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. Author manuscript; available in PMC: 2022 Jan 23.
Published in final edited form as: Cell Rep. 2021 Nov 2;37(5):109955. doi: 10.1016/j.celrep.2021.109955

Figure 6. IL-4-induced and MEK/ERK-mediated PPARγ and RA signaling are required for M2-type macrophage polarization.

Figure 6.

(A) GW9662 reduces IL-4-induced MRC1 promoter-driven luciferase activity in a dose-dependent manner at concentrations that do not affect cell viability in THP-1-derived macrophages. Concentrations of chemicals used indicated in nM on the x axis; light units on y axis are normalized to the control carrier (DMSO). Red: luciferase activity; green: CTG. Graphs represent data as means ± SD. N = 4/group.

(B) THP-1-derived macrophages treated with IL-4 at the indicated time points (in hours). Western blotting for ALDH1A2, TGM2, and as a loading control β-actin or β-tubulin. High ALDH1A2 protein levels are detected at 24 h, but they are completely blocked by trametinib and panobinostat (upper band is the specific ALDH1A2 band; lower band is an unspecific background band). GW9662 reduces and rosiglitazone increases ALDH1A2. Panobinostat, trametinib, and GW9662 inhibit TGM2 levels (peaking at 24 h), whereas rosiglitazone, ATRA, or AM580 increase TGM2.

(C) GW9662 reduces M2-type polarization in primary mouse macrophages with reduction of Tgm2 and Arg1, whereas rosiglitazone, AM580, and ATRA strongly promote M2-type polarization. AM580 and ATRA even induce Arg1 and Tgm2 in macrophages treated with IFN-γ/LPS. 4 days of chemokine treatment.

(D) IL-4-induced TGM2 is reduced with GW9662 but increased with rosiglitazone, AM580, or ATRA. Primary human macrophages after 4 days of treatment with IL-4.

(E) Primary human macrophages treated with IL-4 at the indicated time points (in hours) in the presence or absence of GDC-0879. IL-4 increases ERK1 (T202/Y204) phosphorylation at 2–8 h with a concomitant increase in PPARγ, which then both decline at 24 h. GDC-0879 increases ERK1(T202/Y204) phosphorylation and PPARγ levels, which leads to an increase in MRC1 and TGM2.

(F) Primary human macrophages treated with IL-4 for either 8 h or for 24 h in the presence or absence of trametinib or GDC-0879 (DMSO as controls). The increase in ERK1 (T202/Y204) phosphorylation and PPARγ with GDC-0879 is associated with increased MRC1 and TGM2, whereas the block of ERK1 (T202/Y204) phosphorylation and the low levels of PPARγ with trametinib treatment lead to reduced MRC1 and TGM2.

Western blots with β-actin or β-tubulin loading control. Western blot values indicate normalization to loading control and IL-4 DMSO control sample at 24 h (B), IL-4 DMSO control sample (C) and (D), IL-4 DMSO control sample at 0 h (for MRC1 at 24 h) (E), or DMSO control sample with no IL-4 (F).