Macrophage heterogeneity and spatial localization in granulomas characterize early responses in Mycobacterium tuberculosis infected nonhuman primates.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is defined by granulomas—immune aggregates that either contain or support bacterial replication. Macrophages, fundamental components of these lesions, are crucial to TB pathogenesis, yet their phenotypic and functional diversity remains incompletely understood. Here, we used single-cell RNA sequencing and immunofluorescence to profile macrophages in lung tissue and granulomas from a nonhuman primate model of early TB. We identified transcriptionally distinct subsets, including embryonic-origin tissue-resident alveolar macrophages and monocyte-derived alveolar and interstitial macrophages, each with distinct spatial localization in granulomas. Tissue-resident alveolar macrophages and a subset undergoing epithelial-to-mesenchymal transition accounted for the highest frequency of Mtb-infected cells. Infected cells exhibited differential expression of immune- and migration-associated genes compared to uninfected counterparts, suggesting Mtb either induces or exploits these pathways as a survival strategy. These findings highlight macrophage heterogeneity as a major driver of differential susceptibility to Mtb and provide insights relevant to future immunomodulatory strategies. Overall design: CITE-seq analysis of macrophages from granulomatous and non-granulomatous NHPs lungs infected with mCherry-positive Mtb. Cynomolgues macaques have been infected with mCherry-positve Mtb Erdman. 5 weeks post-infection animals have been necropsied and lung tissue sections from granuloma and non-granuloma regions cryopreserved with CellBanker2. Samples have then been thawed at 37C, processed with the GentleMacs tissue dissociator, stained with TotalSeq antibodies at 4C, sorted to enrich for macrophages and processed for CITE-seq. We used the standard 10X and commercially available Biolegend protocols to generate sequencing libraries.