Mitochondrial superoxide disrupts the metabolic and epigenetic landscape of CD4+ and CD8+ T-lymphocytes

Abstract

While the role of mitochondrial metabolism in controlling T-lymphocyte activation and function is becoming more clear, the specifics of how mitochondrial redox signaling contributes to T-lymphocyte regulation remains elusive. Here, we examined the global effects of elevated mitochondrial superoxide (O₂^-) on T-lymphocyte activation using a novel model of inducible manganese superoxide dismutase (MnSOD) knock-out. Loss of MnSOD led to specific increases in mitochondrial O₂^- with no evident changes in hydrogen peroxide (H₂O₂), peroxynitrite (ONOO^-), or copper/zinc superoxide dismutase (CuZnSOD) levels. Unexpectedly, both mitochondrial and glycolytic metabolism showed significant reductions in baseline, maximal capacities, and ATP production with increased mitochondrial O₂^- levels. MnSOD knock-out T-lymphocytes demonstrated aberrant activation including widespread dysregulation in cytokine production and increased cellular apoptosis. Interestingly, an elevated proliferative signature defined by significant upregulation of cell cycle regulatory genes was also evident in MnSOD knock-out T-lymphocytes, but these cells did not show accelerated proliferative rates. Global disruption in T-lymphocyte DNA methylation and hydroxymethylation was also observed with increased mitochondrial O₂^-, which was correlated to alterations in intracellular metabolite pools linked to the methionine cycle. Together, these results demonstrate a mitochondrial redox and metabolic couple that when disrupted may alter cellular processes necessary for proper T-lymphocyte activation.

Keywords: Adaptive immunity; Apoptosis; Cytokines; Hydroxymethylation; Immune; Manganese superoxide dismutase; Metabolism; Methylation; Oxidative stress; Proliferation; Redox.

Graphical abstract

Fig. 1

MnSOD in T-lymphocytes is efficiently removed using an inducible knock-out strategy. Tamoxifen was administered to MnSOD knock-out (Cre^+/-) and control (Cre^−/-) mice for five consecutive days followed by two weeks to allow full recombination and loss of MnSOD. Splenic T-lymphocytes were isolated from mice and analyzed in either their naïve or activated (cultured with anti-CD3 and anti-CD28 beads for 96 h) states. A. Left, MnSOD gene and right, CuZnSOD gene mRNA levels assessed by quantitative real-time RT-PCR. B. Left, representative western blot of MnSOD and CuZnSOD. Right, quantification of respective proteins. C. Left, representative activity gel of MnSOD and CuZnSOD. Right, quantification of respective proteins. #p < 0.01 versus naïve Cre^−/-; *p < 0.01 versus activated Cre^−/- by parametric Student's t-test or nonparametric Mann-Whitney U test where appropriate.

Fig. 2

Loss of MnSOD specifically increases steady-state O₂^•-in T-lymphocytes. Tamoxifen was administered to MnSOD knock-out (Cre^+/-) and control (Cre^−/-) mice for five consecutive days followed by two weeks to allow full recombination and loss of MnSOD. Splenic T-lymphocytes were isolated from mice activated by anti-CD3 and anti-CD28 beads in culture for 96 h. A. Representative electron paramagnetic resonance spectrums from Cre^−/- and Cre ^± mice. Quantification located in inset. Also, flow cytometric fluorescent quantification of dihydroethidium staining (DHE) for total cellular O₂^•- (B), MitoSOX Red staining for mitochondrial O₂^•- (C), PY1 staining for total cellular H₂O₂ and ONOO⁻ (D), and MitoPY1 staining for mitochondrial H₂O₂ and ONOO⁻ (E). *p < 0.01 versus activated Cre^−/- by parametric Student's t-test or nonparametric Mann-Whitney U test where appropriate.

Fig. 3

Mitochondrial O₂^•-disrupts mitochondrial and glycolytic metabolism in T-lymphocytes. Tamoxifen was administered to MnSOD knock-out (Cre^+/-) and control (Cre^−/-) mice for five consecutive days followed by two weeks to allow full recombination and loss of MnSOD. Splenic T-lymphocytes were isolated from mice and analyzed in either their naïve or activated (cultured with anti-CD3 and anti-CD28 beads for 96 h) states. A. Upper, representative oxygen consumption rate (OCR) curves of a mitochondrial stress test. Colored lines correspond to color coding in quantifications below. Lower, quantification of metabolic states. Numbers correspond to respective time points or calculations on representative curves above. B. Upper, representative extracellular acidification rate (ECAR) curves of a glycolysis stress test. Colored lines correspond to color coding in quantifications below. Lower, quantification of metabolic states. Numbers correspond to respective time points or calculations on representative curves above. #p < 0.01 versus naïve Cre^−/-; *p < 0.01 versus activated Cre^−/- by 2-way ANOVA with multiple comparisons and Bonferroni post-hoc analysis.

Fig. 4

Loss of MnSOD increases T-lymphocyte apoptosis while increasing proliferative capacity. Tamoxifen was administered to MnSOD knock-out (Cre^+/-) and control (Cre^−/-) mice for five consecutive days followed by two weeks to allow full recombination and loss of MnSOD. Splenic T-lymphocytes were isolated from mice and analyzed in either their naïve or activated (cultured with anti-CD3 and anti-CD28 beads for 96 h) states. A. Growth curves of T-lymphocytes at various time points. N = 6. B. Flow cytometric quantification of apoptotic fraction by annexin V (AV) and propidium iodide (PI) staining. AV-/PI- indicates healthy, viable cells; AV+/PI- indicates early apoptotic cells; AV+/PI + indicates late apoptotic/necrotic cells. C. Flow cytometric quantification of CFSE staining. Open histogram represents cells stained pre-activation, while shaded histogram shows cells after 96 h of activation. Y-axis set to modal view for comparison. D. Flow cytometric quantification of the cell cycle utilizing intracellular propidium iodide staining. N = 6. E. Heat map representation of quantitative real-time RT-PCR analysis of various cell cycle genes. Each individual square represents one biological replicate. N = 5. *p < 0.01 versus activated Cre^−/- by parametric Student's t-test, nonparametric Mann-Whitney U test, or 2-way ANOVA with Bonferroni post-hoc analysis where appropriate.

Fig. 5

Global cytokine and chemokine dysregulation occurs with increased steady-state mitochondrial O₂^•-in T-lymphocytes. Tamoxifen was administered to MnSOD knock-out (Cre^+/-) and control (Cre^−/-) mice for five consecutive days followed by two weeks to allow full recombination and loss of MnSOD. Splenic T-lymphocytes were isolated from mice activated by anti-CD3 and anti-CD28 beads in culture. Culture media was utilized for cytokine and chemokine analysis. A, B, C, D, F, G, H. Cytometric bead array analysis of various secreted cytokines at 48 and 96 h post-activation. E, I, J. Mesoscale multiplex array of various cytokines and chemokines at 96 h post-activation. Black circles indicate Cre^−/-, while blue triangles indicate Cre^+/-. Data are shown normalized to cell number at the respective time points. *p < 0.01 versus activated Cre^−/- by parametric Student's t-test, nonparametric Mann-Whitney U test, or 2-way ANOVA with Bonferroni post-hoc analysis where appropriate.

Fig. 6

Elevated mitochondrial O₂^•-disrupts the T-lymphocyte metabolic-epigenetic axis. Tamoxifen was administered to MnSOD knock-out (Cre^+/-) and control (Cre^−/-) mice for five consecutive days followed by two weeks to allow full recombination and loss of MnSOD. Splenic T-lymphocytes were isolated from mice and analyzed in either their naïve or activated (cultured with anti-CD3 and anti-CD28 beads for 96 h) states. A. Global DNA cytosine methylation (5mdC) and hydroxymethylation (5hmdC) shown as percent of total nucleotides in naïve T-lymphocytes. B. Global DNA cytosine methylation (5mdC) and hydroxymethylation (5hmdC) shown as percent of total nucleotides in activated T-lymphocytes. C. TET gene mRNA levels as assessed by quantitative real-time RT-PCR. D. Schematic of the methionine cycle in reference to DNA methylation. Targeted intracellular metabolomics analysis of S-adenosyl methionine (SAMe; E), S-adenosyl homocysteine (SAH; F), SAMe/SAH ratio (G), total glutathione (GSH; H), methionine sulfoxide (Met-Ox; I), and adenosine (J). *p < 0.01 versus activated Cre^−/- by parametric Student's t-test, nonparametric Mann-Whitney U test, or 2-way ANOVA with Bonferroni post-hoc analysis where appropriate.