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Necroptosis in immunity, inflammation and autoimmunity induced by nucleic acid sensors

Periodic Reporting for period 1 - NECROPTOSIS (Necroptosis in immunity, inflammation and autoimmunity induced by nucleic acid sensors)

Reporting period: 2018-10-01 to 2020-03-31

Cell death plays an important role in host defence to virus infections, but also contributes to tissue damage, inflammation and disease. Necroptosis is a recently characterised type of regulated necrotic cell death that has been implicated in host defence as well as in the pathogenesis of inflammatory diseases. The overall objective of this project is to understand the mechanisms regulating necroptosis and its function in the pathogenesis of inflammatory and autoimmune diseases and identify novel therapeutic targets. ZBP1 is a protein that induces necroptosis by activating the kinase RIPK3. ZBP1-mediated necroptosis and has been shown to regulate anti-viral immunity, tissue homeostasis and inflammation. ZBP1 senses Z-form DNA and RNA, which are left-handed double helix nucleic acid structures. Although the existence of Z-DNA and Z-RNA is known for decades, their physiological role remains unknown largely because of the lack of experimental methodologies allowing studying the mechanisms regulating their formation and biological function in the context of an organism. Studying the proteins that sense them is currently the only means to interrogate the function of Z nucleic acids in vivo. One of the key aims of this project is to study the role of Z-DNA and Z-RNA sensing by ZBP1 in the regulation of cell death and inflammation and in the pathogenesis of autoimmune and inflammatory diseases.
During the reporting period, we studied the role of Z-nucleic acid sensing by ZBP1 in the regulation of necroptosis and inflammation. We found that mutation of the Z domains of ZBP1, which are the protein domains sensing Z-nucleic acids, prevented skin inflammation in mouse models with mutations in the protein kinase RIPK1. In addition, we identified a previously unknown function of ZBP1 in triggering intestinal epithelial cell death and causing intestinal inflammation by sensing Z-nucleic acids. 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 and this is necessary for the activation of 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. Moreover, 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 addition, we could show that sensing of Z-nucleic acids by ZBP1 induces the activation of RIPK3 and the phosphorylation of its substrate MLKL within the nucleus, resulting in necroptosis. Collectively, the 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 triggering necroptosis, resulting in the pathogenesis of chronic inflammatory pathologies.
Our findings showed that sensing of endogenous Z-nucleic acids by ZBP1 causes necroptosis and inflammation in vivo, providing the first experimental evidence that endogenous Z-nucleic acids have important functions in regulating immune homeostasis and contributing to disease pathogenesis. Our ongoing studies aim to unravel the role of necroptosis induced by endogenous Z-nucleic acids and other pathways in the pathogenesis of inflammatory and autoimmune diseases, focusing on understanding the underlying mechanisms and identifying novel therapeutic targets.