GM-CSF differentiation of human monocytes stabilizes macrophage state via oxidative signaling

Macrophages are central mediators of immunity that integrate diverse signals derived from differentiation cues, tissue location, and disease. Controlling macrophage state and function is an appealing therapeutic objective across many diseases including cancer, atherosclerosis, and tuberculosis. Despite the growing appreciation for the in vivo complexity of macrophage state, existing in vitro models of human monocyte-derived macrophages have used a limited number of individual perturbations to explore the complex phenotypic space that macrophages can occupy. Here, we leverage a tiered differentiation, activation, and stimulation strategy to generate libraries of in vitro monocyte-derived macrophages and examine the in vitro state space of macrophage function using high-dimensional technologies. Our tiered experimental approach further revealed a striking relationship between GM-CSF differentiation and IL-10 production. Cells that were differentiated with GM-CSF produced very low or undetectable levels of IL-10 independent of activation or stimulation condition. To nominate candidate regulators of this IL-10 response, we leverage unbiased single-cell mRNA sequencing to identify transcriptional networks associated with GM-CSF-derived cells. Using these data, we identify oxidative signaling pathways as upregulated in GM-CSF derived cells and demonstrate that scavenging of oxidative radicals can enhance IL-10 production in these cells. Collectively, these data underscore the complexity of monocyte-derived macrophage state over time and highlight a dominant role for GM-CSF in tuning macrophage inflammatory phenotype, metabolic state, and plasticity. ### Competing Interest Statement A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Orche Bio, and Dahlia Biosciences.