Oxidative stress is a shared characteristic of ME/CFS and Long COVID

Abstract

Over 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition marked by fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). With no clinically approved treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in 25 healthy controls, 27 ME/CFS, and 20 LC donors, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, especially in the memory subset. Using a combination of flow cytometry, RNA-seq, mass spectrometry, and systems chemistry analysis, we observed aberrations in reactive oxygen species (ROS) clearance pathways including elevated glutathione levels, decreases in mitochondrial superoxide dismutase protein levels, and glutathione peroxidase 4-mediated lipid oxidative damage. Strikingly, these redox pathways changes show sex-specific trends. While ME/CFS females exhibit higher total ROS and mitochondrial calcium levels, males have normal ROS levels, with pronounced mitochondrial lipid oxidative damage. In females, these higher ROS levels correlate with T cell hyperproliferation, consistent with the known role of elevated ROS in initiating proliferation. This hyperproliferation can be attenuated by metformin, suggesting this Food and Drug Administration (FDA)-approved drug as a possible treatment, as also suggested by a recent clinical study of LC patients. Moreover, these results suggest a shared mechanistic basis for the systemic phenotypes of ME/CFS and LC, which can be detected by quantitative blood cell measurements, and that effective, patient-tailored drugs might be discovered using standard lymphocyte stimulation assays.

Keywords: ME/CFS; fatigue; long COVID; metabolism; oxidative stress.

Fig. 1.

Comparison of total ROS levels in lymphocytes of HC, ME/CFS, and LC donors. (A) Peripheral blood mononuclear cells were characterized from 25 HC, 20 LC, and 27 ME/CFS samples. Age and gender distributions are shown. (B) Representative flow cytometry plots (normalized to the mode) for ROS indicator DCFDA are shown for CD4 T cells for a single HC, LC, and ME/CFS donor. For (C–D), * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 based on a two-sided t test. (C) Comparing 16 HC, 15 LC, and 15 ME/CFS donors, DCFDA staining shows higher total MFI ROS levels in ME/CFS (CD4T P = 5.60 × 10⁻⁴, CD8T P = 1.91 × 10⁻⁴, CD19B P = 3.04 × 10⁻⁴) and LC (CD4T P = 0.104, CD8T P = 0.0210, CD19B P = 0.0727) lymphocytes compared to HCs. (D) ROS levels are uniquely elevated in ME/CFS and LC females, compared to controls (females: ME/CFS vs. HC: CD19B P = 0.00065, CD4T P = 0.00045, CD8T P = 9.5 × 10⁻⁵; LC vs. HC: CD19B P = 0.144, CD4T P = 0.062, CD8T P = 0.01065). In contrast, males show no significant differences (males ME/CFS vs. HC: CD19B P = 0.357, CD4T P = 0.469, CD8T P = 0.516; males LC v. HC: CD19B P = 0.278, CD4 T P = 0.643, CD8T P = 0.583).

Fig. 2.

Summary of identified sex-specific redox pathway differences in ME/CFS and LC donors, compared to controls. ME/CFS and LC donor lymphocytes exhibit oxidative stress and mitochondrial damage, leading to proliferation defects that drive lymphocytes to consume excess energy. ROS generation and clearance pathways are shown, including mitochondrial superoxide, calcium, and superoxide dismutase mediated-conversion to hydrogen peroxide, and hydrogen peroxide breakdown by catalase and glutathione. Sex-specific alterations in these pathways among ME/CFS, LC donors are shown (females in purple, males in gray). The upward arrow corresponds to elevation of parameter in ME/CFS and LC donors, compared to control counterparts.

Fig. 3.

Comparison of intermediates and proteins involved in ROS generation and clearance within lymphocytes between HC, LC, and ME/CFS donors. (A) Rhod-2 AM flow cytometric staining identifies significantly elevated mitochondrial calcium levels in ME/CFS and LC female donors (females: ME/CFS vs. HC CD19B P = 0.00238, CD4T P = 0.0063, CD8T P = 0.0137; LC v. HC CD19B P = 0.0097, CD4T P = 0.0172, CD8T P = 0.240; males: ME/CFS vs. HC CD19B P = 0.791, CD4T P = 0.514, CD8T P = 0.825; LC vs. HC CD19B P = 0.851, CD4T P = 0.241, CD8T P = 0.358). (B) Flow cytometric staining shows significantly lower mitochondrial superoxide dismutase 2 (SOD2) protein levels in LC donors (HC vs. ME/CFS CD19 B cells P = 0.326, CD4 T cells P = 0.638, CD8 T cells P = 0.384; HC vs. LC CD19 B cells P = 0.073, CD4 T cells P = 0.023, CD8 T cells P = 0.023). (C) ME/CFS lymphocytes show significantly higher glutathione (GSH) levels (females ME/CFS vs. HC CD19B cells P = 0.00048, CD4T cells P = 0.00014, CD8T cells P = 0.00159; LC vs. HC CD19B P = 0.580, CD4T P = 0.827, CD8T P = 0.958; males ME/CFS vs. HC CD19B P = 0.0327, CD4T P = 0.0354, CD8T P = 0.0894; LC vs. HC CD19B P = 0.196, CD4T P = 0.093, CD8T P = 0.278). (D) Based on the reaction between hydrogen peroxide (H₂O₂) and glutathione (GSH) in Fig. 2 (H₂O₂ + 2GSH ←→ 2H₂O + GSSG), the association between GSH and total ROS levels (Fig. 1C) is shown for both females (Top row) and males (Bottom row). With each point corresponding to a donor, the plot shows a statistically significant positive correlation between total ROS and GSH levels (females CD19B R = 0.56, P = 0.0039, CD4T R = 0.63 P = 6.9 × 10⁻⁴; CD8T R = 0.52 P = 7.8 × 10⁻³; males CD19B R = 0.5 P = 0.025, CD4T R = 0.56 P = 0.01, CD8T R = 0.43 P = 0.057).

Fig. 4.

Mass-spectrometry and immunofluorescence analysis suggests lipid oxidative damage in ME/CFS and LC patients. (A) Using extracted metabolites from 200,000 sorted CD3 T cells from ME/CFS and HC donors, hydrophilic interaction liquid chromatography-mass spectrometry and Turium-based systems chemistry analysis were conducted to map metabolic pathway differences between ME/CFS donor and HC T cells. From 747 identified metabolites, the top five mapped pathway differences are shown. Size of the nodes corresponds to magnitude of the difference in relative abundance of detected metabolites from mass spectrometry between ME/CFS and HC samples. (B) The reactions encoded by the large red circles (A) correspond to reactions significantly up-regulated in ME/CFS donors compared to HCs. The phospholipid synthesis reactions involve the production of lysoPE metabolites and are catalyzed by PLA2. (C) Reanalysis of bulk RNA-seq data (45) between 11 sedentary controls and 14 ME/CFS patients identifies significantly higher PLA2 transcript levels in ME/CFS donors (P = 0.044, two-sided t test). FPKM refers to fragments per kilobase of transcript per million mapped reads. (D) Representative images from immunofluorescence staining are shown for GPX4 in CD3 T cells from 1 HC, 1 LC, and 1 ME/CFS donor (blue corresponds to DAPI, red to CD3, magenta to MitoTracker Far Red, green to GPX4). (E) Quantification of average GPX4 pixel intensity from 1 HC, 1 LC, and 1 ME/CFS donor. Each point corresponds to a CD3 T cell, with comparisons conducted across 426 HC, 603 ME/CFS, and 239 LC T cells from maximum projection images. Significant increases in average pixel intensity indicate higher GPX4 levels in ME/CFS and LC donors, compared to HCs (P < 2.2 × 10⁻¹⁶ for both ME/CFS and LC from two-sided t test).

Fig. 5.

Sex-specific lipid oxidative damages in ME/CFS and LC donor lymphocytes, especially in mitochondria. (A) From Phetsouphanh et al. single cell RNA-seq data (Nat. Commun. 2024; GSE262861 (46)), GPX4 transcript levels are uniquely elevated in female LC donors central memory T cells, compared to recovered controls. Comparisons conducted at 4 (13F, 12M), 8 (11F, 11M), 24 (11F, 11M) months after infection (Female: 4 mo P = 0.0024, 8 mo = 0.00077, 24 mo P = 0.0032; Male: 4 mo P = 0.59, 8 mo P = 0.63, 24 mo P = 0.3, two-sided t test). (B) Flow cytometric comparison of lipid peroxide levels, using ratiometric lipid peroxidation sensor, shows both male ME/CFS and LC donor lymphocytes have significantly higher lipid peroxidation, compared to controls (males ME/CFS vs. HC: CD19B P = 0.0027, CD4T P = 0.0037, CD8T P = 0.0041, LC vs. HC CD19B P = 0.003, CD4T P = 0.0033, CD8T P = 0.0029; females ME/CFS vs. HC: CD19B P = 0.076, CD4T P = 0.448, CD8T P = 0.131, LC vs. HC: CD19B P = 0.0365, CD4T P = 0.236, CD8T P = 0.0977). Lower y-axis values correspond to higher lipid peroxidation. (C) Flow cytometric analysis of 10 females (5 HC, 5 ME/CFS) and 9 males (5 HC, 4 ME/CFS) identified elevated mitochondrial lipid peroxidation, particularly in males (CD4T P = 0.0178, CD8T P = 0.034; female: ME/CFS vs. HC CD4T P = 0.348, CD8T P = 0.085). Lower y-axis values correspond to higher mitochondrial lipid peroxidation. (D) Reanalysis of bulk RNA-seq data (GSE224615) from 13 recovered controls (5F, 8M), 23 LC donors (15F, 8M) from 8-mo after COVID infection identifies statistically significant suppression of PLA2G6 in LC male donors (P = 0.028, two-sided t test) with no significant differences among female donors (P = 0.078). (E) Flow cytometric comparison of lipid droplet levels identifies lower lipid droplet levels in ME/CFS donors compared to HC across both males and females (males ME/CFS vs. HC: CD19B P = 1.52 × 10⁻⁴, CD4T P = 5.92 × 10⁻⁴, CD8T P = 7.14 × 10⁻⁴, LC vs. HC: CD19B P = 7.31 × 10⁻³, CD4T P = 9.39 × 10⁻³, CD8T P = 0.0114; females ME/CFS vs. HC CD19B P = 0.0242, CD4T P = 0.0205, CD8T P = 0.00876; LC vs. HC CD19B P = 0.622, CD4T P = 0.826, CD8T P = 0.974). (F) Negative association between lipid droplet (E) and GSH levels (Fig. 3C) (females: CD19 B cells R = −0.63, P = 0.0072, CD4 T cells R = −0.67 P = 0.003, CD8 T cells R = −0.54 P = 0.025; males: CD19 B cells R = −0.9, P = 2.5*10⁻⁵, CD4 T cells R = −0.75 P = 0.0032, CD8 T cells R = −0.78, P = 0.0015).

Fig. 6.

Effects of ROS on adaptive immune response. (A) Representative glutathione (GSH) profile from flow cytometry staining for one HC, LC, and ME/CFS donors’ CD4 T cells shows a long-tailed distribution for both HC and LC donors and a shifted distribution for ME/CFS. (B) To study long-tails, the GSH 95th percentile fluorescence intensity distribution is plotted across HC, LC, and ME/CFS donors. Each group demonstrates distinct tail-decaying behavior, where HCs decay quickly (light-tailed distribution), LC show heavy-tailed decay, and ME/CFS show a shifted and bounded tail distribution. (C) To understand this tail decaying behavior, we compared GSH levels on an additional set of 5 HC, 4 LC, and 5 ME/CFS donors between total, naïve (CCR7⁺CD45RO⁻), and memory CD4 T cell populations (CD45RO⁺). The long tails are found predominantly in memory and not naïve CD4 T cells, indicating that memory T cells are the main contributors to the heterogeneity in oxidative stress responses. For (D–F), PBMCs were labeled with CellTraceViolet and stimulated with anti-CD3/CD28 beads and IL-2. Based on a dye dilution assay, the proportion of proliferating T cells as a function of total ROS levels are shown at 5 d poststimulation. Experiments for (D–F) were conducted separately. (D) Proliferation data from 5 control and 5 ME/CFS females show female ME/CFS T cells hyperproliferate and exhibit higher ROS levels compared to control counterparts. Regression lines also show that control female CD4 and CD8 T cell proliferation has 10× greater slope with respect to ROS, compared to ME/CFS females. Compared to controls, the flat slope and higher line for ME/CFS T cells highlights hyperproliferation, suggesting a potential source of patient fatigue. (E) Data for 4 control and 5 LC females shows hyperproliferation among LC CD4 and CD8 T cells. The regression lines also show that control female CD4 and CD8 T cell proliferation has 10× greater slope with respect to ROS, compared to LC females. (F) Data from 7 control and 5 ME/CFS males shows a distinct pattern from females. CD4 and CD8 T cells from ME/CFS males do not hyperproliferate compared to controls, as ME/CFS males with similar ROS levels to controls do not show higher proliferation. As T cell proliferation among controls show 3.5× and 2.3× greater slope compared to ME/CFS CD4 and CD8 T cell counterparts, these results show greater insensitivity to ROS levels among ME/CFS male donors.

Fig. 7.

Metformin can redress T cell hyperproliferation in ME/CFS donors. (A) Study of fPOP subjects flagged patients 27 and 31, along with the father of 27, based on measured total ROS levels (DCFDA) in lymphocytes. Patient clinical information (table) identifies 27 and 31 as ME/CFS patients, based on the National Academy of Medicine ME/CFS diagnostic criteria (8). (B) Using CellTraceViolet staining, the proportion of proliferating T cells 5-d after stimulation were assessed for fPOP donors. ME/CFS donor CD4 and CD8 T cells with higher ROS are also associated with higher proportion of proliferation, consistent with Fig. 6. (C) To reduce proportion of proliferating T cells, PBMCs were treated at day 0 with ROS-modulating drugs, including N-acetylcysteine, metformin, and liproxstatin-1. Effects of drug on proliferation and ROS levels are shown 5-d after stimulation, where drugs reduce the proportion of high ROS proliferating T cells in Q1. (D) Comparison of drug effects between donor 27 and donor 27’s mother and father shows tested ROS-lowering drugs selectively modulate T cell proliferation in the ME/CFS donor 27 and not in the parents. (E) In-vitro metformin treatment in 5 HC and 6 ME/CFS donors finds a statistically significant reduction in the proliferation of ME/CFS CD4 T cells (P = 0.041). Proportion of proliferating T cells is estimated by proportion of (Q1+Q4)/(total CD4 T cells) as shown in (C). (F) Summary of Precision Medicine methodology for identifying ME/CFS patients and ROS-modulating drugs to address oxidative stress and hyperproliferation of T cells.