The Retroelement LINE1 and the DNA Sensor IF16 in Sjogren's Syndrome

项目总结/摘要 该奖项将促进寻求获得技能和知识的申请人的发展 成为一名专注于人类风湿性疾病的独立医生科学家。研究 这项资助的重点是逆转录因子LINE1，及其与细胞质DNA传感器IFI16的潜在相互作用， 在自身免疫性疾病舍格伦综合征（SS）的背景下。免疫反应的持续激活 抗含核酸自身抗原的抗体是几种风湿性疾病的标志，包括SS，其中 在血液和受影响的组织中发现"干扰素（IFN）信号"。先天核酸传感器的激活 是干扰素信号传导的关键驱动因素，与SS和相关疾病有关。在本提案中，我们将 研究逆转录因子LINE1作为先天性和体液免疫的潜在内源性驱动因子 反应，并检查自噬在调节IFN对LINE1的反应中所起的作用。这些 研究将建立在我们最近的观察基础上，即DNA传感器和SS自身抗原IFI16在一个细胞内被激活， 一些SS唾液导管上皮细胞中的丝状形式-这一过程似乎与 SS血清中的抗IFI16抗体。初步数据显示LINE1蛋白ORF 1p似乎是一种SS， 自身抗原，并且在这些患者中与抗IFI16抗体显著相关。此外，LINE1 在SS中携带活化的IFI16的同一导管上皮细胞层中可以检测到蛋白质表达 提示LINE1在这些细胞中的表达可能是一个潜在的核酸来源， 对于在该位点观察到的IFI16细丝形成。基于这些初步发现，我们假设IFI16 与上皮细胞胞质中LINE1核酸的结合可以解释抗- 部分SS患者存在IFI16和抗ORF1p抗体。使用培养的细胞，我们还观察了共- IFI16-DNA复合物与自噬蛋白的定位，并观察到自噬的证据 细胞质DNA传感后的激活。我们认为，自噬介导的稳态处置， 干扰原性IFI16-LINE1复合物，因此可能代表这种疾病的一种可增强途径。 将使用人体组织、细胞试验和体外研究的组合来检验这些假设。这 这项工作将受益于两位关键合作者的专业知识：Kathy Burns博士，一位复古元素专家， 博士先天免疫传感器方面的生物制药专家孙正山（Jungsan Sohn）说。除了完成拟议的 实验工作，申请人将执行正式的课程，接受技术培训，参加 相关的会议，并从一个多学科的成功的调查团队获得认真的指导 在这个奖项的期限内。完成这项工作将促进候选人的发展成为一个 独立的医生研究员，在人类风湿性疾病的机制研究方面具有专业知识， 特别关注逆转录因子与先天免疫之间的关系。

Project Summary/Abstract This award will promote the development of the applicant who seeks to acquire the skills and knowledge necessary to become an independent physician scientist focused on human rheumatic diseases. The research focus of this grant is the retroelement LINE1, and its potential interaction with the cytoplasmic DNA sensor IFI16, in the context of the autoimmune disease Sjögren’s syndrome (SS). Persistent activation of immune responses against nucleic acid-containing autoantigens is a hallmark of several rheumatic diseases, including SS, where an “interferon (IFN) signature” is found in the blood and affected tissues. Activation of innate nucleic acid sensors is a key driver of IFN signaling that has been implicated in SS and related diseases. In this proposal, we will examine the retroelement LINE1 as a potential endogenous driver of both the innate and humoral immune response in SS, and examine the role that autophagy plays in regulating the IFN response to LINE1. These studies will build upon our recent observation that the DNA sensor and SS autoantigen IFI16 is activated in a filamentous form in some SS salivary ductal epithelial cells – a process which seems linked to the presence of anti-IFI16 antibodies in SS sera. Preliminary data reveal that the LINE1 protein ORF1p appears to be an SS autoantigen as well, and is strikingly associated with anti-IFI16 antibodies in these patients. Moreover, LINE1 protein expression can be detected in the same ductal epithelial cell layer that harbors activated IFI16 in SS glands, suggesting that LINE1 expression in these cells could be a potential source of nucleic acid responsible for IFI16 filament formation observed at that site. Based on these preliminary findings, we hypothesize that IFI16 binding to LINE1 nucleic acids in the cytoplasm of epithelial cells could explain the association between anti- IFI16 and anti-ORF1p antibodies in some SS patients. Using cultured cells, we have also visualized co- localization of IFI16-DNA complexes with autophagy proteins, and have observed evidence of autophagy activation following cytoplasmic DNA sensing. We propose that autophagy mediates homeostatic disposal of interferogenic IFI16-LINE1 complexes, and could therefore represent an augmentable pathway in this diseases. These hypotheses will be tested using a combination of human tissues, cellular assays, and in vitro studies. This work will benefit from the expertise of two key collaborators: Dr. Kathy Burns, an expert in retroelements, and Dr. Jungsan Sohn, a biophysicist expert in innate immune sensors. In addition to completion of the proposed experimental work, the applicant will perform formal coursework, receive hands on technical training, attend relevant conferences, and obtain careful mentorship from a multi-disciplinary team of accomplished investigators over the duration of this award. Completion of this work will foster the development of the candidate into an independent physician researcher with expertise in the mechanistic study of human rheumatic disease, with particular focus on the relationship between retroelements and innate immunity.