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 are unclear. Here, we use single-cell RNA sequencing to explore how individual macrophages respond when co-stimulated with inflammatory stimuli LPS and IFN-gamma and the resolving cytokine IL-4. These co-stimulated macrophages display a distinct global transcriptional program. However, variable negative cross-regulation between some LPS + IFN-gamma-specific and IL-4-specific genes results in cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation leads to mutually exclusive expression of the T-cell-polarizing cytokine genes 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 h. This study suggests that increasing functional diversity in the population is one strategy macrophages use to respond to conflicting environmental cues.

Automated summary

The study using single-cell RNA sequencing to explore co-stimulated macrophages reveals cell-to-cell heterogeneity in transcription and mutually exclusive expression of specific genes, supporting the maintenance of specialized functions for at least 48 hours. This suggests that increasing functional diversity in the macrophage population is an effective strategy to respond to conflicting environmental cues.