Integration of single-cell and bulk RNA sequencing reveals divergent T-cell immune circuits and a shared osteo-immune pathway in spinal tuberculosis and brucellar spondylitis.

BACKGROUND: Spinal tuberculosis (STB) and Brucella spondylitis (BS) are the two most common infectious diseases of the spine. Despite overlapping clinical features, their immunopathological mechanisms remain poorly defined.

METHODS: We performed integrated transcriptomic profiling of infected nucleus pulposus tissues from STB and BS patients, combining single-cell RNA sequencing (STB n=3; BS n=3) with bulk RNA sequencing (STB n=7; BS n=5). Single-cell analysis resolved immune heterogeneity and T-cell subset architecture, whereas bulk RNA-seq defined global transcriptional signatures. Cross-platform integration identified concordant differentially expressed genes and shared pathways. AUCell was applied to quantify T-cell functional programmes. Pseudotime trajectory reconstruction, regulon inference, and cell-cell communication analyses were performed to delineate immune circuit differences.

RESULTS: A total of 67,274 high-quality cells were analysed, identifying nine major cell populations, with T cells as a dominant immune component. STB tissues were enriched in inflammatory cells, including monocytes/macrophages, neutrophils, and osteoclasts, whereas BS showed higher proportions of B and T cells. In STB, CD4 T cells were skewed toward IL26-CD4 T, Cytotoxic CD4 T, Proliferating CD4 T, and Treg subsets, while CD8 T cells were enriched in IFNG-AS1-CD8 T and exhaustion-like populations, reflecting sustained IFN-driven activation with proliferative and apoptotic stress. In contrast, BS exhibited expansion of GZMB-CTLs and IFNG-CTLs subsets, consistent with a focused cytotoxic programme with preserved memory features. Integrated single-cell and bulk analyses demonstrated convergence of overlapping genes on TCR, NF-κB, cytokine signalling, and osteoclast differentiation pathways. Functional scoring and pseudotime analysis indicated broader Th1/Th17 activation and progression toward terminal exhaustion in STB, whereas BS T cells maintained intermediate effector-memory states. Regulatory network analysis revealed a STAT1/IRF-dominated interferon axis in STB and an IRF4/EOMES/RFX3-centred effector-memory programme in BS. Cell-cell communication mapping showed an MHC-II-CD4 axis predominating in STB and an MHC-I-CD8 axis in BS. Despite divergent immune circuits, both conditions converged on osteoclast differentiation pathways, suggesting immune-bone metabolic coupling as a shared mechanism of vertebral destruction.

CONCLUSIONS: STB and BS exhibit pathogen-specific T-cell immune circuits that converge on a common osteo-immune pathway, providing mechanistic insight and potential targets for host-directed therapy.