Co-stimulation with opposing macrophage polarization cues leads to orthogonal secretion programs in individual cells

Macrophages are innate immune cells that contribute to fighting infections, tissue repair, and maintaining tissue homeostasis. To enable such functional diversity, macrophages resolve potentially conflicting cues in the microenvironment via mechanisms that remain unclear. Here, we use single-cell RNA sequencing to explore how individual macrophages respond when co-stimulated with the inflammatory stimuli, LPS+IFN-γ, and the resolving cytokine, IL-4. We find that co-stimulated macrophages display a distinct global transcriptional program. However, variable negative cross-regulation between some LPS+IFN-γ- and IL-4-specific genes results in significant cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation leads to mutually exclusive expression of the T-cell-polarizing cytokines Il6 and Il12b versus the IL-4-associated factors Arg1 and Chil3 in single co-stimulated macrophages, and single-cell secretion measurements show that these specialized functions are maintained for at least 48 hours. Overall, our study suggests that increasing functional diversity in the population is one strategy macrophages use to respond to conflicting environmental cues.