Background: Severe eosinophilic asthma (SEA) presents with eosinophilia, driven by cytokines IL-3, IL-5 and GM-CSF and is reduced by anti-IL-5 (mepolizumab) therapy. Mitochondrial dysfunction contributes to asthma pathology. However, the role of mitochondria in abnormal eosinophil function in SEA is unknown.

Aim: To investigate the role of mitochondrial function in SEA eosinophils.

Methods: Eosinophils were isolated from whole blood of healthy volunteers (HV) (n=15), SEA patients (n=11), SEA patients on mepolizumab therapy (n=4) and moderate asthmatics (MA) (n=4), via Percoll density centrifugation and magnetic bead negative selection. Metabolic activity was assessed using the Seahorse XF Cell Mito Stress test. Mitochondrial ROS (mtROS) and mitochondrial membrane potential (??m) were measured by MitoSOX and TMRM staining, and survival using annexin-V/PI staining.

Results: Baseline metabolic activity was increased in SEA compared to HV, particularly basal (2-fold; p=0.007 ) and ATP-linked mitochondrial respiration (2-fold; p=0.008), and glycolytic activity (2.5-fold; p=0.02). Eosinophils from SEA patients on mepolizumab and MA, had a similar metabolic profile to HV. Stimulation with IL-3, IL-5 and GM-CSF increased metabolic activity in eosinophils from all groups, except SEA patients on mepolizumab. In HV and SEA, IL-5 had no effect on mtROS, but decreased ??m and increased cell survival.

Conclusions: SEA eosinophils display an altered metabolic phenotype with increased mitochondrial respiration and glycolysis that is further enhanced by cytokines. This phenotype is not apparent in SEA patients on mepolizumab. Metabolic re-programming may contribute to eosinophil survival and persistence in disease.