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

Abstract

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-γ 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-γ-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.

Uncovering the Gene Regulatory Networks Underlying Macrophage Polarization Through Comparative Analysis of Bulk and Single-Cell Data

“…are less likely to express M2 markers ( Figure 4F). When it comes to M2 cells (Path 6), the pair-wise expression correlations are not as strong ( Figure S3C), as previously reported (Muñoz-Rojas et al, 2021). This suggests that terminally polarized M1 present a stronger phenotype and are less likely to express M2 markers.…”

Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state

“…Importantly, we provide experimental evidence that MFs launch biased polarization programs in the different phases of CC, using two of the main polarization signals (IL-4 and IFNG). MF polarization by these and many other cytokines have been extensively studied, yet these studies have not implicated CC as a factor that can alter MF plasticity or immune functions [22, 23, 26, 27, 29, 55]. Strikingly, we report that the M1 polarization program is strongly restricted to the G1-phase of CC.…”

A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression

“…Reciprocal suppression of enhancers of conflicting programs thus ensures mutual exclusion of incompatible biological properties and preserves the identity of costimulated cells. On the other hand, groups of genes induced by antagonistic pathways, such as those triggered by LPS or PGE 2 , can co-exist in single cells, as also shown recently (Muñ oz-Rojas et al, 2021). The ''mixed'' phenotypes of costimulated macrophages are incompatible with dogmatic definitions of polarization states.…”