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RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis

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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Figure 1: Death and spontaneous gut inflammation in mice with RIPK1 deletion in IECs.
Figure 2: Ripk1IEC-KO phenotype in conditions of antibiotic treatment, MYD88 deficiency, or TNFR1 deficiency.
Figure 3: Lethality of Ripk1iIEC-KO in mice requires Casp8 but not RIPK3 or RIPK1 kinase activity.
Figure 4: Ripk1IEC-KO intestinal organoids are sensitized to TNF-induced apoptosis independently of NF-κB and kinase activity.

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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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Peter Vandenabeele.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of conditional Ripk1−/− mice using Cre–loxP technology.

a, Schematic representation of the targeting vector along with wild-type and mutant loci. Double homologous recombination between the wild-type Ripk1 allele and the targeting vector leads to entry of the neomycin positive selection marker and of extra ScaI (*) and PvuII (**) restriction sites in the Ripk1 allele and exclusion of the thymidine kinase (TK) negative selection marker, generating the Ripk1Nfl allele. Southern blot analysis reveals a 4.0-kb DNA fragment instead of a 10.3-kb fragment with the 5′ probe and a 5.3-kb instead of a 10.0-kb fragment with the 3′ probe for the Ripk1Nfl allele compared to the wild-type Ripk1. The Frt-flanked neomycin cassette was removed by crossing Ripk1Nfl mice with a Flp-deleter strain to generate a Ripk1 floxed allele (Ripk1fl). To ablate RIPK1, Ripk1fl/fl mice were crossed with Cre-deleter transgenic mice to induce germline recombination of the loxP sequences or with cell-type-specific Cre transgenic lines to induce tissue-specific recombination. The loxP interstitial exon 3 is looped out, disabling Ripk1 transcription. b, Southern blot analysis of five positive embryonic stem cell clones using a 5′ probe and a 3′ probe. MW, molecular weight; WT, wild type; Rec, recombinant. c. Ripk1+/+ and Ripk1Nfl/+ genotypes were distinguished by PCR using the primers indicated by arrows in a. d, MEF cells were prepared from Ripk1+/+, Ripk1+/− and Ripk1−/− embryos on embryonic day 14.5, and RIPK1 protein levels were analysed in lysates by western blot. β-Actin protein levels were used as a loading control.

Extended Data Figure 2 RIPK1 deficiency in MEFs results in defective MAPK and NF-κB activation associated with apoptosis induction upon TNF stimulation.

Ripk1−/− MEFs and control Ripk1+/+ MEFs were stimulated with 10 ng ml−1 of mTNF. a, Western blot analysis of NF-κB and MAPK activation in lysates sampled at different time points. b, RNA was analysed by qPCR for TNF, A20, IκBα and MCP1 expression (n = 3). Data are represented by mean ± s.e.m.

Extended Data Figure 3 Characterization of Ripk1IEC-KO mice.

a, Birth and survival rates of all genotypes of offspring from selected breeding couples (Ripk1fl/fl × Ripk1fl/+ villin-creTg). *P < 0.05 by Fischer’s exact test. b, Genomic PCR analysis of Ripk1 alleles on DNA extracted from various tissues of Ripk1IEC-KO mice and control Ripk1fl/fl mice. c, Western blot detection of RIPK1 protein in small intestine and colon tissue of Ripk1IEC-KO and control Ripk1fl/fl mice. KO, knockout; bp, base pairs. d, PAS/AB staining of colons of Ripk1IEC-KO and Ripk1fl/fl mice (the same sample set as in Fig. 1c). e, Immunohistochemistry of activated CASP3 in the same sample set as in d.

Extended Data Figure 4 Characterization of older Ripk1IEC-KO mice.

a, Macroscopic images showing 22-week-old Ripk1fl/fl and Ripk1IEC-KO mice, and corresponding splenomegaly and colon shortening in Ripk1IEC-KO mice. b, Haematoxylin and eosin staining of Ripk1fl/fl (n = 3) and Ripk1IEC-KO (n = 3) mice at the ages of 6 weeks and 22 weeks. A representative image is shown.

Extended Data Figure 5 Rescue of the inflammatory phenotype of Ripk1IEC-KO mice by antibiotic treatment.

a, Birth and survival rates (at weaning age) of all genotypes either treated or untreated with broad-spectrum antibiotics (ABX). The interaction between the genotype and the antibiotic treatment was assessed by analysis of deviance. b, Histopathological analysis by haematoxylin and eosin and immunohistochemical staining of Lysozyme P (LysP, Paneth cells) in small intestine samples from mice with or without antibiotic treatment: Ripk1fl/fl mice (n = 5), Ripk1IEC-KO mice without antibiotics (n = 5) and Ripk1IEC-KO mice with antibiotics (n = 7). c, TNF level detected by qPCR of RNA isolated from small intestine of antiobiotic-treated and non-treated Ripk1fl/fl and Ripk1IEC-KO mice (all n = 5). ***P = 0.0001 by two-tailed, two-way ANOVA. Data represent mean ± s.e.m. WT, wild type; KO, knockout. d, Antibiotic-treated Ripk1fl/fl (n = 3) and Ripk1iIEC-KO (n = 7), and non-treated Ripk1fl/fl (n = 3) and Ripk1iIEC-KO (n = 5) mice were subjected to RIPK1 deletion by daily treatment with 1 mg 4-OHT, and survival rate was monitored. ***P = 0.0007.

Extended Data Figure 6 Rescue of the lethal phenotype of Ripk1IEC-KO mice by MYD88 deficiency or TNFR1 deficiency.

a, Birth and survival rates (at weaning age) of offspring of all genotypes from selected breeding couples (Ripk1fl/fl Myd88−/− × Ripk1fl/+ villin-creTg Myd88−/−); Pearson’s χ2 test. b, Birth and survival rates (at weaning age) of offspring of all genotypes from selected breeding couples (Ripk1fl/fl Tnfr1−/− × Ripk1fl/+ villin-creTg Tnfr1−/−); Pearson’s χ2 test. c, Haematoxylin and eosin staining and TUNEL assay of Ripk1fl/fl Myd88−/− mouse and a littermate Ripk1IEC-KO Myd88−/− mouse small intestine. d, Haematoxylin and eosin staining and TUNEL assay of Ripk1fl/flTnfr1−/− mouse and a littermate Ripk1IEC-KOTnfr1−/− mouse small intestine. e, TNF levels of small intestine lysate from Ripk1fl/fl (n = 5), Ripk1IEC-KO(n = 5), Ripk1IEC-KO Myd88−/− (n = 4) and Ripk1IEC-KO Tnfr1−/− mouse (n = 6) determined by qPCR analysis.

Extended Data Figure 7 Ripk1IEC-KO phenotype involves CASP8 but not RIPK3.

a, Survival rate of offspring of all genotypes from selected breeding couples (Ripk1fl/fl Casp8fl/fl × Ripk1fl/+ Casp8fl/+ villin-creTg) at weaning. b, An image of a 26-week-old Ripk1IEC-KO Casp8IEC-KO mouse and a littermate Ripk1fl/fl Casp8fl/fl mouse showing no detectable difference. All Ripk1IEC-KO Casp8IEC-KO mice at different ages (n = 6) were indistinguishable from littermate Ripk1fl/fl Casp8fl/fl mice (n = 8). c, Survival rate of offspring all of genotypes from selected breeding couples (Ripk1fl/flRipk3−/− × Ripk1fl/+ Ripk3−/− villin-creTg) at weaning showing significantly fewer Ripk1fl/fl Ripk3−/− mice surviving at weaning. d, An image of a 9-week-old Ripk1IEC-KO Ripk3−/− mouse and a littermate Ripk1fl/fl Ripk3−/− mouse. All Ripk1IEC-KORipk3−/− mice at different ages (n = 5) were smaller than littermate Ripk1fl/fl Casp8fl/fl mice (n = 14). e, Haematoxylin and eosin staining and TUNEL assay of small intestine of Ripk1IEC-KO, Ripk1fl/+ Casp8IEC-KO (indicated as Casp8IEC-KO), Ripk1IEC-KO Casp8IEC-KO, Ripk1IEC-KO Casp8IEC-KO and Ripk1IEC-KO Ripk3−/− mice. f, The number of necrotic cells per crypt was counted based on haematoxylin and eosin staining.

Extended Data Figure 8 RIPK1 kinase activity is not required for intestinal homeostasis.

Haematoxylin and eosin staining and TUNEL assay and immunohistochemical staining of active CASP3 (C3*) in small intestine samples from Ripk1fl/fl mice (n = 5), Ripk1IEC-KO mice (n = 5) and RIPK1-kinase-dead knock-in mice (Ripk1KD-KI, n = 5) at the age of 6−14 weeks. The images of 12-week-old mice are shown. Scale bars, 50 µm; black arrows indicate active CASP3 positivity. A magnified view is inserted in the left corner of the immunohistochemistry images.

Extended Data Figure 9 TNF-independent death of organoids and no direct cell death induction by TLR4 or TLR2 ligand.

Intestinal organoids were derived from small intestine crypt cells isolated from wild-type (WT) and Ripk1iIEC-KO mice. RIPK1 deletion (iKO) was induced in vitro by 4-OHT treatment (200 nM) for 20−24 h. a, Organoids were observed for up to 4 days after removal of 4-OHT. b, Organoids were treated with 10 ng ml−1 mTNF, Gram-negative bacterial lipopolysaccharide (TLR4 ligand, 100 µg ml−1), or Gram-positive bacterial lipoteichoic acid (TLR2 ligand, 100 µg ml−1) after removal of 4-OHT. Time-lapse confocal microscopic imaging of propidium iodide (PI)-stained organoids (n = 2). Scale bar, 100 µm. c, Image analysis of the percentage of propidium-iodide-positive nuclei in an experiment resembling those in b.

Extended Data Figure 10 Degradation of cFLIP in TNF-treated Ripk1−/− MEFs and Ripk1iIEC-KO organoids.

a, Protein lysates were prepared at designated times from Ripk1−/− MEFs treated with mTNF (10 ng ml−1) and analysed by western blot for NF-κB-inducing kinase (NIK), TRAF2, cIAP1 and cFLIP. b, Protein lysates were prepared at designated times from Ripk1iIEC-KO organoids treated with 4-OHT (200 nM) for 20 h before stimulation with mTNF (10 ng ml−1), and analysed by western blot for cIAP1. c, Protein lysates isolated from Ripk1KD-KI organoids were stimulated with mTNF (10 ng ml−1) in an independent experiment and analysed for RIPK1 and cFLIP. WT, wild type.

Supplementary information

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. (MP4 2192 kb)

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. (MP4 6126 kb)

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. (MP4 10633 kb)

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. (MP4 14749 kb)

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. (MP4 10171 kb)

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 (MP4 11909 kb)

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. (MP4 9296 kb)

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Takahashi, N., Vereecke, L., Bertrand, M. et al. RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis. Nature 513, 95–99 (2014). https://doi.org/10.1038/nature13706

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