ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1
Margaret Solon1, Nianfeng Ge1, Shannon Hambro1
1Department of Pathology, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.
Abstract:
Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period. Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.
Insights
Necroptosis, a cell death pathway involving RIPK1, RIPK3, and MLKL, is characterized in mouse tissues. Z-form nucleic acid sensor ZBP1 plays a critical role in embryonic lethality and perinatal demise, revealing new insights into cell death regulation.
Area of Science:
- Cellular Biology
- Immunology
- Developmental Biology
Background:
- Necroptosis is a regulated form of lytic cell death.
- It is mediated by RIPK3 and MLKL kinases, particularly when caspase-8 is inhibited.
- Key upstream activators include death receptors, TLR3, TLR4, and ZBP1.
Purpose of the Study:
- To create a comprehensive tissue atlas of RIPK1, RIPK3, MLKL, and ZBP1 expression in mice.
- To investigate the role of ZBP1 and other factors in embryonic lethality and perinatal mortality.
- To elucidate the pathways driving necroptosis after birth in the absence of caspase-8 and TNFR1.
Main Methods:
- Immunohistochemistry (IHC) and in situ hybridization (ISH) assays were employed.
- Expression patterns of RIPK1, RIPK3, MLKL, and ZBP1 were analyzed across various mouse tissues.
- Genetic knockout mouse models (Casp8-/-, Tnfr1-/-, Zbp1-/-, Trif-/-) were utilized to study lethality and necroptosis pathways.
Main Results:
- RIPK1, RIPK3, and MLKL showed co-expression in immune cells, endothelial cells, and epithelia.
- ZBP1 expression was prevalent in immune cells but more variable in epithelia.
- Elevated ZBP1 in Casp8-/- Tnfr1-/- embryos preceded lethality, with ZBP1 deficiency improving survival.
- TRIF-mediated necroptosis contributed to perinatal death in Casp8-/- Tnfr1-/- Zbp1-/- pups.
- Postnatal lethality in Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice suggested RIPK1-dependent necroptosis via alternative death receptors.
Conclusions:
- A detailed expression map of key necroptosis mediators was established in mouse tissues.
- ZBP1 is crucial for embryonic lethality in the absence of caspase-8 and TNFR1.
- TRIF-dependent necroptosis contributes to perinatal lethality.
- Postnatal lethality is likely driven by RIPK1-dependent necroptosis mediated by death receptors other than TNFR1.
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