ROS scavenging mitigates TNFα-induced endoplasmic reticulum stress and mitochondrial fragmentation in human airway...

The proinflammatory cytokine tumor necrosis factor alpha (TNFα) mediates airway responses to acute inflammation. Previously, we demonstrated that, in human airway smooth muscle (hASM) cells, TNFα increases reactive oxygen species (ROS) formation. TNFα also selectively activates the inositol-requiring enzyme 1α (pIRE1α^S724 autophosphorylation) endoplasmic reticulum (ER) stress pathway involving splicing of X-box binding protein 1 (XBP1s) and transcriptionally activates cyclin-dependent kinases 1 and 5 (CDK1 and CDK5), promoting dynamin-related protein 1 (DRP1) phosphorylation at serine 616 (pDRP1^S616) and mitochondrial fragmentation. In the present study, we hypothesized that in hASM cells, TNFα-induced ROS triggers pIRE1α^S724/XBP1s ER stress pathway. To test this hypothesis, we examined the impact of the ROS scavenger tempol on TNFα-induced pIRE1α^S724/XBP1s ER stress pathway and downstream signaling mediating mitochondrial fragmentation. Bronchiolar tissue samples were obtained from six patients with no history of smoking or chronic pulmonary disease. The smooth muscle layer was dissected, and hASM cells were dissociated and randomly assigned to four treatment groups: 1) vehicle, 2) vehicle + TNFα (20 ng/mL, 6 h), 3) tempol (500 μM) only, and 4) tempol (500 μM) + TNFα (20 ng/mL, 6 h). ROS formation was determined by confocal imaging using MitoSox Red. Mitochondria were labeled with MitoTracker Green and imaged using confocal microscopy. Using Western blot, we demonstrated that tempol reduced cellular ROS formation and mitigated the TNFα-induced increase in pIRE1α^S724, XBP1s, CDK1/5, pDRP1^S616 protein levels and reduced mitochondrial fragmentation. These findings support our hypothesis and indicate a role of ROS in mediating TNFα-induced ER stress and mitochondrial fragmentation.NEW & NOTEWORTHY In human airway smooth muscle cells, TNFα induces reactive oxygen species (ROS) generation and promotes endoplasmic reticulum (ER) stress and mitochondrial fragmentation. Importantly, treatment with the ROS scavenger tempol effectively attenuated TNFα-induced oxidative stress, suppressed ER stress and mitochondrial fragmentation, and improved the intrinsic respiratory capacity and functional efficiency of individual mitochondria. These findings identify oxidative stress as a central mediator linking TNFα signaling to ER stress and mitochondrial fragmentation, highlighting tempol as a potential therapeutic strategy.