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.

Project Narrative This project seeks to determine whether the retroelement LINE1 activates the immune system in a way that contributes to autoimmunity in Sjögren’s Syndrome. This work could help explain how dysregulated immune surveillance of endogenous genetic material could promote autoimmunity in some people. Completion of this work may help direct study of new targeted therapies for Sjögren’s Syndrome and related rheumatic diseases.