Immune cell heterogeneity in multiple sclerosis reveals myeloid-specific dysfunction and distinct regulatory programs across CD14+, CD4+, and CD8+ immune cells

Abstract Background Multiple Sclerosis (MS) is a neuroinflammatory and neurodegenerative disorder driven by dysregulated peripheral immune responses. Among immune cells, myeloid cells (CD14 + monocytes) are key players in initiating and sustaining CNS inflammation, yet their cell‑specific molecular dysfunction remains incompletely understood. Methods We performed an integrated systems biology analysis on bulk RNA‑seq data from CD14+ monocytes, as well as CD8+ and CD4+ T cells, from MS patients versus healthy controls. The pipeline included differential expression, pathway enrichment, protein‑protein interaction network clustering, hub gene identification, and upstream regulator analysis (transcription factors, kinases, lncRNAs). Independent datasets were used for validation. Results Myeloid cells (CD14+) displayed a distinct pro‑inflammatory and metabolically stressed signature, including upregulation of RNA150, CTLA4, KIF5A, THBS1, EGR2, KCNA2, OCLN, FLT4 and downregulation of IL13, FGF8. Functional enrichment revealed overactive NF‑κB, chemokine, and Toll‑like receptor signaling, alongside suppressed anti‑inflammatory and lipoprotein metabolism pathways. Three significant subnetworks (clusters) within the myeloid protein-protein interactions (PPIs) network uncovered dysregulated Wnt and calcium signaling associated with CACNA1H, LEF1, and SOST. Upstream regulatory analysis identified EGR3, FOXJ1, ID1, LEF1 as key transcription factors controlling myeloid dysfunction. In T cells, we observed complementary alterations, but the most pronounced transcriptional and regulatory alterations were found in the myeloid compartment. Conclusion Our integrated transcriptomic and network analysis reveals substantial immune-cell heterogeneity across CD14+ monocytes, CD4+ T cells, and CD8+ T cells in multiple sclerosis. Among these populations, CD14+ monocytes exhibited the most pronounced transcriptional, functional, and regulatory alterations, characterized by dysregulated inflammatory, metabolic, Wnt-signaling, and calcium-signaling programs. The identified cell-type-specific hub genes and regulatory factors highlight distinct immune-cell states and suggest that myeloid-centered dysfunction represents a major component of MS immunopathology. These findings provide a systems-level framework for understanding immune-cell heterogeneity and neuroimmune interactions in multiple sclerosis.