Figure 3. Chemical screens identify pharmacologic blockers of M2-type macrophage polarization.

(A) Scatterplot shows results of small-molecule chemical screen performed in duplicate (plates 1 and 2) with MRC1 promoter-driven luciferase activity as a readout. Axes show log₂ FC (fold change) of luciferase activity. Select HDAC inhibitors, pan-Raf inhibitors, MEK inhibitors, proteasome/immunoproteasome inhibitors, or HSP90 inhibitors are highlighted in the plots.

(B) Luciferase assays show a dose-dependent inhibition of MRC1 promoter-driven luciferase activity by the MEK inhibitor trametinib, the pan-Raf inhibitor AZ628, the HDAC inhibitor panobinostat, and the HSP90 inhibitor NMS-E973 at concentrations that do not affect cell viability (CTG). Concentrations of chemicals used indicated in nM on the x axis; values indicated on the y axis normalized to the control carrier (DMSO). Red: luciferase activity; green: cell viability assay (CTG). Graphs represent data as means ± SD. N = 4/group.

(C) Inhibitors of IL-4-induced MRC1 expression in the chemical screens were tested for their effects on expression of M2-type markers MRC1 and CD209 and M1-type markers CXCL9, CCR7, and iNOS in THP-1-derived macrophages that were treated with either IL-4 or IFN-γ/LPS for 4 days in the presence or absence of these inhibitors. Values were normalized to CD68 expression levels and the IL-4-treated or IFN-γ/LPS-treated control group. Expression fold changes based on semiquantitative RT-PCR shown in y axes. Graphs represent data as means ± SEM. N = 3/group (each in triplicate).

(D) HDAC inhibitors attenuate M2-type polarization of IL-4-treated THP-1-derived macrophages with diminished MRC1 and TGM2 without affecting phospho-STAT1 (Y701) levels in IFN-γ/LPS-treated macrophages. Cytokine treatment for 4 days.

(E) Effects of inhibitors on Arg1 and phospho-STAT1 (Y701) protein levels in mouse BMDMs treated with IL-4. Panobinostat and the JAK½ inhibitor S-ruxolitinib potently block Arg1 expression, and MEK inhibitors (trametinib, PD0325901) reduce Arg1 protein. GW9662 has only a moderate inhibitory effect at the concentration tested. The Src inhibitor KX2–391 and the B-Raf inhibitor GDC-0879 increased Arg1 protein. Cytokine treatment was for 24 h.

(F) Semiquantitative RT-PCR shows that four different MEK inhibitors strongly reduce MRC1 expression in IL-4-treated THP-1-derived macrophages, whereas they increase expression of CXCL9 in IFN-γ/LPS-treated macrophages (normalized to CD68 and to IL-4 or IFN-γ/LPS value). Cytokine treatment for 4 days. Expression fold changes shown in y axes. Graphs represent data as means ± SEM. N = 3/group (each in triplicate). p values are shown (t test).

Western blot values indicate normalization to β-actin loading control and DMSO control sample with no inhibitors (D) or IL-4 DMSO control sample (IL-4 with DMSO vehicle but no inhibitors) (E). See also Figure S5; Table S3.