High-dimensional flow cytometry analysis reveals temporal dynamics of lung macrophages during Mycobacterium tuberculosis infection 2261143

Abstract Introduction Mycobacterium tuberculosis (Mtb) spends most of its lifespan within macrophages. During early infection, alveolar macrophages (AMs) facilitate rapid Mtb growth, whereas recruited monocyte-derived interstitial macrophages (IMs) restrict replication. However, macrophage subsets harboring Mtb in the presence of protective T cell—mediated adaptive immunity at the later stage of infection remain poorly defined. Many studies have relied on scRNA-seq to characterize macrophages without detailed phenotypic or functional analysis. Methods Our study aims to establish consensus on identifying lung macrophage subsets and analyze their dynamics and permissiveness across infection stages using high-dimensional flow cytometry and lineage tracing with scRNA-seq datasets. Results At 2 weeks post infection, CD11bhi AMs highly expressed CD63, CD38, and iNOS, consistent with a pro-inflammatory AM signature. Lineage tracing further confirmed the presence of monocyte-derived AMs. Four IM subsets were identified, with CCR2loCD11chiSca-1+ IMs being the predominant subset infected with Mtb. At 4 weeks post infection, six subsets of IMs were identified, and only three of them harbored Mtb. Notably, all three Mtb-infected IM subsets expressed high levels of CD11c, CD38, and iNOS. Conclusion These data provide a high-resolution analysis on heterogeneous lung macrophages and highlight shifts of their phenotypes during infection. It lays the foundation for future studies on how these macrophage alterations affect bacterial control. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)