Salivary Proteomics Reveals Disease- and Therapy-Associated Molecular Patterns in Relapsing-Remitting Multiple Sclerosis

Multiple sclerosis (MS) is a chronic immune-mediated disease for which non-invasive biomarkers, able to detect disease- and treatment-associated molecular changes, are needed. We characterized the whole-saliva proteome of patients with relapsing-remitting (RR) MS at baseline before treatment initiation (MS-T0; n = 66) and after six months of disease-modifying therapy (MS-T1; n = 40), in comparison with healthy controls (HC; n = 31). Nano-liquid chromatography-high-resolution tandem mass spectrometry identified 1305 proteins, of which 209 differed among the main comparisons. Pairwise and paired analyses distinguished persistent RRMS-associated alterations, baseline-attenuation patterns with changes shifting towards HC levels, treatment-associated differences emerging at MS-T1, and longitudinal-only alterations. The identified patterns are descriptive and cannot be interpreted as evidence of pharmacological treatment effects or clinical response. The existence of specific RRMS alterations was supported by exploratory Random Forest, which classified HC, MS-T0 and MS-T1 samples using a panel of 50 proteins, most of which (96%) also showed significant changes in univariate tests. Reactome enrichment evidenced that proteins pertaining to the identified patterns are mainly involved in innate immunity, neutrophil degranulation, cell-death pathways, cytokine signalling, and protein folding. These findings indicate that six months of therapy was associated with substantial salivary proteome changes and support saliva as a candidate biofluid for longitudinal molecular monitoring and non-invasive detection of treatment-associated salivary proteome variations in RRMS.