Abstract
The RNA-editing enzyme ADAR1 is essential for the suppression of innate immune activation and pathology caused by aberrant recognition of self-RNA, a role it carries out by disrupting the duplex structure of endogenous double-stranded RNA species1,2. A point mutation in the sequence encoding the Z-DNA-binding domain (ZBD) of ADAR1 is associated with severe autoinflammatory disease3,4,5. ZBP1 is the only other ZBD-containing mammalian protein6, and its activation can trigger both cell death and transcriptional responses through the kinases RIPK1 and RIPK3, and the protease caspase 8 (refs. 7,8,9). Here we show that the pathology caused by alteration of the ZBD of ADAR1 is driven by activation of ZBP1. We found that ablation of ZBP1 fully rescued the overt pathology caused by ADAR1 alteration, without fully reversing the underlying inflammatory program caused by this alteration. Whereas loss of RIPK3 partially phenocopied the protective effects of ZBP1 ablation, combined deletion of caspase 8 and RIPK3, or of caspase 8 and MLKL, unexpectedly exacerbated the pathogenic effects of ADAR1 alteration. These findings indicate that ADAR1 is a negative regulator of sterile ZBP1 activation, and that ZBP1-dependent signalling underlies the autoinflammatory pathology caused by alteration of ADAR1.
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Data availability
RNA-seq and NanoString data are available through the Gene Expression Omnibus of the National Institutes of Health, accession numbers GSE200854 (RNA-seq) and GSE200985 and GSE200986 (NanoString). Source data are provided with this paper.
Code availability
The R analysis was carried out using publicly available code, described in the Methods section; custom R scripts are available at https://github.com/OberstLab/Hubbard-et-al-2022-Nature.
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Acknowledgements
This work is supported by the grants R01 AI153246 (to A.O. and D.B.S.), R01 CA228098 (to A.O.), R01 AI084914 (to D.B.S.), R01 AI143227 and R01 AI147177 (to R.S.), a Titus Fellowship (to N.W.H.), T32 T32AR7108-41 (to J.M.A.), and a Helen Hay Whitney Foundation Postdoctoral Fellowship (to N.S.G.). Extended Data Fig. 9a was created with BioRender.com. We thank P. Jain, I. Silva and R. Lee for technical assistance.
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Conceptualization: N.W.H., M.M., D.B.S., A.O.; methodology: N.W.H., M.M., R.S., L.H.C., N.S.G., D.B.S., A.O.; analysis: N.W.H. (all), J.M.A. (NanoString data), K.Y.S. (RNA-seq data analysis), J.M.S. (histopathological analysis and scoring), A.O.; investigation: N.W.H. (all), J.M.A. (NanoString, tissue culture and cell death assays), N.S.G. (Supplementary Fig. 1 co-immunoprecipitation experiments), L.H.C. (tissue culture, western blot and co-immunoprecipitation), K.L. (flow cytometry), M.W. (Ripk3/Trex1A.O.; writing original draft: N.W.H., A.O.; writing, review and editing: all authors.
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D.B.S. is a co-founder and shareholder of Danger Bio, LLC, and a scientific advisor for Related Sciences LLC. A.O. is a co-founder and shareholder of Walking Fish Therapeutics.
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Extended data figures and tables
Extended Data Fig. 1 Interaction of ZBP1 with RNA and ADAR1.
A. Co-precipitation ADAR1 with WT or mutant Zαβ (mZαβ) Flag-tagged ZBP1 (FLAG-ZBP1) after FLAG immunoprecipitation. Β. Immunoprecipitation and IR-CLIP analysis for RNA binding by of WT or mZαβ FLAG-ZBP1. C. Co-precipitation of ADAR1 and FLAG-tagged ZBP1 after UV-crosslinking. These experiments were performed in HEK293T cells.
Extended Data Fig. 2 Immunopathology in ADAR-mutant mice is ZBP1 dependent.
Survival proportions observed upon cross of Adarp150null/WT::Zbp1-a+/− mice to AdarP195A/P195A::Zbp1-+/−, **** p < 0.0001 (Mantel-Cox Log-Rank test) (A) or Adarp150null/WT::Zbp1-a−/− mice to AdarP195A/P195A::Zbp1-a−/−, not significant (Mantel-Cox Log-Rank test) (B). (C) Histopathological analysis of liver and kidney from affected, rescued and unaffected mice (genotypes indicated.) For liver samples, regions of cytoplasmic vacuolation indicated with asterisk. Original magnification 20x, HE staining. For kidney, glomeruli are indicated with arrows, from original magnification 40x HE staining.
Extended Data Fig. 3 Cross of AdarP195A/p150null mice to a separately derived, fully congenic Zbp1−/−-g strain.
A–B. SNP typing analysis of ZBP1-g (A) and ZBP1-a (B) mice. C–D. Zbp1−/−-g::AdarP195A/p150null survival proportions(C) and observed weight (D) at 21 days (weaning). Combined male & female, Zbp1+/+::AdarP195A/WT (n = 17), Zbp1+/−::AdarP195A/WT (n = 21), Zbp1−/−::AdarP195A/WT (n = 10), Zbp1+/+::AdarP195A/p150null (n = 9), Zbp1+/−::AdarP195A/WT (n = 25), Zbp1−/−::AdarP195A/WT (n = 7).
Extended Data Fig. 4 ZBP1 is IFN dependent and partially dependent on MDA5.
A. Twelve-hour stimulation of LET1 and SVEC cells with varying concentrations of IFN-β followed by Western Blot analysis for ZBP1 protein. B. Quantitative PCR analysis for Zbp1 and Ifnb after ADAR1 depletion in wild-type (scramble gRNA) or MDA5 knockout LET1. Gene was normalized against Gapdh. Significance determined by individual student t-tests. Each group (Zbp and Ifnb) contains 3 biologic replicates, each comprising the average of 4 technical replicates). This experiment is representative of two independent repeats. Whisker bars are presented as mean +/− SD. C. Survival of Zbp1−/−::MDA5−/−::ADARp150null/p150null mice. Zbp1-a+/+::Ifih1−/−::Adarp150null/+ n = 6, Zbp1-a+/+::Ifih1−/−::Adarp150null/p150null n = 4, Zbp1-a−/−::Ifih1−/−::Adarp150null/+ n = 5, Zbp1-a−/−::Ifih1−/−::Adarp150null/p150null n = 3. Statistical significance determined by Mantel-Cox (Log-Rank) test. D. Survival proportions of Zbp1−/−-a::Adarp150/WT intercross. Chi square power analysis performed, indicating significance at p = 1.82x10−5.
Extended Data Fig. 5 Identification of ZBP1 dependent and independent aspects of the ADAR1 inflammatory signature.
A–B: Cleveland plots indicating changes in the ADAR1 dependent gene signature observed in the spleens of 23 day old mice, indicating most- (A) and least- (B) changed genes upon ZBP1 ablation in AdarP195A/p150null mice. Gene selection is the top 30 largest contributors to the ZBP1 dependent (A) and independent (B) signature from ADARP195A/p150null mice, in comparison to WT mice. Gene Ontology analysis was performed on the signatures from A and B. C–D: GO-terms analysis for ZBP1-independent signature (C), and the ZBP1 dependent signature analysis (D).
Extended Data Fig. 6 Flow cytometry analysis of splenic cellular subsets.
B cell, T cell, monocyte, macrophage percentages from day 23 spleens of Zbp1::Adarp150/P195A mice.
Extended Data Fig. 7 ZVAD treatment induces phosphorylation of RIPK3 in ADAR mutant MEFs in a ZBP1 dependent fashion.
A. Analysis of phospho-RIPK3 in ADAR1 mutant MEFs after 4 h ZVAD treatment. B. Confirmation of ZBP1 gRNA knockout in ADAR1 mutant MEFs.
Extended Data Fig. 8 MLKL or RIPK1kinase dead mutations do not rescue ADARP195A/p150null mutation.
Three-week-old weights (weaning) of male or female AdarP195A/p150null mice crossed to animals lacking, A: MLKL. Mlkl+/−::AdarP195A/WT (m/f n = 9/7), Mlkl+/−::AdarP195A/p150null (m/f n = 8/2), Mlkl−/−::AdarP195A/WT (m/f n = 8/5), Mlkl−/−::AdarP195A/p150null (m/f n = 6/3). or B: carrying a point mutation abrogating the kinase activity of RIPK1 (Ripk1kd). Ripk1kd/+::AdarP195A/WT (m/f n = 3/11), Ripk1kd/+::AdarP195A/p150null (m/f n = 7/5), Ripk1kd/kd::AdarP195A/WT (m/f n = 11/8), Ripk1kd/kd::AdarP195A/p150null (m/f n = 5/5). Statistical differences determined by individual student t-tests (two tailed). All genotypes are littermates from mixed litters.
Extended Data Fig. 9 Oligomerization of ZBP1 triggers necroptotic cell death.
A. Schematic indicating the replacement of ZBP1’s Z-DNA binding domain with a tandem FKBP domain. B–D: Cell death following addition of B/B homodimerizer with indicated combinations of ZVAD and GSK843 in LET1s (each group, n = 4 biologic replicates) (B), SVECs (each group n = 4 biologic replicates) (C) or MEFs (each group n = 3 biologic replicates) (D). Statistical significance was determined by unpaired t tests (two-tailed). Experiments B and C are representative of two independent experiments, and D is representative of 3 independent experiments. All whisker bars are presented as mean +/− SD.
Extended Data Fig. 10 Molecular analysis of cell death induced by ZBP1-2xFV homodimerization.
A. Phospho-MLKL analysis of ZBP1-2xFV MEFs (wild-type, Ripk3−/−, MLKL−/−) after 1 h stimulation with B/B homodimerizer. B. Cleaved-caspase 3 analysis of ZBP1-2xFV MEFs (wild-type, Ripk3−/−, MLKL−/−) after 3 h stimulation with B/B homodimerizer. C. Representative image depicting Caspase-8 deficiency exacerbation of disease phenotype in ADARP195A/p150null::RIPK3−/− mice. Image of 20-day old littermates from the cross depicted in Fig. 4b.
Extended Data Fig. 11 Pathologic analysis of Mlkl−/−::Casp8−/−::Adarp150null/P195A mice. A–B.
Immunohistochemical staining (A) and quantification (B) in liver for cleaved-caspase 3. (n = 3 d.0 pups, each group) Original magnification 10x. C. Additional histological images of other tissue sites (kidney, liver and small intestine). Kidney (20x) and liver (10x) PAS staining, small intestine HE staining (original magnification as stated). D–E. Immunohistochemical staining, with 2.5x and 10x images (D) and quantification (E) in brain for Iba1 (n = 3 d.0 pups, each group). B & E use tissues from matched animals.
Extended Data Fig. 12 Immunoprecipitation of RIPK3 and RIPK1 by ZBP1 is enhanced by Casp8 deficiency.
A. Pulldown of ZBP1-2xFV (FKBP) and co-precipitation of RIPK1 in Ripk3−/−::Casp8−/− MEFs. B. Pulldown of ZBP1-2xFV (FKBP) and co-precipitation of RIPK1 and RIPK3 in Mkl−/−::Casp8−/− MEFs. C. Pulldown of ZBP1 and co-precipitation of RIPK3 and in Mlkl−/−::Adarp150null/P195A MEFs.
Extended Data Fig. 13 ZBP1 activation results in RIPK1 dependent, RIPK3 independent gene transcription which is enhanced by knockout of Casp8.
A–B Differential transcript analysis of Mlkl−/−::Casp8+/+ and Mlkl−/−::Casp8−/− MEFs (n = 3) following ADAR depletion, by (A) Heatmap analysis and (B) individual quantification of the top 5 differentially expressed genes. Statistical significance determined by individual unpaired t tests (two-tailed). Where not indicated: **** p < 0.0001. C. QPCR analysis of top 5 differentially expressed genes identified (B) in MEFs expressing ZBP1-2xFV after B/B activation after depletion of RIPK1. n = 6 biologic replicates for each treatment group, each representing the average of three technical replicates. Data is compiled from two independent experiments. Statistical significance determined by individual unpaired t tests (two-tailed). Where indicated: **** p < 0.0001. Whisker bars represent the mean +/− SD.
Extended Data Fig. 14 ZBP1A64P mutation attenuates ZBP1 activity in ADAR deficiency model and during influenza infection.
A. Previously-reported structures of the ZBDs of ZBP1 (left) and ADAR1 (right), with A64 and P195A (respectively) highlighted in green. PDB accession #s: 1J75 and 3F21. B. Survival proportions of Adar1P195A/p150null::ZBP1A64P mice (n = 9 animals) compared with previous survival statistics of AdarP195A/p150null::ZBP1-a+/− animals. C. Cell death analysis following influenza (X31, MOI 2) infection of primary MEFs derived from Wild Type (B6/J), Zbp1−/− or ZBP1A64P/A64P mice. Each group, n = 2 biologic replicates. Whisker bars represent the mean +/− SD. Experiment was independently replicated using MEFs derived from a different embryo. D. Expression of ZBP1 in wild-type, Zbp1−/−, and Zbp1A64P/A64P MEFs following 24 stimulation with 1000 IU/mL murine IFN-β.
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Hubbard, N.W., Ames, J.M., Maurano, M. et al. ADAR1 mutation causes ZBP1-dependent immunopathology. Nature 607, 769–775 (2022). https://doi.org/10.1038/s41586-022-04896-7
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DOI: https://doi.org/10.1038/s41586-022-04896-7
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