Multimodal temporal mapping of macrophage transcriptome remodeling during Salmonella infection

Macrophages are equipped to eliminate invading pathogens, yet several intracellular bacteria exploit them as replicative niches. Salmonella enterica serovar Typhimurium subverts host immunity by injecting effector proteins that remodel macrophage functions. While macrophages typically induce a pro-inflammatory program upon bacterial invasion, Salmonella can redirect them toward an anti-inflammatory and replication-permissive state via manipulation of the NF-{kappa}B and STAT3 host transcription factors. How the integration of the effects on these two transcription factors and potentially others underpins this reprogramming remains poorly charted. Here, we use a multipronged approach combining a bacterial reporter, temporal single-cell RNA-seq with RNA metabolic labeling, transcription factor (TF) footprinting, and single-cell CRISPR perturbations to dissect macrophage polarization dynamics during early infection. We catch the bifurcation during infection, where a subset of macrophages transition toward the anti-inflammatory phenotype. This shift involves the activation of Salmonella pathogenicity island 2 (SPI2) leading to both the dampening of the initial NF-{kappa}B-driven inflammatory program and the induction of specific transcriptional modules beyond NF-{kappa}B and STAT3, with possible contributions from AP-1 and Maf family members. Together, our study uncovers host decision points in macrophage polarization circuitry and reveals a vulnerability exploited by Salmonella to modulate host immunity. HIGHLIGHTSO_LITemporal scRNA-seq defines the emergence of inflammatory and anti-inflammatory macrophage states concomitant with Salmonella Typhimurium SPI2 induction. C_LIO_LIA critical decision point for infection outcome is reached by 6 hours post-infection. C_LIO_LIMacrophage reprogramming is associated with widespread chromatin remodeling and dynamic transcription factor binding. C_LIO_LISingle-cell perturb-seq experiment uncovers Cybb and Foxo1 as modulators of infection outcome in Hoxb8 macrophages. C_LI