In vivo single-cell high-dimensional mass cytometry analysis to track the interaction betweenKlebsiella pneumoniaeand myeloid cells
Authors: Calderon-Gonzalez Ricardo , Dumigan Amy , sa Pessoa Joana , Kissenpfennig Adrien , Bengoechea Jose A. 
Published: December 2023

ABSTRACTIn vivo single-cell approaches have transformed our understanding of the immune populations in tissues. Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interaction with bacteria, a method we term Bac-CyTOF. We focus on the pathogenKlebsiella pneumoniaeinterrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with aK. pneumoniaevirulent strain. The atlas is characterized by a decrease in the populations of alveolar macrophages and monocyte-derived macrophages, whereas neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers characteristic of less active cells such as the immune checkpoint PD-L1. These cells are those with associated bacteria. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shiftingKlebsiellafrom alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. Lack of T6SS results in cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By infecting withKlebsiella, andAcinetobacter baumanniistrains cleared by mice, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of infected mice clearing infections. Lastly, we leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING inKlebsiellainfections.sting−/−infected mice present features of mice clearing the infection including the reduced levels of PD-L1. STING absence facilitatesKlebsiellaclearance.AUTHOR SUMMARYHost-pathogen interactions are vital to our understanding of infectious disease, as well as its treatment and prevention. Mass cytometry and high-dimensional single-cell data analysis have enabled to resolve the tremendous diversity of immune cell subsets, and their heterogeneous functionality. Here we take this technology one step further to immuno profile cells in vivo while tracking their interaction with bacteria, a method we term Bac-CyTOF. Using this technology, we unveil the atlas of lung immune cells following infection with the human pathogenKlebsiella pneumoniae. The atlas is characterized by an increase in the populations expressing markers characteristic of less active cells such as the immune checkpoint PD-L1. The pathogen interacts with these cells. We uncover the contribution of the antimicrobial nanoweapon T6SS to shape the immune landscape, highlighting its potential in host defence. By probing two pathogens effectively cleared by mice, we reveal features of infections successfully cleared by mice. Lastly, we leverage this knowledge platform to investigate the role of the DNA sensor STING inKlebsiellainfections. There was no prior knowledge on the role of STING inKlebsiellainfection biology. Our findings suggestKlebsiellamay utilize STING signalling for its own benefit because absence of STING facilitatesKlebsiellaclearance.