Integration of M. tuberculosis phenotype with single cell RNA-seq to interrogate host macrophage heterogeneity in vivo.

Over the last several years, scRNA-seq has proven an invaluable tool for dissecting the role of the immune environment in many diseases. However, for infectious disease studies, the absence of information regarding the transcriptional status of the infecting agent reduces our functional appreciation of the roles of different immune effector cells in determining control versus progression of disease. In this study we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis-infected alveolar (AMs) and interstitial macrophages (IMs) in vivo and identifies clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. Our approach is readily transferable to other infectious disease agents with the potential for a better understanding of the role that different host-cell populations play during the course of an infection. scRNA-seq analysis of Mtb-infected and uninfected mice lungs at 3 wpi (weeks post-infection); scRNA-seq of BAL (bronchoalveolar lavage) recovered from the lung of 3 healthy human volunteers; Bulk Dual RNA-seq analysis of M.tuberculosis transcriptome from hspx-high and hspx-low bacteria at 3 wpi; Bulk ATAC-seq analysis of BCG infected and uninfected mouse AM and IM populations 4 wpi.