Norepinephrine‐Mediated Suppression of T‐lymphocyte Activation is Regulated by Mitochondrial Redox Mechanisms

Hypertension is a disease associated with increased sympathoexcitation and norepinephrine (NE) outflow. Within the past decade, studies have suggested that this increase in sympathetic drive mediates T‐lymphocyte activation, which is required for the complete hypertensive response in various experimental animal models of hypertension. However, we recently reported that NE has direct suppressive effects on naïve splenic T‐lymphocyte activation including decreased pro‐inflammatory cytokine production, decreased proliferation, and suppressed cyclin expression. Moreover, we showed that these effects were mediated in part by superoxide (O 2 •− ) as evidenced by increased dihydroethidium (DHE) oxidation; however, the source of this O 2 •− remains elusive. We have recently observed that NE does not elevate O 2 •− flux acutely, but requires chronic stimulation over the course of the T‐lymphocyte activation period. Herein, we tested the hypothesis that mitochondria are a key source of NE‐mediated O 2 •− production in T‐lymphocytes and that chronic stimulation with NE disrupts oxidative metabolism in T‐lymphocytes. We examined T‐lymphocyte mitochondrial O 2 •− levels during NE‐stimulation utilizing the mitochondrial‐targeted superoxide probe MitoSOX Red. NE caused an approximate 2‐fold increase (p<0.01 vs. vehicle) in MitoSOX oxidation in splenic CD4 and CD8 T‐lymphocytes, suggesting an increase in mitochondrial O 2 •− levels. Furthermore, NE led to an approximate 30% decrease (p<0.05 vs. vehicle) in mitochondrial reserve respiratory capacity in both CD4 and CD8 T‐lymphocytes as measured by a Seahorse Flux Bioanalyzer, which suggests the potential for uncoupling and electron leak leading to mitochondrial O 2 •− production. With the observation that mitochondria appear to be a primary source of T‐lymphocyte O 2 •− with NE treatment, we next attempted to assess how NE was provoking this effect. Using agonists specific for unique α or β isoforms of adrenergic receptors, we revealed that NE appears to be working through a combination of adrenergic receptors on T‐lymphocytes as no single agonist could replicate the magnitude of NE‐mediated O 2 •− production. Adrenergic receptors were additionally implicated in the mechanism of NE, as the use of a NE‐transporter inhibitor ( i.e. atomoxetine), which prohibits NE uptake into the cell, exacerbated NE‐suppression of T‐lymphocyte proliferation 40% (p<0.05 vs. NE alone) as well as augmented O 2 •− production 50% (p<0.01 vs. NE alone). Interestingly, the increase in mitochondrial O 2 •− was shown to be partially‐causal to the inhibitory effects of NE on T‐lymphocytes as the addition of MitoTempol ( i.e. mitochondria‐targeted O 2 •− scavenger) could completely attenuate NE‐driven increases in O 2 •− (p<0.01) as well as significantly restore T‐lymphocyte growth (45%; p<0.05 vs. NE alone). Overall, our data suggest the potential for a new paradigm involving mitochondrial redox‐dependent inhibition of T‐lymphocyte activation by NE. These findings may have significant relevance to lymphatic‐positioned naïve T‐lymphocytes in hypertension as the increased sympathetic activation observed in the disease may have chronic suppressive effects on these immune cells. Support or Funding Information This work supported by NIH K99HL123471 (to AJC) and NIH R01HL103942 (to MCZ).