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

项目摘要/摘要 该奖项将促进寻求获得技能和知识的申请者的发展 成为一名专注于人类风湿病的独立内科科学家是必要的。这项研究 这项资助的重点是逆转录元件LINE1，以及它与细胞质DNA传感器IFI16的潜在相互作用， 在自身免疫性疾病Sjögren综合征(SS)的背景下。持续激活免疫反应 抗含有核酸的自身抗原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了“干扰素(干扰素)标志”。先天核酸感受器的激活 是干扰素信号的关键驱动因素，与SS和相关疾病有关。在这项提案中，我们将 研究反转录元件LINE1作为先天免疫和体液免疫的潜在内源性驱动因素 在SS中的反应，并检查自噬在调节干扰素对LINE1的反应中所起的作用。这些 研究将建立在我们最近观察到的DNA传感器和SS自身抗原IFI16在 一些SS唾液管上皮细胞中的丝状形态--这一过程似乎与 SS患者血清中的抗IFI16抗体。初步数据显示，LINE1蛋白ORF1p似乎是一个SS 自身抗原也是如此，并且与这些患者中的抗IFI16抗体显著相关。此外，LINE1 在SS中可检测到含有激活的IFI16的同一导管上皮细胞层中的蛋白质表达 提示LINE1在这些细胞中的表达可能是一个潜在的核酸来源。 对于在该地点观察到的IFI16灯丝形成。根据这些初步发现，我们假设IFI16 与上皮细胞胞浆中的LINE1核酸结合可以解释抗- 部分SS患者存在IFI16和抗ORF1p抗体。利用培养的细胞，我们还可视化了联合- 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感应后的激活。我们认为，自噬介导的动态平衡处置 产生干扰性的IFI16-LINE1复合体，因此可能是该病的一条可扩大的途径。 这些假说将结合人体组织、细胞分析和体外研究进行验证。这 这项工作将受益于两位关键合作者的专业知识：逆转录元素专家凯西·伯恩斯博士和 生物物理学家孙正山博士说，他是天生免疫感应器方面的专家。除了完成拟议的 实验工作，申请者将进行正式的课程作业，接受动手技术培训，参加 相关会议，并从经验丰富的多学科调查团队中获得仔细的指导 在本奖项的有效期内。这项工作的完成将促进候选人发展成为 独立内科研究员，在人类风湿病的机制研究方面具有专业知识， 特别关注逆转录因子和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.