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. 2024 May;31(5):672-682.
doi: 10.1038/s41418-024-01286-6. Epub 2024 Mar 28.

ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1

Margaret Solon  1 , Nianfeng Ge  1 , Shannon Hambro  1 , Susan Haller  1 , Jian Jiang  1 , Miriam Baca  1 , Jessica Preston  1 , Allie Maltzman  2 , Katherine E Wickliffe  2 , Yuxin Liang  3 , Rohit Reja  2   4 , Dorothee Nickles  4   5 , Kim Newton  6 , Joshua D Webster  7
Affiliations

ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1

Margaret Solon et al. Cell Death Differ. 2024 May.

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Assays for assessing RIPK1, Ripk1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues.
a RIPK1 IHC in E11.5 Ripk1+/+Ripk3−/− (n = 1) and Ripk1−/−Ripk3D161N/− (n = 1) embryos. Scale bar, 100 μm. b RIPK1 IHC (left column) and Ripk1 ISH (right column) in WT small intestines. Scale bar, 50 µm. Results representative of 5 mice. c RIPK3 IHC in lung. Note the non-specific labeling of Ripk3−/− striated muscle and granulocytes with the Abcam polyclonal antibody, which is not seen with the Genentech 1G6 antibody. Scale bar, 100 µm. Results representative of 5 WT and 3 Ripk3−/− mice. d Mlkl ISH in small intestine with RNAscope probes targeting the full transcript (left) and a Basescope probe (center and right) that targets exon 3, which is deleted in Mlkl−/− mice. Scale bar, 25 µm. Results representative of 5 WT and 3 Mlkl−/− mice. e ZBP1 IHC in small intestine. Scale bar, 50 µm. Results representative of 5 WT and 4 Zbp1−/− mice.
Fig. 2
Fig. 2. Expression of RIPK1, RIPK3, and Mlkl in mouse tissues.
a, b RIPK1 IHC, RIPK3 IHC, and Mlkl ISH in WT mouse tissues. Scales bars, 25 µm (Mlkl: large intestine), 50 µm (RIPK1, RIPK3, and Mlkl: lymph node, liver, small intestine, and eye; Mlkl: urinary bladder and pancreas), or 100 µm (RIPK1 and RIPK3: large intestine and urinary bladder). Results representative of 5 mice. #, denotes germinal centers in the lymph nodes; >, denotes epithelial labeling; *, denotes endothelial labeling; and +, highlights immune cell labeling. c Ripk1 ISH in WT mouse tissues. Scale bars, 50 µm. Results representative of 5 mice.
Fig. 3
Fig. 3. ZBP1 IHC in mouse tissues.
ZBP1 IHC in mouse tissues. Scale bars, 100 μm (spleen, large intestine) or 50 µm (liver, skin). Results representative of 5 WT and 4 Zbp1−/− mice.
Fig. 4
Fig. 4. Casp8−/−Tnfr1−/− embryos have skin and liver defects.
a E15.5 embryos. Casp8−/− Tnfr1−/− embryos from 4 different litters appeared grossly normal (n = 4, middle panel) or lacked normal yolk sac vasculature (n = 1, right panel). A representative Tnfr1−/− littermate is shown (n = 5; left panel). b E16.5 liver sections stained with hematoxylin and eosin (H&E). Scale bars, 50 μm. Myeloid aggregates (#) were observed in all Casp8−/− Tnfr1−/− embryos (n = 5). Foci of hepatocellular death (+) were observed in 3 out of 5 Casp8−/− Tnfr1−/− embryos. A representative section from a Tnfr1−/− littermate (n = 3) is shown. c Representative H&E-stained skin sections from the E16.5 embryos in b. Scale bars, 50 μm. d E18.5 embryos. Results representative of 4 Tnfr1−/− and 5 Casp8−/− Tnfr1−/− embryos.
Fig. 5
Fig. 5. ZBP1 is expressed in myeloid cells and the vasculature of E15.5 Casp8−/−Tnfr1−/− embryos.
E15.5 skin (a), liver (b), and placenta (c) sections with ZBP1 immunolabeling (brown). Scale bars, 50 μm (a, b) or 100 μm (c). Images representative of WT (n = 5), Tnfr1−/− (n = 7), Casp8−/− Tnfr1−/− (n = 4), Casp8−/− Mlkl−/− (n = 3), and Casp8−/− Tnfr1−/− Zbp1−/− (n = 7) embryos. Arrows highlight vascular labeling in Casp8−/− Tnfr1−/− skin. d ZBP1 IHC scores for the embryos in a–c and their placentas. Lines indicate the mean. See methods for scoring criteria. e E15.5 liver sections with immunolabeling of F4/80 (brown). Scale bars, 100 μm. Images representative of WT (n = 5), Casp8−/− Tnfr1−/− (n = 5), Casp8−/− Tnfr1−/− Zbp1−/− (n = 7), and Casp8−/− Mlkl−/− (n = 3) embryos. f Heatmap shows differentially expressed genes in E15.5 Casp8−/− Tnfr1−/− fetal livers (n = 3) when compared with WT (n = 3), Tnfr1−/− (n = 2), and Casp8−/− Mlkl−/− (n = 3) fetal livers.
Fig. 6
Fig. 6. Combined loss of ZBP1 and TRIF delays lethality in Casp8−/−Tnfr1−/− mice.
a E17.5 skin sections stained with H&E or immunolabeled with phospho-RIPK3 T231, S232 (pRIPK3, brown). Scale bars, 100 μm. Graphs show dermatitis histology scores and pRIPK3 IHC scores for Tnfr1−/− Zbp1−/− (n = 5), Casp8−/− Tnfr1−/− Zbp1−/− (n = 4), Tnfr1−/− Trif−/− (n = 2), and Casp8−/− Tnfr1−/− Trif−/− (n = 4) embryos. Lines indicate the mean. b Hepatocellular vacuolation histology scores for the embryos in a. Lines indicate the mean. See methods for scoring criteria used in a and b. c Representative H&E-stained skin sections from E18.5 Casp8−/− Tnfr1−/− Trif−/− Zbp1−/− (n = 3) and Tnfr1−/− Trif−/− Zbp1−/− (n = 3) embryos demonstrating epidermal thickening and increased dermal cellularity in the Casp8−/− Tnfr1−/− Trif−/− Zbp1−/−embryo. Scale bar, 50 μm. d Kaplan–Meier curves of mouse survival. P-value determined by 2-sided log-rank test. e Body weights of Casp8−/− Tnfr1−/− Trif−/− Zbp1−/− (red bars; females, n = 17 aged 3 weeks, n = 12 aged 5 weeks; males, n = 11 aged 3 wks, n = 10 aged 5 weeks) and Tnfr1−/− Trif−/− Zbp1−/− (white bars; females, n = 14 aged 3 weeks, n = 11 aged 5 weeks; males, n = 13 aged 3 wks, n = 11 aged 5 weeks) mice. Bars indicate the mean ± s.e.m. P-values determined by 2-sided student t-test with Welch’s correction. f Representative liver, lung, and spleen sections from male Casp8−/− Tnfr1−/− Trif−/− Zbp1−/− mice (n = 3) with increased cellular infiltrates in the lung and liver and increased splenic hematopoiesis and male littermate controls (Casp8+/- Tnfr1−/− Trif−/−, n = 2 and Casp8+/- Tnfr1−/− Trif+/- Zbp1+/-, n = 1) aged 9-10 weeks. Scale bars, 100 μm (lung, spleen) or 50 μm (liver). g Peripheral blood cell counts in Casp8−/− Tnfr1−/− Trif−/− Zbp1−/− (n = 4) and Tnfr1−/− Trif−/− Zbp1−/− (n = 4) mice aged 4 weeks. Bars indicate the mean ± s.e.m. P-values are shown if P < 0.05 by 2-sided t-test with Welch’s correction. h Spleen weight as a percentage of body weight for mice aged 4-5 weeks. Red bars, Casp8−/− Tnfr1−/− Trif−/− Zbp1−/− mice (n = 4; 1 male, 3 females). White bars, littermate Tnfr1−/− Trif−/− Zbp1−/− (n = 2; 1 male, 1 female) or Tnfr1−/− Trif−/− (n = 1, female) mice. Bars indicate the mean ± s.e.m. P-value determined by 2-sided student t-test with Welch’s correction. i Splenic leukocyte subsets for the mice in h. Bars indicate the mean ± s.e.m. P-values are shown if P < 0.05 by 2-sided t-test with Welch’s correction. j Representative spleen flow cytometry contour plots of the mice in h.
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