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.