Periodic Reporting for period 3 - NECROPTOSIS (Necroptosis in immunity, inflammation and autoimmunity induced by nucleic acid sensors)
Período documentado: 2021-10-01 hasta 2023-03-31
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 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 Zα 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 Zα 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, 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 inducing necroptosis and inflammation, which is relevant for the better understanding of the mechanisms controlling the pathogenesis of chronic inflammatory diseases.
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.