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E-type prostanoid receptor 4 drives resolution of intestinal inflammation by blocking epithelial necroptosis

Abstract

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.

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Fig. 1: PGE2–EP4 signalling is associated with resolution of experimental colitis and EP4 expression correlates with improved flare-free survival in ulcerative colitis.
Fig. 2: Selective agonism of EP4 induces transcriptomic changes akin to the resolution of colitis that can be exploited therapeutically.
Fig. 3: The activation of the PGE2–EP4 axis inhibits non-apoptotic cell death in experimental colitis.
Fig. 4: EP4 agonism blocks non-resolving inflammation and non-apoptotic cell death in Casp8ΔIEC mice.
Fig. 5: EP4 receptor activation suppresses intestinal epithelial necroptosis via the inhibition of MLKL oligomerization and membrane translocation.
Fig. 6: EP4 agonism does not protect from TAK1-inhibition-induced IEC death.
Fig. 7: EP4 agonism inhibits necroptosis in human intestinal crypts and patient-derived epithelial organoids.

Data availability

The RNA-seq data that support the findings of this study have been deposited in the Array Express service of the Molecular Biology Laboratory–European Bioinformatics Institute under accessions E-MTAB-9850 and E-MTAB-9820. Previously published sequencing data that were re-analysed here are available under accession codes GSE1122360, GSE5794561, GSE10914262, GSE12868263, GSE1687964, GSE5907165, GSE11693666, GDS518214 and E-MTAB-647667. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

Funding: DFG projects SFB1181 (C02, C05), TRR241 (A02, A03, A08, B04, B05, C02, C04 and INF: IBDome), KFO257 (TP01) and individual grant BE3686/2. Further support: Interdisciplinary Center for Clinical Research (IZKF: J68, A76). S.K. and R.N.Y. received funding from Crohn’s and Colitis Canada. J.V.P. received a post-doctoral fellowship from the Alexander von Humboldt foundation. We thank S. Reid from the Sonnewald laboratory for help with array scanning.

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Study concept, literature search, experimentation, analysis, interpretation of data and critical revision of manuscript: J.V.P., R.N.Y. and C.B. Experimentation and analysis: J.P., T.M.M., S.K., M.G.A., F.M., K.S., M.M., C.H., Y.Y., W.L., M.J.M. and S.Z. Material support: K.J., B.R,. C.G., M.L., S.W., C.N. and A.A.K. Manuscript drafting: J.V.P. and C.B. Intellectual contributions, manuscript editing and material support: A.A.K., B.R., M.F.N., K.J., R.A. and C.B.

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Correspondence to Christoph Becker.

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Peer review information Nature Cell Biology thanks Shigekazu Nagata, Arthur Kaser and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended data Fig. 1 EP4 expression is associated with improved IBD outcome.

a, Expression levels (normalized counts) of PTGER4 from the indicated publicly available RNA-Seq studies from IBD patient gut tissues. b, mRNA expression levels (log2 fold change) of the inflammation marker S100A9 by qPCR at various inflammation grades (scores) of intestinal tissues derived from patients with Crohn’s disease (CD) and ulcerative colitis (UC). (n, CD = score 0 = 32; 1 = 19; 2 = 21; 3 = 18) (n, UC = score 0 = 15; 1 = 11; 2 = 20; 3 = 16, geometric mean ± geometric SD). c, Flare-free survival in CD patients with a high versus a low expression levels of PTGER4 d, change in swelling response of mouse small intestinal organoids induced by the EP4 selective agonist, EP4D, upon treatment with increasing concentrations of either LCL161,982 (EP4 receptor antagonist) or TG6–10–1 (EP2 receptor antagonist).

Extended data Fig. 2 Transcriptional signature of PGE2 synthesis is associated with resolution of colitis.

a, KEGG pathway gene ontologies (-log10 p values) of the up- and down-regulated genes in C57BL/6 mice challenged with DSS and receiving EP4D versus those that received vehicle. b, KEGG pathway gene ontologies (-log10 p values) of the up and down-regulated genes in C57BL/6 mice at the high inflammation (Inf_High) and fully resolved (Res_Ful) time points of DSS-induced colitis. c, KEGG pathway gene ontologies (-log10 p values) of the up and down-regulated genes in ulcerative colitis patients from the indicated publicly available dataset. Bars for the pathways of arachidonic acid and linoleic acid metabolism are hashed.

Extended data Fig. 3 IEC-specific ablation of Casp8 increases non-apoptotic death.

a, TUNEL positive cells per 10 crypts, dots represent biological replicates of C57BL/6 mice that were challenged with DSS and received either Vehicle or two different doses of EP4-D (n = 10, mean ± SD, *** = p < 0.001, one way ANOVA, followed by Dunnett’s multiple comparisons test). b, representative micrographs of colonic tissue sections immunostained for phosphorylated MLKL from C57BL/6 mice that were challenged with DSS and received either Vehicle or EP4-D. c, body weight excursions in Casp8fl/fl and Casp8ΔIEC mice challenged with the indicated percentages of DSS. (scale bars = 250 µm) d, survival of Casp8fl/fl and Casp8ΔIEC mice challenged with 1.5% DSS. e, representative micrographs of colonic tissue sections from Casp8ΔIEC and Casp8ΔIEC X Mlkl-/- mice co-stained for phosphorylated MLKL and TUNEL. (scale bars = 100 µm) f, representative micrographs of colonic tissue sections from Casp8fl/fl and Casp8ΔIEC mice challenged with DSS immunostained for caspase 8. (scale bars = 150 µm) g, tamoxifen treatment scheme to induce the deletion of Casp8 specifically in the IEC, followed by DSS a challenge and body weight excursions in the inducible Casp8iΔIEC mice, challenged either with vehicle (Veh) or with EP4-D (n = 3, mean ± SD, at day 4 p = 0.04, at day 5 p = 0.01, two way ANOVA, followed by Sidak’s multiple comparisons test).

Extended data Fig. 4 Selective EP4 activation suppresses Casp8-independent, TNF-induced IEC death.

a, intestinal organoids from Casp8fl/fl and Casp8ΔIEC mice treated as indicated and stained with PI to reveal dead cells followed by quantification of PI intensity (n = 4, mean ± SD, *** = p 0.0002, one way ANOVA, followed by Tukey’s multiple comparisons). b, intestinal organoids from Casp8ΔIEC mice treated as indicated and stained with PI to reveal dead cells followed by quantification of PI intensity (n = 4, mean ± SD, *** = p < 0.001 compared versus TNF, one way ANOVA, followed by Dunnett’s multiple comparisons). c, gating scheme for HT-29 cells subjected to cell death treatments followed by flow cytometric evaluation. d, densitometric quantification of the blots for pRIPK1 from Fig. 5h (n = 3, mean ± SD, ** = p 0.003, *** = p 0.0003, compared versus TSz, one way ANOVA, followed by Dunnett’s multiple comparisons).

Extended data Fig. 5 EP4 is the primary E-type prostanoid receptor on IEC.

a, Micrographs of PI and caspase 3/7 activity probe stained, biopsy-derived colonic organoids from human volunteers treated as indicated. b, normalized counts (fpkm) of Ptger2 and Ptger4 from C57BL/6 mouse intestinal organoids subjected to RNA-Sequencing and normalized counts (log2 TPM) from the indicated publicly available single-cell RNA sequencing dataset from the mouse small intestine.

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Patankar, J.V., Müller, T.M., Kantham, S. et al. E-type prostanoid receptor 4 drives resolution of intestinal inflammation by blocking epithelial necroptosis. Nat Cell Biol 23, 796–807 (2021). https://doi.org/10.1038/s41556-021-00708-8

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