Metabolic reprogramming of NK cells in chronic brucellar arthritis: role of the HIF-1α/mTOR axis.

BACKGROUND

Brucellar arthritis is the most common complication of brucellosis, and its chronic progression is closely associated with immune cell dysfunction. Natural killer (NK) cells play a central role in anti-infective immunity; however, their functional status and regulatory mechanisms in brucellar arthritis remain poorly understood.

METHODS

Peripheral blood and synovial tissue samples were collected from healthy controls, as well as from patients with acute-phase and chronic-phase brucellar arthritis. Single-cell RNA sequencing (scRNA-seq), flow cytometry, quantitative real-time PCR (qPCR), JC-1 mitochondrial membrane potential assay, Seahorse metabolic analysis, and Western blotting were employed to systematically evaluate NK cell phenotypes, functions, metabolic profiles, and HIF-1α/mTOR pathway activity. In vitro, heat-killed Brucella melitensis 16M (HKBA) was used to stimulate NK-92 cells and HIF-1α-knockout (KO) cell lines, with mTOR inhibitor intervention, to dissect the regulatory mechanism of the HIF-1α/mTOR axis in NK cell metabolic reprogramming.

RESULTS

scRNA-seq revealed enrichment of NK cells in the synovium of brucellar arthritis patients, with aberrant expression of oxidative phosphorylation (OXPHOS)-related genes. Clinical sample analyses showed that, compared with healthy controls, the proportion of peripheral blood NK cells increased in both acute- and chronic-phase patients; however, NK cells from chronic-phase patients exhibited significantly reduced IFN-γ, Granzyme B, and CD107a degranulation levels. HIF-1α protein was consistently overexpressed in patient NK cells, whereas phosphorylated mTOR-pathway proteins (p-PI3K, p-AKT, p-mTOR) were markedly suppressed. In vitro, HKBA treatment inhibited mTOR pathway activity in NK-92 cells in a dose- and time-dependent manner and reduced mitochondrial membrane potential as shown by JC-1 staining. HIF-1α knockout significantly restored NK cell cytotoxic function, upregulated OXPHOS- and glycolysis-related gene expression, and improved mitochondrial membrane potential. Transcriptomic sequencing confirmed enrichment of mTOR signaling and energy metabolism gene sets after knockout. Importantly, mTOR pathway inhibition completely blocked the HIF-1α-knockout-mediated restoration of NK cell function and mitochondrial fitness, indicating that HIF-1α primarily drives NK cell metabolic and functional abnormalities through mTOR suppression.

CONCLUSIONS

Brucella infection induces HIF-1α overexpression, which suppresses mTOR signaling and drives metabolic reprogramming of NK cells characterized by impaired OXPHOS and functional exhaustion, thereby promoting chronicity of brucellar arthritis. Targeting the HIF-1α/mTOR axis may offer a novel therapeutic strategy for brucellar arthritis.