Deciphering macrophage molecular responses to Porphyromonas gingivalis outer membrane vesicles through combined immunoassay and Raman spectroscopic analyses.

Outer membrane vesicles released by Porphyromonas gingivalis (Pg-OMVs) are emerging as critical mediators of periodontal and systemic pathology. Published literature has so far associated the presence of OMVs to an increased risk of cerebrovascular and neurodegenerative diseases, but the molecular-scale mechanisms underlying their interactions with immune cells remain insufficiently understood and characterized. Here, we employ high-resolution Raman spectroscopy in addition to established immunoassays to quantitatively decode the real-time chemical reprogramming induced by Pg-OMVs in macrophages. Our results reveal early and pronounced alterations in lipid composition, including significant increases in sphingomyelin- and ceramide-associated vibrational signatures, paralleled by perturbations in cholesterol-rich membrane domains. These lipidomic changes coincide with Raman markers of mitochondrial dysfunction, as characterized by reduced cytochrome c oxidation signals, indicating a shift toward a more reduced redox state. We further identify time-resolved modifications of glycogen-associated bands, implicating metabolic rewiring and inflammatory activation. Together, these spectral fingerprints demonstrate how Pg-OMVs orchestrate a coordinated disruption of membrane architecture, intracellular lipid homeostasis, and mitochondrial function. Raman findings provide a non-destructive, label-free molecular atlas of OMV-driven macrophage dysregulation and establish Raman spectroscopy as a powerful platform for quantitatively elucidating host-pathogen interactions with direct relevance to oral-systemic health and chronic inflammatory disease.