Mitochondrial bioenergetic remodeling underlies fingolimod-induced immunometabolic adaptation in multiple sclerosis

Mitochondrial dysfunction and immune cell metabolic reprogramming are central mechanisms driving the pathogenesis and progression of multiple sclerosis (MS). Fingolimod (FTY720), a sphingosine-1-phosphate analog widely used in MS, limits lymphocyte egress from lymphoid organs; however, its effects on mitochondrial function and immunometabolic pathways remain incompletely understood. We evaluated mitochondrial bioenergetics and metabolic reprogramming in lymphocytes from healthy controls incubated with FTY720 and from MS patients treated with fingolimod, compared with healthy controls and patients receiving interferon-beta or glatiramer acetate. Mitochondrial respiration and glycolytic activity were assessed by extracellular flux analysis under basal conditions and following phytohaemagglutinin (PHA) stimulation, while mitochondrial parameters and metabolic markers were analyzed by flow cytometry and immunoblotting. Fingolimod exhibited a blunted metabolic response to PHA in lymphocytes from acutely-treated controls and patients receiving chronic therapy. In both unstimulated and PHA-stimulated conditions, acute incubation with FTY720 in control lymphocytes displayed decreased basal, maximal, and ATP-linked respiration; and basal and maximal glycolytic activity that resulted in reduced glycolytic reserve. In lymphocytes from in vitro acutely exposed controls and from chronically treated MS patients, fingolimod treatment was associated with decreased mitochondrial mass and membrane potential after PHA-stimulation, with reduced expression of lactate transporters (MCT1 and MCT4). FTY720-treatment in control lymphocytes was associated to lower expression of ETC complex proteins and of those involved in mitochondrial dynamics, and increased ROS and PFKFB3 levels under basal and stimulated conditions. These findings suggest that mitochondrial metabolism of lymphocytes is a potential component of fingolimod activity, promoting an altered bioenergetic state.