Single-cell transcriptome analysis reveals aberrant low-density neutrophil differentiation and immune signaling dysregulation in myasthenia gravis

Background Myasthenia gravis (MG) is an antibody-mediated autoimmune neuromuscular disorder. Although dysregulation of adaptive immunity is well recognized, the single-cell landscape and functional role of low-density neutrophils (LDNs) in MG remain largely uncharacterized. Methods We conducted single-cell RNA sequencing on peripheral blood immune cells from five anti-acetylcholine receptor antibody-positive MG patients and five age- and sex-matched healthy controls (HCs) using the BD Rhapsody platform. After quality control, we used Seurat for cell clustering, Presto for differential gene analysis, Monocle2 for pseudotime analysis, SCENIC for transcription factor networks, and CellChat for intercellular communication analysis. Results Twelve distinct immune cell populations were identified, among which LDNs were significantly expanded, whereas CD4 + T cells were reduced in MG ( P < 0.01). LDN-specific upregulated genes—including S100A8/A9 , CSF3R , CD55 , and CLEC12A —were enriched for pathways related to neutrophil chemotaxis and neutrophil extracellular trap (NET) formation. LDN subclustering revealed nine subsets, with pro-inflammatory CXCR2 + and NET-forming PADI4 + subsets (characterized by elevated expression of NET-associated transcripts) expanded, while immunoregulatory CD74 + and LTF + subsets were reduced in MG. Pseudotime analysis delineated a disease-specific differentiation trajectory (CCND3 + → CXCR2 + → PADI4 + ), suggesting aberrant LDN maturation in MG. The transcription factor ELF2 exhibited enhanced activity and regulated 342 target genes, 129 of which were pseudotime-dependent and associated with cell division and protein phosphorylation. Intercellular communication analysis demonstrated weakened ICAM1-mediated signaling and enhanced ADGRE5–CD55/LPAR2 axis activity in MG LDNs, indicating a shift toward pro-inflammatory and autoimmunity-promoting interactions. Conclusions Peripheral blood in MG exhibits an immune microenvironment characterized by LDN-subset imbalance, ELF2-driven aberrant differentiation, and dysregulated intercellular communication, offering novel cellular markers and potential therapeutic targets for MG.