Elevated LDL-C induces T-cell metabolic dysfunction and increases inflammation and oxidative stress in midlife adults

Abstract

T-cells may contribute to chronic, low-grade, sustained inflammation and oxidative stress commonly observed with aging and chronic disease. T-cell metabolic alterations impact T-cell differentiation, inflammation, and oxidative stress in animal models. Low-density lipoprotein cholesterol (LDL-C) has been identified as a novel antigen that activates T-cells via a canonical pathway. However, in humans, little is known about the direct effect of LDL-C on T-cells. Endogenous LDL-C concentration peaks during midlife in humans and may contribute to midlife chronic disease risk by inducing T-cell dysfunction. Thus, this study investigated the effects of exogenous LDL-C exposure on CD4⁺ and CD8⁺ T-cells from midlife adults. Compared with a physiologically "low" LDL-C concentration, we hypothesized that exposure to "borderline high" LDL-C would induce activation, alter metabolism, and increase mitochondrial reactive oxygen species and inflammatory cytokine production in T-cells from midlife adults. T-cell metabolism was assessed using extracellular flux analysis, and all other outcomes were assessed using flow cytometry. Our findings indicate that exposure to a borderline high concentration of LDL-C induced CD4⁺ and CD8⁺ T-cell activation, impaired mitochondrial respiration, and increased glycolytic metabolism. Further, we observed exogenous LDL-C exposure induced T-cell differentiation toward activated effector memory and effector memory re-expressing CD45RA subpopulations and increased inflammatory cytokine and mitochondrial reactive oxygen species production. These data suggest that borderline high LDL-C induces T-cell dysfunction that may increase the risk for age-related diseases. Future observational and clinical research should investigate the effects of endogenous LDL-C and other blood lipids on in vivo T-cell function and the implications for disease risk.NEW & NOTEWORTHY We evaluated the effect of low-density lipoprotein cholesterol (LDL-C) exposure on human T-cells isolated from midlife adults. T-cells were exposed to physiologically low and borderline high concentrations of LDL-C. We observed that high LDL-C exposure increased intracellular lipids, activated T-cells, and induced metabolic dysfunction. Additionally, high LDL-C exposure induced T-cell differentiation, a senescent-like phenotype, and induced inflammatory cytokine and mitochondrial reactive oxygen species production.

Keywords: T-cells; inflammation; low-density lipoprotein cholesterol; mitochondrial function; oxidative stress.

Figure 1.. Representative data for CD4⁺ and CD8⁺ T-cell mitochondrial respiration and glycolysis following low and high LDL-C exposure.

Pan CD4⁺ and CD8⁺ T-cell data from a representative participant exposed to a low and high concentration of LDL-C for 20-hours are presented. The high LDL-C condition (133 mg/dL) is displayed in a dark color and the low LDL-C condition is displayed in a light color (66 mg/dL). Pan CD4⁺ and CD8⁺ oxygen consumption rate (OCR, representing mitochondrial respiration, A-B) and extracellular acidification rate (ECAR, representing glycolysis, C-D) during the mitochondrial stress test are graphed over time. Each data point is a measurement of OCR or ECAR at a specific timepoint. Three measurements are made prior to each of the injections of oligomycin (oligo), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), or rotenone and antimycin A (ROT/AA). Three additional measurements are also made following the injection of ROT/AA.

Figure 2.. Flow Cytometry Gating Strategy.

Pan CD3⁺ T-cell were isolated from peripheral blood mononuclear cells using negative magnetic bead selection prior to performing flow cytometry. T-cell phenotype experiment gating strategy is presented (A). Cells were first identified by plotting forward scatter area (FSC-A) vs. side scatter area (SSC-A). Doublets were then removed by plotting FSA vs. forward scatter height (FSC-H). Live CD3⁺ cells were then identified as ghost dye^- CD3⁺. CD4⁺ and CD8⁺ T-cells were identified from live CD3⁺ T-cells. Contour plots were used to identify CD4⁺ and CD8⁺ CD69^HI and T-cell phenotype (CD45RA vs. CD27), and CD8⁺ MitoSOX™^HI cells. Histograms were used to measure the median fluorescence intensity (MFI) of CD69, MitoSOX™, and BODIPY™ 493/503 in CD4⁺ and CD8⁺ T-cells. Intracellular cytokine experiment gating strategy is presented (B). Cells were identified by plotting FSC-A vs. SSC-A, and the doublets were removed by plotting FSC-A vs. FSC-H. Live CD3⁺ cells were identified as zombie dye^- CD3⁺. Histograms were used to measure the MFI of the cytokines IL-10, IL-6, IFN-γ, IL-17A, TNF-α, and IL1β in CD4⁺ and CD8⁺ T-cells. The effect of 142 mg/dL low-density lipoprotein (LDL-C) (dark color) vs. 71 mg/dL (light color) LDL-C on CD4⁺ (blue) and CD8⁺ (green) T-cells was assessed by comparing differences in contour plots and histograms in both experiments.

Figure 3.. The Effect of LDL-C on CD4⁺ and CD8⁺ Lipid Uptake and Activation.

CD3⁺ T-cells were incubated with a low (71 mg/dL) and high (142 mg/dL) of low-density lipoprotein-cholesterol (LDL-C), and flow cytometry was used to measure CD4⁺ intracellular lipid uptake and CD8⁺ intracellular lipid uptake via BODIPY™ 493/503 median fluorescence intensity (MFI) (A-B). Additionally, CD4⁺ and CD8⁺ T-cell activation was assessed as the percentage of CD69^HI cells gated from contour plots (C-D) and CD69 MFI (E-F). The high LDL-C condition (142 mg/dL) is displayed in a dark color and the low LDL-C condition is displayed in a light color (71 mg/dL). CD4⁺ and CD8⁺ T-cells were displayed in blue and green colors, respectively. Wilcoxon signed rank tests were used for statistical analysis. Data are presented as mean ± SD or median and IQR. Other abbreviation: A.U., arbitrary units. Statistical significance was set at *P<0.05 compared to low LDL-C condition.

Figure 4.. The Effect of LDL-C on CD4⁺ and CD8⁺ Metabolism.

CD4⁺ and CD8⁺ T-cells were isolated from peripheral blood mononuclear cells and incubated separately with a low-density lipoprotein cholesterol (LDL-C) (66 mg/dL) or high LDL-C (133 mg/dL) concentration for 20 hours. Oxygen consumption rate and extracellular acidification rate were measured using extracellular flux analysis. CD4 and CD8 basal oxygen consumption rate (OCR) (A-B), ATP-linked OCR (C-D), basal extracellular acidification rate (ECAR) (E-F), and the ratio of basal OCR/ECAR (G-H). The high LDL-C condition (133 mg/dL) is displayed in a dark color and the low LDL-C condition is displayed in a light color (66 mg/dL). CD4⁺ and CD8⁺ T-cells were displayed in blue and green colors, respectively. Wilcoxon signed rank tests were used for statistical analysis. Data are presented as mean ± SD. Statistical significance was set at *P<0.05 compared to low LDL-C condition.

Figure 5.. The Effect of LDL-C on CD4⁺ and CD8⁺ T-cell Differentiation.

The effect of physiologically high low-density lipoprotein (LDL-C) exposure on CD4⁺ and CD8⁺ T-cells was assessed using flow cytometry following 20-hour exposure to a physiologically low (71 mg/dL) and high (142 mg/dL) concentration of LDL-C. CD4⁺ and CD8⁺ gating plots (A-B), population percentages (C-D), and naïve (E-F), central memory (G-H), effector memory (I-J), and terminally differentiated (effector memory T-cells re-expressing CD45RA, T_EMRA) (K-L) T-cell subpopulations are presented. The high LDL-C condition is displayed in a dark color and the low LDL-C condition is displayed in a light color. CD4⁺ and CD8⁺ T-cells were displayed in blue and green colors, respectively. Wilcoxon signed rank tests were used for statistical analysis. Data are presented as mean ± SD. Statistical significance was set at *P<0.05 compared to low LDL-C condition.

Figure 6.. The Effect of LDL-C on CD4⁺ and CD8⁺ T-cell Inflammatory Cytokine Production.

Pan CD3⁺ T-cells were exposed to a physiologically low (71 mg/dL) and high (142 mg/dL) concentration of low-density lipoprotein (LDL-C). The median fluorescence intensity (MFI) of CD4⁺ and CD8⁺ production of anti-inflammatory IL-10 (A-B), and pro-inflammatory IL-6 (C-D), IL-1β (E-F), IFN-γ (G-H), IL-17A (I-J), and TNF-α (K-L) are displayed. The high LDL-C condition is displayed in a dark color and the low LDL-C condition is displayed in a light color. CD4⁺ and CD8⁺ T-cells were displayed in blue and green colors, respectively. Wilcoxon signed rank tests were used for statistical analysis. Data are presented as mean ± SD. Statistical significance was set at *P<0.05 compared to low LDL-C condition.

Figure 7.. The Effect of LDL-C on CD4⁺ and CD8⁺ T-cell mtROS Production and Mitochondrial Damage.

The production of mitochondrial reactive oxygen species (mtROS) was assessed following exposure to a physiologically low (71 mg/dL) and high (142 mg/dL) concentration of low-density lipoprotein (LDL-C). The median fluorescence intensity (MFI) (A-B) of CD4⁺ and CD8⁺ T-cells was assessed. CD4⁺ and CD8⁺ T-cell proton leak was also measured as an assessment of mitochondrial damage in response to exposure to a physiologically low (66 mg/dL) and high (133 mg/dL) concentration of LDL-C using extracellular flux analysis (C-D). The high LDL-C condition is displayed in a dark color and the low LDL-C condition is displayed in a light color. CD4⁺ and CD8⁺ T-cells were displayed in blue and green colors, respectively. Wilcoxon signed rank tests were used for statistical analysis. Data are presented as mean ± SD or median and IQR. Statistical significance was set at *P<0.05 compared to low LDL-C condition.

Figure 8.. Correlation Matrix between LDL-C-Induced Changes in CD4⁺ and CD8⁺ T-cell Function.

The absolute change in measures of T-cell function were calculated if that measure was significantly affected by the high low-density lipoprotein-cholesterol (LDL-C) exposure. The absolute change for each of these measures was correlated with one another for (A) CD4⁺ and (B) CD8⁺ T-cells. Other abbreviations: MFI, median fluorescence intensity; OCR, oxygen-consumption rate; ECAR, extracellular acidification rate; A.U., arbitrary units; and mtROS, mitochondrial reactive oxygen species. Statistical significance was set at *p<0.05.