Nur77 serves as a molecular brake of the metabolic switch during T cell activation to restrict autoimmunity

Abstract

T cells critically depend on reprogramming of metabolic signatures to meet the bioenergetic demands during activation and clonal expansion. Here we identify the transcription factor Nur77 as a cell-intrinsic modulator of T cell activation. Nur77-deficient T cells are highly proliferative, and lack of Nur77 is associated with enhanced T cell activation and increased susceptibility for T cell-mediated inflammatory diseases, such as CNS autoimmunity, allergic contact dermatitis and collagen-induced arthritis. Importantly, Nur77 serves as key regulator of energy metabolism in T cells, restricting mitochondrial respiration and glycolysis and controlling switching between different energy pathways. Transcriptional network analysis revealed that Nur77 modulates the expression of metabolic genes, most likely in close interaction with other transcription factors, especially estrogen-related receptor α. In summary, we identify Nur77 as a transcriptional regulator of T cell metabolism, which elevates the threshold for T cell activation and confers protection in different T cell-mediated inflammatory diseases.

Keywords: Nur77; T cells; autoimmunity; immunometabolism.

Fig. 1.

Nur77 restricts proliferation and activation of T cells. (A) Appearances of spleens from 30-wk-old C57BL/6J and Nur77^KO mice. The graph displays total cell numbers; n = 6/group. (B) Frequency of splenic CD4⁺ T cells was determined in 6- or 30-wk-old C57BL/6J or Nur77^KO mice by flow cytometry; n = 6/group. (C) Homeostatic T cell proliferation of eFluor670-labeled Nur77^WT-OTII and Nur77^KO-OTII T cells in Rag1^KO mice; n = 7–8/group. The mean percentage of proliferated splenic T cells was determined by flow cytometric analysis on day 10 after transfer. (D) Nur77^WT-OTII and Nur77^KO-OTII T cells were stimulated in an antigen-specific manner in the presence of 10 ng/mL IL-7 as well as blocking antibodies against MCH-II and IL-7 (both 5 µg/mL). After 6 d, T cells were labeled with eFluor670 and injected into Rag1^KO mice; n = 2/group. The mean fluorescence intensity (MFI) of proliferated T cells was determined by flow cytometric analysis on day 6 after transfer. (E) Surface expression of CD25 or CD69 (markers of early activation; Left) or CD44 and CD62L (markers of memory phenotype; Right) on splenic CD4⁺ T cells from 30-wk-old C57BL/6J and Nur77^KO mice was analyzed by flow cytometry. The percentage of positive T cells or MFI is shown; n = 6/group. (F) Nur77 expression of αCD3/αCD28-stimulated WT CD4⁺ T cells was determined by flow cytometry at the indicated time points. (G) CD4⁺ T cell (C57BL/6J or Nur77^KO) proliferation on αCD3 stimulation was analyzed by flow cytometry after 72 h. Shown are representative histograms and division index. (H) WT and Nur77^KO CD4⁺ T cells were subjected to Th1 (IFN-γ, Left) and Th17 (IL-17A, Right) conditions. Cytokine production on restimulation was assessed by flow cytometry after 72 h. (I) Nur77^WT-OTII or Nur77^KO-OTII T cells were cocultured with OVA_323–339–loaded DCs. After 72 h, proliferation (Left) and cytokine secretion (Right) were analyzed. (J) Nur77 expression was analyzed in αCD3-stimulated human CD4⁺ T cells by immunohistochemistry after 4 h and flow cytometry at the indicated time points, respectively. (Scale bar: 200 µm.) (K) Human CD4⁺ T cells were cotransfected with control-siRNA or Nur77-siRNA. Nur77 expression was analyzed in transfected CD4⁺ T cells after αCD3 stimulation for 3 h. Representative dot plots are shown. (L) Cytokine production of transfected and αCD3 ± αCD28-stimulated CD4⁺ T cells was analyzed by ELISA after 72 h. Graphs display mean ± SEM of one representative experiment of three experiments, unless stated otherwise. Statistical analysis was performed using Student’s t test. *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.001.

Fig. 2.

Nur77 restricts T cell-mediated autoimmunity. (A) Clinical monitoring of Nur77^WT-2D2 (n = 15) and Nur77^KO-2D2 (n = 16) mice. Statistical analysis is described in Methods. (B) Spatial frequency was recorded in 15-wk-old Nur77^WT-2D2 and Nur77^KO-2D2 mice and is shown based on clinical score; n = 4–6/group. (C) Histological analysis was performed in 25-wk-old mice from A with regard to infiltration of myeloid cells (MAC3) and demyelination [luxol fast blue (LFB)]. (Scale bars: 200 µm.) Graph depicts mean inflammatory index; n = 14/group. (D) Cell numbers (Left) and IFN-γ-production (Right) of CNS infiltrating CD4⁺ T cells in 15-wk-old mice from A were determined by flow cytometry; n = 8/group. (E) Active EAE was induced by MOG_35–55 immunization in WT and Nur77^KO mice. Mean clinical score of two pooled independent experiments is depicted; n = 15/group. (F) On day 13 of EAE, mice from E were histologically analyzed for infiltration of myeloid cells (MAC3), T cells (CD3), and demyelinated area (LFB). (Scale bars: 200 µm.) Graphs show the mean inflammatory index and number of CD3⁺ T cells in the white matter; n = 7/group. (G) Numbers of IL-17A– and IFN-γ–producing CD4⁺ T cells were determined on day 15 of EAE in lymph nodes and CNS by ELISpot analysis after ex vivo restimulation with MOG_35–55 for 24 h; n = 10/group. (H) On day 15 postimmunization, the frequency of Treg cells was determined in draining lymph nodes; n = 6/group. (I) Splenocytes were isolated from MOG_35–55–immunized mice on day 10 of EAE and then ex vivo stimulated with MOG_35–55. Cytokine secretion was determined by ELISA; n = 6/group. (J and K) Adoptive transfer EAE experiments were performed with either transfer of WT and Nur77^KOcells into WT recipients (J) or transfer of WT cells into WT and Nur77^KO recipients (K). Shown is the clinical score. Graphs depict mean ± SEM. Additional information on all EAEs is provided in SI Appendix, Table S6. Shown is one representative experiment out of at least two experiments. Statistical analysis was performed using Student’s t test and two-way ANOVA with Bonferroni posttest (E, I, and J). *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.001.

Fig. 3.

Nur77 limits T cell responses by restriction of T cell metabolism and cell cycle progression. (A and B) Frequencies of early apoptotic (Caspase3/7⁺, PI⁻) and late apoptotic (Caspase3/7⁺, PI⁺) CD4⁺ T cells were determined in spleens of WT or Nur77^KO mice (A) and in Nur77^WT-2D2 or Nur77^KO-2D2 mice (B), respectively. Frequency of early and late apoptotic T cells was assessed either ex vivo or after 72 h of stimulation with αCD3/αCD28 (A and B) or addition of MOG_35–55 (B); n = 5 mice/group. (C) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) by CD4⁺ T cells were determined after 72 h of αCD3/αCD28 stimulation. Graphs depict mean basal and maximal respiration (Left) or mean glycolysis and glycolytic capacity (Right). (D) Basal respiration and glycolysis of activated WT and Nur77^KO CD4⁺ T cells analyzed at the indicated time points. In parallel, Nur77 expression of stimulated WT CD4⁺ T cells was determined. (E) CD4⁺ T cells were stimulated with αCD3/αCD28 for 3 d. Rotenone (100 nM) and/or 2-deoxy-d-glucose (2-DG) (5 mM) were initially added during stimulation as indicated. T cell proliferation was assessed by flow cytometry, and the percentage of inhibition was calculated by (% proliferation [without inhibitor])/(% proliferation [± Rotenone ± 2-DG]). Graphs depict mean ± SEM. Shown is one representative experiment out of three experiments, unless stated otherwise. Statistical analysis was assessed with Student’s t test. *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.001.

Fig. 4.

Nur77 regulates genes required for metabolic functions. (A and B) Gene expression analysis of mitochondrial energy metabolism and glucose metabolism genes was performed with αCD3/αCD28-activated Nur77^KO and WT CD4⁺ T cells; n = 6 mice/group. (A) Venn diagram of all significant differential expressed genes (false discovery rate, <0.05). The orange color in the Venn diagram indicates the overlap between genes regulated by T cell stimulation (stim-dep genes) and genes regulated by Nur77 (Nur77-reg genes) (stim-dep WT genes, n = 68; stim-dep Nur77^KO genes, n = 78; Nur77-reg genes, n = 21). (B) Functional gene expression network of stimulated Nur77^KO vs. WT CD4⁺ T cells (Pearson correlation coefficient ≥0.9), showing analysis of all investigated OXPHOS genes (rectangle) and glycolytic genes (circle), indicated as increasedly (red) or decreasedly expressed (blue). iRegulon-based analysis led to the identification of five transcription factors (hexagon) with enriched binding sites among network genes. (C–E) Unbiased RNA-Seq analysis was performed with αCD3/αCD28-activated Nur77^KO and WT CD4⁺ T cells; n = 4 mice/group. (C) Venn diagram of all significant differentially expressed genes (false discovery rate, <0.05). Orange indicates the overlap between genes regulated by T cell stimulation (stim-dep genes) and genes regulated by Nur77 (Nur77-reg genes) (stim-dep WT genes, n = 8,357; stim-dep Nur77^KO genes, n = 10,549; Nur77-reg genes, n = 3,725). (D) Heatmap depicting the normalized and transformed read counts of all significant genes from the activated Nur77^KO and WT CD4⁺ T cells. (E) Principal component analysis of data from D including Nur77^KO and WT CD4⁺ T cells under control conditions (unstimulated).

Fig. 5.

ERRα inhibition partially reverses Nur77-mediated effects on T cell metabolism and T cell-mediated CNS autoimmunity. (A) ChIP-seq analysis of WT CD4⁺ T cells activated with αCD3/αCD28 for 4 h. Nur77 binds to target genes encoding for transcription factors that regulate T cell metabolism. The red line displays a narrow peak region from peak calling with MACS2 (q < 0.05; n = 6/group). (B) ERRα expression by WT and Nur77^KO CD4⁺ T cells after 72 h of αCD3/αCD28 stimulation. Shown is a representative histogram; the graph depicts the mean fluorescence intensity (MFI) of ERRα expression; n = 4/group. (C) WT or Nur77^KO CD4⁺ T cells were activated with αCD3/αCD28 for 12 h in the presence of ERRα inhibitors XCT790 (XCT; 10 µg/mL) or Compound A (CompA; 10 µg/mL) before T cells were subjected to RNA-Seq. Both plots depict the natural logarithm of the mean of normalized read counts from ERRα target genes; n = 4 mice/group. (D) WT or Nur77^KO CD4⁺ T cells were αCD3/αCD28-activated and treated with 2.5 µg/mL XCT. After 72 h, the mean cytokine production was determined by ELISA. (E) Maximal respiration and glycolytic capacity of αCD3/αCD28-stimulated WT and Nur77^KO CD4⁺ T cells was determined on day 3 of αCD3/αCD28 stimulation in the presence or absence of XCT (2.5 µg/mL). Shown is a representative run. Bar graphs depict OCR and ECAR; n = 3 mice/group. (F) MOG_35–55 EAE was induced in WT or Nur77^KO mice. In parallel, 5 mg/kg body weight XCT or vehicle only (DMSO) were administered daily by oral gavage. Shown is the mean clinical score; the graph depicts the mean ± SEM cumulative clinical score; n = 7/group. (G) On day 13 postimmunization, numbers of CNS-specific Th1 and Th17 cells were determined by flow cytometry; n = 6/group. (H) On day 10 of EAE, T cells were isolated from draining lymph nodes, and basal respiration as well as glycolysis was determined ex vivo; n = 10–13/group. (I and J) Nur77^KO or WT CD4⁺ T cells were lentivirally transfected with control-shRNA (scr) or Esrra-shRNA (shA) and subsequently stimulated with αCD3/αCD28 for 72 h. (I) Then IFN-γ production was assessed by flow cytometry; representative dot plots are shown. (J) Alternatively, qRT-PCR analysis of metabolic genes was performed. Graphs display mean ± SEM. Statistical analysis was performed using Student’s t test and two-way ANOVA with Bonferroni posttest (F–H). Shown is one representative experiment out of three experiments, unless stated otherwise. *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.001. Oligo, oligomycin; Rot, rotenone; AA, antimycin A; 2-DG, 2-deoxy-d-glucose.

Fig. 6.

Nur77 restricts T cell responses in contact dermatitis and autoimmune arthritis. (A–D) Allergic contact dermatitis (ACD) was induced in C57BL/6J or Nur77^KO mice. (A) Relative ear thickness of mice was determined; n = 7–9/group. (B) On day 2 of ACD, ear tissue was stained with hematoxylin and eosin; n = 7 mice/group. (Scale bars: 200 µm.) (C) Numbers of CD4⁺ and CD8⁺ T cells (Left) and IFN-γ⁺ CD4⁺ and CD8⁺ T cells (Right) were determined in auricular and axillar lymph nodes by flow cytometry; n = 6/group. (D) In addition, expression of CD69 on T cells was determined by flow cytometry; the mean fluorescence intensity (MFI) is depicted. Shown is one representative out of two experiments. (E and F) Collagen-induced arthritis (CIA) was induced in WT and Nur77^KO mice by Collagen type II immunization. (E, Left) On day 59 of CIA, cell numbers of CD4⁺ and CD8⁺ T cells and intracellular cytokine production (IFN-γ) of both cell types were determined in inguinal and popliteal lymph nodes by flow cytometry. (E, Right) Mean MFI of CD69 in CD4⁺ and CD8⁺ T cells; n = 6/group. (F) Clinical evaluation of CIA induced in WT and Nur77^KO (n = 8–10/group) (SI Appendix, Table S6). Data were generated in one experiment. Infl., inflamed. Data are depicted as mean ± SEM. *P ≤ 0.05; **P ≤ 0.001; ***P ≤ 0.001.