A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection

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. Overall design: LCMV-specific CD8+ T cells were isolated from the spleen and pooled lymph nodes of mice 14 days post infection with LCMV docile. Paired-end RNA-seqeuencing data was then generated. Of these samples, 4 are wild type (WT) and 7 are Ripk3 knock-out (KO). Differential analyses were performed, identifying differentially expressed genes between the KO and WT groups.