Serum context shapes macrophage baseline state and LPS responsiveness in human monocyte-derived macrophages

Background Human monocyte-derived macrophages (MDMs) are central to translational immunology, yet most differentiation protocols rely on xenogeneic fetal calf serum (FCS), which may alter macrophage functional states. Autologous serum (AS) maintains the donor-specific immunometabolic milieu, but its impact on macrophage baseline programming and stimulus responsiveness has not been systematically evaluated. Methods Monocytes from 32 healthy donors were differentiated in parallel with 10% AS or FCS for five days with M-CSF, followed by 24-hour stimulation with LPS, IL-4/IL-10/TGF-β, or vehicle. Cytokine secretion was quantified by multiplex assay, activation markers by qPCR, and baseline transcriptional programs by bulk RNA sequencing. Results AS- versus FCS-differentiated MDMs showed significantly greater LPS-induced secretion of TNF-α, IL-6, IL-1β, IP-10, IL-23, TARC and IL-1RA together with higher CD86 and CCR7 expression, whereas these differences were not observed following IL-4/IL-10/TGF-β stimulation or in unstimulated cells. Transcriptomic analysis of unstimulated MDMs identified distinct serum-dependent baseline programs, with FCS associated with increased expression of inflammatory cytokines, chemokines, and innate immune activation genes, whereas AS was associated with genes involved in lipid metabolism, extracellular matrix remodeling, and immune surveillance. Conclusions Serum context is a major determinant of macrophage baseline state and inflammatory responsiveness. FCS is associated with a more inflammatory baseline and attenuated LPS-induced activation, while AS shows a metabolically and immunologically homeostatic baseline with greater LPS responsiveness. These findings demonstrate that serum composition shapes basal macrophage programs and differentially modulates responses to inflammatory versus alternative stimulation, supporting consideration of autologous serum when modeling donor-specific innate immune responses.