Single-cell spatial-temporal understanding of the role of macrophage subsets in maintenance of lesion biology and local Mycobacterium tuberculosis infection outcome - PROJECT SUMMARY/ABSTRACT Lung macrophages serve as primary host cells for Mycobacterium tuberculosis (Mtb) during infection and are recognized as one of the most crucial cell types in determining the outcome of Mtb infection. We have previously reported that two types of lung macrophages, namely embryonic-derived alveolar macrophages (AMs) and monocyte-derived interstitial macrophages (IMs), display different permissiveness to Mtb at the early stages of infection in C57BL/6 mice. Such distinct permissiveness of macrophages to Mtb infection is determined by their unique metabolic status. However, most of the studies performed to date on the interaction between lung macrophages and Mtb have utilized in vitro models and/or primarily focused on the early stage of in vivo infection. The widely used “resistant” C57BL/6 mice do not form caseous necrotic granulomas (lesions) that are a hallmark of human tuberculosis (TB) disease, which additionally hinders the translation of our knowledge from the mouse model to human disease. Further, while significant work has been put into understanding the immune drivers of lesion formation, the role of immune cells in maintaining lesion structure and control of Mtb growth after lesions have formed remains poorly understood. To this end, how different lung macrophage subsets differ in their capacity to control or promote localized growth of Mtb, and how Mtb responds to the local environment in the context of necrotic lesions during the later stages of infection, is almost completely unknown. Utilizing the C3HeB/FeJ mouse Mtb infection model that forms the hallmark caseous necrotic lesions observed in human TB, we have revealed that manipulation of different lung macrophage subsets after lesion formation has distinct influence on Mtb growth in the necrotic lesions during the later stages of infection, depending on lesion sublocation. We thus hypothesize that the function of lung macrophage subsets is spatially distinct and contributes to the heterogeneity in local environment experienced by Mtb, and consequently bacterial physiological status, within the lesions. We will test this hypothesis with two complementary aims. In Aim 1, we will utilize novel metabolic analysis approaches together with spatial transcriptomics on the host side to elucidate the role of AMs versus IMs in Mtb lesion biology in spatiotemporal context. In Aim 2, we will delineate the relationship between macrophage subsets and local Mtb environment and activity at the single bacterium level, adapting methods to visualize Mtb transcripts in situ. With both Aims, depletion of macrophage subsets, including IMs, will enable elucidation of how macrophages act in the maintenance of lesion structure and local Mtb growth control. Together, this work will provide a conceptual framework for understanding how permissiveness of lung macrophage subsets relates to their spatial distribution within granulomas, and how this spatiotemporal relationship impacts on local Mtb environment and growth.
