Single-Cell and Spatial Transcriptomic Analyses Deciphering the Three-Layer Architecture of Human Tuberculosis Granulomas

Abstract Background Granulomas (defining tuberculosis histopathological feature) are central to the host’s defense against Mycobacterium tuberculosis , critically influencing patient outcomes. However, knowledge of human granulomas’ structure and function are incomplete. This study employs single-cell and spatial transcriptomics to dissect human granuloma’s cellular composition, structure, communication and function from 19 pulmonary, lymphatic and skeletal samples. Results Our study identified nine key immune-activated/signaling-active cell clusters. Notably, we delineated a three-layered granuloma structure: a core with macrophages (Macro-c09, Macro-c10) and occasional fibroblasts (Fib-c03); a fibroblast-rich (Fib-c01) periphery; and an immune-infiltrated intermediate layer comprising diverse immune-cells recruited by strong signaling-molecules (SPP1/MIF) from core/periphery cells. This study also shows granuloma heterogeneity across individuals and tissues. Conclusions By merging scRNA-seq with ST-seq, we offer an intricate single-cell perspective of granulomas’ spatial-structure and formation mechanisms, identify signaling-molecules and significantly changed genes as potential targets for host-directed tuberculosis immunotherapy, highlight fibroblasts’ crucial role in granuloma formation, and provide an important reference/improved understanding of TB.