Fluoxetine Reshapes Macrophage Membrane Sphingolipids and Inflammatory Response Without Affecting Extracellular Vesicle Biogenesis upon Inactivated SARS-CoV-2 Stimulation

Sphingolipids (SL) are essential structural and bioactive components of cell membranes, remarkably involved in inflammatory signaling and membrane dynamics. Dysregulation of SL metabolism contributes to pathological inflammation and cellular stress. Selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine (FXT), are known inhibitors of acid sphingomyelinase (aSMase), although their impact on macrophage SL remodeling and inflammatory responses remains unclear. Here, we investigated the modulation of FXT on SL species composition and inflammatory activation in THP-1-derived macrophages stimulated with inactivated SARS-CoV-2 particles, which is a model of viral-induced inflammation. Sphingolipidomic profiling revealed that FXT pre-treatment markedly reduced ceramide (Cer) species while increasing sphingomyelin (SM) and sphingosine-1-phosphate (S1P) levels, consistent with inhibition of the aSMase-Cer axis. These changes were accompanied by attenuation of proinflammatory components, including interleucin (IL)-6, IL-1β, and matrix metalloproteinase (MMP)-9, indicating that SL remodeling correlates with reduced macrophage activation. Despite pronounced alterations in membrane lipid composition, the quantification of extracellular vesicles (EVs) released by FXT-treated macrophages remained unchanged, however the EVs size distribution was smaller compared to non-treated cells. Altogether, our findings demonstrate that FXT reshapes SL metabolism and lipid membrane composition, thereby diminishing macrophage activation without affecting EVs biogenesis. This study emphasizes the immunometabolic role of SL on membrane reprogramming as a mechanism by which pharmacological aSMase inhibition modulates viral inflammation responses.