In this project, we studied how Z-nucleic acids may be implicated in the pathogenesis of inflammatory conditions. In particular, we focused on the role of Z-nucleic acid sensing by ZBP1 in the regulation of cell death and inflammation in vivo in mouse models of inflammatory diseases. We could show that ZBP1 is activated by binding to endogenous Z-nucleic acids and triggers keratinocyte necroptosis and inflammation in mice lacking RIPK1 in the epidermis or expressing RIPK1 with mutated RIP homotypic interaction motif (RHIM). In addition, we found that Z-nucleic acid-induced ZBP1-mediated necroptosis is under negative regulation by caspase-8 and that inhibition of caspase-8 triggers ZBP1-dependent inflammation in the intestine. These results provided the first experimental evidence that sensing of endogenous Z-nucleic acids by ZBP1 causes inflammation in vivo in relevant models of inflammatory diseases. Furthermore, we found that ZBP1 binds cellular dsRNA via its Z domains, identifying endogenous Z-RNA as a key ligand triggering ZBP1-mediated necroptosis. Computational analysis revealed that endogenous retroelement-derived transcripts constitute the majority of putatively dsRNA species in mouse skin, suggesting that endogenous retroelement-derived Z-RNA is the most likely ligand activating ZBP1 in vivo. Furthermore, we could show that ZBP1 acts in a Zalpha domain-dependent manner to induce the activation of RIPK3 and its substrate MLKL within the nucleus, suggesting that sensing of nuclear Z-RNA provides a potent trigger activating ZBP1-mediated cell death. Collectively, these results obtained during the first period of the project revealed an important role of ZBP1 in sensing endogenous Z-RNA likely derived from endogenous retroelements and inducing necroptosis and inflammation, which is relevant for the better understanding of the mechanisms controlling the pathogenesis of chronic inflammatory diseases.
In a separate line of experiments, we studied how Zalpha-dependent sensing of endogenous Z-RNA regulates the function of ADAR1 and its capacity to edit endogenous ERE-derived dsRNA to prevent their recognition by MDA5 and the induction of pathogenic type I interferon responses. We could show that hemizygous mutation of the ADAR1 Zalpha domain resulted in severe, early postnatally lethal, pathology in mice, which was driven by MDA5-MAVS-mediated signalling. In this mouse model of type I interferonopathy, we found that Zalpha-dependent ZBP1 signalling also contributes to the severe pathology induced by mutation of the ADAR1 Zalpha domain, revealing a critical interplay between ADAR1 and ZBP1 in the regulation of Z-RNA-dependent pathology. An unexpected finding in our studies was the ZBP1 drives the pathology of mice with hemizygous ADAR1 mutant Zalpha expression not by inducing cell death but by promoting type I interferon responses. Together, these results revealed a novel role of ZBP1 in sensing Z-RNA and promoting pathogenic type I interferon responses in a mouse model of type I interferonopathy induced by ADAR1 Zalpha mutation, suggesting that ZBP1 may also contribute to the pathology in human patients suffering from type I interferonopathies caused by ADAR1 mutations.
