PROJECT SUMMARY/ABSTRACT Cutaneous inflammation is a prevalent and often difficult-to-treat manifestation of systemic lupus erythematosus (SLE) that is driven in part by elevated type I IFN signaling that leads to increased myeloid cell recruitment and activation, increased keratinocyte cell death, repression of regulatory cell responses, and increased chemokine and cytokine production. The sources of type I IFN production in cutaneous lesions include recruited immune cells and skin-intrinsic sources of type I IFNs, including keratinocyte production of IFNκ. The mechanisms that drive and regulate increased keratinocyte production of type I IFNs remain unknown yet are important novel targets for therapeutic intervention. Our preliminary data support a critical role for mitochondrial-triggered STING signaling in the upregulation of IFNB and IFNK transcription that is enhanced by type I IFN production of Z-nucleic acid binding protein 1 (ZBP1). Upregulation of type I IFNs is paralleled by increased expression of type III IFNs (IFNλ family) in a similar fashion, and their interplay with type I IFNs may lead to inflammatory responses not targeted by anti-IFNAR treatment approaches. Our preliminary data support UVB-driven IFNλ 1 and 3 expression in human KCs, with regulation downstream of UV activation in a manner like IFNK and IFNB respectively. The contributions of IFNλ to type I IFN-driven immune responses remain unknown. Thus, the next 5 years of funding of this grant will focus on dissecting detailed mechanisms of keratinocyte IFN regulation with a new focus on mitochondrial upregulation of type I IFNs in keratinocytes, the interplay with type III IFNs, and the study of type I IFN signaling blockade in SLE patient skin. Our overall hypothesis is that in the majority of SLE patients, chronic over-production of IFNκ is perpetuated through an IFN-primed Z-DNA binding protein (ZBP1) and mitochondrial oxidation-dependent activation of STING that requires IFNβ and potentially type III IFNs to sustain and or amplify inflammatory signaling. Patients refractory to IFN blockade may have activation of additional signals downstream of mitochondrial stress that drive persistent inflammation. Three aims are proposed: Aim 1: Dissect the role of the mitochondria in keratinocyte IFN regulation. Aim 2: Determine the SLE-relevant interactions between type I and type III IFNs in keratinocytes. Aim 3: Determine how type I IFN blockade changes inflammatory phenotypes in SLE skin and determine the critical pathways that remain in refractory disease. When completed this project will identify novel ways of targeting skewed interferon production and uncover novel, non- type I IFN pathways that contribute to ongoing disease.