Myalgic encephalomyelitis/chronic fatigue syndrome patients exhibit altered T cell metabolism and cytokine associations

Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease with no known cause or mechanism. There is an increasing appreciation for the role of immune and metabolic dysfunction in the disease. ME/CFS has historically presented in outbreaks, often has a flu-like onset, and results in inflammatory symptoms. Patients suffer from severe fatigue and postexertional malaise. There is little known about the metabolism of specific immune cells in patients with ME/CFS. To investigate immune metabolism in ME/CFS, we isolated CD4+ and CD8+ T cells from 53 patients with ME/CFS and 45 healthy controls. We analyzed glycolysis and mitochondrial respiration in resting and activated T cells, along with markers related to cellular metabolism and plasma cytokines. We found that ME/CFS CD8+ T cells had reduced mitochondrial membrane potential compared with those from healthy controls. Both CD4+ and CD8+ T cells from patients with ME/CFS had reduced glycolysis at rest, whereas CD8+ T cells also had reduced glycolysis following activation. Patients with ME/CFS had significant correlations between measures of T cell metabolism and plasma cytokine abundance that differed from correlations seen in healthy control subjects. Our data indicate that patients have impaired T cell metabolism consistent with ongoing immune alterations in ME/CFS that may illuminate the mechanism behind this disease.

Keywords: Glucose metabolism; Immunology; Metabolism; Mitochondria; T cells.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. ME/CFS CD8+ T cell proton leak and ATP production are reduced compared with healthy control samples.

(A) Resting mitochondrial respiration parameters for healthy control and ME/CFS CD4+ T cells, including nonmitochondrial respiration (n = 24 healthy control samples; n = 23 ME/CFS samples [n = 24/23]), basal respiration (n = 24/23), maximal respiration (n = 24/23), proton leak (n = 11/10), ATP production (n = 11/10), and spare respiratory capacity (n = 24/23). (B) Mitochondrial respiration parameters for healthy control and ME/CFS CD4+ T cells after overnight stimulation with anti-CD3/anti-CD28 and IL-2, including nonmitochondrial respiration (n = 12/11), basal respiration (n = 12/11), maximal respiration (n = 11/11), proton leak (n = 7/7), ATP production (n = 7/7), and spare respiratory capacity (n = 11/11). (C) Resting mitochondrial respiration parameters for healthy control and ME/CFS CD8+ T cells, including nonmitochondrial respiration (n = 20/22), basal respiration (n = 20/22), maximal respiration (n = 19/21), proton leak (n = 8/12), ATP production (n = 8/12), and spare respiratory capacity (n = 19/21). (D) Mitochondrial respiration parameters for healthy control and ME/CFS CD8+ T cells after stimulation, including nonmitochondrial respiration (n = 15/11), basal respiration (n = 15/11), maximal respiration (n = 15/11), proton leak (n = 8/8), ATP production (n = 8/8), and spare respiratory capacity (n = 15/11). Box plots represent the median (middle line) and 25th and 75th quartiles (bottom and top edges of box). Whiskers represent 1.5 times the IQR and outliers are defined as values beyond whiskers. *P < 0.05 by Wilcoxon rank-sum test.

Figure 2. Mitochondrial mass and membrane potential do not differ between healthy control and ME/CFS CD4+ T cells.

(A) MTG, MTR CMXRos, and Hoechst 33342 staining of representative resting and activated control and ME/CFS CD4+ T cells. The experiment was conducted 4 times for each condition. Scale bars: 5 μm. (B) MTG and MTR CMXRos MFI as determined by flow cytometry in healthy control and ME/CFS CD4+ T cells at rest and after overnight activation (n = 15 healthy control samples at rest; n = 14 healthy control samples after activation; n = 17 ME/CFS samples at rest; n = 16 ME/CFS samples after activation). Data represent the mean ± SEM. MTG, MitoTracker Green; MTR, MitoTracker Red.

Figure 3. Mitochondrial membrane potential is decreased in CD8+ T cells from patients with ME/CFS.

(A) MTG, MTR CMXRos, and Hoechst 33342 staining of representative resting and stimulated control and ME/CFS CD8+ T cells. The experiment was conducted 4 times for each condition. Scale bars: 5 μm. (B) MTG and MTR CMXRos MFI as determined by flow cytometry in healthy control and ME/CFS CD8+ T cells at rest and after overnight activation (n = 15 healthy control samples at rest; n = 15 healthy control samples after activation; n = 17 ME/CFS samples at rest; n = 14 ME/CFS samples after activation). Data represent the mean ± SEM. **P < 0.01 by Kruskal-Wallis followed by Dunn’s test with FDR-based multiple testing correction. MTG, MitoTracker Green; MTR, MitoTracker Red.

Figure 4. Basal glycolysis is reduced in ME/CFS CD4+ T cells.

(A) Resting glycolysis measurements from Seahorse extracellular flux analysis of healthy control and ME/CFS CD4+ T cells, including basal glycolysis (n = 28), compensatory glycolysis (n = 15 healthy control samples; n = 16 ME/CFS samples [n = 15/16]), and post-2DG acidification (n = 22/17). (B) Glycolysis measurements in stimulated healthy control and ME/CFS CD4+ T cells, including basal glycolysis (n = 10/11), glycolytic capacity (n = 10/11), and post-2DG acidification (n = 7/10). (C) Percentage of GLUT1+ cells in resting and activated CD4+ T cells from patients with ME/CFS and healthy controls (n = 14 healthy control samples at rest; n = 14 healthy control samples after activation; n = 16 ME/CFS samples at rest; n = 13 ME/CFS samples after activation). Box plots represent the median (middle line) and 25th and 75th quartiles (bottom and top edges of box). Whiskers represent1.5 times the IQR) and outliers are defined as values beyond the whiskers. For dot plots, data represent the mean ± SEM. *P < 0.05; **P < 0.01, by Wilcoxon rank-sum test (A and B) and Kruskal-Wallis followed by Dunn’s test with FDR-based multiple testing correction (C).

Figure 5. Basal glycolysis is reduced in ME/CFS CD8+ T cells.

(A) Resting glycolysis measurements from Seahorse extracellular flux analysis of healthy control and ME/CFS CD8+ T cells, including basal glycolysis (n = 21 healthy control samples; n = 20 ME/CFS samples [n = 21/20]), compensatory glycolysis (n = 13/12), and post-2DG acidification (n = 20/18). (B) Glycolysis measurements in stimulated healthy control and ME/CFS CD8+ T cells, including basal glycolysis (n = 14/11), glycolytic capacity (n = 14/11), and post-2DG acidification (n = 13/9). (C) Percentage of GLUT1+ cells in resting and activated CD8+ T cells from patients with ME/CFS and healthy controls (n = 14 healthy control samples at rest; n = 13 healthy control samples after activation; n = 14 ME/CFS samples at rest; n = 14 ME/CFS samples after activation). Box plots represent the median (middle line) and 25th and 75th quartiles (bottom and top edges of box). Whiskers represent 1.5 times the IQR and outliers are defined as values beyond the whiskers. For dot plots, data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by Wilcoxon rank-sum test (A and B) and Kruskal-Wallis followed by Dunn’s test with FDR-based multiple testing correction (C).

Figure 6. Plasma cytokines are uniquely correlated with T cell metabolism in patients with ME/CFS.

Significant correlations between plasma cytokines and cellular metabolism in patients and nonsignificant correlations in healthy controls. Correlations between resting CD8+ T cell (CD8+ T) basal glycolysis and (A) IL-2 (n = 19 patients; n = 21 controls), (B) IL-8 (n = 18 patients; n = 19 controls), (C) IL-10 (n = 19 patients; n = 21 controls), (D) IL-12 p70 (n = 19 patients; n = 21 controls), (E) SCGF-β (n = 19 patients; n = 20 controls), (F) and IL-9 (n = 19 patients; n = 21 controls); between resting CD8+ T cell compensatory glycolysis and (G) M-CSF (n = 12 patients; n = 12 controls) and (H) TNF-α (n = 12 patients; n = 12 controls); (I) between activated CD8+ T cell post-2DG acidification and M-CSF (n = 9 patients; n = 13 controls); and (J) between activated CD8+ T cell glycolytic capacity and M-CSF (n = 10 patients; n = 14 controls). All correlations were evaluated using a Spearman’s correlation test with FDR-based multiple testing correction, where a q value of less than 0.01 was considered significant.

Figure 7. Plasma cytokines correlate with T cell metabolism in healthy controls.

Significant correlations between plasma cytokines and cellular metabolism in healthy control subjects and nonsignificant correlations in patients. Correlations between (A) activated CD4+ T cell basal respiration and IL-17 in control subjects (n = 12) and patients (n = 10); (B) between activated CD4+ T cell maximal respiration and IL-17 in control subjects (n = 15) and patients (n = 10); (C) between activated CD4+ T cell basal respiration and IL-9 in control subjects (n = 12) and patients (n = 10); and (D) between resting CD8+ T cell spare respiratory capacity and G-CSF in control subjects (n = 19) and patients (n = 20). All correlations were evaluated using a Spearman’s correlation test with FDR-based multiple testing correction, where a q value of less than 0.01 was considered significant.