Mitochondrial O₂^●- is increased in T-lymphocytes treated with NE.

<p>T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of 1 μM NE. <b>A</b>. Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes 96 hours post-activation. Cells were incubated with DPI 1 hour prior to and during the incubation with DHE. N = 3. *p<0.05 vs. vehicle with 0 μM NE by 2-way ANOVA followed by Bonferroni post-hoc analysis. <b>B</b>. Quantification of MitoSOX red oxidation in CD4+ and CD8+ T-lymphocytes at various time points post-activation. N = 6. *p<0.05 vs. 0 μM NE by Student’s t-test at respective time points. <b>C</b>. Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes at 96 hours post-activation. 1 μM Tempol (Temp) or MitoTempol (MT) were supplemented at time of plating and every 24 hours post-activation. N = 4. *p<0.05 vs. vehicle with 0 μM NE; ^†p<0.05 vs. vehicle with 1 μM NE by 2-way ANOVA followed by Bonferroni post-hoc analysis.</p

T-lymphocyte mitochondrial reserve respiratory capacity is decreased with NE.

<p>T-lymphocytes were isolated and purified. Cells were either immediately analyzed in a Seahorse Bioscience XFp extracellular flux analyzer with a 30-minute acute treatment of 1 μM NE, or activated via CD3/CD28 stimulation in the presence of 1 μM NE for 96 hours prior to analysis. <b>A</b>. Cumulative average data of oxygen consumption rate (OCR) measured over-time with acute treatment of NE. N = 9. <b>B</b>. Cumulative average data of OCR measured over-time in activated T-lymphocytes cultured in the presence of NE. N = 9. *p<0.05 vs. 0 μM NE by Student’s t-test at respective time points.</p

Mitochondrial O₂^●- partially regulates CD8+ T-lymphocyte cytokine profiles.

<p>CD8+ T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE, Tempol (1 μM), and/or MitoTempol (1 μM) for 96 hours. Media was harvested for cytokine analysis and results normalized to cell number. N = 5. *p<0.05 vs. vehicle with 0 μM NE; ^†p<0.05 vs. vehicle with 1 μM NE by 2-way ANOVA followed by Bonferroni post-hoc analysis.</p

NE provokes inverse effects on T-lymphocyte growth and O₂^●- levels.

<p>T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of 1 μM NE. <b>A</b>. T-lymphocyte growth curves at various time points of ex vivo culture. N = 4. <b>B</b>. Quantification of DHE oxidation in CD4+ and CD8+ T-lymphocytes at various time points post-activation. N = 4. <b>C</b>. Representative EPR spectra showing amplitude (Amp) for T-lymphocytes stimulated in the presence or absence of 1 μM NE for 96 hours. N = 3. *p<0.05 vs. 0 μM NE by Student’s t-test at respective time points.</p

NE-mediated changes in O₂^●- are mediated via various adrenergic receptors.

<p>T-lymphocytes were isolated, purified, and cells were activated via CD3/CD28 stimulation in the presence of 1 μM NE or the respective antagonist for 96 hours prior to analysis. Quantification of DHE oxidation in CD4+ (upper) and CD8+ (lower) T-lymphocytes activated in the presence of the treatments for 96 hours. N = 4. *p<0.05 vs. vehicle with 0 μM NE; ^†p<0.05 vs. vehicle with 1 μM NE by 2-way ANOVA followed by Bonferroni post-hoc analysis.</p

Inhibiting NE transport increases intracellular O₂^●- levels.

<p>T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE and/or the norepinephrine transporter inhibitor atomoxetine. Quantification of DHE oxidation in CD4+ (upper) and CD8+ (lower) T-lymphocytes 96 hours post-activation. N = 4. *p<0.05 vs. vehicle with 0 μM NE; ^†p<0.05 vs. vehicle with 1 μM NE by 2-way ANOVA followed by Bonferroni post-hoc analysis.</p

Mitochondrial O₂^●- partially regulates CD4+ T-lymphocyte cytokine profiles.

<p>CD4+ T-lymphocytes were isolated, purified, and activated via CD3/CD28 stimulation in the presence of NE, Tempol (1 μM), and/or MitoTempol (1 μM) for 96 hours. Media was harvested for cytokine analysis and results normalized to cell number. N = 5. *p<0.05 vs. vehicle with 0 μM NE; ^†p<0.05 vs. vehicle with 1 μM NE by 2-way ANOVA followed by Bonferroni post-hoc analysis.</p

Proposed NE signaling cascade in T-lymphocytes.

<p>T-lymphocytes have been shown to express all adrenergic receptors, and NE has binding affinity to each isoform. In both CD4+ and CD8+ cells, NE appears to reduce proliferation via β-receptor canonical signaling (solid arrows) independent of redox events. CD4+ cells utilize the α2 receptor while CD8+ cells use all four adrenergic receptors to initiate redox signaling (dashed arrows) to affect specific subsets of cytokines. In both cells, the addition of MitoTempol can decrease this increase in mitochondrial O₂^●- and reverse the changes in specific cytokine expression.</p