Estrogen-related receptor-α is a metabolic regulator of effector T-cell activation and differentiation

Abstract

Stimulation of resting CD4(+) T lymphocytes leads to rapid proliferation and differentiation into effector (Teff) or inducible regulatory (Treg) subsets with specific functions to promote or suppress immunity. Importantly, Teff and Treg use distinct metabolic programs to support subset specification, survival, and function. Here, we describe that the orphan nuclear receptor estrogen-related receptor-α (ERRα) regulates metabolic pathways critical for Teff. Resting CD4(+) T cells expressed low levels of ERRα protein that increased on activation. ERRα deficiency reduced activated T-cell numbers in vivo and cytokine production in vitro but did not seem to modulate immunity through inhibition of activating signals or viability. Rather, ERRα broadly affected metabolic gene expression and glucose metabolism essential for Teff. In particular, up-regulation of Glut1 protein, glucose uptake, and mitochondrial processes were suppressed in activated ERRα(-/-) T cells and T cells treated with two chemically independent ERRα inhibitors or by shRNAi. Acute ERRα inhibition also blocked T-cell growth and proliferation. This defect appeared as a result of inadequate glucose metabolism, because provision of lipids, but not increased glucose uptake or pyruvate, rescued ATP levels and cell division. Additionally, we have shown that Treg requires lipid oxidation, whereas Teff uses glucose metabolism, and lipid addition selectively restored Treg--but not Teff--generation after acute ERRα inhibition. Furthermore, in vivo inhibition of ERRα reduced T-cell proliferation and Teff generation in both immunization and experimental autoimmune encephalomyelitis models. Thus, ERRα is a selective transcriptional regulator of Teff metabolism that may provide a metabolic means to modulate immunity.

Fig. 1.

ERRα expression regulates CD4⁺ T-cell homeostasis and function. (A) Immunoblot of resting or 1-d anti-CD3+/− anti-CD28–stimulated WT CD4⁺ T cells. (B and C) Representative histogram and quantitation of CD4⁺ CD44^high and CD4⁺ Foxp3⁺ T cells in 1-y-old sex-matched ERRα^−/− (−/−) and WT (+/+) littermates. (D) CD44 expression was measured flow cytometrically in 6- to 8-wk-old ERRα^−/− and WT resting and stimulated CD4⁺ T cells at 24 h. (E) Cytokine levels were measured by ELISA after 3 d of stimulation. (F) Frequency by FACS analysis of cytokine expressing CD4⁺ T cells from ERRα^−/− and WT mice after 3 d stimulation and acute restimulation. Results are representative of a minimum of three independent experiments, and graphs are displayed as the average and SD. *P ≤ 0.05.

Fig. 2.

ERRα contributes to activation-induced changes in CD4⁺ T-cell metabolism. (A and B) Gene expression analyses were performed on WT CD4⁺ T cells stimulated for 12 h in the presence or absence of XCT790. (A) Quantitative RT-PCR of mitochondrial energy metabolism and glucose metabolism genes. Probes exhibiting a greater than twofold change compared with stimulated T cells are indicated as increased (red) or decreased (green) with XCT790 treatment. (B) Representative graph of genes of interest confirmed by independent quantitative RT-PCR analysis. (C–E) WT and ERRα^−/− CD4⁺ T cells were examined ex vivo or activated for 24 h, and (C) cytochrome c expression, (D) glucose uptake, and (E) O₂ respiration were assessed in the presence or absence of XCT790 (XCT) or compound A (CA). Results are representative of a minimum of three independent experiments, and graphs are displayed as the average and SD. *P ≤ 0.05.

Fig. 3.

Reduced CD4⁺ T-cell growth and proliferation after acute ERRα inhibition. (A) Flow cytometric analyses of CD4⁺ T cells stimulated in the presence or absence of XCT790 or compound A for 24 h. (B) Cell proliferation was assessed by CFSE fluorescence in WT, Glut1-transgenic, or ERRα^−/− CD4⁺ T cells stimulated for 72 h in the presence or absence of XCT790 (XCT) or compound A (CA) in the presence or absence of IL-2 (10 ng/mL). (C) WT and ERRα^−/− CD4⁺ T cells were stimulated for 1 d in the presence or absence of XCT790 (XCT) or compound A (CA) and assessed for glycolytic flux. (D and E) Ovalbumin (OVA) -specific CD45.2⁺ OTII⁺ T cells were CFSE-labeled and adoptively transferred into congenic CD45.1⁺ recipient mice that were not immunized (No Stim) or immunized (Stim) with OVA and treated with vehicle or XCT790. Splenocytes were analyzed by flow cytometry on day 3. (F and G) WT CD4⁺ T cells were stimulated with XCT790 and lipids as indicated. (F) ATP levels were measured after 1 d, and (G) proliferation by CFSE dilution was measured by flow cytometry after 3 d. Results are representative of two or more independent experiments, and graphs displayed average and SDs. *P ≤ 0.05.

Fig. 4.

Exogenous lipids rescue Treg but not Teff differentiation in the absence of ERRα. (A and B) Teff (Th1, Th2, Th17) and Treg cells were generated in vitro from WT CD4⁺ T cells in the presence or absence of XCT790 (XCT) and in the presence or absence of fatty acid (FA) on day 0. (A) After 5 d, T-cell lineages were assayed by intracellular flow cytometry for cytokine production. (B) CFSE-labeled CD4⁺ T cells were differentiated in the presence or absence of XCT790 or FA and assessed by flow cytometry 3 d poststimulation. (C) On day 5 after T-cell lineages were established, XCT790 was added to cultures, and cytokine production was assessed after an additional 48 h by intracellular flow cytometry for cytokine production. Results are representative of three independent experiments.

Fig. 5.

Reduced Teff function and severity of EAE in the absence of ERRα. (A–D) EAE was induced in WT and ERRα^−/− mice with or without XCT790 (XCT) treatment as indicated. T cells from draining lymph nodes were analyzed on day 9 by flow cytometry; (A) representative plots and (B) averages of each group for cytokine production and (C) FoxP3⁺ cells are shown. Graphs are displayed as the average and SD (*P ≤ 0.05). (D) Mean clinical score and SEM of EAE disease severity in each population over 35 d (*P ≤ 0.01). Results are compiled from two independent experiments.