Integrated Single-cell RNA Sequencing Atlas of White Matter Microglial Functional Diversity in Chronic Multiple Sclerosis

Background: In multiple sclerosis (MS), chronic “smoldering” lesions are driven by microglial activation, which promotes demyelination and disease progression. However, microglia exhibit a functional dichotomy by performing both neurotoxic and reparative roles. Precisely modulating these functions remains a major therapeutic challenge, because non-specific approaches can inadvertently impair beneficial microglial functions. Delineating microglial subtypes in chronic MS is therefore essential for targeted therapy. Methods: Single-cell RNA sequencing, spatial transcriptomics, deconvolution, and pseudotime trajectory mapping were used to characterize cell subtypes. Subsequent investigations encompassed functional annotation, differential gene expression profiling, cell-cell communication pathways, and transcription factor network analysis. Results: We identified six distinct microglial subtypes with varied functions and focused on the highly disease-relevant C2 microglial subtype. This subtype simultaneously exhibited enhanced phagocytic and pro-inflammatory functions potentially associated with the fatty acid metabolic pathway. Pseudotime trajectory analysis suggested that this subtype is likely to represent a late disease stage. Cell-cell communication analysis revealed interactions between this subtype and both oligodendrocytes and T cells, as further supported by spatial transcriptomics demonstrating co-localization of the C2 microglial subtype with T cells. Finally, transcriptional regulatory network analysis identified an association between the FOSL2 regulon and both the C2 microglial subtype and chronic MS. Conclusion: Through integrated single-cell sequencing approaches, we identified a C2 microglial subtype in chronic MS that might contribute to disease progression via pro-inflammatory functions. We propose the transcription factor FOSL2 as a promising hypothesis-generating candidate for future studies aimed at modulating microglial differentiation toward a less harmful state. Our findings provide mechanistic insights into the pathology of white matter lesions in chronic MS and suggest novel subtype-specific directions for the development of next-generation disease-modifying therapies.