Fig 1.
Time depended changes in the expression of macrophage polarization markers at mRNA level.
Primary human MDMs (M0) were left unstimulated or were polarized to M1 (100 ng/mL LPS, 20 ng/mL IFNγ), M2a (20 ng/mL IL-4), or M2c (20 ng/mL IL-10) for 4, 8, 12, 24, 48, or 72 h. The expression of (A-F) CXCL9, CXCL10, TNF, IL-1β, IL-12, and IDO1 mRNA for M1 polarization; of (G-K) MRC1, TGM2, CCL17, CCL22 and IL-10 mRNA for M2a polarization; and of (L-N) IL-10, CD163 and TGFβ mRNA for M2c polarization were analyzed by qPCR. Data shown are mean ± SEM of biological replicates of 3 independent donors. Polarized macrophages (M1, M2a, or M2c) at all time points were compared with unstimulated (US) M0 macrophages. Trends for individual donors are depicted in S1 Fig. Statistical analyses were performed with repeated measures ANOVA, * p < 0.05; ** p < 0.01; *** p < 0.001 (Statistical comparison of all time points are shown in the S1 Table).
Table 1.
Suggested stimulation times for the detection of M1, M2a, and M2c polarization markers in human monocyte-derived macrophages.
Fig 2.
Time depended changes in the expression of macrophage polarization markers at protein level.
Primary human MDMs (M0) were left unstimulated or were polarized to M1 (100 ng/mL LPS, 20 ng/mL IFNγ) or M2a (20 ng/mL IL-4) for 4, 8, 12, 24, 48, or 72 h. Surface marker expression (A-C) of CD86, CD64 and HLA-DR for M1 polarization; (D-E) of CD200R and CD206 (MRC1) for M2a polarization, and (F) of CD163 for M2c polarization was analyzed by flow cytometry. Shown are representative histograms (left) and summary data shown are mean ± SEM of biological replicates of 3 independent donors (right). Polarized macrophages (M1, M2a, or M2c) at all time points were compared with unstimulated (US) M0 macrophages. Trends for individual donors are depicted in S2 Fig. Statistical analyses were performed with repeated measures ANOVA, * p < 0.05; ** p < 0.01; *** p < 0.001 (Statistical comparison of all time points are shown in the S2 Table).
Fig 3.
Time depended changes in cytokine production by M1, M2a, and M2c macrophages.
Primary human MDMs (M0) were left unstimulated or were polarized to M1 (100 ng/mL LPS, 20 ng/mL IFNγ), M2a (20 ng/mL IL-4), or M2c (20 ng/mL IL-10) for 4, 8, 12, 24, 48, or 72 h. Production of (A-C) TNF, IL-12 and IL-1β for M1 polarization; of (D) IL-10 for M2a polarization, and of (E) TGFβ for M2c polarization was analyzed by ELISA. Data shown are mean ± SEM of biological replicates of 3 independent donors. Polarized macrophages (M1, M2a, or M2c) at all time points were compared with unstimulated (US) M0 macrophages. Trends for individual donors are depicted in S3 Fig. Statistical analyses were performed with repeated measures ANOVA, * p < 0.05; ** p < 0.01; *** p < 0.001 (Statistical comparison of all time points are shown in the S3 Table).