Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression

Abstract

CD4⁺ effector T cells (T_eff cells) and regulatory T cells (T_reg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for T_eff cell proliferation and inflammatory function, the mechanisms that regulate T_reg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote T_reg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired T_reg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of T_reg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of T_reg cells.

Figure 1

T_reg cell proliferation is associated with increased glycolysis and anabolic signaling. (a,b) Flow cytometry analyzing the expression of Glut1 (a) and phosphorylated S6 (p-S6) (b) in Ki67_high and Ki67_low populations of CD4⁺Foxp3⁺ tT_reg cells from the spleens of wild-type mice (gating strategy, Supplementary Fig. 1a). MFI, mean fluorescent intensity. *P < 0.05 (two-tailed Student’s t test). Data in a and b are each representative of three independent experiments (left) or are pooled and normalized to Ki67^low from three independent experiments (right; mean + s.d.).

Figure 2

Signaling via TLR1 and TLR2 drives T_reg cell glycolysis and proliferation, yet reduces suppressive capacity. (a) Glut1 expression (as in Fig. 1) of Ki67^high and Ki67^low tT_reg cells derived from the spleens of wild-type mice an of iT_reg cells derived from CD4⁺CD25⁻ T cells isolated from the spleens of wild-type mice and polarized for 5 d under T_reg cell–skewing conditions and treated with H₂O vehicle (Veh) or Pam₃CSK₄ (PAM; 5 μg/ml) for the final 24 h. (b) Immunoblot analysis of HK2, Glut1 and actin (loading control) in iT_reg cells as in a. (c,d) ECAR of vehicle or Pam₃CSK₄ treated iT_reg (c), and Ki67 expression and phosphorylated S6, determined by flow cytometry (d), in iT_reg cells as in a. (e) Inhibition of the proliferation of T_eff cells by T_reg cells treated for 24 h with DMSO (vehicle (−)) or 20 nM rapamycin (Rap) along with Pam₃CSK₄ (5 μg/ml) (left margin), then repurified by magnetic selection and functionally assessed an in vitro suppression assay with various ratios (above plots) of T_reg cells to T_eff cells. *P < 0.05 (Two-tailed Student’s t-test). Data are representative of three independent experiments (a,b,d,e) or two independent experiments with four technical replicates per group (c; mean + s.d.).

Figure 3

Foxp3 reduces aerobic glycolysis and promotes mitochondrial oxidative metabolism. (a) Gene-expression microarray analysis and pathway analysis (with the DAVID bioinformatics database) of RNA from primary mouse CD4⁺CD25⁻ T cells stimulated and transduced with control or Foxp3-expressing retrovirus, presented as enrichment for selected pathways (left and right margins) among genes upregulated (top) or downregulated (bottom) by Foxp3. (b–g) Immunoblot analysis (b,c), glucose uptake (d), glycolysis (as ECAR after glucose injection) (e) basal OCR (f) and basal OCR/ECAR (g) of primary mouse CD4⁺CD25⁻ T cells stimulated and transduced with control (NGFR) or Foxp3-expressing retrovirus. Numbers above lanes (b,c) indicate band intensity relative to that of actin. CPM, counts per minute. *P < 0.05 (Two-tailed Student’s t-test). Data are representative of an experiment with biological triplicates for each group (a), three independent experiments (b,c) or two independent experiments with technical triplicates (d–g; mean + s.d.).

Figure 4

Foxp3 expression is sufficient to suppress glycolysis and promote oxidative metabolism. (a–i) Glucose uptake (a), glycolytic rate (assessed by a radiolabeled-glucose-tracer assay) (b), ECAR (c), flux of glucose into the pentose phosphate pathway (PPP) (d), OCR (e), OCR/ECAR (f), pyruvate oxidation (g), fatty-acid oxidation of palmitate (h) and glutamine oxidation (i) of vector-control-transduced FL5.12 pro-B cells or FL5.12 pro-B cells transduced with tamoxifen-inducible expression of Foxp3, treated with 4-hydroxytamoxifen. (j) Quantification of live cells as in a–i to assess cell accumulation over time. *P < 0.05 (two-tailed Student’s t test). Data are representative of three (a,b,j) or two (c–i) independent experiments with three independent clones per group (mean + s.d. in a–i or (mean ± s.d. in j).

Figure 5

Glut1 expression increases the number and size of T_reg cells but reduces their suppressive capacity. (a–c) Number (a), frequency (b) and size (by forward scatter (FSC)) (c) of Foxp3⁺ tT_reg cells from the spleen of control and Glut1-tg mice, assessed by flow cytometry. (d) RNA-sequencing analysis of T_reg cells from age-matched control and Glut1-tg mice (two independent cohorts), presented as normalized data. (e,f) Flow cytometry analyzing the expression of CD25, Foxp3, ID3 and EZH2 (e) or IFN-γ (f) by CD4⁺CD25⁻ T cells isolated from the spleen of control and Glut1-tg mice and polarized under T_reg cell–skewing conditions; results presented as the geometric mean fluorescent intensity (MFI) in e, right. Numbers adjacent to outlined areas (f) indicate percent IFN-γ⁺ cells. (g) Inhibition of T_eff cell proliferation by control and Glut1-tg T_reg cells as in e,f (presented as in Fig. 2e). *P < 0.05 (two-tailed Student’s t test). Data are representative of five independent experiments (a,b; mean + s.d.), four independent experiments (c; mean + s.d.), an experiment with two independent cohorts (two control mice with three Glut1-tg mice, and three control mice with one Glut1-tg mouse) (d), an experiment with four biological replicates per genotype (e,f; mean + s.d. in e) or three independent experiments (g).

Figure 6

Increased glucose uptake impairs the suppressive ability of T_reg cells. (a) Body weight of T cell– and B cell–deficient Rag1^−/− host mice given injection of sorted naive T_eff cells (CD4⁺CD25⁻CD45RB^hi) for the induction of colitis, then (after weight loss indicated active disease), given injection of no additional cells (T_eff alone) or control or Glut1-Tg CD4⁺CD25⁺CD45RB^lo T_reg cells (downward arrow), assessed by weighing three times per week and presented as change from initial weight. (b–e) Frequency of CD4⁺Foxp3⁺ T cells (b), number of CD4⁺ T cells (c) in each T_reg cell recipient group, and expression of Foxp3 and the MFI of Foxp3 expression of CD4⁺Foxp3⁺ T cells in the mesenteric lymph nodes (d,e) of mice as in a (with naive effector cells expressing Thy1.1 and T_reg cells expressing Thy1.2 in e, to allow distinction of cells by these congenic markers). *P < 0.05 (b–d; two-tailed Student’s t test, a; two-way ANOVA followed by Tukey’s post hoc test). Data are representative of at least three independent experiments with over four mice per group in each (a; mean ± s.e.m.), two independent experiments with n = 6 mice (T_eff cells alone), n = 9 mice (control T_reg cells) or n = 8 mice (Glut1-Tg T_reg cells) (b,c; mean + s.d.), two experiments with at least four mice per group (c; mean + s.d.), or one experiment with n = 4 mice (T_eff cells alone), n = 4 mice (control T_reg cells) or n = 5 mice (Glut1-Tg T_reg cells) (e).