ABSTRACT Type I interferon (IFN) pathway signaling is a primary pathogenic factor in systemic lupus erythematosus. Despite the importance of type I IFN in SLE, it is not currently known how the pathway is activated in a given patient. While 70 to 80% of SLE patients have an IFN signature in peripheral blood, we consistently find that only 40 to 50% of SLE patients have high levels of functional circulating type I IFN. Thus approximately 30% of SLE patients have an IFN signature without a significant elevation of type I IFN in blood. This disconnect is important, because clinical trials of type I IFN antagonists have used the IFN signature as a biological indicator of response. Recently, we identified a common genetic polymorphism in the purine nucleotide phosphatase (PNP) gene that results in an IFN signature that does not require type I IFN receptor signaling. The lupus- associated variant results reduced enzyme activity, and increased S phase block. In preliminary studies, we find that PNP variant cells have increased cytosolic DNA inclusions. Cytosolic DNA inclusions can form following S phase block or DNA damage, and these inclusions can activate the cytosolic anti-viral response via STING. We find that purified B cells in culture carrying the PNP variant have increased IFN-induced gene expression, but do not make type I IFN. We frequently use drugs that induce S phase block, such as mycophenolate mofetil and azathioprine. It is possible that some of the IFN signature observed in SLE is related to cytosolic DNA inclusions in response to these agents, with or without the PNP variant. Whether this IFN-induced gene expression would then limit the efficacy of these drugs or contribute to side effects is not known. Even if this IFN-induced gene expression does not worsen inflammation in SLE, it would reduce the clinical utility of an IFN signature. We hypothesize that S phase block related to the PNP polymorphism contributes to IFN pathway activation in SLE via cytosolic DNA inclusions and activation of cytosolic receptors, and that PNP deficiency worsens lupus and may complicate treatment with S phase blockers. In Aim 1, we will dissect the influence of PNP deficiency and cytosolic DNA inclusions upon the IFN signature in human cells. In Aim 2, we will determine the impact of PNP deficiency on lupus phenotype and treatment in vivo in murine models. This high-risk, high-reward proposal would establish a new paradigm for the type I IFN signature in SLE, that endogenous cytosolic DNA inclusions resulting from inefficient cell cycle contribute to type I IFN pathway activation, and could suggest a role for commonly used SLE medications in this process. These results could help to personalize treatment in SLE and to refine our biomarkers related to type I IFN.