Figure 3

Macrophages convert between polarized states in response to cytokine treatment. (A) Schematic of experimental design, where each point represents a collected sample, and each colored trajectory represents a different treatment condition. Murine bone marrow-derived macrophages were treated with cytokines to induce polarization (IFN-γ + LPS for M1, IL-4 for M2); M0 was maintained in base media as an unpolarized control. After 24 h, cells were washed to remove cytokines and then cultured with the alternate set of polarizing cytokines (M1 switched to M2, and vice versa) (n = 3). (B) Multidimensional scaling (MDS) plot of RNA-seq expression (averaged across three replicates for each sample); points representing each sample are connected in order of collection, with point size representing the time spent (in hours) in culture. After switching polarizing cytokines, the expression profiles of previously polarized macrophages follow trajectories that trend towards the new polarized state. (C) Gene expression in log(FPKM) of iNOS (a M1 marker gene) and Arg1 (a M2 marker gene) over time. Switching the polarizing cytokines induces reduced expression of the previous marker gene and increased expression of the marker associated with the new polarization state. (D) Jensen–Shannon distance (JSD) between the RNA expression profiles of M2 → M1 repolarized macrophages compared to 24 h M1-polarized macrophages (top) and M1 → M2 macrophages compared to 24 h M2-polarized macrophages (bottom). As a baseline for expected global expression differences between M1-polarized samples over time, the purple line in the top panel shows the average JSD between both later M1 controls (collected at 48 h and 96 h) and the 24 h M1 sample. Similarly, the red line in the bottom panel shows the average JSD between both later M2 controls (48 h and 96 h) and the 24 h M2 sample. After switching media, the JSD for repolarized macrophages decreases as M1 → M2 macrophages become more similar to M2-polarized macrophages and M2 → M1 macrophages become more similar to M1-polarized macrophages.