Role of the L1 retrotransposon in interferon-positive SLE

项目摘要/摘要 我们研究的总体目标是阐明近端分子驱动因素和机制。 系统性红斑狼疮(SLE)，使新的和更好的治疗方法的发展成为可能 以这些过程为目标，但不影响更广泛的免疫系统。这项提案将探索 长分布核素-1(LINE-1或L1)在I型干扰素阳性中的作用 SLE。我们发现，大多数系统性红斑狼疮患者都有高滴度的自身抗体。 L1、ORF1p编码的蛋白质以及这些自身抗体的滴度与疾病相关 通过SLEDAI评分、补体消耗量、肾炎的存在、抗- DsDNA等自身抗体，以及I型干扰素基因诱导。 我们的工作假设是L1的生物学与L1的发病机制密切相关。 通过两种机制：i)驱动对病毒样L1蛋白复合体的自身免疫 含有RNA和DNA；以及ii)通过逆转录RNA(包括 它自己)，并触发DNA传感器信号，以诱导I型IFN。 目的1.研究L1在系统性红斑狼疮(SLE)中的表达。表达最高水平的细胞类型 SLE患者L1水平为中性粒细胞和低密度粒细胞。L1-表达 中性粒细胞有激活迹象，中性粒细胞网织红细胞增多症生物标志物升高 血清。通过RNA-Seq，我们鉴定了一个显性的(chr4q22.1)和另外六个不同的 人类特异的L1元件作为上调转录本和ORF1p蛋白的来源。我们会 巩固这些观察结果，检测并分析它们对患者性别和疾病的依赖性 参数。我们将研究影响这些基因座转录的因素，我们将询问 如果含有ORF1p的应激颗粒从经历网织或其他疾病的中性粒细胞中释放出来 细胞死亡的形式。这些实验将通过询问是否存在其他蛋白质来补充 在应激颗粒中，SLE中也有自身抗原。 目的2.探讨L1ORF2p在系统性红斑狼疮(SLE)I型干扰素诱导中的作用。最近的几个 顶级期刊上的论文已经证明了母语表达和诱导之间的联系 干扰素在细胞衰老、克罗恩病和癌细胞中的表达。为了确定这些 发现适用于SLE，我们将分析L1表达的中性粒细胞激活的DNA传感器， 这些传感器的信号通路，以及I型干扰素本身。我们将使用 用超灵敏的方法定量检测ORF2p，然后使用其RT活性的抑制剂 确定它是否驱动I型干扰素的产生。最后，我们会问如果让DNA传感器静音 DAI/ZBP1可阻断这种干扰素的产生。

Project Summary/Abstract The overall goal of our research is to elucidate the proximal molecular drivers and mechanisms of systemic lupus erythematosus (SLE) to enable the development of new and better therapies that target these processes but spare the broader immune system. This proposal will explore the role of the Long Interspersed Nuclear Element-1 (LINE-1, or L1) in type I interferon (IFN)-positive SLE. We have discovered that most SLE patients have high-titer autoantibodies against the first protein encoded by L1, ORF1p and that the titers of these autoantibodies correlate with disease activity as measured by SLEDAI score, complement consumption, presence of nephritis, anti- dsDNA and other autoantibodies, as well as with type I interferon gene induction. Our working hypothesis is that the biology of L1 is intimately connected with the pathogenesis of SLE by two mechanisms: i) driving autoimmunity to the virus-like L1 protein complexes that contain RNA and DNA; and ii) generating aberrant DNA by reverse-transcribing RNAs (including its own) and triggering DNA-sensor signaling to induce type I IFNs. AIM 1. To characterize the expression of L1 in SLE. The cell types that express the highest levels of L1 in SLE are the neutrophil and the low-density granulocyte. The L1-expressing neutrophils show signs of activation, and biomarkers of neutrophil NETosis are elevated in patient serum. By RNA-Seq we have identified one dominant (chr4q22.1) and six additional distinct human-specific L1 elements as the origins of the elevated transcripts and ORF1p protein. We will solidify these observations, detect and analyze their dependence on patient gender and disease parameters. We will examine factors that influence the transcription of these loci, and we will ask if stress granules containing ORF1p are released from neutrophils undergoing NETosis or other forms of cell death. These experiments will be complemented by asking if other proteins present in stress granules are also autoantigens in SLE. AIM 2. To elucidate the role of L1 ORF2p in type I IFN induction in SLE. Several recent papers in top journals have demonstrated a connection between L1 expression and the induction of IFN in cellular senescence, Crohn's disease, and in cancer cells. To determine how these findings apply to SLE, we will analyze L1-expressing neutrophils for activated DNA sensors, the signaling pathways from these sensors, and the type I IFN themselves. We will use an ultrasensitive method to quantitate ORF2p and then use inhibitors of the its RT activity to determine if it drives type I IFN production. Lastly, we will ask if silencing the DNA sensor DAI/ZBP1 blocks this IFN production.