Abstract
A20 is an anti-inflammatory protein linked to multiple human diseases; however, the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We found that A20-deficient T cells and fibroblasts were susceptible to caspase-independent and kinase RIPK3–dependent necroptosis. Global deficiency in RIPK3 significantly restored the survival of A20-deficient mice. A20-deficient cells exhibited exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 underwent physiological ubiquitination at Lys5 (K5), and this ubiquitination event supported the formation of RIPK1-RIPK3 complexes. Both the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex required the catalytic cysteine of A20's deubiquitinating motif. Our studies link A20 and the ubiquitination of RIPK3 to necroptotic cell death and suggest additional mechanisms by which A20 might prevent inflammatory disease.
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Change history
21 May 2015
In the version of this article initially published, the filled circles in Figure 3b were incorrectly labeled 'A20+/flCD4-Cre'. The correct label is 'A20fl/flCD4-Cre'. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank X. Wang for Ripk3−/− mice; J.C. Patarroyo and N. Molnarfi for assistance with EAE experiments; and S. Oakes for discussions. Mass spectrometry analysis was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at the University of California, San Francisco, supported by funding from the Biomedical Technology Research Centers program of the National Institute of General Medical Sciences of the US National Institutes of Health (8P41GM103481) and the Howard Hughes Medical Institute. Supported by the US National Institutes of Health (DK071939 and DK095693 (A.M.), and AI073737 and NS063008 (S.S.Z.)), the Kenneth Rainin Foundation (A.M.), the National Multiple Sclerosis Society (RG 4768 and RG 5180 (S.S.Z.); and U.S.), the Guthy Jackson Charitable Foundation (S.S.Z.), the Maisin Foundation (S.S.Z.) and The Crohn's and Colitis Foundation of America (M.O. and S.O.).
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M.O. performed necroptosis experiments and assisted with the manuscript; S.O. performed most of the T cell studies and assisted with the manuscript; U.S.-T. designed and performed most of the EAE experiments; J.O.-P. designed and performed mass spectrometry studies; T.L. performed initial experiments of necroptosis in MEFs; R.T. and M.I.W. performed initial T cell studies; T.P. performed EAE experiments; B.D. provided mice and cell lines for necroptosis experiments; R.A. assisted with breeding and mouse experiments; A.A. and J.B. provided technical assistance; H.W. analyzed RIPK3 structure; A.B. supervised mass spectrometry studies; B.A.M. supervised T cell and necroptosis studies and helped write the manuscript; S.S.Z. supervised EAE experiments; and A.M. supervised the overall study and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Acute deletion of A20 from A20fl/flROSA26-ER-Cre T cells in vitro.
Quantitative real-time PCR analyses of A20 mRNA expression in purified CD4+ T cells from A20fl/fl ROSA-ER-Cre and A20+/fl ROSA-ER-Cre mice using the indicated doses of 4-OH-tamoxifen (4-OHT). Naïve CD4+ T cells were purified from the indicated mice, stimulated with agonist anti-CD3 and anti-CD28 antibodies and the indicated doses of 4-OHT for three days, and then stimulated with PMA/ionomycin for the indicated times (0, 1, or 3 h). Cells were harvested for qPCR analyses of A20 and Actin mRNA expression. Data represent ratios of A20/Actin mRNA expression. Note virtually complete deletion of A20 mRNA from A20fl/fl ROSA-ER-Cre cells at both doses of 4-OHT.
Supplementary Figure 2 Active Nec-1 is more effective than methylated (inactive) Nec-1 in rescuing A20-deficient T cells.
Naïve (CD62Lhi CD25lo) CD4+ T cells were FACS sorted from A20fl/fl CD4-Cre (CD45.1) and congenic A20+/fl CD4-Cre (CD45.2) mice, mixed 1:1 in co-cultures in vitro, and stimulated with anti-CD3 and anti-CD28 antibodies plus Z-VAD for the indicated time periods. Data reflect percentages of live cells after treatment with the RIPK1 kinase inhibitor, Nec-1 (10 μM), or an inactive (methylated) form of Nec-1 (10 μM). Data are displayed as a ratio of each genotype at each time point, normalized to the 1:1 ratio at the experiment’s initiation. Note that active Nec-1 selectively rescues A20fl/fl CD4-Cre T cells better than the inactive form of Nec-1.
Supplementary Figure 3 A20 supports the differentiation of both TH1 and TH17 CD4+ T cells.
Naïve CD4+ T cells were FACS-purified from the indicated genotypes of mice, stimulated with agonist anti-CD3 and anti-CD28 antibodies in conjunction with either IL12 and anti-IL4 to polarize T cells toward TH1 cells or TGFβ, IL6, anti-IL4 and anti-IFNγ to polarize toward TH17 cells. Theumbers of CD4+ T cells expressing IFNγ or IL17A as well as the total numbers of live T cells were quantitated after 3 days. Note that the total numbers of A20fl/fl CD4-Cre T cells were markedly reduced under both TH1 and TH17 polarizing conditions compared to A20+/fl CD4-Cre T cells (as they are under neutral conditions, see Fig. 1d). Moreover, the specific numbers of TH1 (IFNγ+) T cells in TH1 polarizing cultures and the numbers of TH17 (IL17A+) T cells in TH17 polarizing conditions are also reduced in A20fl/fl CD4-Cre T cell cultures compared with A20+/fl CD4-Cre T cell cultures. Finally, the reduced numbers of total live CD4+ T cells, TH1 cells, and TH17 cells in A20fl/fl CD4-Cre T cell cultures are all significantly restored in A20fl/fl CD4-Cre Ripk3-/- T cell cultures. Increased T cell survival by RIPK3 deficiency occurs in A20fl/fl CD4-Cre T cells but not in A20+/fl CD4-Cre T cells, suggesting that A20fl/fl CD4-Cre T cells are selectively susceptible to RIPK3 dependent necroptosis.
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Onizawa, M., Oshima, S., Schulze-Topphoff, U. et al. The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis. Nat Immunol 16, 618–627 (2015). https://doi.org/10.1038/ni.3172
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DOI: https://doi.org/10.1038/ni.3172