Apoptotic nucleosomal DNA and its relevance in SLE

描述（由申请人提供）： 这个R 03拨款提案旨在为我们的假设驱动项目提供“原则证明”，因此是NIH R 01申请的坚实基础。 系统性红斑狼疮（SLE）的特点是一个独特的谱致病性自身抗体，主要针对无处不在的核目标。最常见的自身抗原是核小体，染色质的基本结构，及其组分：双链DNA（dsDNA）和组蛋白。为了了解SLE的病因，了解核小体DNA的来源及其如何引发自身免疫反应是很重要的。由于凋亡细胞或其碎片的清除受损可导致某些小鼠模型中的SLE样疾病，由于SLE自身抗原集中于凋亡泡和体，并且由于核小体DNA的产生是凋亡过程的最显著特征之一，因此凋亡细胞已被提出作为自身抗原的关键储库。 我们打算使用一种基因工程小鼠，其中DNA片段化的能力被破坏，因此细胞凋亡起源的核小体DNA是不存在的，作为一种工具，在体内研究，如果凋亡细胞是SLE中核小体的主要来源。我们提出了一个三年的时间表，其中将实现2个目标。在AIM I中，我们将研究染色质再分布，浓缩成小体和泡，以及在体内细胞凋亡过程中的释放，在没有半胱天冬酶激活的DNA酶（CAD）的情况下，最重要的已知DNA酶参与凋亡核小体DNA的产生。在AIM II中，我们提出产生几种小鼠狼疮模型，其“缺乏”凋亡起源的核小体DNA，因为它们是CAD缺陷的，因此在凋亡过程中不能使DNA片段化，以直接证明凋亡核小体是SLE中的驱动自身抗原，并且它们是发展和/或维持狼疮中的自身免疫应答所必需的。 AIM I和II中的实验组将为R 01提案应用提供最初的重要步骤，其长期目标是遗传操纵染色质片段化以操纵狼疮疾病，并最终使用新技术来沉默体内CAD或其他基因，从而抑制凋亡核小体的产生，作为这种危及生命的疾病的潜在治疗。

DESCRIPTION (provided by applicant): This R03 grant proposal is designed to provide the "proof of principle" of our hypothesis-driven project and is therefore the solid ground for an NIH R01 application. Systemic lupus erythematosus (SLE) is characterized by a unique spectrum of pathogenic autoantibodies, mainly directed at ubiquitous nuclear targets. The most frequent autoantigens are nucleosomes, the fundamental structure of chromatin, and their components: double stranded DNA (dsDNA) and histones. To understand the etiology of SLE, it is important to know the source of nucleosomal DNA and how it incites the autoimmune response. Because impaired clearance of apoptotic cells or their debris can lead to SLE-like disease in certain mouse models, because SLE autoantigens concentrate on apoptotic blebs and bodies, and because generation of nucleosomal DNA is one of the most distinct characteristics of the apoptotic process, apoptotic cells have been proposed as a critical reservoir of autoantigens. We intend to use a genetically engineered mouse in which the ability to fragment DNA is disrupted, and therefore nucleosomal DNA of apoptotic origin is absent, as a tool to investigate in vivo if apoptotic cells are the main source of nucleosomes in SLE. We propose a time line of three years in which 2 AIMs will be attained. In AIM I, we will investigate chromatin redistribution, concentration into bodies and blebs, and release during apoptosis in vivo, in the absence of caspase activated DNase (CAD), the most important known DNase involved in the generation of apoptotic nucleosomal DNA. In AIM II we propose to generate several murine lupus models, which "lack" nucleosomal DNA of apoptotic origin because they are CAD deficient, and therefore cannot fragment DNA during apoptosis, to directly demonstrate that apoptotic nucleosomes are the driving autoantigen in SLE, and that they are necessary to develop and/or sustain the autoimmune response in lupus. The sets of experiments in AIM I and II will provide the initial important steps toward an R01 proposal application, which long term goals are to genetically manipulate chromatin fragmentation in order to manipulate lupus disease, and ultimately to use novel technologies to silence in vivo CAD, or other genes and so inhibit the generation of apoptotic nucleosomes as a potential treatment of this life threatening disease.