Increased Mitochondrial Biogenesis and Reactive Oxygen Species Production Accompany Prolonged CD4+ T Cell Activation

Abstract

Activation of CD4⁺ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following activation, mouse CD4⁺ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Moreover, prolonged activation of CD4⁺ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4⁺ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

Figure 1.

Activated CD4⁺ T cells remodel their glucose uptake machinery in order to meet increased energy demand. For all experiments purified naïve and 96 h ex vivo activated CD4⁺ T cells were used. A-B) Cells were FACS sorted for viability and 5×10⁵ viable cells were immobilized onto each well of a 96 well Seahorse analyzer where they were starved of glucose for 30 min and basal ECAR levels were measured. Glucose, A/R and 2DG were added consequently after this period and changes in ECAR values were recorded. A) Arrow heads indicate the time points treatments were added. Symbols and error bars refer to mean and SD of triplicates. B) ECAR values pooled from four independent glycolysis stress tests (Glycolysis= ECAR_post-glucose-ECAR_basal, glycolytic capacity=ECAR_post-A/R-ECAR_post-2DG). Symbols demonstrate the means of individual experiments, lines mark the mean of the pooled data. C-F) The expression levels of GLUT-1 (C,D) and GLUT-3 (E,F) as measured by flow cytometry. Representative histograms (C,E) and bar graphs (D,F) demonstrating the MFI values are shown. Bars and error bars represent mean and standard deviation of triplicates. G) Cells were stained with Live-DEAD and incubated in the presence of 10 µM 2-NBDG for up to 90 min. At each time point aliquots were harvested and run on flow cytometer. MFI values were then normalized by subtracting the background MFI. Symbols and error bars represent mean and standard deviation of quadruplicates respectively. H) Expressions of Slc2a1 and Slc2a3 genes encoding GLUT-1 and GLUT-3 respectively are quantified using RNA isolated from naïve and activated T cells. Bars and error bars represent mean and SEM of five independent experiments respectively. Data in (A,C-G) represent four independent experiments. Statistical significance was measured with Welch’s t-test (B,D,F,H) or Two-way ANOVA with Sidak’s multiple comparisons analysis (G). (0.01<P≤0.05=*, 0.001<P≤0.01=**, P ≤0.0001= ****)

Figure 2.

T cells continuously activated in vitro, also increase their mitochondrial respiration despite greatly accelerated glycolysis. For all experiments purified naïve and 96 h ex vivo activated CD4⁺ T cells were used A-C) Cells were FACS sorted for viability and 5×10⁵ viable cells were immobilized onto each well of a 96 well Seahorse analyzer. Upon basal OCR measurements cells were sequentially treated with oligomycin, 2,4 DNP and A/R on time points indicated with arrow heads. A) Graph represents the changes in OCR values. Symbols and error bars refer to mean and SD of each recording. B) OCR values pooled from four independent mitochondrial stress tests (Basal: OCR_initial - OCR_post-A/R, maximal respiration=OCR_post-DNP - OCR_post-A/R, ATP-coupled respiration= OCR_Basal - OCR _{post-Oligomycin}). Symbols demonstrate means of individual experiments, lines mark the mean of the pooled data. C) Ratio of the basal OCR to basal ECAR values, pooled from the mitochondrial stress tests above. D-F) Mitochondrial membrane potentials were measured using TMRM either alone or in combination with oligomycin or FCCP. Representative histograms (D), MFI bar graphs (E) and percent of the maximum potential graphs (F) are shown. Data represent four independent experiments each carried out with triplicates. Statistical significance was calculated using Welch’s t-test. (P>0.05=ns; 0.01<P≤0.05=*, 0.0001<P≤0.001=***)

Figure 3.

Increased ROS production following T cell stimulation persists during prolonged activation without causing oxidative stress induced mitochondrial dysfunction. For all experiments purified naïve and 96 h ex vivo activated CD4⁺ T cells were used. A,B) Cells were stained with SYTOX Blue viability dye together with CellROX (A) or MitoSOX (B). Representative histogram overlays (top panels) and graphs of MFI values (bottom panels) are shown. Bars and error bars represent mean and standard deviation respectively. Data represents three independent experiments each carried out with quadruplicates. C) Transcription of genes coding for anti-oxidant enzymes glutathione reductase (Gsr), glutathione peroxidase 1 (Gpx1) superoxide dismutase (Sod1 and Sod2) and catalase (Cat) as measured by qPCR are shown for both activated and naïve T cells. Bars and error bars refer to mean of individual samples obtained from five independent experiments and standard error of the mean respectively. D) TEM electron micrograms of naïve and activated T cells. Images represent sections obtained from 10 individual cells. N refers to nucleus (Scale bars = 400 nm). Statistical significance was calculated using Welch’s t-test. (P>0.05=ns, 0.001<P≤0.01=**, 0.0001<P≤0.001=***)

Figure 4

Prolonged T cell activation leads to an increase in total mitochondrial content. For all experiments purified naïve and 96 h ex vivo activated CD4⁺ T cells were used A,B) Cells were stained with a viability dye together with various mitochondria specific markers and analyzed in flow cytometry. Representative histogram overlays (A) and MFI graphs (B) are shown. Bars and error bars represent mean of triplicates and standard deviation respectively. C) Live cells were FACS sorted and equal numbers of naïve and activated cells were lysed, separated in protein gel and immunoblotted for mitochondrial markers as well as H3 for loading control. Representative western blot image is shown. Data represent two independent experiments. D) Total DNA was isolated from naïve and FACS sorted live activated CD4⁺ T cells. Graphs show mitochondrial DNA copy numbers relative to genomic DNA that were measured using qPCR. Bars and error bars refer to mean of individual samples obtained from five independent experiments and standard error of the mean respectively. Statistical significance was calculated using Welch’s t-test. (*=0.01<P≤0.05; ***=0.0001<P≤0.001; P ≤0.0001= ****)

Figure 5

Metabolic remodeling due to activation can be prevented by the inhibition of NO or mTOR pathways. Purified naïve CD4⁺ T cells were activated up to 96 h ex vivo. A-B) Time dependent changes in activation status (CD44), GLUT1 expression and mitochondrial content (TOM20 and COXIV). Representative histogram overlays (A) and MFI graphs (B) are shown. Symbols and error bars represent mean of triplicates and standard deviation respectively. Time points were compared against 0 h. C-E) Naïve CD4⁺ T cells were activated for 24 h ex vivo in the presence of Rapamycin or Carboxy-PTIO where indicated. Representative histogram overlays and MFI graphs for Nitric Oxide levels (C), mTOR dependent surface markers (D) and metabolic markers (E) are shown. Bars and error bars represent mean of triplicates and standard deviation respectively. Data are representative of two independent experiments. Freshly isolated naïve CD4⁺ T cells were used as control. Statistical significance was measured using one-way ANOVA with Dunnett’s (B) or Tukey’s (C-E) multiple comparisons analysis (P>0.05=ns, *=0.01<P≤0.05, 0.001<P≤0.01=**, ***=0.0001<P≤0.001, P ≤0.0001= ****).

Figure 6

In vivo activation of CD4⁺ T cells leads to changes that are similar to those observed in vitro. A) Schematic outline of the adoptive transfer experiment. B) Gating strategy used to discriminate adoptively transferred OT-II and polyclonal T cells in recipient mouse spleens. C) Histogram overlays showing the levels of CD44 expression (left) and the extent of proliferation (right) in adoptively transferred polyclonal and OT-II T cells four days post stimulation with DCs pulsed with control GP peptide (top) or OVA peptide (bottom). D-G) T cells were activated in vivo for four days as illustrated in (A). Histogram overlays (left) and MFI graphs (right) show the levels of GLUT1 (D), TOM20 (E), COXIV (F) and MitoSOX (G). Each circle is an individual mouse. Data represent two independent experiments. Statistical significance was calculated using Two-way ANOVA with Sidak’s multiple comparisons test. (P>0.05=ns; P ≤0.0001= ****)

Figure 7

High-resolution multicolor imaging of lymphocytes reveals mitochondrial changes in CD4⁺ T cells induced upon prolonged activation. Cells activated for four days in vitro or freshly isolated from spleens of C57BL/6 mice were stained with Live/Dead and MitoTracker CMXROS red, then mounted on Poly-L lysine coated coverslips. Cells were then fixed, permeabilized and stained for TOM20 and DAPI as described in methods. A) Microscope image demonstrating viable (white arrow heads) and non-viable (yellow arrow heads) cells in the same area of interest (Scale bar 4 µm). B) Outline of the sequential image processing strategy used for distinguishing mitochondrial boundaries in lymphocytes. Naïve CD4⁺ T cells, stained as outlined above, were imaged for TOM20, Mitotracker, and DAPI in STED system Images left to right show microscope images of TOM20, Mitotracker, their merged view with DAPI, combined channel showing sum of both mitochondrial markers in one channel, computer generated surface of the combined channel and individual mitochondria estimates based on the watershed splitting algorithm applied to the combined surface (Scale bars: 1 µm). C-F) Naïve and activated T cells were imaged and analyzed in groups of five cells per area of interest as described above. Representative STED microscope images (C) and data derived from image analysis showing average total mitochondrial volumes per cell (D), average number of mitochondria per cell (E) and volume distribution of individual mitochondria (F) are given. Lines represent mean values, symbols represent averages of five cells (D, E) or individual mitochondrion (F). Data represent two independent experiments each with at least 25 cells analyzed per condition. Statistical significance was calculated using Welch’s t-test. (P ≤0.0001= ****)