ABSTRACT Collaborative Project; PI – Lyden, D. Systemic autoimmune diseases are heterogeneous both clinically and molecularly. Among them, systemic lupus erythematosus (SLE) is relatively frequent, affecting ~1:5000 people, with a higher prevalence in women. Lupus phenotypes are heterogeneous, but robust biomarkers that predict SLE progression, organ involvement and response to treatment are lacking. One of the fundamental criteria for SLE diagnosis is the presence of anti-nuclear antibodies (ANA), which comprise nucleic acids as well as proteins. Lupus presents as an interferonopathy, though IL1-b activation is also broadly observed. Given the central role of ANA in activating and propagating chronic IFN responses, understanding the sources of immunogenic DNA and mechanisms of IFN activation are critical. Despite their great clinical relevance major questions remain including how anti-DNA responses are prevented in healthy individuals, whether differences in structure or levels of DNA are responsible for immunogenicity in SLE and potentially other SAD, and how these mechanisms could be exploited for therapeutic purposes. We have recently discovered that activated T cell- derived exosomes (ATexo) carry large amounts of double stranded DNA complexed with histones (exoDNA), and that ATexo are efficient activators of IFN responses in a DNA-dependent manner. Thus, we hypothesize that, in SAD patients (SLE, scleroderma), due to anti-DNAse antibodies and DNASE1, DNASE1L3 mutations, combined with chronic T and B, plasma cell activation and expansion, the amount of circulating exoDNA exceeds DNA-degrading capacity and DNA-laden exosomes accumulate, chronically activating anti-DNA responses and leading to chronic inflammation. We propose to isolate and characterize DNA and protein cargo of T and B cells from healthy controls and SAD subjects, defining the function of activated T, B cell and plasma cell exoDNA in activating anti-DNA responses in antigen presenting cells and identifying the enzymes controlling circulating exoDNA levels. Testing this hypothesis will provide a systems-level understanding of relationships between the clinical, molecular, and functional heterogeneity in SLE and other SADs. By profiling the cargo of adaptive immune cell exosomes, and investigating, for the first time, the roles of activated immune cell-derived exoDNA in driving SAD chronic inflammation, these studies will help dissect the pathogenic mechanisms of the disease and designing novel, personalized treatments. Since exosomes reflect the systemic status of a disease, and, unlike circulating immune cells, can reflect pathologies associated with distant organs, often before these changes can be detected by other approaches, they are the ideal biomarker candidate for heterogeneous, multifactorial, systemic diseases such as autoimmune diseases in general and SLE in particular.