Normalization of CD4+ T cell metabolism reverses lupus

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4(+) T cells play an essential role. CD4(+) T cells rely on glycolysis for inflammatory effector functions, but recent studies have shown that mitochondrial metabolism supports their chronic activation. How these processes contribute to lupus is unclear. We show that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4(+) T cells from lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to non-autoimmune controls. In vitro, both the mitochondrial metabolism inhibitor metformin and the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) reduced interferon-γ (IFN-γ) production, although at different stages of activation. Metformin also restored the defective interleukin-2 (IL-2) production by TC CD4(+) T cells. In vivo, treatment of TC mice and other lupus models with a combination of metformin and 2DG normalized T cell metabolism and reversed disease biomarkers. Further, CD4(+) T cells from SLE patients also exhibited enhanced glycolysis and mitochondrial metabolism that correlated with their activation status, and their excessive IFN-γ production was significantly reduced by metformin in vitro. These results suggest that normalization of T cell metabolism through the dual inhibition of glycolysis and mitochondrial metabolism is a promising therapeutic venue for SLE.

Figure 1

CD4⁺ T cells from TC mice show an enhanced metabolism. ECAR (A), OCR (B, C), and SRC (D) measured in total CD4⁺ T cells from 2 and 9 month old B6 and TC mice. (B) Representative OCR in 9 month old B6 and TC CD4⁺ T cells. (E) Extracellular lactate production from 3 month old B6 and TC CD4⁺ T cells. (F) ATP production by B6 and TC CD4⁺ T cells stimulated with PMA/ionomycin or anti-CD3/CD28. ECAR (G) and OCR (H) in Tn and Tem from 9 month old B6 and TC mice. ECAR (I) and OCR (J) in B6 and TC Tn after 24 h stimulation with anti-CD3/CD28. N = 3-6.

Figure 2

CD4⁺ T cells from TC mice show an increased mTORC1 activity. (A) S6 and 4E-BP1 phosphorylation and expression of CD98 and CD71 in total CD4⁺ T cells as well as Tn, Tem and Tcm subsets from 2 month old mice. N = 3-4. (B) ECAR and OCR in B6 CD4⁺ T cells stimulated with anti-CD3/CD28 with or without rapamycin (100 nM) for 24 h. Representative graphs of 2 independent assays each performed with N =7 technical replicates.

Figure 3

Metabolic modulators normalized TC CD4⁺ T cell effector functions in vitro. ECAR (A, C) and OCR (B, D) in B6 CD4⁺ T cells stimulated with anti-CD3/CD8 for 24 h in the presence of 2DG or Met. Representative graphs of 3 assays each performed with technical replicates. (E) IFNγ production in CD4⁺ T cells stimulated with PMA/Ionomycin/GolgiPlug for 6 h (Ctrl), in the presence of Met (2 mM), 2DG (5 mM), antimycin A/retonone (both 0.5 uM), or oligomycin (1 uM). IFNγ production in CD4⁺ T cells cultured under Th1 condition for 3 d with Met (0-5 mM in F or 1 mM in G) or 2DG (1 mM) added from d0 (F, G) or d2 (H). IL-2 production in CD4⁺ T cells stimulated with anti-CD3/CD28 for 3 d with 0 – 1 mM (I) or 1 mM (J). Panel G shown the statistical significance of a 2-way ANOVA between B6 and TC. N=3-8.

Figure 4

Treatment with Met+2DG for 3 months reversed disease in 7 month old B6 and TC mice. (A) ECAR and (B) OCR in CD4⁺ T cells. (C) Spleen weight (representative spleens on the right of 14 for TC and 5 for B6) (D). Serum anti-dsDNA IgG in TC mice (2-way ANOVA). (E) Initial (I) and terminal (T) serum ANA from TC mice. Representative images of 14 sera per group and ANA intensity quantification, with each linked symbol representing a mouse. Untreated B6 mice are shown as control (paired t-tests). (F) Autoantibody microarray analysis of terminal sera (IgG). (G) Immune complex deposition in TC glomeruli. Representative images of 14 kidneys per group with C3 and IgG2a deposits (left) and C3 intensity (3-6 glomeruli per mouse). (H) Renal pathology assessed by severity rank (median and interquantile range, 2-tailed Mann-Whitney test) and GN score distribution (χ² test). (I) Representative glomeruli (PAS stain) from untreated mice (left) showing large subendothelial deposits (arrows), and from treated mice (right) showing open capillaries and reduced hypercellularity (stars) (scale bars = 25 μm). N=14 TC and 5 B6 mice per group.

Figure 5

A 3-month Met+2DG treatment normalized CD4⁺ T cell phenotypes in aged TC mice. Percentage of total splenic CD4⁺ T cells (A), CD69⁺ (B) and Tem (C) CD4⁺ T cells in B6 and TC treated and control mice. (D) Representative CD4⁺-gated FACS plots showing the CD62L⁺ CD44⁻ Tn and CD62⁻ CD44⁺ Tem subsets. Frequency of Tfh (E) and Tfr (F) CD4⁺ T cells, and representative CD4⁺-gated FACS plots showing the PD-1^hi CXCR5^hi BCL6⁺ Foxp3⁻ Tfh and PD-1^hi CXCR5^hi BCL6⁺ Foxp3⁺ Tfr subsets (G). (H) Frequency of GC CD19⁺ B cells. (I) IL-2 production in CD4⁺ T cells stimulated with anti-CD3/CD28 for 24 h. (J-M) Effect of treatment on phosphorylation of S6 (J) and 4E-BP1 (K), as well as expression of CD98 (L) and CD71 (M) in total, Tn, and Tem T cells. N = 4-14.

Figure 6

Met+2DG treatment for one month reversed immunophenotypes in NZB/W mice. ECAR (A) and OCR (B) in splenic CD4⁺ T cells. (C) Change between the terminal and the initial serum anti-dsDNA IgG for each mouse. (D) Serum ANA intensity with each linked symbol representing the initial and final value for each mouse. Change between the terminal and initial total serum IgM (E) and IgG (F). Frequency of total CD4⁺ T cells (G), CD69⁺ (H), Tem (I), Tfh (J) and Tfr (K) CD4⁺ T cells and GC B cells (L). (M-P) Effect of treatment of mTORC1 targets: Phosphorylation of S6 (M) and 4E-BP1 (N) and expression of CD98 (O) and CD71 (P) in total CD4⁺ T cells. Renal pathology assessed by severity rank (Q, median and interquantile range) and distribution of GN mesangial (Mn 2-3 and 4) scores (R). N = 4-5.

Figure 7

CD4⁺ T cells from SLE patients have an enhanced metabolism. Representative ECAR (A) and OCR (B) graphs of human CD4⁺ T cells during a mitochondrial stress test. Anti-CD3/CD28 or isotope controls, oligomycin, FCCP and antimycin A/retonone were added to the cells as indicated. (B) ECAR (C), OCR (D) and SRC (E) in HC and SLE CD4⁺ T cells, with and without anti-CD3/CD28 activation. N=19 HC and 20 SLE. Correlations between Tn percentages and activated ECAR (F) or basal OCR (G), and between Treg percentages and activated ECAR (H). For F-H, the significance and correlation coefficient of Pearson tests are shown. (I) IFNγ production in Th1-polairized CD4⁺ T cells with or without Met. 2-tailed paired t-tests compared the effect of treatment within each cohort. N = 6.