Single-cell multi-omics analysis identifies SPP1+ macrophages as key drivers of ferroptosis-mediated fibrosis in ligamentum flavum hypertrophy

Abstract Background Ligamentum flavum hypertrophy (LFH) is a primary contributor to lumbar spinal stenosis. However, a thorough understanding of the cellular and molecular mechanisms driving LFH fibrotic progression remains incomplete. Methods Single-cell RNA sequencing (scRNA-seq) was performed to construct the single-cell map of human ligamentum flavum (LF) samples. An integrated multi-omics approach, encompassing scRNA-seq, bulk RNA sequencing (bulk RNA-seq), and Mendelian randomization (MR), was applied to conduct comprehensive functional analysis. Clinical tissue specimens and animal models were employed to further confirm the multi-omics findings. Results ScRNA-seq provided a single-cell level view of the fibrotic microenvironment in LF, revealing significantly increased proportions of fibroblasts, myofibroblasts, and macrophages in LFH. Using transmission electron microscopy, single-cell gene set scoring, and MR analysis, ferroptosis was identified as a critical risk factor and pathway within LFH. Subcluster analysis of fibroblasts revealed functional heterogeneity among distinct subpopulations, highlighting the functional characteristics and the metabolic dynamics of fibroblast with a high ferroptosis score (High Ferro-score FB). The quantification of gene expression at single-cell level revealed that ferroptosis increased along with fibrosis in LFH specimens, a finding further validated in both human and mice tissue sections. Consistently, bulk RNA-seq confirmed increased proportions of fibroblasts and macrophages in LFH specimens, underscoring a strong correlation between these cell types through Spearman correlation analysis. Notably, subcluster analysis of the mononuclear phagocytes identified a specific subset of SPP1 + macrophages (SPP1 + Mac) enriched in LFH, which exhibited activation of fibrosis and ferroptosis-related metabolic pathways. Cell-cell communication analysis highlighted that SPP1 + Mac exhibited the strongest outgoing and incoming interactions among mononuclear phagocytes in the LFH microenvironment. Ligand-receptor analysis further revealed that the SPP1-CD44 axis could serve as a key mediator regulating the activity of High Ferro-score FB. Multiplex immunofluorescence confirmed substantial Collagen I deposition and reduced Ferritin Light Chain expression in regions with SPP1-CD44 co-localization in LFH specimens. Conclusions Our findings indicated that SPP1 + Mac may contribute to LFH fibrosis by regulating ferroptosis in High Ferro-score FB through the SPP1-CD44 axis. This study enhances our understanding of the cellular and molecular mechanisms underlying LFH progression, potentially improving early diagnostic strategies and identifying new therapeutic targets.