Biallelic loss of function mutations in the linear chain specific deubiquitinase (DUB) OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) result in OTULIN Related Autoinflammatory Syndrome (ORAS). To date all reported ORAS patients have had homozygous or compound heterozygous loss of function mutations, however we identified a patient with a monoallelic heterozygous mutation p.Cys129Ser. Consistent with the ORAS phenotype, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF induced cell death and dysregulation of inflammatory signalling in both patient cells and in vitro exogenous expression models. Levels of the mutant OTULIN protein were consistent with wild type OTULIN in patient cells and exogenous expression systems and maintained binding capacity to both LUBAC and linear ubiquitin chains. However, even in a heterozygous context this mutant DUB promoted the global accumulation of linear ubiquitin chains. Furthermore, it allowed accumulation of ubiquitin on the linear ubiquitin chain assembly complex (LUBAC). Altered ubiquitination of LUBAC leads to a dysregulation of NF-κB signalling and promotion of TNF induced cell death. By reporting the first dominant negative mutation driving ORAS this study expands our clinical understanding of Otulin mediated pathology.

Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.

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