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RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation

Nature volume 540, pages 124–128 (2016)Cite this article

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Abstract

Receptor-interacting protein kinase 1 (RIPK1) regulates cell death and inflammation through kinase-dependent and -independent functions1,2,3,4,5,6,7. RIPK1 kinase activity induces caspase-8-dependent apoptosis and RIPK3 and mixed lineage kinase like (MLKL)-dependent necroptosis8,9,10,11,12,13. In addition, RIPK1 inhibits apoptosis and necroptosis through kinase-independent functions, which are important for late embryonic development and the prevention of inflammation in epithelial barriers14,15,16,17,18. The mechanism by which RIPK1 counteracts RIPK3–MLKL-mediated necroptosis has remained unknown. Here we show that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3–MLKL-dependent necroptosis mediated by Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM1). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 (RIPK1mRHIM) caused perinatal lethality that was prevented by RIPK3, MLKL or ZBP1 deficiency. Furthermore, mice expressing only RIPK1mRHIM in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 in cells expressing RIPK1mRHIM, suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Collectively, these results show that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis. Furthermore, these findings identify ZBP1 as a critical mediator of inflammation beyond its previously known role in antiviral defence and suggest that ZBP1 might be implicated in the pathogenesis of necroptosis-associated inflammatory diseases.

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Figure 1: ZBP1 induces keratinocyte necroptosis and skin inflammation in RIPK1E-KO mice.
Figure 2: Mutation of the RIPK1 RHIM domain causes perinatal lethality and inflammatory skin hyperplasia in mice.
Figure 3: Mutation of the RIPK1 RHIM domain prevents TNF-induced association of RIPK1 with RIPK3 and necroptosis.
Figure 4: RHIM-dependent RIPK1 function prevents MLKL–ZBP1-mediated necroptosis and skin inflammation.

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Acknowledgements

We thank Genentech and V. Dixit for Ripk3−/− mice, S. Akira for Zbp1−/− mice, and J. Han for lentiviral vectors. We thank B. Zevnik, P. Jankowski and S. Assenmacher at the CECAD Transgenic Core Facility for CRISPR/Cas9 mutagenesis in mouse zygotes and C. Uthoff-Hachenberg, J. Buchholz, E. Mahlberg and B. Kühnel for excellent technical assistance. Research reported in this publication was supported by funding from the ERC (grant agreement no. 323040) and the DFG (SFB829 and SFB670). J.L. was supported by a Humboldt research fellowship and C.K. was supported by a Humboldt research fellowship and an EMBO long-term fellowship.

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Author notes

  1. Juan Lin, Snehlata Kumari and Chun Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Genetics, Centre for Molecular Medicine (CMMC), and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, 50931, Germany

    Juan Lin, Snehlata Kumari, Chun Kim, Trieu-My Van, Laurens Wachsmuth, Apostolos Polykratis & Manolis Pasparakis

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Contributions

J.L. designed and generated the Ripk1mRHIM mice. J.L. and C.K. analysed Ripk1mRHIM/mRHIM and RIPK1mRHIM/E-KO mice and performed genetic crosses to address the role of RIPK3 and ZBP1 in these mice. J.L. and C.K. carried out all immunoblots and immunoprecipitation experiments. S.K. generated and characterized RIPK1E-KO Zbp1−/− and FADDE-KO Zbp1−/− mice and made the initial discovery that ZBP1 is required for keratinocyte necroptosis in RIPK1E-KO mice. S.K. and T-M.V. conducted immunostainings and qRT–PCR assays in skin samples from RIPK1E-KO and RIPK1mRHIM/E-KO mice. A.P. generated the Ripk1fl/fl mice and L.W. generated Mlkl−/− mice. M.P. supervised the study, interpreted data and wrote the manuscript together with J.L., C.K. and S.K. J.L., C.K. and S.K. contributed equally and their order of appearance in the author list is random.

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Correspondence to Manolis Pasparakis.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks H. Walczak and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 ZBP1 deficiency strongly delays and ameliorates skin inflammation in RIPK1E-KO mice.

a, Photographs of mice with the indicated genotypes at the age of 4 weeks. Images shown are representative of n ≥ 60 RIPK1E-KO and n ≥ 40 RIPK1E-KO Zbp1−/− mice. b, Photographs of mice with the indicated genotypes and age. Images shown are representative of n ≥ 4 RIPK1E-KO mice at the age of 5–7 weeks and n ≥ 20 RIP1E-KO Zbp1−/− mice at the age of 17–35 weeks. c, Table summarizing the macroscopically observed skin lesions and time of sacrifice of 21 aged RIPK1E-KO Zbp1−/− mice. d, Representative images of skin sections from RIPK1E-KO Zbp1−/− mice and their respective controls stained with H&E (n ≥ 18) or immunostained with the indicated antibodies (n ≥ 4) at the age of 17-35 weeks. Nuclei stained with DAPI. Scale bars, 50 μm. e, Representative images of skin sections from 4–5 week old RIPK1E-KO (n ≥ 6) and RIPK1E-KO Zbp1−/− (n ≥ 3) and their respective control mice stained with TUNEL or immunostained with anti-CC3 antibodies. Nuclei stained with DAPI. Scale bars, 50 μm. f, Microscopic quantification of CC3 and TUNEL positive cells on skin sections from 4–5 week old mice with the indicated genotypes. Epi, epidermis; der, dermis. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

Source data

Extended Data Figure 2 ZBP1 is not required for keratinocyte necroptosis and skin inflammation in mice with epidermis-specific FADD deficiency.

a, Representative photographs depicting the macroscopically observed phenotype of FADDE-KO (n ≥ 10) and FADDE-KO Zbp1−/− (n = 5) mice at the age of 6 days. b, Representative images of skin sections from 6 day old mice with the indicated genotypes stained with H&E (n = 6) or immunostained with the indicated antibodies (n = 3). Nuclei stained with DAPI. Scale bars, 50 μm. c, qRT–PCR analysis of the mRNA expression of the indicated cytokines and chemokines in total skin RNA from 6-day-old mice with the indicated genotypes. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

Source data

Extended Data Figure 3 CRISPR–Cas9-mediated generation of Ripk1mRHIM and Mlkl−/− mice.

a, Schematic depiction of the generation of Ripk1mRHIM/mRHIM mice indicating the sequence of the sgRNA and the single stranded oligonucleotide used for mutating the RHIM domain that were introduced by pronuclear injection into mouse zygotes and the sequencing result of one of the two obtained founders. b, Small intestinal sections from E18.5 pups were stained with H&E or TUNEL or immunostained with anti-CC3 antibodies. Representative images shown (wild type n = 6 for H&E, n = 3 for TUNEL and anti-CC3; Ripk1mRHIM/mRHIM n = 5 for H&E, n = 3 for TUNEL and anti-CC3; Ripk1−/− n = 3 for H&E, TUNEL and anti-CC3). Nuclei stained with DAPI. Scale bars, 50 μm. c, Microscopic quantification of CC3+ and TUNEL+ cells on gut sections from E18.5 pups with the indicated genotypes. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. d, Diagram indicating the sgRNA target sequence (capital letters) used to generate a mutation in exon 2 downstream of the ATG of the Mlkl gene. The PAM sequence is indicated in red. Sequencing showing the 2 base pair deletion found in 97 at position chr8:111,333,648–111,333,649 (mm10), which results in a frameshift after amino acid 34 and a premature stop codon at amino acid position 55 of MLKL. This Mlkl knockout allele was used throughout this study.

Source data

Extended Data Figure 4 Rescue of perinatal lethality of Ripk1mRHIM/mRHIM mice by deficiency of ZBP1, MLKL or RIPK3.

a, Representative photographs and body weights of the indicated mice. b, Representative H&E stainings of skin, liver, spleen, colon and small intestine sections from 5-month-old Ripk3WT/−(n = 4), Ripk1mRHIM/mRHIMRipk3−/− (n = 4) and Ripk1mRHIM/mRHIMZbp1−/− mice (n = 3).

Source data

Extended Data Figure 5 MLKL or ZBP1-deficiency prevents skin inflammation in RIPK1mRHIM/E-KO mice.

a, Representative photographs of RIPK1mRHIM/E-KO (n = 9), RIPK1mRHIM/E-KO MlklWT/− (n = 11), RIPK1mRHIM/E-KO Mlkl−/− (n = 16) and RIPK1mRHIM/E-KO Zbp1−/− (n = 7) at the age of 9–11 weeks. b, Representative images of skin sections from 9–11-week-old RIPK1mRHIM/E-KO MlklWT/− (n = 11) and the respective control mice stained with H&E or immunostained with the indicated antibodies. Nuclei stained with DAPI. Scale bars, 50 μm. c, Representative images of skin sections from 9–11-week-old RIPK1mRHIM/E-KO (n ≥ 6), RIPK1mRHIM/E-KO Mlkl−/− (n = 3), RIPK1mRHIM/E-KO Zbp1−/− (n = 3) and their respective control mice stained with TUNEL or immunostained with anti-CC3 antibodies. Nuclei stained with DAPI. Scale bars, 50 μm. d, Microscopic quantification of CC3+ and TUNEL+ positive cells on skin sections from 9–11 week old mice with the indicated genotypes. Epi, epidermis; der, dermis. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

Source data

Extended Data Figure 6 Expression of ZBP1 in primary keratinocytes.

a, Immunoblot analysis of lysates of primary keratinocytes derived from mice with the indicated genotypes. Each lane represents keratinocytes from individual mice. Cell lysates of wild-type FLM was used as a positive control. For gel source data, see Supplementary Fig. 1. b, Immunoblot analysis of primary keratinocytes derived from mice with the indicated genotypes were left untreated (‘medium’) or stimulated with TNF or IFN-β for 18 h. For gel source data, see Supplementary Fig. 1.

Extended Data Table 1 Numbers of progeny obtained at weaning age from rescue crosses of Ripk1mRHIM/WT mice with Ripk3−/−, Mlkl−/−, Zbp1−/− and Trif−/− mice
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Lin, J., Kumari, S., Kim, C. et al. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature 540, 124–128 (2016). https://doi.org/10.1038/nature20558

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