目标。特定目标1：测定细胞外DNA的释放 在不同形式的体外细胞死亡中。我们将在体外诱导不同形式的细胞死亡 确定mtDNA和nDNA在可溶性和颗粒状中的表现形式；具体目的2.用于 活体模型来阐明DNA进入血液的释放。我们将使用小鼠体内的系统来 探索细胞死亡后mtdna和ndna的释放；我们还将对血液中的dna进行高密度测序。 吞吐量技术；和具体目标3：使用狼疮患者和小鼠模型的血液，我们将 确定各种来源的细胞外DNA抗体在SLE中的存在和特异性。至 开发新的血清学方法，我们将确定不同抗体的存在和特异性 DNA的抗原性形式，包括游离态和颗粒态，并通过评估一大群井的结果 确定患者的特征，开发新的生物标志物。这些实验的成功完成将提供新的 深入了解致病机制，为临床和研究提供新的生物标志物 目的。

Abstract Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antinuclear antibodies (ANAs) in association with severe multisystem inflammatory disease manifestations. ANAs target a wide array of nuclear macromolecules and can mediate disease by the formation of immune complexes that deposit in the tissue to promote inflammation and damage; these complexes can also stimulate the production of cytokines including type 1 interferon to drive generalized immune system disturbances. Among ANAs forming immune complexes, antibodies to DNA (anti-DNA) are the serological hallmark of SLE and markers of diagnosis and prognosis. These antibodies bind to both single and double stranded DNA and form immune complexes that deposit in the tissue, especially the kidney, to promote inflammation; in addition, these immune complexes can stimulate the production of cytokines by plasmacytoid dendritic cells, triggering internal sensors of DNA. While the properties of anti-DNA antibodies have been extensively studied, much less is known about the origin of antigenic DNA, its access to the immune system and its unique molecular properties. In general, the source of this DNA has been considered to be nuclear in origin and the product of dead and dying cells. Our studies have provided a new perspective on this issue by demonstrating two important features of extracellular DNA: 1) its existence in the form of microparticles and 2) the presence in these particles of mitochondrial (mt) as well as nuclear DNA (nDNA). This presence of mtDNA is important since mtDNA is intrinsically immunostimulatory because of structural features that differ from those of nDNA. These involve CpG motifs in mtDNA, a consequence of the origin of mitochondria from bacteria, and oxidation of guanosine residues. Furthermore, we have shown that mitochondria released from cells have properties similar to those of microparticles, suggesting that mitochondria may serve as self-antigens in lupus, contributing to immune activities attributed to microparticles and providing a nidus for immune complex formation. Building on preliminary work, the proposed studies will focus on three main hypotheses: 1) DNA autoantigen can be released from cells undergoing various death forms and exist in both a free and particle form; 2) the metabolism of DNA during cell death influences the amount in the blood, its size and its representation in particle or soluble form; and 3) anti-DNA antibodies can bind to various antigenic forms of DNA, both free and particulate, with assay of antibodies to these forms providing more informative biomarkers. We will pursue three specific aims. Specific Aim 1: To determine the extracellular release of cellular DNA during different forms of in vitro cell death. We will induce different forms of cell death in cell lines in vitro and determine the representation of mtDNA and nDNA in soluble and particulate forms; Specific Aim 2. To use in vivo models to elucidate the release of DNA into the blood. We will use in vivo systems in the mouse to explore the release of mtDNA and nDNA following cell death; we will also sequence DNA in the blood by high throughput techniques; and Specific Aim 3: Using blood from patients with lupus and murine models, we will determine the presence and specificity of antibodies to various sources of extracellular DNA in SLE. To develop new serological approaches, we will determine the presence and specificity of antibodies to different antigenic forms of DNA, both free and particulate and, by assessing results from a large panel of well characterized patients, develop new biomarkers. Successful completion of these experiments will provide new insights into the mechanisms of pathogenicity as well as provide new biomarkers for clinical and research purposes.