Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease

Abstract

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1,2,3,4,5,6,7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy—a condition we term ‘cleavage-resistant RIPK1-induced autoinflammatory syndrome’. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1−/− mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

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Fig. 1: Heterozygous mutations of the RIPK1 caspase-8 cleavage site cause autoinflammatory disease.
Fig. 2: Homozygous mutation of the RIPK1 caspase-8 cleavage site in mice causes early embryonic lethality.
Fig. 3: Ripk1D325A/D325A and Ripk1D325A/+ cells are hypersensitive to TNF-induced death.
Fig. 4: RIPK1 cleavage limits inflammation in vivo.

Data availability

The original RNA sequencing data are uploaded and available at the Gene Expression Omnibus (GEO) under accession GSE127572. All other data are available from the corresponding authors upon reasonable request.

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Acknowledgements

This study was funded by the Intramural Research Programs of the National Human Genome Research Institute, the Intramural Research Program of NIH, NIH Clinical Center, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute, by European Research Council Advanced Grant 787826, by NHMRC grants 1025594, 1046984, 1145788, 1162765 and 1163581, NHMRC fellowships 1081421 and 1107149, by the Stafford Fox Foundation and was made possible through Victorian State Government Operational Infrastructure Support, Australian Government NHMRC IRIISS (9000433) and Australian Cancer Research Fund. N.L. is supported by project grant 1145588 from the Cancer Australia and Cure Cancer Australia Foundation and a Victorian Cancer Agency Mid-career Fellowship 17030. This work used the sequencing resources at the NIH Intramural Sequencing Center and the computational resources of the Biowulf Linux cluster at NIH (http://biowulf.nih.gov). We thank the families for their participation, D. Follmann for statistical advice, T. Uldrick and D. Fajgenbaum for assistance procuring samples, and D. Adams, A. Negro, A. Walts and Y. Yang for clinical and technical assistance, C. Liegeois for IT assistance and the staff of the WEHI Bioservices facility for mouse husbandry. The generation of Ripk1D325A and Ripk1D138N,D325A mice used in this study was supported by the Australian Phenomics Network (APN) and the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program.

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Contributions

N.L., S.E.B. and H.O. designed and performed experiments and interpreted data. G.M.W., D.C., L.L., M.S., T.K., K.E.L., K.J.M.Z., N.E., K.S.-A., C.B., W.L.T., M.D.B., H.S.K., D.Y., H.A., N.S., L.W., L.Z., N.S.M., D.B.B., G.G.-C., C.H., H.W., J.J.C., N.I.D, M.M., A.L., Q.Z., I.A., J.C.M., A.K.V. and J.S. performed experiments. A.J.K., M.J.H., L.W. and M.P. generated the CRISPR mice. D.L.S., P.M.H., A.K.O., G.P.P.-P., B.K.B., A.J., T.M.R., A.J.G. and A.K.S. provided the clinical data. E.D.H., S.L.M., M.J.L., M.B., S.D.R. and M.G. contributed reagents, analysis and interpretation. N.L., S.E.B., D.L.K. and J.S. conceived the project and wrote the paper with input from all authors.

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Correspondence to Najoua Lalaoui or Steven E. Boyden or Daniel L. Kastner or John Silke.

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Extended data figures and tables

Extended Data Fig. 1 Inflammatory gene signature in P7 whole-blood RNA.

a, MA plot between two P7 samples and two unrelated adolescent healthy controls, both sequenced with technical duplicates. TCC-edgeR package of R followed by adjustment for multiple comparisons detected 1,394 differentially expressed genes (false discovery rate < 0.05), with 903 genes upregulated in P7, and 491 genes downregulated in P7. b, Representative Gene Ontology terms associated with immune signalling.

Extended Data Fig. 2 Exome reads in family 1.

Excerpts of coverage histograms and aligned exome sequence reads for the proband and her parents in family 1, displayed using the integrative genomics viewer, demonstrate de novo occurrence of the c.970G>A (p.D324N) missense mutation in the LXXD caspase-6/8 cleavage motif preceding the cleavage site (arrow). Paternity and maternity were confirmed using Mendelian inheritance error rates from the same exome data.

Extended Data Fig. 3 ‘Kinase-dead’ RIPK1 or combined loss of Ripk3 and Casp8 rescue Ripk1D325A/D325A lethality.

a, b, Observed numbers of offspring from Ripk1D325A/+ intercrosses and numbers expected from Mendelian ratios at the indicated stage of development. Ripk1D325A/D325A mice are E10.5. All observed E11.5 Ripk1D325A/D325A embryos were dead and most of the E10.5 Ripk1D325A/D325A embryos were abnormal, as described in Fig. 2a, b. Loss of Ripk3 rescued to E12.5; however, 50% of the embryos were abnormal. None of the Ripk1D325A/D325ARipk3−/− mice were born. All observed E11.5 Ripk1D325A/D325AMlkl−/− embryos were dead, showing that loss of Mlkl did not provide any protection. All Ripk1D325A/D325ARipk3−/−Casp8−/− mice were born and developed ALPS owing to loss of Casp8. c, Kaplan–Meyer survival curves of the indicated genotypes. d, Cervical lymph nodes (LN), spleen and thymus of 17-week-old mice of the indicated genotypes. Pictures are representative of five mice per genotype. e, Tissue sections of 18-day-old Ripk1D138N,D325A/+, Ripk1D138N,D325A/D138N,D325A and control mice stained with H&E (left) and anti-CC3 (brown; right). Pictures are representative of two mice per genotype.

Extended Data Fig. 4 Ripk1D325A/+ cells are hypersensitive to TNF-induced death.

ac, MDFs (a), BMDMs (b) and MEFs (c) of the indicated genotypes were treated with either a high dose of TNF (T100; 100 ng ml−1) or a low dose of TNF (T; 10 ng ml−1) combined with SMAC mimetic (S; 100 nM), caspase inhibitor (I; 5 µM), RIPK3 inhibitor (R; 1 µM), necrostatin (N; 10 µM), TAK1 inhibitor (TAKi; 100 nM), IKK inhibitor (IKKi; 100 nM), MK2 inhibitor (MK2i; 2 µM) or cycloheximide (1 µg ml−1) for 16 h. Cell death was quantified by propidium iodide uptake and time-lapse imaging every 30–45 min using IncuCyte. Duplicates are shown for each genotype. Graphs are representative of three (MEFs and MDFs) and two (BMDMs) biologically independent cell lines per genotype repeated independently. d, MEFs were treated as in Fig. 3d for 2 h. e, f, MDFs (e) and MEFs (f) were treated as in Fig. 3d for the indicated times. Results in df are representative of two independent experiments. β-Actin was used as a loading control. g, BMDMs were treated with TNF (100 ng ml−1) combined with SMAC mimetic (500 nM) with or without caspase inhibitor (5 µM) for 90 min, and lysates were immunoprecipitated with anti-FADD. Results are representative of two independent experiments. For gel source data, see Supplementary Fig. 2.

Extended Data Fig. 5 Alternative cleavage of RIPK1.

a, MEFs were treated with 10 ng ml−1 TNF combined with 500 nM SMAC mimetic for 2 h. b, Doxycycline-inducible caspase-8-gyrase41, wild-type and mutant mouse RIPK1 constructs or GFP were co-expressed in 293T cells. Cells were treated for 2 h with 1 μg ml−1 doxycycline to induce caspase-8-gyrase expression and then for 2 h with 700 nM coumermycin to dimerize caspase-8-gyrase. Antibody recognizing the N-terminal end of RIPK1 was used. Results are representative of four (a) and two (b) independent experiments. For gel source data, see Supplementary Fig. 2.

Extended Data Fig. 6 RIPK1 cleavage limits inflammation in an NF-κB-independent manner.

a, Serum cytokine levels in wild-type and Ripk1D325A/+ mice treated for 3 h with 50 μg of poly(I:C). Each dot represents a mouse. Data are mean ± s.e.m., n = 3 mice. b, TNF levels in the supernatant (S/N) of two unrelated adolescent controls (Ctl RIPK1+/+) and P7 RIPK1D324Y/+ PBMCs treated for 3 h with 5 µg ml−1 poly(I:C). Data are mean of triplicates. c, Body temperature of mice of the indicated genotypes after injection of 2 mg kg−1 LPS. Each line represent a mouse; n = 5 mice per genotype. d, BMDMs of the indicated genotypes were treated for 24 h with 25 ng ml−1 LPS or with 2.5 μg ml−1 poly(I:C). Cell death was quantified by propidium iodide staining and flow cytometry. Each dot represents a biological repeat. Graph shows mean; n = 1 for Ripk1+/+ and n = 2 for Ripk1D325A/+. e, f, BMDMs (e) and MDFs (f) were treated with 100 ng ml−1 of TNF for the indicated times. Results are representative of two independent experiments. β-Actin was used as a loading control. For gel source data, see Supplementary Fig. 2. g, NF-κB activation in fibroblasts derived from patient skin biopsies was assessed by measuring nuclear translocation of subunit p65. Each dot represents the median of more than 1,000 single-cell measurements of nuclear mean p65 fluorescent intensities for one individual subject. Data are mean ± s.d., n = 4 patients and 4 controls. P values determined by unpaired one-tailed (a) or unpaired two-tailed (g) t-tests.

Extended Data Fig. 7 Proposed model for RIPK1(D325A)-induced cell death.

Left, TNF binding to TNFR1 triggers the formation of complex I, and subsequent ubiquitylation and phosphorylation of RIPK1. These post-translational modifications (PTMs) inhibit the cytotoxic activity of RIPK1. Complex I formation activates NF-κB- and MAPK-dependent survival genes such as CFLAR, which encodes cFLIP. Subsequently, a cytosolic complex II containing FADD, caspase-8, RIPK1 and cFLIP is formed. In this complex, cFLIP inhibits caspase-8 activity so that a restricted number of substrates (such as RIPK1) are cleaved, but others (such as pro-caspase-3) are not. Cleavage of RIPK1 dismantles complex II. Activation of the NF-κB and MAPK signalling pathways PTM of RIPK1 prevent TNF from inducing cell death, resulting in cell survival (top left). Inhibition of the NF-κB or MAPK signalling pathways reduces levels of cFLIP and accelerates formation of complex II, resulting in cell death via apoptosis (middle left). When NF-κB or MAPK signalling is disrupted in caspase-8-deficient conditions, RIPK1 is not cleaved and autophosphorylates, which triggers the recruitment of RIPK3 and its autophosphorylation. RIPK3 phosphorylates MLKL and necroptosis occurs (bottom left). Right, according to this model, lack of RIPK1 cleavage could result in several distinct outcomes, as follows. (1) RIPK1 accumulation could stabilize complex II, and the presence of cFLIP and inhibitory PTMs to RIPK1 may prevent caspase-8 from killing, resulting in cell survival. (2) The accumulation of ‘uncleavable’ RIPK1 to complex II could override the inhibitory RIPK1 PTMs, resulting in autophosphorylation of RIPK1 and recruitment of RIPK3, leading to necroptosis. (3) RIPK1 accumulation could result in activated caspase-8 that cleaves RIPK3, resulting in cell survival. (4) Stabilization of complex II could result in recruitment and activation of caspase-8 that induces apoptosis and possibly prevents necroptosis by cleaving RIPK3. (5) Finally, the accumulation of RIPK1 could result in activation of both RIPK3 and caspase-8 and therefore induce both apoptotic and necroptotic cell death. In terms of how these potential outcomes match with our data, in homozygote Ripk1D325A cells, both caspase-8 and RIPK3 are activated after TNF signalling, which suggests that apoptosis and necroptosis occur at the same time (Figs. 2d, 3a, b). However, according to these models, loss of RIPK3 limits caspase-8 activation (Fig. 3a, b). This suggests that the recruitment of RIPK3 to complex II increases the recruitment and activation of caspase-8. A precedent for this observation comes from experiments in which RIPK3 inhibitors promoted RIPK1-dependent caspase-8 activation42,43, in a manner we term ‘reverse activation’. In our experiments, however, RIPK3 activation occurs downstream of TNF signalling, which suggests that reverse activation might represent a physiological amplification loop that increases caspase-8 activation. Yet, this requirement for RIPK3 is not present in all cells, as the embryonic lethality of the RIPK1-cleavage mutant is only partially rescued by loss of Ripk3. In the heterozygote Ripk1D325A cells, caspase-8 cleaves wild-type RIPK1, thus limiting TNF-induced cell death as compared to homozygote cells. However, reduction of cFLIP and/or RIPK1 PTMs by treatment with IAP, TAK1, IKK or translational inhibitors decreases the threshold of TNF sensitivity (Extended Data Fig. 4). This may cause the hyper-inflammatory response observed in patients with CRIA syndrome (Fig. 1).

Extended Data Table 1 Leukocyte surface markers in patients with CRIA syndrome
Extended Data Table 2 Effect of tocilizumab treatment and RIPK1 caspase cleavage site mutations is absent in known autoinflammatory diseases
Extended Data Table 3 Conservation of RIPK1 caspase-8 cleavage site

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Lalaoui, N., Boyden, S.E., Oda, H. et al. Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease. Nature 577, 103–108 (2020). https://doi.org/10.1038/s41586-019-1828-5

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