PROJECT SUMMARY This project is a continuation and further development of a previous INIA project, which was based on studies showing ethanol-induced changes in neuroimmune gene expression in animal models and humans. Those data suggested that ethanol dysregulates Toll-like receptor (TLR) signaling through the myeloid differentiation primary response gene 88 (MyD88) and thereby promotes excessive ethanol intake. Our most recent work showed that genetic and pharmacological manipulation of another branch of TLR signaling via the TIR-domain- containing adapter-inducing interferon-β (TRIF) protein also regulates ethanol intake. In exploring downstream mechanisms by which these signaling disruptions act, we found that chronic alcohol consumption increased several TRIF-dependent signaling components, including type 1 interferons (IFN1s). These findings lead us to the central hypothesis of this renewal application, which is that chronic alcohol exposure activates pathways leading to IFN1 production and expression of interferon-stimulated genes (ISGs), which increase alcohol consumption. To test this hypothesis, we will study alcohol intake and alcohol-related behaviors in mice deficient in critical components of pathways leading to IFN1 production and signaling, and in mice administered compounds that block IFN1 signaling. The proposal has three Specific Aims. Specific Aim 1 will study changes in alcohol consumption in mice undergoing every other day two-bottle choice (EOD-2BC) drinking for at least 4 weeks. To reduce IFN1 production, we will examine these behaviors in mice with genetic deletion of the transcription factors Irf3 or Irf7. To examine the role of IFN1 signaling, we will use Ifnar1 knockout mice which carry a null mutation in the IFN1 receptor. We will also study wild-type mice treated with inhibitors of the kinase TYK2, which mediates IFN1 receptor signaling. Specific Aim 2 will determine if EOD-2BC drinking induces IFN1 responses in specific brain regions by detecting Fos expression in Mx1GFP mice in which the interferon-stimulated response element of the Mx1 gene drives GFP expression. Data from these whole brain imaging studies will be shared with the Harsan-Keiffer project to be incorporated into their multimodal connectome analyses. To determine if IFN1 regulates alcohol responses in certain regions, we will knockout Ifnar1 using local microinjection of Cre recombinase in floxed Ifnar mice and use Fos-Cre-ER (TRAP2) mice to knockout or knockdown Ifnar1 in activated neurons and glia. Specific Aim 3 will identify ISGs induced by EOD-2BC alcohol drinking in brain regions identified in Aim 2. The Mayfield project will help us analyze transcriptomic data to identify ISGs. To investigate causality, groups of ISGs will be knocked down using multiplex CRISPR interference in collaboration with the Farris-Homanics project. We anticipate that some other INIA projects will identify additional proteins that require behavioral testing with pharmaco...