Comprehensive profiling of immune cell infiltration and biomarker identification in intervertebral disc degeneration.

Intervertebral disc (IVD) homeostasis and function depend on the immunological privilege status, while immune cell infiltration constitutes a pivotal pathological driver of intervertebral disc degeneration (IDD). Nevertheless, the immune infiltration landscape and associated molecular targets in IDD remain elusive. We therefore integrated bioinformatic analysis of public datasets and single-cell RNA-sequencing analysis (scRNA-seq) data, flow cytometric (FACS) and experimental validation to delineate immune infiltration dynamics and identify therapeutic targets. CIBERSORT deconvolution analysis revealed significant increases in follicular helper T cells (p < 0.05) and M2 macrophages (p < 0.05) in IDD, with M1 macrophages demonstrating an upward trend. WGCNA showed that the blue module was associated with M1 macrophages infiltration (positive correlation 0.87, P < 0.001), among which VAMP8, JUN and others were mainly enriched in macrophage activation and myeloid leukocyte activation. FACS quantification established postoperative day 14 as the peak of leukocyte infiltration in rat IVDs following puncture. scRNA-seq resolved seven cellular subsets within degenerated rat IVDs: annulus fibrosus cells, smooth muscle cells, fibroblasts, macrophages, monocytes, vascular endothelial cells, and nucleus pulposus (NP) cells, with pronounced expression of Jun and Vamp8. Western blotting confirmed upregulated VAMP8 and JUN expression in TNF-α-stimulated (50 ng/ml, 24 h) NP cells. Immunohistochemistry further demonstrated elevated VAMP8 and JUN levels during rat IDD progression. This work identifies post-puncture day 14 as the critical window for peak immune infiltration in rat IVDs, and reveals VAMP8 and JUN are macrophage-associated regulators of IDD pathogenesis, thereby revealing promising targets for immunomodulatory interventions against disc degeneration.