Abstract
The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR11. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2,3,4,5,6,7,8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9,10,11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1−/− (also known as Rbck1−/−) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3−/−Casp8−/−Hoil-1−/− embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.
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Acknowledgements
V. Dixit and K. Newton provided Ripk3−/− mice, S. Hedrick and R. Hakem provided Casp8fl/fl mice; H. Anderton and U. Nachbur helped with Sharpincpdm analysis; P. Levy and staff and L. Lawrence provided technical and histology services. A. Leister and J. Marinis advised on GSK’457A. T. Marafioti and A. Akarsa helped with phosphorylated MLKL detection. A. Annibaldi provided scientific advice and helpful discussions. B. J. Ferguson provided reagents and advice. The flow cytometry, microscopy and imaging core facilities are supported by Cancer Research UK through the CRUK-UCL Centre (515818) and a Cancer Immunotherapy Accelerator award (CITA, 525877). This work was funded by a Wellcome Trust Senior Investigator Award (096831/Z/11/Z), an ERC Advanced grant (294880) and a Cancer Research UK programme grant (A17341) awarded to H.W., NHMRC grants awarded to P.B., A. St., J.S. and H.W. (602516, 1037321, 1043414, 1080321, 1105209, 461221, 1042629, 1057905), the Leukemia and Lymphoma Society (Specialised Center of Research grant 7015) and a postdoctoral fellowship awarded to N.P. by the Swiss National Science Foundation (P300P3_158509).
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Nature thanks F. Chan and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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H.W. conceived the project. N.P. and M.D. performed most experiments. N.P., M.D. and H.W. designed the research and co-wrote the manuscript. A.M., C.B. and T.E. conceived and contributed to the haematopoietic analyses. H.D. and P.D. contributed to in vitro experiments in Fig. 2 and Extended Data Fig. 3, S.K. generated Mlkl−/− mice, L.T., E.R. contributed to in vivo experiments, T.H performed cytokine arrays and E.L. and Y.S. contributed with biochemistry data. P.B., T.L.H. and H.W. designed the Hoil-1-floxed allele and P.B. generated it. A.F. and W.K. generated and analysed Ripk1−/−Ripk3−/−Casp8−/−Hoil-1−/− mice. H.D. and A.Sa. performed genotyping. A.B. and J.B. provided GSK’547A and Ripk1K45A mice. J.R., S.A.-D., A.St. and J.S. performed the Sharpincdpm studies. C.H. and M.T.A. performed pathological and microfocus computed tomography analyses. T.E., P.B., A.St., J.S. and E.R. provided scientific insight.
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Extended data figures and tables
Extended Data Fig. 1 HOIL-1-deficient mice die at mid-gestation.
a, Schematic representation of the Hoil-1-knockout strategy. Solid boxes represent Hoil-1 exons and grey boxes with a star indicate the targeted exons. Boxes with diagonal and horizontal strips represent loxP and Frt sites, respectively. b, Specificity of gene recombination was assessed by Southern blotting with 5′ and 3′ probes external to the construct in four clones (14B8, 14F6, 20D7 and 21F7). Digest of the DNA with ApaI, followed by hybridization with the 3′ probe was expected to show a 5,700-bp band for the wild-type allele and a 7,700-bp band for the mutant allele. All four clones appeared to have the correct recombination on the 3′ side. Digest of the DNA with SphI and hybridization with the 5′ probe was expected to show a 4,500-bp wild-type band and a 6,200-bp band for the mutated allele. Clones 14B8, 14F6 and 21F7 appeared to be correctly recombined on the 5′ side. Finally, cutting the DNA with ApaI and hybridizing with a hygromycin probe showed a single band in all clones, indicative of a single integration of the construct in all four ES clones. Clones 14B8 and 14F6 were selected for generation of the two Hoil-1−/− strains. c, PCR analysis of Hoil-1 wild-type, heterozygous and knockout mice. d, Protein levels of HOIL-1, HOIP and SHARPIN in whole embryo lysates (n = 3 for Hoil-1+/− and Hoil-1−/− embryos and n = 1 for Hoil-1+/+ embryos). For gel source data (c, d), see Supplementary Fig. 1. e, Quantification of genotypes of animals obtained from intercrossing C20Hoil-1+/− mice. Asterisk indicates dead embryo. f, Representative images of C20Hoil-1+/− and C20Hoil-1−/− embryos from E9.5 to E11.5 as quantified in e. Scale bars, 2 mm. g, Single staining showing vascularization (PECAM-1, top) and apoptosis (cleaved CASP3, bottom) of yolk sacs. Merged image is shown in Fig. 1c. h, Whole-mount TUNEL staining of embryos at the indicated stages (embryo per genotype n = 2 at E10.5, n = 8 for Hoil-1+/− and n = 5 for Hoil-1−/− at E11.5). Scale bar, 2 mm. i, Quantification of genotypes of animals obtained from inter-crossing Hoil-1fl/wtTie2-cre+ with Hoil-1fl/flTie2-cre− mice. Asterisk indicates dead embryo. j, Representative images of embryos with conditional deletion of Hoil-1 in Tie2-Cre-expressing cells as quantified in i. Scale bar, 2 mm. Asterisks denote poorly vascularized yolk sac.
Extended Data Fig. 2 TNFR1 signalling drives cell death and lethality of HOIL-1-deficient mice at mid-gestation.
a, d, Quantification of genotypes of animals obtained from intercrosses of Tnf−/−Hoil-1+/− (a) and Tnfr1−/−Hoil-1+/− (d) mice. Asterisk denotes dead embryo. b, Representative images of embryos quantified in a at E10.5 and E15.5. Asterisk denotes poor yolk sac vascularization. c, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E10.5 (n = 3 embryos per genotype). e, Single staining showing vascularization (PECAM-1, top) and apoptosis (cleaved CASP3, bottom) of yolk sacs. Merged image is shown in Fig. 1g. Scale bar, 50 µm. f, Representative images of embryos at E16.5 (n = 2 for Tnfr1−/−Hoil-1+/− and n = 4 for Tnfr1−/−Hoil-1−/−).
Extended Data Fig. 3 HOIL-1 is required for optimal TNF-induced NF-κB activation independently of its RBR domain.
a, b, d, Western blot analysis of the indicated proteins in whole-cell lysates from MEFs of the indicated genotypes after they had been stimulated with TNF (or left untreated) for the indicated time points (a), after overexpression of the different LUBAC components (b; HOIL-1, HOIP, SHARPIN) or after expression of the indicated mutant forms of HOIL-1 (d) (n = 2 independent experiments). c, SHARPIN immunoprecipitation was performed in Tnf−/−Hoil-1−/− MEFs reconstituted with HOIL-1 or a combination of HOIP and SHARPIN and analysed by western blotting (n = 2 independent experiments). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Ablation of the kinase activity of RIPK1 in HOIL-1- or HOIP-deficient embryos prevents cell death and lethality at mid-gestation but not at late gestation.
a, b, Quantification of genotypes of animals obtained after intercrossing Ripk1K45AHoil-1+/− (a) and Ripk1K45AHoip+/− (b) mice. Asterisk indicates dead embryo. c, Representative images of embryos quantified in b. Asterisks denote poor yolk sac vascularization. Scale bar, 2 mm. d, Whole-mount TUNEL staining of embryos (n = 2 embryos). Scale bar, 2 mm. e, Single staining showing vascularization (PECAM-1, top) and apoptosis (cleaved CASP3, bottom) of yolk sacs. Merged image is shown in Fig. 3b. f, g, Representative images of cell death in different organs (f) and quantification (g) as detected by TUNEL staining at E14.5 (n = 3 embryos per genotype). Scale bar, 50 μm (f). Mean ± s.e.m. (n = 3 embryos per genotype). P values from one-way ANOVA are reported. h, Representative images of H&E staining on whole embryo paraffin sections (n = 3 embryos per genotype). Asterisk denotes pericardial effusion. n, necrotic area. Scale bar, 200 µm. i, Cell death was analysed by propidium iodide (PI) staining in MEFs stimulated with TNF for 24 h plus the indicated cell death inhibitors. Mean ± s.e.m. (n = 3 independent experiments). P values from two-way ANOVA are reported.
Extended Data Fig. 5 Individual deletion of mediators of apoptosis or necroptosis does not prevent cell death and lethality at mid-gestation of HOIL-1- or HOIP-deficient embryos.
a, Western blot analysis of MLKL expression in the indicated organs derived from Mlkl−/− mice (n = 2 mice per genotype), as control. For gel source data, see Supplementary Fig. 1. b, d–f, Representative images of embryos at different stages of gestation (E10.5: n = 7 for Ripk3−/−Hoil-1+/− and n = 5 for Ripk3−/−Hoil-1−/−; E11.5: n = 5 for Ripk3−/−Hoil-1+/− and n = 2 for Ripk3−/−Hoil-1−/−; E12.5: n = 9 for Ripk3−/−Hoil-1+/− and n = 2 for Ripk3−/−Hoil-1−/− (b), E10.5: n = 16 for Mlkl−/−Hoip+/− and n = 6 for Mlkl−/−Hoip−/−; E11.5: n = 8 for Mlkl−/−Hoip+/− and n = 6 for Mlkl−/−Hoip−/−; E12.5: n = 10 for Mlkl−/−Hoip+/− and n = 5 for Mlkl−/−Hoip−/− (d), E10.5: n = 5 for Casp8+/−Hoip+/− and n = 4 for Casp8+/−Hoip−/−; E11.5: n = 6 for Casp8+/−Hoip+/− and n = 3 for Casp8+/−Hoip−/−; E12.5: n = 3 for Casp8+/−Hoip+/− and n = 2 for Casp8+/−Hoip−/− (e), E10.5: n = 2 for Casp8+/−Hoil-1+/− and n = 4 for Casp8+/−Hoil-1−/−; E11.5: n = 2 for Casp8+/−Hoil-1+/− and n = 5 for Casp8+/−Hoil-1−/−; E12.5: n = 6 for Casp8+/−Hoil-1+/− and n = 3 for Casp8+/−Hoil-1−/− (f)). Asterisks denote poor yolk sac vascularization. Scale bars, 2 mm. c, Representative images of yolk sac vascularization and cell death at E10.5 as detected by PECAM-1 (red) and cleaved CASP3 staining (green) (top) and whole-mount TUNEL staining (bottom) (n = 4 per genotype). Scale bar, 50 µm.
Extended Data Fig. 6 Combined deletion of RIPK3 and caspase-8 prevents cell death but not embryonic lethality at late gestation that is caused by the loss of HOIL-1.
a, Quantification of genotypes of animals obtained from inter-crosses of Ripk3−/−Casp8+/−Hoil-1+/− with Ripk3−/−Casp8−/−Hoil-1+/− mice (left) or Ripk3−/−Casp8−/−Hoil-1+/− mice (right). b, Health status of Ripk3−/−Casp8+/−Hoil-1−/− and Ripk3−/−Casp8−/−Hoil-1−/− embryos at different developmental stages. c, Single staining showing vascularization (PECAM-1, top) and apoptosis (cleaved CASP3, bottom) of yolk sacs. Merged image is shown in Fig. 3f. Scale bar, 50 µm. d, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E14.5 (left) and respective quantification (right). Mean ± s.e.m. (n = 3 embryos per genotype). P values from one-way ANOVA are reported. e, f, Representative images (e) and quantification (f) of cell death in different organs as detected by TUNEL staining at E13.5 (n = 3 embryos per genotype) and E14.5 (n = 5 for Ripk3−/−Casp8−/−Hoil-1+/−, n = 2 for Ripk3−/−Casp8−/−Hoil-1−/− and Ripk3−/−Casp8−/−Hoil-1−/− lung and liver and n = 3 Ripk3−/−Casp8−/−Hoil-1−/− heart). Scale bars, 50 µm. Data are mean ± s.e.m. g, Cell death was analysed by propidium iodide (PI) staining in MEFs stimulated or not with the indicated ligands for 24 h. Data are mean ± s.e.m. (n = 3 independent experiments). P values from two-way ANOVA are reported. h, Representative images of H&E staining on E13.5 whole embryo paraffin embedded sections (n = 3 for Ripk3−/−Casp8−/−Hoil-1+/− and Ripk3−/−Casp8−/−Hoil-1−/− and n = 2 for Ripk3−/−Casp8+/−Hoil-1−/−). Asterisks denote pericardial effusion. Arrows denote congested vessels. Scale bar, 200 µm. i, Representative images of microfocus computed tomography scan images of whole E13.5 embryos (n = 3 embryos per genotype). Asterisks denote pericardial effusion.
Extended Data Fig. 7 Combined deletion of MLKL and caspase-8 promotes survival of LUBAC-deficient mice.
a, Quantification of genotypes of animals obtained from intercrosses of Mlkl−/−Casp8+/−Hoip+/− with Mlkl−/−Casp8−/−Hoip+/− mice. Asterisk denotes dead embryo. b, Representative images of adult mice as quantified in a. c, Kaplan–Meier plot of mouse survival (n = 6 for Mlkl−/−Casp8−/−Hoip−/− and n = 9 for Mlkl−/−Casp8−/−Hoil-1−/− mice). d, Representative images of H&E staining of the indicated organs (n = 3 mice per genotype). Scale bars, 200 µm. e, Representative images of yolk sac vascularization (PECAM-1, red) and apoptosis (cleaved CASP3, green) (top) at E13.5 and respective quantifications (bottom). Data are mean ± s.e.m. (n = 5 for Mlkl−/−Casp8−/−Hoil-1+/− and Mlkl−/−Casp8−/−Hoil-1−/− and n = 2 for Mlkl−/−Casp8+/−Hoil-1−/−). Statistical significance was determined with unpaired two-tailed t-tests comparing Mlkl−/−Casp8−/−Hoil-1+/− and Mlkl−/−Casp8−/−Hoil-1−/− embryos. f, Representative H&E staining images of the indicated organs (n = 3 embryos per genotype). Scale bars, 200 µm. g, Epidermal thickness quantification of mice of the indicated genotypes in f. Data are mean ± s.e.m. (n = 3 mice per genotype). Statistical significance was determined with unpaired two-tailed t-tests. h, Western blot analysis of lysates from whole E13.5 embryos of the indicated genotypes and L929 cells treated with/without TNF plus zVAD-fmk (TZ) for 2 h as antibody validation (n = 4 embryos per genotype performed twice). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 8 Combined deletion of RIPK3 and caspase-8 causes haematopoietic defects and RIPK1-dependent embryonic lethality in HOIL-1-deficient mice.
a, Venn diagram depicting genes differentially expressed by RNA-seq analysis between E13.5 embryos of the indicated genotypes. b, Gene Ontology (GO) enrichment analysis of differentially expressed genes (85 low and 35 high in a). FDR, false discovery rate. c, Representative FACS profile of E13.5 fetal liver cells with different erythroblast populations gated according to their CD71 and TER119 expression levels (R1–R5) and quantification. R1 contains immature red blood cell progenitors, including primitive and later-stage erythroid progenitor cells (erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E), respectively); R2 comprises mainly pro-erythroblasts and early basophilic erythroblasts; R3 contains both early and late basophilic erythroblasts; R4 is composed of chromatophilic and orthochromatophilic erythroblasts; and R5 consists of late orthochromatophilic erythroblasts and reticulocytes. Data are mean ± s.e.m. (n = 14 Ripk3−/−Casp8−/−Hoil-1+/−, n = 8 Ripk3−/−Casp8−/−Hoil-1−/−, n = 5 for Mlkl−/−Casp8−/−Hoil-1−/− and n = 3 for Mlkl−/−Casp8−/−Hoil-1−/− fetal livers). P values from two-way ANOVA are reported. d, h, k, Representative FACS profile of E13.5 fetal liver cells for the indicated haematopoietic populations (sample size specified in e–g, i, j). e, f, j, Total cell number of the different haematopoietic cell subsets in fetal liver cell suspensions from E13.5 embryos of the indicated genotypes gated as in d, h and k, respectively. Total number of multipotent progenitors (LSK and LK cells) (e), mature CD45+ blood cells, including granulocytes (GR-1+) and macrophages (F4-80+) (f) and myeloid progenitors (common myeloid progenitor (CMP), granulocyte–monocyte progenitor (GMP) and megakaryocyte–erythrocyte progenitor (MEP)) (j). Data are mean ± s.e.m. P values from unpaired two-tailed t-tests are shown. g, i, Percentages of mature CD45+ leucocytes, GR-1+ and F4-80+ cells (g) and CMP, GMP and MEP cells (i). Data are mean ± s.e.m. P values from unpaired two-tailed t-tests are shown. l, Differentiation of E13.5 fetal liver (c-KIT+) progenitors into CFU-granulocytes and macrophages (GM), BFU-E and/or CFU-granulocyte, erythroid, macrophage, megakaryocyte (GEMM). Mean ± s.e.m. (n = 2 fetal livers). m, Micrographs of differentiated macrophages (n = 3 Ripk3−/−Casp8−/−Hoil-1+/− and Ripk3−/−Casp8−/−Hoil-1−/−, n = 5 Mlkl−/−Casp8−/−Hoil-1+/− and n = 4 Mlkl−/−Casp8−/−Hoil-1−/− fetal livers) and percentage viability of macrophages from E13.5 fetal liver cell suspensions from embryos of the indicated genotypes in the presence or absence of the indicated inhibitors. Data are mean ± s.e.m. (n = 3 Ripk3−/−Casp8−/−Hoil-1+/− and Ripk3−/−Casp8−/−Hoil-1−/−, n = 5 Mlkl−/−Casp8−/−Hoil-1+/− and n = 4 Mlkl−/−Casp8−/−Hoil-1−/− fetal livers). P values from two-way ANOVA are shown. o, Microfocus computed tomography scan images of Ripk3−/−Casp8−/−Hoil-1−/− embryos showing maximum intensity projections, with windowing applied to highlight vasculature (high contrast). No anatomical defects that would explain destruction of red blood cells or poor distribution of blood to the peripheries were found (n = 3 embryos). In the left image, yellow star denotes distal aorta, green star denotes umbilical vessels, and red star indicates descending thoracic aorta. In the right image, yellow star denotes carotid artery, red star denotes descending thoracic aorta, white star denotes ductus arteriosus, and blue star denotes ascending thoracic aorta. p, Representative FACS profile of a pool of three E11.5 dorsal aortas, containing the AGM region, per indicated genotype and quantification. This experiment was performed once with three embryos per genotype.
Extended Data Fig. 9 Concomitant deletion of RIPK1 prevents embryonic lethality of Ripk3−/−Casp8−/−Hoil-1−/− mice.
a, Kaplan–Meier plot of mouse survival (n = 17 for Ripk1−/− Ripk3−/−Casp8−/−Hoip−/− and n = 2 for Ripk+/−Ripk3−/−Casp8−/−Hoip−/− mice). b, Quantification of genotypes of animals obtained from intercrosses of Ripk1+/−Hoil-1+/− mice. For simplicity not all possible genotypes are represented. c, Percentage viability of macrophages from E13.5 fetal liver cell suspensions from embryos of the indicated genotypes. Data are mean ± s.e.m. (n = 5 fetal livers/genotype). Statistical significance was determined with unpaired two-tailed t-tests. d, Cytokine arrays from Ripk3−/−Casp8−/−Hoil-1+/− and Ripk3−/−Casp8−/−Hoil-1−/− embryos (left) and table listing the altered cytokines (right). Red squares highlight the differences (n = 1 for each genotype). For gel source data, see Supplementary Fig. 1. e, Cytokine analysis in homogenates from embryos of the indicated genotypes. Data are mean ± s.e.m. (n = 3 embryos per genotype). P values from one-way ANOVA are reported. f, Representative images of E16.5 embryos from control mothers or mothers fed with the RIPK1 kinase inhibitor GSK’457A from mating and throughout gestation (embryos treated with GSK’457A n = 5 for Ripk3−/−Casp8−/−Hoil-1+/− and n = 7 Ripk3−/−Casp8+/−Hoil-1−/− and n = 3 for Ripk3−/−Casp8−/−Hoil-1−/−). Scale bar, 5 mm.
Extended Data Fig. 10 Schematic representation of findings in this study.
a, Diagram indicating extent of viability and phenotypes of single, double, triple and quadruple knockout mice. Red lines indicate cell death and loss of yolk sac vascularization phenotype. Green line indicates mild cell death phenotype without loss of yolk sac vascularization. Asterisk indicates that heart defects were observed. b, Proposed model of LUBAC function during embryogenesis. At mid-gestation (left), LUBAC maintains vascular tissue integrity by preventing aberrant TNF/LT-α-mediated caspase-8- and RIPK3/MLKL-induced cell death. At late gestation, LUBAC is required not only to prevent aberrant cell death but also to prevent severe defects in haematopoiesis that are driven by RIPK1 but can be prevented by RIPK3 (middle). Genetic ablation of LUBAC and of different components of the cell death machinery indicates that (right): (1) in the absence of LUBAC, caspase-8 and RIPK3, RIPK1 provokes lethality, probably by depleting multipotent progenitors in the haematopoietic compartment; (2) in the absence of caspase-8 and MLKL, cell death induced by loss of LUBAC is prevented and RIPK3 is present to exert its protective role on fetal haematopoiesis by precluding aberrant RIPK1 signalling; and (3) in the absence of caspase-8 and RIPK3, the presence of LUBAC is sufficient to prevent RIPK1 from causing severe defects in haematopoiesis and lethality since Ripk3−/−Casp8−/− mice are viable14,15,24. This indicates that RIPK3 and LUBAC can compensate for each other to block aberrant RIPK1 signalling.
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A list of differentially expressed genes by RNA seq
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Peltzer, N., Darding, M., Montinaro, A. et al. LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis. Nature 557, 112–117 (2018). https://doi.org/10.1038/s41586-018-0064-8
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DOI: https://doi.org/10.1038/s41586-018-0064-8
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