K27-linked RORγt ubiquitination by Nedd4 potentiates Th17-mediated autoimmunity

Abstract

Background: The HECT E3 ubiquitin ligase Nedd4 has been shown to positively regulate T cell responses, but its role in T helper (Th) cell differentiation and autoimmunity is unknown. Th17 cells are believed to play a pivotal role in the development and pathogenesis of autoimmune diseases. Nevertheless, the regulation of RORγt activation during Th17 cell differentiation by TCR signaling is yet to be elucidated. These uncharted aspects inspire us to explore the potential role of Nedd4 in Th17-mediated autoimmunity.

Methods: We evaluated the impact of Nedd4 deficiency on mouse T cell development and differentiation using flow cytometry and siRNA transfection, and subsequently validated these findings in T cells from patients with multiple sclerosis (MS). Furthermore, we investigated the influence of Nedd4 deficiency on Th17-mediated autoimmunity through experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Subsequently, we elucidated the molecular mechanism underlying the interaction between Nedd4 and RORgt through immunoprecipitation, mass spectrometry analysis, and lentiviral transduction. Additionally, we identified Nedd4 as an E3 ubiquitin ligase for RORγt. Moreover, we characterized the tyrosine residue sites and polyubiquitination patterns involved in RORγt ubiquitination.

Results: In this study, we report that loss of Nedd4 in T cells specifically impairs pathogenic and non-pathogenic Th17 responses, and Th17-mediated EAE development. At the molecular level, Nedd4 binds to the PPLY motif within the ligand binding domain of RORγt, and targets RORγt at K112 for K27-linked polyubiquitination, thus augmenting its activity.

Conclusion: Nedd4 is a crucial E3 ubiquitin ligase for RORγt in the regulating Th17 cell development and offers potential therapeutic benefits for treating Th17-mediated autoimmune diseases.

Keywords: Experimental autoimmune encephalitis; HECT E3 ubiquitin ligase; K27 polyubiquitination; Nedd4; RORγt; T helper cell 17.

Fig. 1

Nedd4 potentiates Th17 cell differentiation in vitro and in vivo. A–C Th1, Th2, and iTreg cell differentiation assays using naïve CD4⁺ T cells from Nedd4^CreER mice pretreated with tamoxifen or corn oil (n = 3 mice per group). D Pathogenic vs. non-pathogenic Th17 cell differentiation assays using naïve CD4⁺ T cells from in Nedd4^CreER mice pretreated with tamoxifen or corn oil (n = 3 mice per group). ***p < 0.001; student t test. E Th1 and Th17 cells in the lamina propria of Nedd4^CreER mice small intestinal tissue pretreated with tamoxifen or corn oil (n = 3 mice per group). **p < 0.01; student t test. F Immunoblot analysis of NEDD4, NEDD4-2, and RORγT in CD4⁺ T cells from MS patients (n = 6) and age- and sex-matched healthy controls (n = 6). *p < 0.05, **p < 0.01; student t test. r = 0.737, Pearson correlation coefficient. The relationship between two variables is generally considered strong when their r value is larger than 0.7. G Expression of NEDD4 and RORγt ex vivo in total CD4⁺ T cells, vs. naïve and memory CD4 + T cells from MS patients and healthy controls (HC) by flow cytometry. Cumulative data from multiple donor samples (10 MS patients and 13 healthy donors). *p < 0.05, **p < 0.01, ***p < 0.001; unpaired student t test. H Flow cytometry evaluating Th17 cell differentiation of human naive CD4⁺ T cells from six healthy blood donors nucleofected with Ctr siRNA or Nedd4 siRNA at the temporal peak of differentiation conditions. **p < 0.01, ***p < 0.001; paired student t test. I NEDD4 knock down efficiency was determined by immunoblot analysis of lysates of T cells transfected with NEDD4 siRNA or control siRNA on day 3

Fig. 2

Nedd4 Expression in T cells confers the susceptibility of mice to EAE induction and is required for antigen-specific Th17 response. EAE was induced by MOG_35-55 in CFA, and the disease activity was monitored for 21–25 days after immunization. For ex vivo T cell responses, Cd4 Cre and Cd4 Cre-Nedd4^f/f mice, or Tcrb^–/– mice adoptively transferred with naïve CD4⁺ T cells from Nedd4^f/f and Rorc Cre-Nedd4^f/f mice were immunized with MOG_35-55 in CFA, and mice were analyzed at day 8 after immunization for Th1/Th17 in draining lymph nodes (dLNs) and at day 14–16 for total infiltrating cells and Th1/Th17 in the spinal cords. A EAE scores of Nedd4^CreER mice pretreated with tamoxifen or corn oil, or B6 mice (n = 5 mice per group) pretreated with or without tamoxifen. WT, WT-TAM, or Nedd4^CreER−oil vs. Nedd4^CreER−TAM. *p < 0.05, **p < 0.01; Mann–Whitney U test. B EAE scores of Nedd4^f/f (n = 6), Cd11c Cre-Nedd4^f/f (n = 5), LysM Cre-Nedd4^f/f (n = 6) mice. C EAE scores of Cd19 Cre (n = 7) and Cd19 Cre-Nedd4^f/f (n = 6) mice. D EAE scores of Cd4 Cre (n = 8) and Cd4 Cre-Nedd4^f/f (n = 5) mice. *p < 0.05, **p < 0.01; Mann–Whitney U test. E Flow cytometric analysis of CD4⁺IFN-γ⁺ Th1 cells and CD4⁺IL-17⁺ Th17 cells of Cd4 Cre and Cd4 Cre-Nedd4^f/f mice (n = 3 per group) at day 8 after immunization. **p < 0.01; student t test. F Flow cytometric analysis of I-A^b-MOG_35-55⁺IL-17⁺ T cells of Cd4 Cre and Cd4 Cre-Nedd4^f/f mice (n = 3 mice per group) at day 8 after immunization. *p < 0.05; student t test. G Numbers of infiltrating cells in CNS from Cd4 Cre and Cd4 Cre-Nedd4^f/f mice (n = 3 mice per group) at day 14–16 after immunization. ***p < 0.001, student t test. H Flow cytometric analysis of CD4⁺IFN-γ⁺ Th1 cells and CD4⁺IL-17⁺ Th17 cells in spinal cords of Cd4 Cre and Cd4 Cre-Nedd4^f/f mice (n = 3 per group). *p < 0.05, student t test. I EAE scores of Tcrb^–/– mice (n = 7 mice per group) adoptively transferred with CD4⁺ T cells from Nedd4^f/f and Rorc Cre-Nedd4^f/f mice upon immunization with MOG_35-55 in CFA. *p < 0.05, **p < 0.01; Mann–Whitney U test. J Flow cytometric analysis of Th1/Th17 cells in the dLNs of Tcrb^–/– mice (n = 3 mice per group) received CD4⁺ T cells (5 × 10⁶) from Nedd4^f/f and Rorc Cre-Nedd4^f/f mice at day 8 after immunization. Data are representative of two to three independent experiments

Fig. 3

RORγt PPLY Motif Binds to the WW Domains of Nedd4. A Identification of a conserved Nedd4-binding motif in RORγt ligand-binding domain. Schematic diagram of the domain structure of RORs. Conserved sequence (PPLYKEL) of the AF2 domain among members of the ROR family (upper). Schematic diagram of the domain structure of Nedd4 (lower). B Immunoblot analysis of RORγt immunopreciptates of lysates of CD4⁺ T cells stimulated under Th17-biased condition (anti-CD3, anti-CD28, TGF-β, and IL-6) for three days, and restimulated with anti-CD3 and anti-CD28, with antibodies against Nedd4 and Nedd4-2. The relative expression of Nedd4 and Nedd4-2 to RORγt were shown under the WB bands. **p < 0.01; student t test. C Immunoblot analysis of Myc immunoprecipitates of HEK293T cells transfected with Flag-tagged Nedd4, Nedd4 WW domains, Nedd4 C2 domain, Nedd4∆C2, or Nedd4∆WW, together with Myc-tagged RORγt, with anti-Flag. D GST-Nedd4 and GST-Nedd4 WW domain pull-down assay using Th17 cell lysates. E Immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4, and Myc-tagged RORγt or RORγt Y479F mutant, with anti-Flag. F EAE scores of Tcrb^–/– mice (n = 7 mice per group) receiving CD4⁺ T cells from WT and Rorc^Y479F mice immunized with immunization with MOG_35-55 in CFA. ***p < 0.001; Mann-Whitney U test. G Flow cytometric analysis of Th1/Th17 cells in the dLNs of Tcrb^–/– mice (n = 3 mice per group) received CD4⁺ T cells (5 × 10⁶) from WT and Rorc^Y479F mice at day 8 after immunization. ***p < 0.001; student t test. Data are representative of three independent experiments for B-E, and two independent experiments for F and G

Fig. 4

Nedd4 Targets RORγt for K27-Linked Polyubiquitination, thus Augmenting Its activity. A Anti-HA immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4 or Nedd4 C867A mutant, together with Myc-tagged RORγt and HA-tagged ubiquitin. B Anti-ubiquitin immunoblot analysis of RORγt immunoprecipitates of lysates of CD4⁺ T cells from WT and Nedd4^C854A mice stimulated under Th17-biased condition for three days, and restimulated with anti-CD3 and anti-CD28 for 15 min or left unstimulated. C EAE scores and Th1/Th17 responses of WT and Nedd4^C854A mice (n = 3 mice per group) immunized with MOG_35-55 in CFA. **p < 0.01; Mann–Whitney U test. D Anti-HA immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt, and HA-tagged K48, K48R, K63, or K63R ubiquitin mutants. E Anti-HA immunoblot analysis of Myc immunoprecipitates of lysates of HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt, and HA-tagged K6, K11, K27, K29, or K33 ubiquitin mutants. F Anti-ubiquitin and anti-K27 ubiquitin immunoblot analyses of RORγt immunoprecipitates of Th17 cells from Cd4 Cre and Cd4 Cre-Nedd4^f/f mice restimulated with anti-CD3 and anti-CD28 for 15 min or left unstimulated. G RORγt reporter assay using HEK293T cells transfected with RORγt reporter gene, Flag-tagged Nedd4 or Nedd4 C867A together with HA-tagged ubiquitin (left panel), or alternatively, transfected with RORγt reporter gene, Flag-tagged Nedd4, HA-tagged K27, or K27R ubiquitin mutant (right panel). H ROR binding to the RORE at CNS2 using nuclear lysates of CD4⁺ T cells stimulated under Th17-biased condition for three days, and restimulated with anti-CD3 and anti-CD28, assessed by EMSA. Data are representative of three independent experiments for A–F, and two independent experiments for G

Fig. 5

Lysine 112 Is the Ubiquitination Site in T Cells Mediated by Nedd4. A Mass spectrometric analysis of RORγt ubiquitination site(s) using purified Myc-tagged proteins from HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt, and HA-tagged ubiquitin. B Anti-HA immunoblot analysis of Myc immunoprecipitates of HEK293T cells transfected with Flag-tagged Nedd4, Myc-tagged RORγt or RORγt KXR (K99R, K112R, K169R, K288R and K495R), and HA-tagged ubiquitin. C Anti-ubiquitin immunoblot analysis of RORγt immunoprecipitates of CD4⁺ T cells from Rorc^–/– mice reconstituted with GFP-tagged RORγt or RORγt K112R, cultured under Th17-biased condition for two days, and restimulated with anti-CD3 and anti-CD28 for 15 min. D Th17 cell differentiation assay using naïve CD4⁺ T cells from Rorc^–/– mice (n = 3 mice per group) reconstituted with GFP-tagged RORγt or RORγt K112R. **p < 0.01; student t test. Data are representative of three independent experiments for B, and two independent experiments for C and D