Caffeine-mediated CD39⁺ Treg activation via the CD39-adenosine receptor pathway is a novel risk factor for pulmonary tuberculosis.

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Background: The global impact of pulmonary tuberculosis (PTB) is compounded by a limited understanding of modifiable risk factors. While caffeine is the most consumed psychoactive substance, its causal relationship with PTB and the underlying immunological mechanisms remain unknown.Methods: A three-tiered approach was used: 1) two-sample Mendelian randomization (TSMR) was used to analyze 486 metabolites and 731 immune cells for PTB causality (inverse variance weighting was the primary method with reverse MR and Bonferroni correction), 2) single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq were integrated (Seurat, Gene set enrichment analysis, and pseudotime analysis) to characterize CD39⁺ Tregs traits in lungs with PTB using, 3) core genes (LASSO) regression and eQTL-based genetic analyses uniquely) were validated in THP-1 macrophages, C3HeB/FeJ mice, and patients with PTB via FCM, WB, RT-qPCR, and multiplex immunohistochemistry.Results: Based on TSMR, eight metabolites (including caffeine) and nine immune subsets (including activated CD4⁺ Tregs) were linked to PTB (P < 0.05). Caffeine increased the risk of PTB via CD39⁺CD4⁺ Tregs (mediated proportion = 10.4%, P = 0.046). ScRNA-seq analysis of PTB lungs revealed elevated CD4⁺ Tregs with high caffeine responsiveness and CD39/ADORA2A overexpression. Validation in models revealed that core genes (PSMC5, BAG1, and AGPAT5) exhibited differential expression with PTB (P < 0.05) and good diagnostic efficacy (AUC > 0.7).Conclusions: We first identified a causal association between genetically predicted caffeine levels and PTB risk at the genetic level. We further uncovered a CD39-adenosine-based Treg activation mechanism underlying this association, and identified PSMC5 as a potential therapeutic target for host-directed therapy. These findings inform PTB pathogenesis and host-directed therapy.