Figure 2

Polarized macrophage phenotypes are transient. (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 in base media with no polarizing cytokines (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 the removal of polarizing cytokines, the expression profiles of previously polarized macrophages follow trajectories that trend towards the M0 baseline. (C) Gene expression in log(FPKM) of iNOS (a M1 marker gene) and Arg1 (a M2 marker gene) over time. While marker gene expression increases with initial polarization, it drops sharply upon the removal of polarizing cytokines and falls to near baseline levels after 72 h in base media. (D) Jensen–Shannon distance (JSD) between the RNA expression profiles of polarized and depolarized macrophages compared to the 0 h M0 control. As a baseline for expected global expression differences between samples, the teal line shows the average JSD between all later M0 controls (collected at 24 h, 48 h, and 96 h) and the original 0 h M0 control sample. While JSD initially increases as polarizing macrophages develop distinct mRNA expression profiles and diverge from the unpolarized baseline, removal of polarizing cytokines triggers a reversion towards the baseline.