PROJECT SUMMARY Cell-to-cell variability provides a strategy by which cells can explore diverse states; little is known, however, about the regulatory motifs that stabilize particular states into cell subsets that enable diverse functions within a population. Macrophages, cells of the innate immune system, provide an ideal experimental system to study the regulation of cell subsets, because they transiently adopt polarization states (i.e., tailored sets of molecules) to meet changing functional demands in tissues. However, the polarization state adopted to perform one task is often suboptimal for–or even in opposition to–performing other tasks, raising the question of how macrophages respond to conflicting microenvironmental cues. Our recent work suggests that cell-to-cell variation in mutually inhibitory transcription factor (TF) networks increases macrophage functional diversity and enables them to meet functional demands encoded by conflicting cues. The overall objective of this proposal is to define intracellular and extracellular signaling–TF networks that regulate heterogeneous pro-inflammatory (e.g., in response to LPS+IFN-γ) vs. anti-inflammatory (e.g., in response to IL-4) macrophage subsets and ultimately coordinate a coherent functional response. Our central hypothesis is that mutually inhibitory networks establish macrophage pro- vs. anti- inflammatory subsets that are coordinated via context-dependent mechanisms. To test this hypothesis, we will integrate single-cell measurements with mathematical modeling to determine if mutual inhibition and collective sensing are important network motifs for harnessing cell-to-cell variability and organizing appropriate polarization states to meet functional demands in tissues. Achieving the proposed aims would provide an alternative way to describe the macrophage polarization spectrum in innate immunity, by conceptualizing it as a set of functional cell subsets that emerge from multiple competing network motifs. The proposed research is innovative because we will measure single-cell responses after co-stimulation with conflicting environmental cues–specifically, cues that have known mechanisms of regulatory cross talk and cell-cell communication–in order discover new regulatory mechanisms and establish new frameworks describing macrophage functional diversity. The proposed research is significant because it is expected to have broad translational potential to identify therapeutic targets that more specifically alter macrophage functional states in vivo to treat conditions characterized by dysregulation of macrophage functions.