Network Analysis Reveals a Distinct Axis of Macrophage Activation in Response to Conflicting Inflammatory Cues

Macrophages are subject to a wide range of cytokine and pathogen signals in vivo, which contribute to differential activation and modulation of inflammation. Understanding the response to multiple, often-conflicting cues that macrophages experience requires a network perspective. In this study, we integrate data from literature curation and mRNA expression profiles obtained from wild type C57/BL6J mice macrophages to develop a large-scale computational model of the macrophage signaling network. In response to stimulation across all pairs of nine cytokine inputs, the model predicted activation along the classic M1-M2 polarization axis but also a second axis of macrophage activation that distinguishes unstimulated macrophages from a mixed phenotype induced by conflicting cues. Along this second axis, combinations of conflicting stimuli, IL-4 with LPS, IFN-γ, IFN-β, or TNF-α, produced mutual inhibition of several signaling pathways, e.g., NF-κB and STAT6, but also mutual activation of the PI3K signaling module. In response to combined IFN-γ and IL-4, the model predicted genes whose expression was mutually inhibited, e.g., iNOS or Nos2 and Arg1, or mutually enhanced, e.g., Il4rα and Socs1, validated by independent experimental data. Knockdown simulations further predicted network mechanisms underlying functional cross-talk, such as mutual STAT3/STAT6-mediated enhancement of Il4rα expression. In summary, the computational model predicts that network cross-talk mediates a broadened spectrum of macrophage activation in response to mixed pro- and anti-inflammatory cytokine cues, making it useful for modeling in vivo scenarios.