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Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote TH17 differentiation

Abstract

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (TFH cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the TH17 subset of helper T cells in the lungs. Roquin inhibited TH17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the TH17 cell–promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance TH17 differentiation.

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Figure 1: Inflammatory phenotypes in the lungs of roquin-mutant mice.
Figure 2: Enhanced TH17 differentiation and gastritis in mice that lack roquin in T cells.
Figure 3: Full-length roquin proteins are degraded after triggering of the TCR and costimulatory receptors.
Figure 4: The paracaspase MALT1 cleaves roquin.
Figure 5: Regnase-1 and roquin-1 regulate shared targets.
Figure 6: Roquin and regnase-1 cooperate in post-transcriptional gene regulation.
Figure 7: IκBNS and IκBζ promote TH17 differentiation and regnase-1 inhibits TH17 differentiation.
Figure 8: TCR signal strength determines the derepression of roquin targets.

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Acknowledgements

We thank H.-M. Jäck and J. Wittmann (Friedrich-Alexander-Universität Erlangen, Germany) for the cloned 3′ UTR of mouse Irf4; and D. Baumjohann for critical reading of the manuscript. Supported by the Deutsche Forschungsgemeinschaft (SFB 1054 TP-A03 and Z02 to V.H.; SFB 1054 TP-B01 to J.R.; SFB 1054 TP-A04 to D.K.; SFB 1054 TP-A02 to M.S.-S; GRK1202 to H.-J.A. and M.L.; SFB TR36 to M.H. and HO1116/5-2 to H.H.) the PCCC (M.H.), the Hofschneider Foundation (M.H.), Boehringer Ingelheim Fonds (G.A.H.) and The Federation of European Biochemical Societies (K.U.V.).

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Authors and Affiliations

Authors

Contributions

K.M.J., D.H. and S.B. did most of the experiments, with J.Z., G.A.H., D.N., K.U.V., N.R., S.C.W., S.L.E., R.G., N.M., C.L., M.L., J.E.S. and A.K. contributing to specific experiments; A.G., E.K., A.W., H.-J.A., I.S., M.S.-S., M.F., H.H., D.K., J.R. and M.H. provided reagents, advice, and design and supervision of experiments; V.H. conceived of the project and designed the experiments; and K.M.J., D.H., S.B. and V.H. wrote the paper.

Corresponding author

Correspondence to Vigo Heissmeyer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparison of lung inflammation and mortality in mice with roquin mutations.

(a) Histological sections of H&E-stained (magnification 20x, scale bar represents 100 μm) and anti-CD3-, anti-MHCII- and anti-Ki67-stained (magnification 40x, scale bar represents 10 μm) lung tissues from 26–30-week-old WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice. (b) Total numbers of CD45+ cells in lung tissue of 26–30-week-old WT control and Rc3h1fl/flRc3h2fl/flCd4-Cre mice were determined by flow cytometry. (c) Relative numbers of CD3+ cells in lung tissue of 26–30-week-old WT control and Rc3h1fl/flRc3h2fl/flCd4-Cre mice were detected by histology. (d) Relative numbers of MHCII+ cells detected as in c. (e) Relative number of Ki67+ cells detected as in c. (a,c-e) Results represent analyses of 5 mice for each genotype or (b) 8 WT and 7 Rc3h1fl/flRc3h2fl/flCd4-Cre mice. Error bars are mean ± SEM, *, P = 0.0435; **, P = 0.0266; ***, P = 0.0008; ****, P < 0.0001 (unpaired, two-tailed t-test). (f) Kaplan-Meier survival curve of Rc3h1fl/flRc3h2fl/flCd4-Cre mice. (g) Titers of anti-DNA antibodies in sera from 4 WT, 5 Rc3h1fl/flRc3h2fl/flCd4-Cre, and 4 Rc3h1san/san mice as detected by ELISA. (h) Titers of rheumatoid factor detected as in g. (g-h) Error bars are mean ± SD, *, P = 0.0135; **, P = 0.0039; ***, P = 0.0012; ****, P = 0.0005 (One way ANOVA with Tukey’s test).

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Supplementary Figure 2 Lack of roquin in T cells does not induce inflammation in skin, intestine and joint.

(a) Lung CD4+ T cells from 12–18-week-old WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice were stimulated and stained for CD4 versus IL-4. (b) Lung CD4+ T cells from 12–18-week-old WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice were stained for CD4 and RORγt. (c) Lung CD45+ cells from 12–18-week-old WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice were stimulated and stained for IL-17A versus CCR6. (a-c) FACS plots are representative of three independent experiments. (d) H&E-stained skin sections from WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice (20x magnification, scale bar represents 100 μm). (e) Quantification of CD3+, CD45+ and p-STAT3+ cells in skin sections. (f) H&E-stained small intestine sections from WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice (20x magnification, scale bar represents 100 μm). (g) Quantification of CD3+, CD45+ and p-STAT3+ cells in small intestine sections. (h) H&E-stained joint sections from WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice (40x magnification, scale bar represents 100 μm). (d, f, h) Representatives of two technical replicates from three 26–30-week-old WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice are shown. (e, g) Results represent mean ± SEM, *, P = 0.0269; NS, not significant (unpaired, two-tailed t test). (i) FACS-analyses of IL-17A versus IFN-γ in PMA and ionomycin-stimulated CD4+ T cells from small and large intestine of 12–18-week-old WT and Rc3h1fl/flRc3h2fl/flCd4-Cre mice. FACS plots are representatives of four independent experiments.

Source data

Supplementary Figure 3 Analysis of roquin cleavage by MALT1.

(a) CD4+ T cells from Rc3h1fl/flRc3h2fl/flCAG-CARstop-flCd4-Cre mice were infected with empty adenovirus or reconstituted with roquin-1 wild-type, roquin-1(R510G,R579G) or roquin-1(aa1–510) using IRES–GFP co-expressing adenoviruses. Equally infected cells were analyzed as documented by the histogram derived from pregated GFP+ cells. (b) Detection of roquin C-terminal and N-terminal cleavage fragments. CD4+ T cells were isolated from wild-type or Rc3h1fl/flCd4-Cre mice and were preincubated for 4 h with leupeptin (200 μM), MG132 (25 μM) or with DMSO as a control and then stimulated for 60 min with PMA and ionomycin (PMA + iono) or left untreated (–). Cell extracts were prepared and analyzed for roquin protein expression in immunoblots by either using the polyclonal roquin-1 antibody (upper panel) or the monoclonal roquin antibody (lower panel). Data are representatives of two (b) or three (a) independent experiments.

Supplementary Figure 4 Induced overexpression and deletion of roquin.

(a) Cells from a stably transduced Rc3h1-/-Rc3h2-/- MEF clone were left untreated or treated with doxycycline for 16-24 h to induce the expression of roquin-1 and mCherry. Cell extracts were analyzed for roquin-1 or tubulin protein expression in immunoblots. (b) Stably transduced cells from Zc3h12a-/- MEF cell cultures were left untreated or treated with doxycycline for 16-24 h to induce the expression of regnase-1 and mCherry. Cell extracts were analyzed for regnase-1 or tubulin protein expression in immunoblots. (c) Rc3h1-/-Rc3h2-/- MEF cell were treated as in a. Cells were fixed, permeabilized and stained intracellularly with an antibody specific for roquin and analyzed by flow cytometry. (d) MEF cells from Rc3h1-2fl/flCAG-CARstop-flCreERT2 mice were stably transduced with retroviruses encoding the ICOS mRNA with its 3′ UTR. The cells were left untreated or were treated with tamoxifen (0.3 μM) for 3 d before roquin protein expression was determined by immunoblot and CAR and ICOS expression were determined by FACS analysis. (f) Immunoblot analysis of protein expression of regnase-1 WT and mutant forms in reconstituted Zc3h12a-/- MEF cells using the commercial anti-regnase-1 antibody. Non-transduced WT MEF cells served as control. (g) Immunoblot analysis of protein expression of roquin-1 WT and mutant forms in reconstituted Rc3h1-/-Rc3h2-/- MEF cells. (h) Immunoblot analysis of Zc3h12a-/- MEF cells analyzing the expression of the ROQ-regnase-1 fusion protein using the newly established anti-regnase1 antibody (15D11). Non-transduced and WT MEF cells served as controls. Immunoblots in (f-h) analyzed extracts from the same cells as analyzed by FACS in Fig. 6c, d and f. The data are representative of at least two independent experiments.

Supplementary Figure 5 Evaluation of knockdown approaches.

(a) CD4+ T cells from Tg(DO11.10); Tg(CARΔ-1) mice were transduced with adenoviruses encoding GFP and an shRNA against Zc3h12a or control, as indicated. The CD4+ T cells were cultured under TH0 conditions for 3 d and protein extracts from FACS-sorted GFPhi cells were analyzed for regnase-1 protein expression by immunoblot. (b) CD4+ T cells were treated as in a and analyzed for IκBζ protein expression by FACS after restimulation of cells with PMA and ionomycin. Cells were gated on GFPhi expression. (c) CD4+ T cells from Tg(DO11.10); Tg(CARΔ-1) mice were transduced with adenoviruses encoding GFP and shRNAs against Nfkbiz or control, as indicated. CD4+ T cells were analyzed for IκBζ protein expression by FACS after restimulation with PMA and ionomycin. Cells were gated on on GFPhi expression. (d) Experiment as in a using adenoviral transduction of cells with shRNAs against Nfkbid and analysis of IκBNS protein expression in immunoblots. The data are representative of at least two independent experiments.

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Jeltsch, K., Hu, D., Brenner, S. et al. Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote TH17 differentiation. Nat Immunol 15, 1079–1089 (2014). https://doi.org/10.1038/ni.3008

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