Mucosal BCG vaccination reprograms lung interstitial macrophages and enhances antimicrobial defense in mice

Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB) but provides inconsistent protection against disease. Alveolar macrophages (AM) are widely considered the primary myeloid mediators of BCG-induced lung immunity, whereas the contribution of lung interstitial macrophages (IM) remains poorly defined. Here, we investigated pulmonary macrophage remodeling following BCG vaccination delivered by three distinct routes in mice, using flow cytometry, single cell RNA sequencing and spatial transcriptomics. We show that the route of vaccine administration dictates which macrophage subset is rewired. Intratracheal (IT) BCG preferentially reprograms IM, which form spatially organized immune hubs with CD4 T cells, while AM retain a transcriptional state closer to homeostasis. In contrast, intravenous (IV) BCG induces transcriptional remodeling of AM. Importantly, IT BCG conferred superior protection against both Mycobacterium tuberculosis and the heterologous pathogen Pseudomonas aeruginosa. Collectively, these findings identify IM as key mediators of mucosal vaccine-induced protection and highlight macrophage subset targeting as a framework for optimizing vaccines against respiratory pathogens.