Mesenchymal stromal cell-derived extracellular vesicles partially restore mitochondrial bioenergetics in inflammatory microglia

Microglia serve as key regulators of neuroinflammation and undergo marked metabolic reprogramming when transitioning from an unstimulated surveillance state to a proinflammatory phenotype. Stimulation with LPS, TNF-α, or IFN-γ disrupts mitochondrial homeostasis, leading to reduced respiratory efficiency and heightened oxidative stress. Extracellular vesicles (EVs) released from mesenchymal stromal cells (MSCs) represent a promising approach to modulate neuroimmune responses, and MSC priming with inflammatory cues can further refine functional potency. The objective of this study was to test whether EVs from primed or unprimed MSCs can “reset” the mitochondrial bioenergetics of stimulated microglia toward an unstimulated phenotype. We hypothesized that inflammatory stimulation would impair microglial respiratory conductance and decrease antioxidant buffering capacity, the balance between produced and emitted reactive oxygen species (ROS); and that these effects would be mitigated by primed MSC-EV intervention thereby restoring mitochondrial function toward an unstimulated microglia state. We utilized the C20 immortalized human microglia cell line and divided these cells into three experimental groups. Microglia either remained unstimulated as a control, stimulated with LPS, TNF-α, and IFN- γ, or stimulated and treated with MSC-EVs primed with the same stimuli (n=4-5/group). Respiratory conductance, the ability of mitochondria to match respiration to energetic demand, was assessed in permeabilized microglial cells using high-resolution respirometry (Oroboros). Emitted ROS, produced ROS, and antioxidant buffering capacity were assessed in permeabilized microglia cells using a fluorometric technique (Horiba). Data were analyzed using a one-way ANOVA and Tukey’s post-hoc analysis. ROS emission was 104.3% greater in stimulated vs. unstimulated microglia (p=0.0095). ROS emission from stimulated microglia treated with MSC-EVs was not significantly different than unstimulated microglia (p=0.9801). There was no difference in ROS production following stimulation (p=0.8564) or with MSC-EV treatment (p=0.7991). Antioxidant buffering capacity was 33.9% greater in stimulated vs. unstimulated microglia (p=0.0076), whereas there was no significant difference in antioxidant buffering capacity between stimulated microglia treated with MSC-EVs and unstimulated microglia (p=0.5646). This shows that stimulated microglia produce similar amounts of ROS, but have a diminished ability to buffer it, whereas treatment with EVs restores this antioxidant buffering capacity. Respiratory conductance was 43.9% less following stimulation of microglia (p=0.0314). Respiratory conductance, however, did not significantly improve following treatment, as the MSC-EV treated group also had 44.7% less compared to unstimulated microglia (p=0.0211). These findings suggest that while microglia bioenergetics do not fully return to baseline, treatment with primed EVs aids in regaining some function of the mitochondria. Future experiments will aim to identify other key changes in mitochondrial bioenergetics both post stimulation as well as with treatment of primed MSC-EVs. In conclusion, stimulating microglia to enter the M1 phenotype results in diminished mitochondrial activity; however, following treatment with primed MSC-EVs, some activity is restored to the basal unstimulated levels of function. Funding: R01NS133739. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.