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Deubiquitinase DUBA is a post-translational brake on interleukin-17 production in T cells

Abstract

T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases1,2. The differentiation of TH17 cells is regulated by transcription factors such as RORγt3,4, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-β signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.

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Figure 1: Post-translational regulation of DUBA expression in T cells.
Figure 2: DUBA limits IL-17A production.
Figure 3: DUBA deficiency in TH17 cells increases RORγt stability.
Figure 4: Screens for substrates of DUBA.
Figure 5: Regulation of RORγt stability by UBR5 in TH17 cells.

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Acknowledgements

We thank A. C. Chan for discussions, Q. Song for statistical analysis, and C. Bakalarski for informatics support.

Author information

Authors and Affiliations

Authors

Contributions

S.R., N.K., Q.T.P., C.E., R.N., X.W., J.L., R.L., O.W.H., J.D. and Z.M. performed experiments designed and analysed by S.R., D.S.K., J.R.L., A.G.C., W.O. and V.M.D.; J.W. and L.D. performed histopathology; M.V. synthesized siRNAs; S.R., W.O. and K.N. wrote the paper.

Corresponding authors

Correspondence to Wenjun Ouyang or Vishva M. Dixit.

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Competing interests

All authors were employees of Genentech, Inc.

Extended data figures and tables

Extended Data Figure 1 Analysis of the T-cell compartment in Dubafl/fl CD4-Cre mice aged 6 weeks.

a, Targeting strategy for the Dubafl allele. The PGK-neo selection cassette was removed with an Flp deleter strain. b, Immunoblots of immune cell subsets from Duba+/+ CD4-Cre (wild-type, WT) and Dubafl/fl CD4-Cre (knockout, KO) mice. CD11c+ dendritic cells (DC) and peritoneal macrophages (MF) were stimulated with 50 ng ml−1 lipopolysaccharide (LPS) overnight. cj, Knockout and wild-type mice were aged 6 weeks. n = 5 mice per genotype. Leukocyte numbers in the thymus, spleen and mesenteric lymph node (c), CD4CD8 (DN) and CD4+CD8+ (DP) cells in the thymus enumerated by cell counting and flow cytometry (d), Numbers of CD4+CD8 and CD4CD8+ cells in thymus (e), in mesenteric lymph nodes (mLN) (f) and spleen (g). h, Percentages of naive (CD45RbhiCD44low) and memory (CD45RblowCD44hi) CD4+ T cells. i, FOXP3+ T cells in the thymus. j, Percentage of FOXP3+ T cells in the spleen and mesenteric lymph nodes. Each circle represents one mouse. Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).

Extended Data Figure 2 Increased IL-17A production by CD4+ and CD8+ T cells from Dubafl/fl CD4-Cre mice.

a, b, Percentage of splenic CD4+ T cells from wild-type (Duba+/+ CD4-Cre) or knockout (Dubafl/fl CD4-Cre) mice that stained positive for intracellular IFN-γ, IL-4 or IL-17A (a) or RORγt (b) by flow cytometry. c, d, Percentage of CD4+ T cells expressing IFN-γ, IL-4 and IL-17A (c) or RORγt (d) after collection on day 8 after immunization with OVA/CFA, and subsequent culture with PMA and ionomycin for 4 h. e, Percentage of splenic CD8+ T cells from knockout and wild-type mice that stained positive for intracellular IL-17A by flow cytometry. Representative contour plots are shown. Each circle represents one mouse. Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).

Extended Data Figure 3 Cytokine production by Duba+/+ CD4-Cre and Dubafl/fl CD4-Cre mice injected with anti-CD3 antibodies.

ac, CD4+ T cells expressing IL-17A (a), RORγt (b) or IFN-γ (c) by intracellular staining and flow cytometry. Cells from the spleen or mesenteric lymph node were isolated from wild-type (Duba+/+ CD4-Cre) or knockout (Dubafl/fl CD4-Cre) mice at 48 h after injection with anti-CD3 antibodies and then stimulated with PMA and ionomycin for 4 h. Representative contour plots are shown. d, Serum cytokines at 48 h after injection of anti-CD3 antibodies. PBS indicates wild-type mice injected with vehicle alone. nd, not detected. e, Intraepithelial lymphocytes from the small intestine producing IFN-γ based on intracellular staining and flow cytometry. Cells were collected at 48 h after injection and stimulated with PMA and ionomycin for 4 h. f, Small intestine weights at 48 h after injection of anti-CD3 antibodies. Each circle represents one mouse. Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test). Data are representative of 2 (ad, f) or 3 (e) independent experiments.

Extended Data Figure 4 DUBA-deficient Treg cells express RORγt and produce IL-17A, but are suppressive in vitro and in vivo.

a, b, Percentage of RORγt+ (a) or IL-17A-producing (b) Treg cells in the spleen or mesenteric lymph node of naive mice. c, IL-17A secretion by CD25+ Treg cells after stimulation with anti-CD3 and anti-CD28 antibodies for 24 h. Data are representative of two independent experiments. d, Proliferation of C57BL/6 CD4+ T cells (responder T cells; Tresp) co-cultured with Treg cells in the ratios indicated. Division index denotes the average number of cell divisions that a cell in the original population has undergone. Data are representative of two independent experiments. e, Weight relative to time zero of Rag2/− mice after transfer of either naive CD4+ T cells alone or together with Duba+/+ or Duba−/− Treg cells. f, Animal clinical scores. g, Colon weights at 10 weeks after T-cell transfer. h, Colon histology scores at 10 weeks. n = 10 (cell transfer groups), n = 5 (PBS). Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test). Data are representative of two independent experiments.

Extended Data Figure 5 Composition of the T-cell compartment in Dubafl/fl CD4-Cre mice aged one year.

a, CD4+ and CD8+ T cells enumerated by cell counting and flow cytometry. bf, Percentages of FOXP3+ Treg cells (b), RORγt+ T cells (c), RORγt+ Treg cells (d), naive (CD45RbhiCD44low) CD4+ T cells (e), and memory (CD45RblowCD44hi) CD4+ T cells (f) in the spleen and mesenteric lymph node. g, Percentages of IFN-γ, IL-4 or IL-17-producing CD4+ T cells in spleen. Each circle represents one mouse. Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired Student’s t-test).

Extended Data Figure 6 Cytokine production and lineage transcription factor expression in DUBA-deficient T cells.

a, Immunoblots of T-helper subsets after 24 h of culture. Graphs indicate relative DUBA abundance from densitometry measurements. Error bars, s.e.m. of three independent experiments. b, Representative contour plots indicating the percentage of IL-17A-expressing TH17 and iTreg cells. c, Expression of Il17f, Il21, Il9, Il22 and Il10 mRNAs by Duba+/+ and Duba−/− T cells that were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β and IL-6. Secreted cytokines were measured on the third day of culture. d, IL-17A secretion by Duba+/+ or Duba−/− naive CD4+ T cells after culture with TGF-β and IL-6 for 6 days and then secondary stimulation with anti-CD3 and anti-CD28 antibodies for 2 days. After a 6-day secondary stimulation, the cells were re-stimulated again. e Percentage of CD8+ T cells expressing IL-17A after 5 days of culture under Tc0, Tc1, Tc2 and Tc17 polarizing conditions by flow cytometry. Representative contour plots under Tc17 conditions are shown. f, Flow cytometric analysis of T-bet, GATA3, RORγt and FOXP3 expression in TH11, TH2 and iTreg cells. Error bars, s.e.m. of triplicate measurements. Data are representative of two independent experiments.

Extended Data Figure 7 Normal expression of TH17 factors in DUBA-deficient TH17 cells.

ac, Expression of TH17 factors by Duba+/+ and Duba−/− CD4+ T cells that were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β and IL-6. Graphs show mRNA expression (a) and immunoblots indicate protein expression (b), immunoblots indication expression of retinoid acid receptors (c). Data are representative of two independent experiments.

Extended Data Figure 8 Normal TGF-β receptor expression and SMAD activation in DUBA-deficient TH17 cells.

a, Expression of TGF-βRI and TGF-βRII in Duba+/+ and Duba−/− TH17 cells after culture for the times indicated. Graphs indicate mRNA expression, whereas immunoblots indicate protein expression. Error bars, s.e.m. of duplicate measurements. Data are representative of two independent experiments. b, Immunoblots of the cells in a. c, Immunoblots of Duba+/+ or Duba−/− TH17 cells after stimulation with TGF-β and IL-6.

Extended Data Figure 9 Identification of DUBA substrates in TH17 cells by K-ε-GG peptide analysis.

a, Volcano plots of ubiquitinated proteins identified after immunoprecipitating K-ε-GG peptides from Duba+/+ and Duba−/− TH17 cells after 4 days of culture. P values are plotted against fold-change in number of peptides for each protein. b, Proteins >1.5-fold differentially ubiquitinated in Duba+/+ and Duba−/− TH17 cells (green and red denote increased and decreased ubiquitylation in the knockout, respectively).

Extended Data Figure 10 The roles of PARP-1 and UBR5 in the regulation of Il-17A production in T cells.

a, Mass spectrometry detection of PARP-1 co-immunoprecipitated with Flag-tagged wild-type DUBA or a DUBA(Cys224Ser) mutant expressed in TH17 cells. Graph indicates the number of unique peptides in experimental samples (black) or control cells expressing GFP (white). b, IL-17A production by TH17 cells or iTreg cells (TGF-β plus IL-2) after 3 days of culture in DMSO vehicle or veliparib. c, Expression of Il17a and Rorc mRNA in TH17 cells treated with veliparib or DMSO vehicle. d, Immunoblots of RORγt in TH17 cells cultured for 2 days with either DMSO or veliparib, and then treated for the indicated times in cycloheximide. Graphs indicate relative RORγt abundance from densitometry measurements. Error bars, s.e.m. of three independent experiments. e, Immunoblot of Duba+/+ and Duba−/− TH17 cells cultured for the times indicated. Error bars, s.e.m. of triplicate measurements. **P < 0.01 (unpaired Student’s t-test). f, Immunoblot of UBR5 co-immunoprecipitated with Flag-tagged DUBA from retrovirally transduced CD4+ T cells. Control cells were transduced with GFP-expressing virus. g, Immunoblot of UBR5 in wild-type TH17 cells transfected with the siRNA indicated. h, IL-17A production by wild-type TH17 cells transfected with the siRNA indicated. Error bars, s.e.m. of two independent experiments. i, Expression of Ubr5 mRNA in CD4+ T cells. Error bars, s.e.m of three independent experiments. j, Immunoblots of wild-type T cells stimulated with anti-CD3 or anti-CD28 antibodies for the times indicated.

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Rutz, S., Kayagaki, N., Phung, Q. et al. Deubiquitinase DUBA is a post-translational brake on interleukin-17 production in T cells. Nature 518, 417–421 (2015). https://doi.org/10.1038/nature13979

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