Single-cell profiling identifies ACE+ granuloma macrophages as a non-permissive niche for intracellular bacteria during persistent Salmonella infection

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures comprised of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent Salmonella enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using a STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin converting enzyme (ACE) defines a granuloma macrophage population that is non-permissive for intracellular bacteria and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked ...,
Spleens from mice were minced with surgical blades No. 22 and incubated in digestion buffer (HBSS + Ca2+ + Mg2+ + 50 μg/mL DNase (Roche) + 25 μg/mL Liberase TL (Sigma)) at 37 °C for 25 min, mixing at 200 rpm. EDTA was added at a final concentration of 5 mM to halt digestion. Single cell suspensions were passed through a 70-μm filter and washed with R5 buffer (RPMI containing 5% FBS and 10 mM Hepes). Red blood cells were lysed with ACK Lysis Buffer (Lonza) for 3 min at room temperature, washed, and resuspended in R5 buffer until they were stained for flow cytometry. 
All samples subjected to scRNA-seq were prepared and FACS-enriched using the following procedures. STm infected spleens were harvested and digested in buffer containing HBSS + Ca2+ + Mg2+ + 50 μg/mL DNase (Roche) + 25 μg/mL Liberase TL (Sigma) at 37 °C for 25 min, mixing at 200 rpm. EDTA was added to 5 mM final concentration to stop digestion reaction and cells were washed with RPMI containing 10% FCS. Following RBC lysis,..., Software package versions used:
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