Abstract

Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation1,2,3,4,5,6,7,8. RIPK1 is implicated in inflammatory and cell death signalling9,10,11,12,13 and its kinase activity is believed to drive RIPK3-mediated necroptosis14,15. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.

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Acknowledgements

We are grateful to V. Dixit for Ripk3−/−, D. Gumucio for Villin-Cre and S. Robine for Villin-CreERT2 mice. We thank C. Uthoff-Hachenberg, J. Buchholz, E. Mahlberg, B. Kühnel, B. Hülser, P. Jankowski, S. Assenmacher and P. Scholl for technical assistance. M.P. acknowledges funding from the European Research Council (2012-ADG_20120314), the German Research Council (DFG; SFB670, SFB829, SPP1656), the European Commission (grants 223404 (Masterswitch) and 223151 (InflaCare)), the Deutsche Krebshilfe, the Else Kröner-Fresenius-Stiftung and the Helmholtz Alliance (PCCC). Research reported in this publication was also supported by the National Institute of Allergy and Infectious Diseases division of the National Institutes of Health under award RO1AI075118 to M.K.

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Author notes

    • Marius Dannappel
    • , Katerina Vlantis
    • , Snehlata Kumari
    •  & Apostolos Polykratis

    These authors contributed equally to this work.

    • Michelle Kelliher
    •  & Manolis Pasparakis

    These authors jointly supervised this work.

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, 50931 Cologne, Germany

    • Marius Dannappel
    • , Katerina Vlantis
    • , Snehlata Kumari
    • , Apostolos Polykratis
    • , Chun Kim
    • , Laurens Wachsmuth
    • , Christina Eftychi
    • , Juan Lin
    • , Teresa Corona
    •  & Manolis Pasparakis
  2. Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

    • Nicole Hermance
    • , Matija Zelic
    •  & Michelle Kelliher
  3. Tierforschungszentrum, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany

    • Petra Kirsch
  4. Institute for Laboratory Animal Science, Hannover Medical School, D-30625 Hannover, Germany

    • Marijana Basic
    •  & Andre Bleich

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Contributions

M.D. together with K.V. performed and analysed the experiments related to the intestine and S.K. performed and analysed the experiments related to the skin. N.H. and M.K. designed and generated the targeting constructs for the Ripk1fl/fl and Ripk1D138N/D138N mice. A.P. performed the gene targeting in embryonic stem cells and generated the Ripk1fl/fl, Ripk1D138N/D138N and Triffl/fl mice. M.Z. contributed to the analysis of intestines from Ripk1−/− neonates. C.K. and J.L. performed biochemical analysis of RIPK1-deficient MEFs, IECs and keratinocytes. C.E. performed FACS analysis of intestinal immune cells. T.C. performed qRT–PCR analysis. L.W. designed and tested the short guiding RNAs for CRISPR/Cas9-mediated targeting of Mlkl. P.K., M.B. and A.B. generated germ-free RIPK1IEC-KO mice. M.P. coordinated the project and together with K.V., M.D. and S.K. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Manolis Pasparakis.

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DOI

https://doi.org/10.1038/nature13608

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