Abstract

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.

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Acknowledgements

We thank B. Hough, E. Magee, M. Garcia and K. Cherry for animal husbandry; I. Peng and the Genentech genetic analysis, histology, necropsy, immunohistochemistry, clinical pathology and FACS laboratories for technical assistance; and J. Lai and S. Stawicki for managing antibody generation.

Reviewer information

Nature thanks C. Griffin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA

    • Klaus Heger
    • , Katherine E. Wickliffe
    • , Ada Ndoja
    • , Debra L. Dugger
    • , Allie Maltzman
    • , Kim Newton
    •  & Vishva M. Dixit
  2. Department of Translational Immunology, Genentech, South San Francisco, CA, USA

    • Juan Zhang
    •  & Wyne P. Lee
  3. Department of Cancer Immunology, Genentech, South San Francisco, CA, USA

    • Aditya Murthy
  4. Department of Molecular Oncology, Genentech, South San Francisco, CA, USA

    • Felipe de Sousa e Melo
  5. Department of Pathology, Genentech, South San Francisco, CA, USA

    • Jeffrey Hung
    • , László G. Kőműves
    •  & Joshua D. Webster
  6. Department of Microchemistry, Proteomics and Lipidomics, Genentech, South San Francisco, CA, USA

    • Yi Zeng
    • , Erik Verschueren
    •  & Donald S. Kirkpatrick
  7. Department of Early Discovery Biochemistry, Genentech, South San Francisco, CA, USA

    • Domagoj Vucic
  8. Department of Molecular Biology, Genentech, South San Francisco, CA, USA

    • Merone Roose-Girma
    • , Robert J. Newman
    •  & Søren Warming
  9. Department of Antibody Engineering, Genentech, South San Francisco, CA, USA

    • Yi-Chun Hsiao

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Contributions

K.H. designed and performed experiments with assistance from K.E.W., A.N., A.Mu., D.L.D., A.Ma., and F.d.S.M.; M.R.-G., R.J.N. and S.W. generated the OtulinC129A/+ and HoipiC879S/+ mice; Y.Z., E.V., and D.S.K. performed mass spectrometry; J.Z. and W.P.L. performed in vivo antibody blocking experiments; J.H. performed quantitative image analysis; Y.-C.H. generated the phospho-RIPK1 Ser166, Thr169 antibody; L.G.K. performed confocal microscopy; J.D.W. analysed histological data; D.V., K.N. and V.M.D. contributed to experimental design; K.H. and K.N. wrote the manuscript with input from all authors.

Competing interests

All authors were employees of Genentech.

Corresponding authors

Correspondence to Kim Newton or Vishva M. Dixit.

Extended data figures and tables

  1. Extended Data Fig. 1 Constitutive and conditional OTULIN inactivation using a Cdh5-Cre transgene causes embryonic lethality.

    a, Organization of the constitutive OtulinC129A knock-in allele. Boxes represent exons. Untranslated regions are shaded grey. b, Kaplan–Meier plot of mouse survival. &, Animal euthanized owing to dermatitis with pruritus. c, Numbers of offspring of OtulinC129A/+ parents at clipping (P0–P10). Strains were derived from independently targeted ES cell clones. d, Numbers of offspring from intercrossing OtulinC129A/+ parents. *Embryos resorbed. e, Representative embryos from d with and without their yolk sacs. fi, E10.5 placenta (f, i) or yolk sac (g, h) stained with haematoxylin and eosin (H&E) or labelled for phospho-RIPK3 Thr231, Ser232 or cleaved caspase 3 by immunohistochemistry (IHC) (n = 4 Otulin+/+, n = 3 OtulinC129A/C129A mice in (f, i); n = 7 Otulin+/+, n = 3 OtulinC129A/C129A mice in g, h). Scale bars, 100 μm (H&E staining and cleaved caspase 3 IHC in f), 50 μm (p-RIPK3 IHC in f, g) or 25 μm (cleaved caspase 3 IHC and p-RIPK3 IHC in h, i). Arrows indicate labelling of vessel walls (h, i). j, Western blots of E10.5 embryos (n = 3 per genotype). k, Organisation of the inducible OtuliniC129A knock-in allele. Boxes represent exons. Untranslated regions are shaded grey. Arrows indicate genotyping primer locations. l, Offspring numbers from intercrossing OtuliniC129A/iC129A with Cdh5-Cre+ OtuliniC129A/+ parents. *Embryos resorbed. m, Representative embryos from l with and without their yolk sacs. n, E10.5 yolk sacs stained for PECAM-1 (red) and cleaved caspase 3 (green) (n = 2 Cdh5-Cre+ OtuliniC129A/iC129A). 3D projection of 17 confocal z stacks. The lower image shows part of an orthogonal projection. Arrows indicate cells containing cleaved caspase 3 and PECAM-1. Source data

  2. Extended Data Fig. 2 Systemic OTULIN inactivation using the R26-CreERT2 transgene causes spontaneous inflammation and immune cell defects.

    Additional characterization of the day 5 tamoxifen-treated mice shown in Fig. 1e. a, Serum cytokine and chemokine levels. b, The percentage of liver tissue positive for cleaved caspase 3 by IHC. c, Liver stained with haematoxylin and eosin (H&E; scale bar, 50 μm; n = 8 per genotype) or labelled with Gr-1 or F4/80 IHC (scale bar, 100 μm; n = 5 per genotype). d, Serum ALT, AST, and total bilirubin levels. e, Small intestine stained with H&E (scale bar, 50 μm; n = 8 per genotype) or labelled by phospho-RIPK3 Thr231, Ser232 IHC (scale bar, 50 μm; n = 5 per genotype) or cleaved caspase 3 IHC (scale bar, 100 μm; n = 5 per genotype). f, The percentage of crypt tissue in the small intestine positive for cleaved caspase 3 by IHC. g, Heart tissue. Scale bar, 100 μm (H&E, n = 8 per genotype; and Gr-1, F4/80 and cleaved caspase 3 IHCs, n = 5 per genotype) or 50 μm (phospho-RIPK3 Thr231, Ser232 IHC; n = 5 per genotype). h, Blood parameters. Neutrophils (CD11b+ Ly6G+) are boxed in representative FACS plots. The mean ± s.d. percentage of neutrophils is indicated (n = 5 per genotype). i, Sternum stained with H&E (scale bar, 50 μm; n = 8 per genotype). Graphs indicate bone marrow cell numbers in two femurs and two tibia, the percentage of viable bone marrow cells not stained by 7-AAD, and numbers of bone marrow LK and LSK cells. LK and LSK cells are indicated in representative FACS plots with their mean ± s.d. frequency (n = 8 per genotype). j, Thymus stained with H&E (scale bar, 100 μm; n = 8 per genotype). Graphs indicate thymus weights and total thymocyte numbers. CD4+CD8+, CD4+ and CD8+ thymocytes are indicated in representative FACS plots with their mean ± s.d. frequency (n = 5 per genotype). Circles represent individual mice. Lines represent means; P values, unpaired, two-tailed t-test (a, b, d, f, h, i, j). Source data

  3. Extended Data Fig. 3 OTULIN inactivation in the haematopoietic compartment causes spontaneous inflammation.

    a, FACS plots indicate the efficiency with which irradiated CD45.1+ mice were reconstituted with bone marrow (BM) from CD45.2+ R26-CreERT2 Otulin+/+ or R26-CreERT2 OtuliniC129A/iC129A mice. B cells (B220+ CD3ε), T cells (B220 CD3ε+) and neutrophils (CD11b+ Ly6G+) in whole blood were examined. Numbers represent the mean percentage ± s.d. (n = 11 R26-CreERT2 Otulin+/+, n = 12 R26-CreERT2 OtuliniC129A/iC129A). b, Body weights of tamoxifen-treated mice plotted as a percentage of initial body weight. Lines represent the mean. P values, repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test. c–i, Characterization of the mice in b on day 5. c, Serum cytokines and chemokines. d, Liver (scale bar, 100 μm; n = 9 R26-CreERT2 Otulin+/+, n = 10 R26-CreERT2 OtuliniC129A/iC129A) and serum ALT, AST, and total bilirubin. e, Small intestine (scale bar, 50 μm; n = 9 R26-CreERT2 Otulin+/+, n = 10 R26-CreERT2 OtuliniC129A/iC129A). f, Heart (scale bar, 100 μm; n = 9 R26-CreERT2 Otulin+/+, n = 10 R26-CreERT2 OtuliniC129A/iC129A). g, Blood parameters. Numbers in representative FACS plots indicate the mean ± s.d. neutrophil frequency (n = 9 R26-CreERT2 Otulin+/+, n = 10 R26-CreERT2 OtuliniC129A/iC129A). h, Sternum (scale bar, 50 μm; n = 9 R26-CreERT2 Otulin+/+, n = 10 R26-CreERT2 OtuliniC129A/iC129A). Graphs indicate bone marrow cell numbers in two femurs and two tibia, the percentage of viable bone marrow cells not stained by 7-AAD, and numbers of bone marrow LK and LSK cells. LK and LSK cells are indicated in representative FACS plots with their mean ± s.d. frequency (n = 9 R26-CreERT2 Otulin+/+, n = 10 R26-CreERT2 OtuliniC129A/iC129A). i, Thymus (scale bar, 100 μm; n = 6 R26-CreERT2 Otulin+/+, n = 6 R26-CreERT2 OtuliniC129A/iC129A). Graphs indicate thymus weights and total thymocyte numbers. CD4+CD8+, CD4+ and CD8+ thymocytes are indicated in representative FACS plots with their mean ± s.d. frequency (n = 3 R26-CreERT2 Otulin+/+, n = 4 R26-CreERT2 OtuliniC129A/iC129A). Circles represent individual mice. Lines represent means; P values, unpaired, two-tailed t-test (c, d, gi). Source data

  4. Extended Data Fig. 4 OTULIN inactivation outside the haematopoietic compartment causes mild inflammation.

    a, FACS plots indicate the efficiency with which irradiated CD45.2+ R26-CreERT2 Otulin+/+ and R26-CreERT2 OtuliniC129A/iC129A mice were reconstituted with BM from CD45.1+ mice. B cells (B220+ TCRβ), T cells (B220 TCRβ+) and neutrophils (CD11b+ Ly6G+) in whole blood were examined. Numbers represent the mean percentage ± s.d. (n = 12 per genotype). b, Body weights of tamoxifen-treated mice plotted as a percentage of initial body weight. Lines represent means; P values, repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test. ci, Characterization of the mice in b on day 5. c, Serum cytokines and chemokines. d, Liver (scale bar, 100 μm; n = 5 per genotype) and serum ALT, AST and total bilirubin. e, Small intestine (scale bar, 50 μm; n = 5 per genotype). f, Heart (scale bar, 100 μm; n = 5 per genotype). g, Blood parameters. Numbers in representative FACS plots indicate the mean neutrophil frequency ± s.d. (n = 5 per genotype). h, Sternum (scale bar, 50 μm; n = 5 per genotype). Graphs indicate bone marrow cell numbers in two femurs and two tibia, the percentage of viable bone marrow cells not stained by 7-AAD, and numbers of bone marrow LK and LSK cells. LK and LSK cells are indicated in representative FACS plots with their mean ± s.d. frequency (n = 5 per genotype). i, Thymus (scale bar, 100 μm; n = 5 per genotype). Graph indicates thymus weights. Circles represent individual mice. Lines represent means; P values, unpaired, two-tailed t-test (c, d, gi). Source data

  5. Extended Data Fig. 5 OTULIN inactivation leads to LUBAC auto-ubiquitination, a reduction in LUBAC components, muted NF-κB activation and enhanced cell death upon stimulation.

    a, PCR on genomic DNA isolated from primary MEFs treated or not treated with 4-OHT. b, c, g, k, m, o, p, Western blots of MEFs (b, m, o, p), immortalized haematopoietic progenitors (c), dermal fibroblasts (g) and fetal liver-derived macrophages (k). d, Western blots of MEF lysates treated or not treated with IsoT (left). Coomassie-stained gel of free linear tetra-ubiquitin chains treated or not treated with IsoT (right). e, Total (unique) peptide counts by mass spectrometry after affinity purification of linear or K48-linked ubiquitin from haematopoietic progenitors. f, Organization of the conditional HoipiC879S knock-in allele. Boxes represent exons. h, Western blots of MEF lysates with or without OTULIN or USP2 treatment and with or without incubation at 32 °C. *Nonspecific band. i, j, Graphs of relative Sharpin, Hoil-1 and Hoip mRNA expression in MEFs (i) or fetal liver-derived macrophages (j). Each circle represents cells from a different embryo. Lines represent means; P values, unpaired, two-tailed t-test (i, j). l, Western blots of MEFs treated for 4 h as indicated. n, q, Graphs indicate the percentage of MEFs that were viable and not stained by PI after overnight treatment. Each circle represents cells from a different embryo. Bars represent means; P values, one-way ANOVA followed by Tukey’s multiple comparison test (n, q). Results are representative of two independent experiments (d, h, l, m, o) or cells from three mice per genotype (ac, g, k, p). Source data

  6. Extended Data Fig. 6 Cell death mediated by TNFR1, RIPK1 catalytic activity, caspase 8 and RIPK3 contributes to the embryonic lethality caused by OTULIN inactivation.

    a, Western blots of MEFs and their immunoprecipitates (IP) with or without USP2 treatment. Results are representative of two independent experiments. b, Offspring numbers from intercrossing OtulinC129A/+ Ripk1D138N/D138N parents. *Embryos resorbed; #embryos grossly abnormal. c, Representative embryos from b with and without their yolk sacs. d, Offspring numbers from intercrossing OtulinC129A/+ Tnfr1−/− parents. *Embryos resorbed; #Embryos grossly abnormal. e, Representative embryos from d with and without their yolk sacs. f, E14.5 embryo numbers from intercrossing OtulinC129A/+ Casp8+/− with OtulinC129A/+ Casp8+/+ parents. *Embryos resorbed. g, Representative embryos from f with and without their yolk sacs. h, E14.5 embryo numbers from intercrossing OtulinC129A/+ Ripk3−/− parents. *Embryos resorbed. i, Representative embryos from h with and without their yolk sacs. j, Offspring numbers from intercrossing OtulinC129A/+ Ripk3−/− Casp8−/− parents. &Not found at wean. k, Representative embryos from j with and without their yolk sacs. l, Western blots of E10.5 embryos (n = 1 embryo per genotype). m, A representative P0 litter from j. Graph indicates newborn body weights. Circles represent individual pups. Lines represent means. P value, unpaired, two-tailed t-test. n, PCR on genomic DNA isolated from primary intestinal organoids treated with 4-OHT or untreated (n = 3 per genotype). Graphs indicate relative Lgr5, Ascl2, Axin2 and Olfm4 mRNA levels in intestinal organoids treated with 4-OHT. Circles represent cells from different mice. Lines represent means. Source data

  7. Extended Data Fig. 7 OTULIN inactivation causes perinatal lethality in the absence of RIPK3 and caspase 8, and TNF signalling-dependent inflammation in adult mice.

    a, b, E18.5 and P0 skin or salivary gland (scale bar, 100 μm; E18.5, n = 8 Otulin+/+ Ripk3−/− Casp8−/−, n = 5 OtulinC129A/C129A Ripk3−/− Casp8−/−; P0, n = 3 Otulin+/+ Ripk3−/− Casp8−/−, n = 3 OtulinC129A/C129A Ripk3−/− Casp8−/−). Oedema was noted in three out of five OtulinC129A/C129A Ripk3−/− Casp8−/− mice at E18.5 and in two out of three mice at P0. c, E18.5 heart and liver stained for nicked DNA by TUNEL assay (scale bar, 200 μm; n = 3 per genotype). d, E18.5 serum cytokine and chemokine levels. Each circle represents one embryo. e, Body weights of tamoxifen-treated mice plotted as a percentage of initial body weight. Lines represent means. P values, repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test. P values comparing R26-CreERT2 OtuliniC129A/iC129A isotype control and TNFR2-Fc treated animals are shown. f, Body weights of tamoxifen-treated mice plotted as a percentage of initial body weight. Lines represent the mean. P values, repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test. P values comparing R26-CreERT2 OtuliniC129A/iC129A and R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/− mice are shown. gm, Characterization of the mice in f on day 5. g, Serum cytokine and chemokine levels. h, Liver (scale bar, 100 μm; n = 5 R26-CreERT2 Otulin+/+, n = 5 R26-CreERT2 OtuliniC129A/iC129A, n = 6 R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/−) and serum ALT, AST, and total bilirubin. i, Small intestine (scale bar, 50 μm; n = 5 R26-CreERT2 Otulin+/+, n = 5 R26-CreERT2 OtuliniC129A/iC129A, n = 6 R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/−). j, Heart (scale bar, 100 μm; n = 5 R26-CreERT2 Otulin+/+, n = 5 R26-CreERT2 OtuliniC129A/iC129A, n = 6 R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/−). k, Blood parameters. Numbers in representative FACS plots indicate the mean neutrophil frequency ± s.d. (n = 6 per genotype). l, Sternum (scale bar, 50 μm; n = 5 R26-CreERT2 Otulin+/+, n = 5 R26-CreERT2 OtuliniC129A/iC129A, n = 6 R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/−). Graphs indicate bone marrow cell numbers in two femurs and two tibia, the percentage of viable bone marrow cells not stained by 7-AAD, and numbers of bone marrow LK and LSK cells. LK and LSK cells are indicated in representative FACS plots with their mean ± s.d. frequency (n = 5 R26-CreERT2 Otulin+/+, n = 5 R26-CreERT2 OtuliniC129A/iC129A, n = 6 R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/−). m, Thymus (scale bar, 100 μm; n = 5 R26-CreERT2 Otulin+/+, n = 5 R26-CreERT2 OtuliniC129A/iC129A, n = 6 R26-CreERT2 OtuliniC129A/iC129A Tnfr1−/−). Graphs indicate thymus weights. Circles represent individual mice. Lines represent means; P values, unpaired, two-tailed t-test (d) or one-way ANOVA followed by Tukey’s multiple comparison test (g, h, km). Source data

  8. Extended Data Fig. 8 RIPK3 and caspase 8 deficiency rescues spontaneous inflammation and most immune cell defects caused by OTULIN inactivation in the adult.

    Additional characterization of the day 5 tamoxifen-treated mice shown in Fig. 3f. a, Graph of the percentage of liver tissue that was positive for cleaved caspase 3 by IHC (n = 5 per genotype). b, Serum cytokines and chemokines. c, Liver (scale bar, 100 μm; n = 8 R26-CreERT2 Otulin+/+, n = 8 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A, n = 7 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8+/−, n = 9 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−) and serum ALT, AST, and total bilirubin. d, Small intestine (scale bar, 100 μm; n = 8 R26-CreERT2 Otulin+/+, n = 8 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A, n = 7 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8+/−, n = 9 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). Lower panels indicate labelling of cleaved caspase 3 (brown, n = 5 per genotype) and the graph shows the percentage of crypt tissue that was positive. e, Sternum (scale bar, 50 μm; n = 8 R26-CreERT2 Otulin+/+, n = 8 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A, n = 7 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8+/−, n = 9 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). Graphs indicate bone marrow cell numbers in two femurs and two tibia, the percentage of viable bone marrow cells not stained by 7-AAD, and numbers of bone marrow LK and LSK cells. LK and LSK cells are indicated in representative FACS plots with their mean ± s.d. frequency (n = 8 R26-CreERT2 Otulin+/+, n = 8 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A, n = 7 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8+/−, n = 9 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). f, Thymus (scale bar, 100 μm; n = 8 R26-CreERT2 Otulin+/+, n = 8 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A, n = 7 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8+/−, n = 9 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). Graph indicates thymus weights. Circles represent individual mice. Lines represent means; P values, one-way ANOVA followed by Tukey’s multiple comparison test (af). Source data

  9. Extended Data Fig. 9 OTULIN inactivation in adults lacking RIPK3 and caspase 8 elicits a type I IFN signature.

    Immune cell phenotypes of the day 5 tamoxifen-treated mice shown in Fig. 3f. a, Blood parameters. Numbers in representative FACS plots indicate the mean ± s.d. neutrophil frequency (n = 8 R26-CreERT2 Otulin+/+, n = 8 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A, n = 7 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/−, n = 8 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8+/−, n = 9 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). b, Western blots of BMDMs. Results are representative of cells from three mice of each genotype. c, IFN-β secretion by BMDMs following 24 h treatment as indicated. Circles represent cells from individual mice. Bars represent means. dg, Characterization of day 10 tamoxifen-treated mice. d, Graph indicates CD11b+ Ly6G Ly6Chigh blood cells. e, Serum cytokines and chemokines. f, Graphs indicate the percentage of LSK bone marrow cells and SCA-1 expression (MFI, median fluorescent intensity) on the indicated bone marrow subsets. g, Serum cytokines and chemokines in the mice shown in Fig. 3k. Circles represent individual mice and lines represent means (a, dg). P values, unpaired, two-tailed t-test (df) or one-way ANOVA followed by Tukey’s multiple comparison test (a, g). Source data

  10. Extended Data Fig. 10 OTULIN inactivation in the combined absence of RIPK3 and caspase 8 causes RIPK1-dependent inflammation.

    ac, Characterization of day 35 tamoxifen-treated mice. a, Representative spleen and lymph nodes (inguinal, brachial/axillary, mandibular/cervical and mesenteric lymph; n = 4 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 5 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). Graphs indicate spleen weight, total spleen or lymph node cellularity, and numbers of different cellular subsets (CD3ε+ B220+ T cells, CD11b+ myeloid cells, CD11b+ Ly6G SiglecF+ SSC-Ahigh eosinophils, CD11b+ Ly6G+ neutrophils and CD11b+ Ly6G SiglecF SSC-Alow Ly6Chigh monocytes). b, Liver (scale bar, 100 μm), small intestine (scale bar, 50 μm) and caecum (scale bar, 50 μm) (n = 4 R26-CreERT2 Otulin+/+ Ripk3−/− Casp8−/−, n = 5 R26-CreERT2 OtuliniC129A/iC129A Ripk3−/− Casp8−/−). c, Serum cytokines and chemokines. d, Offspring numbers at P4–6 from intercrossing OtulinC129A/+ Ripk3−/− Casp8−/− Ripk1+/− parents. e, Kaplan–Meier plot of mouse survival. Note that some mice were obtained independent from intercrossing OtulinC129A/+ Ripk3−/− Casp8−/− Ripk1+/− parents and are not included in d. f, E18.5 serum cytokines and chemokines. Circles represent individual mice (a, c) or individual embryos (f). Lines represent means (a, c, f). P values, unpaired, two-tailed t-test (a, c) or one-way ANOVA followed by Tukey’s multiple comparison test (f). g, Model of how OTULIN limits cell death, embryonic lethality and inflammation. OTULIN counters auto-ubiquitination of the E3 ligase LUBAC. Stimulation of TNFR1 by its cognate ligand TNF initiates assembly of complex I containing TRADD, TRAF2, cIAP1/2 and RIPK1. Ubiquitin chains generated by the E3 ligases cIAP1 and cIAP2 recruit LUBAC but not OTULIN into complex I, leading to the modification of complex I components with linear polyubiquitin and activation of NF-κB and MAPK signalling for gene induction. OTULIN inactivation results in LUBAC auto-ubiquitination, a reduction in total LUBAC, and reduced LUBAC recruitment into complex I. These changes promote formation of cytosolic cell death signalling complexes containing RIPK1, RIPK3, FADD, TRADD and caspase 8. Increased necroptotic and apoptotic cell death causes both embryonic lethality and auto-inflammation in the adult. RIPK1 mediates type I IFN induction in the combined absence of OTULIN function, caspase 8 and RIPK3. Source data

Supplementary information

  1. Supplementary Figures

    This file contains Supplementary Figures 1-2. Supplementary Figure 1 contains the uncropped blots for Figures 2-4, and Extended Data Figures 1, 5, 6 and 9. Supplementary Figure 2 shows the gating strategies for flow cytometric analyses.

  2. Reporting Summary

  3. Supplementary Table

    This table contains the total (unique) peptide spectral matches for the proteomics experiment in Extended Data Figure 5e.

Source Data

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DOI

https://doi.org/10.1038/s41586-018-0256-2

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