Integrated NULISA-based CSF neuroimmune profiling reveals microglial–axonal injury signatures associated with early disability and NAWM damage in new-onset Multiple Sclerosis patients

Background

Multiple sclerosis (MS) is characterized by inflammatory demyelination, neuroaxonal injury and progressive neurodegeneration. The biological processes driving the disease are already active at clinical onset, yet comprehensive characterization of different molecular pathways at diagnosis remains limited. ObjectiveTo integrate high-sensitivity CSF biomarker profiling with clinical disability measures, MRI lesion burden, and microstructural diffusion metrics in normal-appearing white matter (NAWM) in treatmentnaïve new diagnosed Multiple Sclerosis patients.

Methods

CSF samples were collected from 70 treatment-naïve newly diagnosed MS patients and 11 age- and sex-matched healthy controls. A CNS Disease Panel of 120 CSF biomarkers was quantified using ultra-sensitive NULISA technology. Differential expression analysis was performed using false discovery rate (FDR) correction. Multivariable linear regression models adjusted for age, sex, and ethnicity were used to evaluate associations with baseline Expanded Disability Status Scale (EDSS), short-term clinical change after high-dose corticosteroid therapy (ΔEDSS) and MRI white matter lesion volume. Partial correlation analyses were performed to assess associations between CSF and diffusion MRI measures in NAWM regions of interest.

Results

Seven biomarkers were significantly increased in MS compared with controls after FDR correction: REST, IL-16, CHI3L1, NEFL, ICAM-1, TREM2, and CHIT1. Correlation analysis demonstrated clustering among markers of glial and innate immune activation (CHI3L1, TREM2, CHIT1) and moderate associations between NFL and glial markers. REST was strongly correlated with IL16, suggesting early transcriptional and immune pathway interplay.In multivariable models, higher levels of glial activation markers were independently associated with greater baseline disability. CSF NFL levels correlated with white matter lesion burden and were significantly associated with diffusion abnormalities in NAWM, particularly in the genu of the corpus callosum, cingulum, and anterior corona radiata, indicating that neuroaxonal injury reflects both focal inflammatory activity and diffuse microstructural damage beyond visible lesions.

Discussion

This application of ultra-sensitive NULISA multiplex CSF profiling in treatment-naïve MS at diagnosis demonstrates that multiple immunological and neurodegenerative pathways are simultaneously active at disease onset and are associated with early disability and microstructural white matter damage. These findings support the concept that inflammatory and neurodegenerative processes occur in parallel early in MS and suggest that integrated CSF biomarker profiling may improve early disease characterization.ConclusionsMultiplex CSF proteomic profiling using NULISA identifies biologically meaningful biomarker signatures at MS disease onset. Integration of multiplex biomarkers with clinical and imaging measures may improve early disease stratification and support personalized therapeutic strategies.