NET-associated Inflammation Defines a Neutrophil-centered Inflammatory Signature in Multiple Sclerosis

Neutrophil extracellular traps (NETs) have been implicated in autoimmune inflammation, yet their relevance to demyelinating injury and clinical severity in multiple sclerosis (MS) remains insufficiently defined. We integrated bulk transcriptomic datasets to characterize NET-associated inflammatory alterations in MS and used network-based and machine-learning approaches to prioritize candidate genes. Public single-cell RNA sequencing data were reanalyzed to determine the cellular distribution of candidate genes and NET-related activity. Experimental autoimmune encephalomyelitis (EAE) was further used to assess NET formation, demyelinating pathology, and the effects of pharmacological NET-targeting interventions. Clinical relevance was examined in independent MS cohorts. The integrated analyses identified a NET-associated inflammatory program and prioritized four candidate genes: CXCL1, MUC1, FASLG, and CAMP. Single-cell analysis showed that CXCL1 and CAMP were mainly enriched in neutrophils and macrophages, whereas neutrophils exhibited the highest NET-related score. In EAE mice, increased MPO and citrullinated histone H3 were accompanied by reduced myelin basic protein expression and marked spinal cord demyelination. Treatment with the PAD4 inhibitor GSK484, as well as N-acetyl-L-cysteine, alleviated EAE severity, reduced NET markers, and partially restored candidate gene expression patterns. In clinical cohorts, peripheral neutrophil-related indices were associated with Expanded Disability Status Scale scores, while serum MPO-DNA complexes and CXCL1 were elevated in patients with MS and positively correlated with disability severity. These findings support the presence of a NET-associated inflammatory program in MS and identify CXCL1 as a consistently supported inflammatory candidate linked to neutrophil-centered immune dysregulation.