Abstract

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors1,2. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice3. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

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Acknowledgements

We thank the Transgenic Mice Core Facility (IRC, VIB-UGent, Ghent) for their assistance. Villin-Cre, villin-Cre-ERT2 and RIPK3 knockout mice were provided by D. Gumucio (University of Michigan), S. Robine (Paris, France) and K. Newton and V. Dixit (Genentech), respectively. We thank A. Bredan for editing the manuscript, and P. De Bleser and M. Vuylsteke for statistical analysis. P.V. is senior full professor at Ghent University and holder of a Methusalem grant. L.V. is holder of a fellowship from Research Foundation Flanders (FWO). W.D. and G.V.L. have research professor positions at Ghent University. M.B. has a tenure track position in the Multidisciplinary Research Program of Ghent University (GROUP-ID). N.T. and V.G. are paid by the Methusalem grant, S.L. by a VIB grant and S.K. by a grant from the FWO. G.V.L. was supported by an FWO Odysseus Grant and by research grants from FWO, Foundation against Cancer and the Queen Elisabeth Medical foundation. Research in P.V.’s group is supported by Belgian grants (Interuniversity Attraction Poles, IAP 7/32), Flemish grants (Research Foundation Flanders, FWO G.0875.11, FWO G.0973.11, FWO G.0A45.12N, FWO G.0787.13N, G.0544.11N, G0C3114N and Methusalem grant BOF09/01M00709), Ghent University grants (MRP, GROUP-ID consortium), grant from the Foundation against Cancer (F94 and 2010-162) and grants from VIB. C.B. and C.G. received funding from the IZKF of the FAU Erlangen-Nürnberg and the DFG within the projects SPP1656, BE3686/2 and SFB796.

Author information

Affiliations

  1. VIB Inflammation Research Center, Technologiepark 927, B-9052 Ghent, Belgium

    • Nozomi Takahashi
    • , Lars Vereecke
    • , Mathieu J. M. Bertrand
    • , Linde Duprez
    • , Tatyana Divert
    • , Amanda Gonçalves
    • , Mozes Sze
    • , Barbara Gilbert
    • , Stephanie Kourula
    • , Vera Goossens
    • , Sylvie Lefebvre
    • , Wim Declercq
    • , Geert van Loo
    •  & Peter Vandenabeele
  2. Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium

    • Nozomi Takahashi
    • , Lars Vereecke
    • , Mathieu J. M. Bertrand
    • , Linde Duprez
    • , Tatyana Divert
    • , Amanda Gonçalves
    • , Mozes Sze
    • , Barbara Gilbert
    • , Stephanie Kourula
    • , Vera Goossens
    • , Sylvie Lefebvre
    • , Wim Declercq
    • , Geert van Loo
    •  & Peter Vandenabeele
  3. Pattern Recognition Receptor Discovery Performance Unit, Immuno-inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA

    • Scott B. Berger
    • , John Bertin
    •  & Peter J. Gough
  4. VIB Bio Imaging Core Gent, Technologiepark 927, B-9052 Ghent, Belgium

    • Amanda Gonçalves
  5. Department of Medicine 1, Friedrich-Alexander-University, D-91054 Erlangen, Germany

    • Claudia Günther
    •  & Christoph Becker
  6. Methusalem program, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium

    • Peter Vandenabeele

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Contributions

N.T., L.V., M.J.M.B., W.D., G.v.L. and P.V. designed the study. N.T., L.V., M.J.M.B., L.D., T.D., M.S., S.K., V.G., S.L., C.G. and B.G. performed the experiments. N.T., L.V., M.J.M.B., A.G., W.D., G.v.L. and P.V. analysed the data. N.T., M.J.M.B., W.D., G.v.L. and P.V. wrote the manuscript. S.B.B., C.B., J.B. and P.J.G. provided reagents and scientific insight.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Peter Vandenabeele.

Extended data

Supplementary information

Videos

  1. 1.

    Time-lapse confocal microscopic imaging of Ripk1iEC-KO organoids treated with vehicle and 10 ng/ml mTNF

    Intestinal organoids were derived from small intestine crypt cells isolated from Ripk1iECK mice and treated with vehicle for 20–24 h, and subsequently with 10 ng/ml recombinant murine TNF (mTNF) 2 h after removal of vehicle. Cell death was visualized by incorporation of propidium iodide (PI)(3µM). Representative video frame.

  2. 2.

    Time-lapse confocal microscopic imaging of Ripk1iEC-KO organoids treated with 4-OHT and 10 ng/ml mTNF

    Intestinal organoids were derived from small intestinal crypt cells isolated from Ripk1iECK mice and RIPK1 was then deleted in vitro by 4-OHT treatment (200 nM) for 20–24 h. Organoids were subsequently treated with 10 ng/ml recombinant murine TNF (mTNF) 2 h after removal of 4-OHT. Cell death was visualized by incorporation of PI (3μM). Representative video frame. Supplementary videos 1 and 2 originate from the same experiment, and were repeated more than 3 times.

  3. 3.

    Time-lapse confocal microscopic imaging of WT organoids treated with 10 ng/ml mTNF

    Intestinal organoids were derived from small intestinal crypt cells isolated from WT mice, treated with 10 ng/ml recombinant murine TNF (mTNF). Cell death was visualized by incorporation of PI (3μM). Representative video frame.

  4. 4.

    Time-lapse confocal microscopic imaging of Ripk1iEC-KO organoids treated with 4-OHT and 10 ng/ml mTNF.

    Intestinal organoids were derived from small intestinal crypt cells isolated from Ripk1iECKO mice, and RIPK1 was deleted in vitro by 4-OHT treatment (200 nM) for 20–24 h. Organoids were subsequently treated with 10 ng/ml recombinant murine TNF (mTNF) 2 h after removal of 4-OHT. Cell death was visualized by incorporation of PI (3μM). Representative video frame.

  5. 5.

    Time-lapse confocal microscopic imaging of Ripk1KD-KI organoids treated with 10 ng/ml mTNF

    Intestinal organoids were derived from small intestinal crypt cells isolated from Ripk1KD-KI mice, and treated with 10 ng/ml recombinant murine TNF (mTNF). Cell death was visualized by incorporation of PI (3μM). Representative movie frame. Supplementary videos 3-5 were recorded in a single experiment, and repeated 2 times.

  6. 6.

    Time-lapse confocal microscopic imaging of WT organoids treated with 4-OHT

    Intestinal organoids were derived from small intestinal crypt cells isolated from WT mice are treated with 4-OHT (200 nM) for 20–24 h. Imaging started 2h after the removal of 4OHT. Cell death was visualized by incorporation

  7. 7.

    Time-lapse confocal microscopic imaging of Ripk1iEC-KO organoids treated with 4-OHT

    Intestinal organoids were derived from small intestinal crypt cells isolated from Ripk1iEOK mice are treated with 4-OHT (200 nM) for 20–24 h. Imaging started 2h after the removal of 4OHT. Cell death was visualized by incorporation of PI (3μM). Representative movie frame. Supplementary videos 3, 4, 5, 6 and 7 were recorded in a single experiment, and repeated 2 times.

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https://doi.org/10.1038/nature13706

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