Abstract
Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD—which mediate cell death downstream of FAS and FASL—cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4,5,6. Mouse models have corroborated a role for FADD–caspase-8 in promoting inflammatory responses7,8,9,10,11,12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1–TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
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Data availability
The datasets generated during the current study are available from the corresponding authors on reasonable request. The RNA-sequencing data (Extended Data Figs. 6b–e, 7a–d) are available at the Gene Expression Omnibus (GEO) under accession code GSE148066. Proteomics raw data files have been deposited to the UCSD MassIVE database (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=6b442a4570b747b296e25a6b28c94613). Source data are provided with this paper.
Code availability
All custom code can be accessed at https://github.com/rreja/GitlinA_manuscript.
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Acknowledgements
We thank members of the Dixit and D. Vucic laboratories for advice, discussions and reagents—in particular, K. Wickliffe, B. Lee and A. Maltzman; the Genentech genetic analysis, histology, necropsy, immunohistochemistry, clinical pathology and FACS groups for technical assistance; R. Asuncion, B. Torres, B. Hough, M. Long, E. Lu and M. Dempsey for animal husbandry; and B. Pulendran, S. Galli and T. Montine for support.
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Contributions
A.D.G., K.H., K.N., V.M.D., B.H., B.S.M., M.X. and W.P.L. designed, planned and/or supervised the study; A.D.G., K.H., A.F.S., D.Y., E.S., J.Z., Y.C.K., E.C.F. and J.K. performed experiments; V.C.P. and T.H. performed and analysed mass spectrometry experiments, under supervision of J.R.L.; R.R. analysed RNA-sequencing data; A.P., R.C., N.B. and M.R.-G. generated mice; M.D. and J.D.W. generated histological and immunohistochemical data; A.D.G. wrote the paper with edits from K.N. and input from co-authors.
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All of the authors, except for A.D.G., are employees of Genentech, Inc. A.D.G. is a Visiting Scientist at Genentech, Inc.
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Peer review information Nature thanks Igor Brodsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Caspase-8-dependent cleavage of N4BP1.
a, Amino acid sequence of N4BP1 showing peptide sequences detected by mass spectrometry, from experiment depicted in Fig. 1a–c. b, Sequence alignment of mouse and human N4BP1 at indicated amino acid residues. c, Western blot of mouse BMDMs treated with LPS for indicated amounts of time. Representative of two independent experiments. d–f, Western blots of BMDMs of indicated genotypes treated with agonists of TLRs, TNF or FASL. Results representative of three independent experiments for wild-type, Mlkl−/− and Mlkl−/− Casp8−/− genotypes treated with TLR ligands; two experiments with TNF and FASL treatments; one experiment for Mlkl−/− Fadd-/- genotype. g, Western blot of wild-type and Tnfr1−/− BMDMs. Results are representative of three independent experiments. h, Graph depicts cleavage of a proluminogenic caspase-8 substrate in BMDMs of indicated genotypes following LPS stimulation. Dots represent mean fold change in luminescence relative to unstimulated cells of the same genotype from three independent experiments; error bars represent s.e.m. i, Western blots of Mlkl−/−, Mlkl−/−Casp8C362A/C362A and Mlkl−/−Casp8−/− primary mouse embryonic fibroblasts stimulated with LPS for indicated amounts of time. Representative of three independent experiments. j, Western blots of wild-type and Trif−/− BMDMs stimulated with indicated agonists for 1 h. k, Western blots of HEK293T cells transfected with a construct encoding N4BP1 and treated with TNF, z-VAD and emricasan for 4 h, as indicated. l, Western blot of cytoplasmic and nuclear extracts from BMDMs stimulated with LPS for indicated periods of time. m, Western blot of 30-min in vitro cleavage reaction containing 250 ng of recombinant N4BP1 (residues 286–893) and fourfold serial dilutions of recombinant active mouse caspase-8, starting from a highest concentration of 12.5 ng/μl. Co-incubation with 20 μM emricasan was used as a control together with the highest concentration of caspase-8. Arrowhead highlights band corresponding to cleavage at Asp488 (data not shown). Results in j–m represent two independent experiments each. Long form (L), short form (S) and cleavage product of N4BP1 are highlighted by arrowheads.
Extended Data Fig. 2 N4BP1 regulates cytokine responses and is inhibited by caspase-8.
a, Western blot of BMDMs electroporated with Cas9 enzyme complexed with control or N4bp1-targeting (∆N4bp1) gRNA. b–e, Plots show cytokine secretion of BMDMs 24 h after stimulation with poly(I:C) (b), TNF (c), Pam3csk4 (d) or CpG (e) and electroporated as in a. Values for unstimulated BMDMs in b–e are the same as those in Fig. 2a and are replotted separately here for clarity. Bars represent mean values; representative of three independent experiments with three biological replicates each. f, g, Plots show IL-6 (f) and G-CSF (g) secretion at 24 h after stimulation with indicated TLR agonists from human peripheral blood monocyte-derived macrophages that were electroporated with indicated gRNAs. Macrophages derived from three healthy human donors are shown, performed in either duplicate or triplicate wells for each condition per donor. Values for unstimulated cells replotted separately for clarity. Bars represent mean values. h, Western blot of wild-type, Mlkl−/− and Mlkl−/−Casp8−/− BMDMs electroporated with control or N4bp1-targeting gRNAs as in a. Representative of three independent experiments. i, Plots show cytokine secretion 24 h after poly(I:C) stimulation from BMDMs electroporated with indicated gRNAs as in h. Lines represent mean values; representative of three independent experiments with three biological replicates each. Values for unstimulated cells were the same as those in Fig. 2c and are replotted here separately for clarity. P values calculated using unpaired, two-tailed t-test (d–g) or one-way ANOVA with Bonferroni’s multiple comparisons test (i).
Extended Data Fig. 3 Characterization of N4bp1−/− mice.
a, Top, schematic depicting N4bp1fl allele and N4bp1−allele following germline Cre-mediated recombination of loxP sites (red triangles). Boxes represent exons, with translated regions in white and untranslated regions in grey. Green and blue lines represent recognition sequences for flp recombinase (blue, F3; green, FRT). Exons and introns not depicted to scale. Bottom, western blot of BMDMs from N4bp1+/+, N4bp1+/− and N4bp1−/− mice. Each lane represents BMDMs from an individual mouse; representative of three independent experiments. b, Plots show cytokine secretion from BMDMs of indicated genotypes 24 h after stimulation with indicated TLR agonists. Lines represent mean values; representative of three independent experiments with three biological replicates each. c–f, Histological images of liver (c), lung (d), spleen (e) and small intestines (f) from N4bp1+/+ and N4bp1−/− mice at 6–7 weeks of age. Bars represent 200 μm for c–e and 100 μm for f. Representative of n = 7 mice for each genotype. g, Serum cytokines from mice of indicated genotypes at 6–7 weeks of age. Lines represent mean values; n = 9 for N4bp1−/−, n = 9 for N4bp1+/−, and n = 5 for N4bp1+/+ mice. P values calculated using two-sided unpaired t-test (b, g). h–j, Representative flow cytometric gating for analysis of germinal centre B cells (h), T cells (i) or Ly6Chigh monocytes (j), pertaining to experiments depicted in Fig. 3a, Extended Data Figs. 4a–g, 8h–j.
Extended Data Fig. 4 Age-dependent immune dysregulation in N4bp1−/− mice.
a–g, Plots show fraction of CD44+ cells among spleen CD8+ T cells (a); number of Ly6Chigh monocytes among 105 live splenocytes (b); fraction of germinal centre (CD38−FAS+) cells among B cells of mesenteric lymph nodes (LNs) (c) and inguinal lymph nodes (d); fraction of CD44+CD62L− cells among inguinal lymph node CD4+ T cells (e); fraction of CD44+ cells among inguinal lymph node CD8+ T cells (f); and total numbers of indicated spleen cell populations (g). Lines represent mean values. For a, b, d–f, n = 7 for each genotype at 6–7 weeks old and n = 8 for each genotype at 13–14 weeks old. For c, n = 4 for each genotype at 6–7 weeks old and n = 8 for each genotype at 13–14 weeks old. For g, n = 4 for each genotype at 6–7 weeks old, n = 6 for N4bp1+/+ at 13–14 weeks old, and n = 5 for N4bp1−/− at 13–14 weeks old. h, Histological images of spleen at 13–14 weeks old. Bar represents 200 μm. Representative of n = 6 N4bp1+/+ and n = 5 N4bp1−/− mice. i, Serum cytokine levels among N4bp1+/+ and N4bp1−/− mice at 12–14 weeks old. Lines represent mean values; n = 9 for N4bp1+/+ and n = 12 for N4bp1−/− mice for G-CSF, IL-6 and CXCL1; n = 8 for N4bp1+/+ and n = 12 for N4bp1−/− mice for CCL3 measurement. j–l, White blood cell counts and haemoglobin levels (j), spleen weights (k) and serum immunoglobulin levels (l) among 13–14-week-old N4bp1+/+ (n = 6) and N4bp1−/− (n = 5) mice. Lines represent mean values. P values for a–g and i were calculated using two-sided unpaired t-test.
Extended Data Fig. 5 Absence of CD3+B220+ cell population among N4bp1−/− mice, and imiquimod-induced psoriasis model.
a, Representative flow cytometric gating for analysing CD3+B220+ cells in spleen and mesenteric lymph nodes of mice of indicated genotypes. b, Plots show per cent of CD3+B220+ cells among live splenocytes (left) or live mesenteric lymph node cells (right). Bars represent mean values; each dot represents one mouse; n = 3 wild-type mice and n = 4 N4bp1−/− mice, aged 22–27 weeks; and n = 3 Ripk3−/−Casp8−/− mice, aged 12 weeks. c, d, Haemoglobin (c) and serum cytokine levels (d) among N4bp1+/+ (n = 10) and N4bp1−/− (n = 10) mice following 4 days of daily topical imiquimod treatment from experiment depicted in Fig. 3c–e. Lines represent mean values. P values were calculated using two-sided unpaired t-test.
Extended Data Fig. 6 Signal transduction and RNA sequencing of N4bp1+/+ and N4bp1−/− BMDMs.
a, Western blots of BMDMs that were electroporated with control or N4bp1-targeting (∆N4bp1) gRNAs and stimulated with either R837 or LPS for the indicated lengths of time; representative of two independent experiments. b, Volcano plots depict transcriptional profiles of N4bp1+/+ versus N4bp1−/− BMDMs either untreated or stimulated with R837 for the indicated lengths of time. c, Four-way plot; y-axis depicts log2-transformed fold change in gene expression of N4bp1+/+ BMDMs stimulated with R837 for 4 h relative to unstimulated N4bp1+/+ BMDMs; x-axis depicts log2-transformed fold change in gene expression of N4bp1−/− BMDMs stimulated with R837 for 4 h relative to unstimulated N4bp1−/− BMDMs. Blue dots represent NF-κB target genes; red dots represent select cytokine genes. Pearson correlation coefficient shown in top-left corner of plot. d, e, Gene set enrichment analysis of upregulated genes in N4bp1−/− BMDMs relative to N4bp1+/+ BMDMs stimulated with R837 for 8 (d) or 16 (e) hours. RNA-sequencing data represent three independent biological replicates for each genotype and each condition.
Extended Data Fig. 7 Role of N4BP1 in LPS-induced transcriptional program of Mlkl−/−Casp8−/− BMDMs.
a, b, Four-way plots; y-axis shows log2-transformed fold change of Mlkl−/−Casp8−/− BMDMs relative to Mlkl−/− BMDMs, stimulated with LPS for 8 h (a, b); in a, x-axis shows log2-transformed fold change of Mlkl−/− ∆N4bp1 BMDMs relative to Mlkl−/− BMDMs stimulated with LPS for 8 h; in b, x-axis shows log2 fold change of Mlkl−/−Casp8−/− ∆N4bp1 BMDMs relative to Mlkl−/−Casp8−/− BMDMs stimulated with LPS for 8 h. Blue and red dots highlight the indicated groups of genes. c, Pie charts show the fraction of genes increased in Mlkl−/−Casp8−/− ∆N4bp1 relative to Mlkl−/−Casp8−/− BMDMs at 8 h after LPS stimulation among LPS-induced and -noninduced genes that are downregulated by caspase-8-deficiency. LPS-induced genes were those that were significantly increased in expression (P < 0.05) in wild-type BMDMs treated with LPS for 8 h relative to untreated wild-type BMDMs. Per cent of N4BP1-dependent genes and total genes analysed per group are indicated in circle within pie chart. P value calculated using two-tailed Fisher’s exact test. d, Gene set enrichment analysis of genes that are both decreased in LPS-stimulated Mlkl−/−Casp8−/− BMDMs relative to Mlkl−/− and wild-type BMDMs as well as increased in LPS-stimulated Mlkl−/−Casp8−/− ∆N4bp1 relative to Mlkl−/−Casp8−/− BMDMs. BMDMs of all genotypes in a–d were electroporated with control or N4bp1-targeting (∆N4bp1) gRNAs. RNA-sequencing data represent three independent biological replicates per condition and genotype. e, Western blots of BMDMs of indicated genotypes that were electroporated with control or N4bp1-targeting (∆N4bp1) gRNAs and stimulated with LPS for the indicated lengths of time; representative of two independent experiments. f, Protein domains of N4BP1, including its RNA-binding K homology (KH) domain, NYN RNase domain and ubiquitin chain-binding UBA- and CUE-like domains. Protein domains not depicted to size; approximate amino acid boundaries of individual protein domains shown beneath protein schematic. KH and NYN domain boundaries were determined on the basis of Interpro domain prediction (https://www.ebi.ac.uk/interpro/); UBA and CUE-like domain boundaries were determined by secondary structure alignment using Phyre253.
Extended Data Fig. 8 TNF licenses cytokine responses by TRIF-independent TLRs and N4bp1D488A/D488A mice.
a–c, Cytokine secretion among BMDMs stimulated for 24 h with TNF and/or CpG (a), Pam3csk4 (b) or poly(I:C) (c). For clarity, unstimulated and TNF-stimulated values are replotted here separately, and are the same as those from Fig. 4b. Bars represent mean values; representative of three independent experiments with three biological replicates each. P values were calculated using two-sided unpaired t-test. d–g, Representative histological images of lung (d), spleen (e), liver (f) and small intestine (g) from N4bp1D488A/D488A and N4bp1+/+ littermate controls at 10–12 weeks of age. Representative of n = 5 mice of each genotype. Bars represent 90 μm. h, i, Plots show fractions of CD44+CD62L− among CD4+ T cells in spleen or inguinal lymph nodes (h) and fraction of CD38−FAS+ germinal centre cells among B cells in spleen and mesenteric lymph nodes (i). j, Plots show absolute numbers of indicated cell populations in spleens of mice of indicated genotypes. k, l, Plots show spleen weights (k) and white blood cell counts and haemogloblin levels (l) in mice of indicated genotypes. Each dot in h–l represents one mouse; lines or bars in h–l represent mean values; n = 5 mice of each genotype.
Extended Data Fig. 9 Resistance of N4BP1(D488A) protein to TLR3-, TLR4-, TNF- and FASL-induced cleavage and TNF licensing in Mlkl−/− Casp8−/− BMDMs.
a–c, Western blots from N4bp1+/+ and N4bp1D488A/D488A BMDMs stimulated for 1 h with indicated stimuli (a, b) or with TNF for indicated lengths of time (c). Representative of three (a, b) or two (c) independent experiments. d, Plots show IL-6 secretion from BMDMs of indicated genotypes stimulated with indicated ligands. These results are the same as those presented in Fig. 4d and are replotted here separately for clarity; results represent three pooled independent experiments each with three biological replicates per condition. P values were calculated using two-sided unpaired t-test. e, Plots show IL-6 secretion from BMDMs of indicated genotypes that were electroporated with control or N4bp1-targeting (∆N4bp1) gRNAs and stimulated with TLR agonists and/or TNF for 24 h, as indicated. Values for TNF-stimulated BMDMs are the same and are replotted separately for clarity. Bars represent mean values; dots represent biological replicates; representative of two experiments.
Extended Data Fig. 10 FAS licenses cytokine responses by TRIF-independent TLRs.
a, b, Cytokine secretion among wild-type, Mlkl−/− and Mlkl−/−Casp8−/− BMDMs 24 h after stimulation with the indicated TLR agonists in the presence or absence of FASL co-stimulation. c, d, Cytokine secretion among wild-type and N4bp1D488A/D488A BMDMs 24 h after stimulation with the indicated TLR agonists in the presence or absence of FASL co-stimulation. Bars represent mean values; representative of three independent experiments with three biological replicates each. Values for cytokine secretion for BMDMs stimulated with TLR agonists alone are from the same experiments depicted in Fig. 4c, d and are replotted here separately for clarity. P values were calculated using two-sided unpaired t-test (a, b) or one-way ANOVA with Bonferroni’s multiple comparison’s test (c, d).
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Gitlin, A.D., Heger, K., Schubert, A.F. et al. Integration of innate immune signalling by caspase-8 cleavage of N4BP1. Nature 587, 275–280 (2020). https://doi.org/10.1038/s41586-020-2796-5
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DOI: https://doi.org/10.1038/s41586-020-2796-5
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