ROS Scavenging Mitigates TNFα Induced Endoplasmic Reticulum Stress and Mitochondrial Fragmentation in Human Airway Smooth Muscle

The pro-inflammatory 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, 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 6 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 Red 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.