Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors

Abstract

Macrophages undergoing M1- versus M2-type polarization differ significantly in their cell metabolism and cellular functions. Here, global quantitative time-course proteomics and phosphoproteomics paired with transcriptomics provide a comprehensive characterization of temporal changes in cell metabolism, cellular functions, and signaling pathways that occur during the induction phase of M1- versus M2-type polarization. Significant differences in, especially, metabolic pathways are observed, including changes in glucose metabolism, glycosaminoglycan metabolism, and retinoic acid signaling. Kinase-enrichment analysis shows activation patterns of specific kinases that are distinct in M1- versus M2-type polarization. M2-type polarization inhibitor drug screens identify drugs that selectively block M2- but not M1-type polarization, including mitogen-activated protein kinase kinase (MEK) and histone deacetylase (HDAC) inhibitors. These datasets provide a comprehensive resource to identify specific signaling and metabolic pathways that are critical for macrophage polarization. In a proof-of-principle approach, we use these datasets to show that MEK signaling is required for M2-type polarization by promoting peroxisome proliferator-activated receptor-γ (PPARγ)-induced retinoic acid signaling.

Keywords: HDAC inhibitors; M1-type polarization; M2-type polarization; MEK signaling; age-related macular degeneration; kinase enrichment analysis; macrophage metabolism; macrophage polarization; phosphoproteomics; retinoic acid signaling.

Figure 1.. Time-course global quantitative proteomics identify dynamic temporal changes in cellular and metabolic pathways during M1- versus M2-type macrophage polarization

(A) Heatmap shows KEGG pathways upregulated in at least one time point, based on the time-course proteomics data of macrophages undergoing M1- (treated with IFN-γ/LPS) or M2-type polarization (treated with IL-4). Color coding indicates −log₁₀ p values when comparing the M1 or M2 group at the indicated time point with the M0 group (after 24 h PMA treatment only) at the same corresponding time point. *p < 0.05. Select pathways with distinct activation pattern during M1- versus M2-type polarization are indicated in bold. (B) Insulin signaling pathway protein changes and KEGG enrichment plot shown in macrophages after 4 h of treatment with IL-4 (compared with M0 macrophages). Heatmap shows Z scores. See also Figures S1–S4; Tables S1 and S2.

Figure 2.. Time-course global quantitative phosphoproteomics identify distinct kinase activation patterns during the induction phase of M1- versus M2-type macrophage polarization

(A) Heatmap based on KEA of time-course phosphoproteomics data. The activities of kinases are displayed as the mean relative levels of the phosphosites that they phosphorylate (log₂ ratios relative to its mean activity). Kinases with a false discovery rate (FDR) < 1% are shown. (B) Visualization of the KEA data for MEK1. Red lines show individual target phosphosites of MEK1, and black lines show averaged values. (C) ERK1 (T202/Y204) phosphorylation peaks between 2 and 8 h (*) after IL-4 treatment in primary human macrophages, which is blocked by trametinib. This inhibition of ERK1 (T202/Y204) phosphorylation by trametinib is associated with inhibition of induction of MRC1 protein by IL-4. (D) Western blotting with cell lysates (THP-1-derived macrophages after 24 h of treatment with IL-4 or IFN-γ/LPS) used in the phosphoproteomics and proteomics experiments in (E) and (F) confirm that GDC-0879 increases ERK (T202/Y204) phosphorylation and TGM2 in IL-4 treated cells, whereas trametinib blocks ERK activation and reduces TGM2. (E) M1- or M2-type marker protein levels based on quantitative proteomics of THP-1-derived macrophages after 24 h of treatment with either IL-4 or IFN-γ/LPS in the presence or absence of trametinib or GDC-0879. The data for each protein are shown as log₂ ratios relative to its mean level. (F) Quantitative phosphoproteomics of THP-1-derived macrophages treated for 24 h with IL-4 (performed in duplicate); cell lysates were also used in the proteomics experiments in (E). GDC-0879 induces phosphorylation events associated with activation of the MEK/ERK pathway and downstream targets. The y axis shows mass spectrometry (MS) intensity values (arbitrary unit). KEA heatmap shows an increase in signaling activities of MEK1 and its downstream targets ERK1/ERK2 and RSK2 with GDC-0879 treatment. The activities of each kinase are shown as log₂ ratios relative to its mean activity. (G) Top: the MEK inhibitor trametinib and the pan-Raf inhibitor TAK-632 potently reduce IL-4-induced MRC1 levels in THP-1-derived macrophages, whereas GDC-0879 increases MRC1. Bottom: trametinib inhibits IL-4-induced Arg1, whereas GDC-0879 increases Arg1 in murine BMDMs. Cytokine treatment for 4 days. (H) Western blotting shows effects of chemicals on MRC1 and phospho-STAT1(Y701) levels in primary human macrophages treated for 24 h with the chemicals in the presence of either IL-4 or IFN-γ/LPS. Western blot values indicate normalization to β-actin or β-tubulin loading control and DMSO control sample (no IL-4) in (C) and (D) or IL-4 DMSO control sample (IL-4 with DMSO vehicle but no inhibitors) in (G) and (H). Phospho-STAT1 (Y701) normalized to IFN-γ/LPS control (H). See also Figures S1 and S2; Tables S1 and S2.

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.

Figure 4.. HDAC and MEK activities promote PPARγ/RA signaling to drive M2-type macrophage polarization

(A) GO enrichment analysis of highly upregulated genes (lg FC > 2) found in RNA-seq data after 24 h of treatment with IL-4 in THP-1-derived macrophages or in primary human macrophages. In both macrophage groups, RA signaling is significantly associated with M2-type polarization (underlined). (B) GSEA enrichment plots show a high correlation of oxidative phosphorylation and PPAR signaling with M2-type polarization both in the RNA-seq as well as in the proteomics datasets (both at 24 h of treatment with IL-4 [M2] versus vehicle-treated control [M0]). (C) Heatmaps show upregulated genes associated with oxidative phosphorylation, retinol metabolism, or PPAR signaling in M2-type macrophages (lg FC is shown), including ALDH1A2, PPARG, FABP4, and ANGPTL4 (arrows). GSEA-KEGG pathway analysis in THP-1-derived M2-type macrophages shows that among the most significant terms associated with M2-type polarization are oxidative phosphorylation and retinol metabolism. GSEA enrichment plot shown for retinal metabolism comparing M2-type macrophages with macrophages not treated with IL-4 (M0). (D) Top-ranking GSEA KEGG terms in THP-1-derived and primary human macrophages show that panobinostat inhibits IL-4-induced transcriptional programs associated with retinol metabolism, PPAR signaling, and oxidative phosphorylation. (E) Left: Venn diagram shows overlap of IL-4-induced genes in THP-1-derived macrophages inhibited by trametinib and panobinostat greater 2-fold (lg FC > 1). Among those genes are key regulators of PPARγ and RA signaling. Right: heatmap shows effects of trametinib or panobinostat on expression of some of these genes (lg FC, normalized to DMSO). N.D. = not detected. (F) KEGG and GO pathway analyses of M2-type genes inhibited by both trametinib as well as panobinostat identify key pathways associated with M2-type polarization, including PPAR signaling, retinol metabolism, ERK signaling, and carboxylic acid biosynthetic process (same pathways identified in both analyses underlined in same color). (G) Extent of inhibition of expression of PPAR signaling genes inhibited by panobinostat or trametinib (lg FC). MMP1, ANGPTL4, and PPARG are among those genes with the greatest inhibition. GeneRatio shows percentage of differentially expressed genes (DEGs) in the given GO term. “Count” shows number of DEGs in the given term. Unless otherwise indicated, data refers to THP-1-derived macrophages. lg = log2. See also Figures S2 and S6–S11; Tables S2, S3, S4, and S5.

Figure 5.. MEK signaling links IL-4 signaling with PPARγ and RA signaling during M2-type polarization

Quantitative time-course proteomics and phosphoproteomics show temporal activation events after initiating treatment with either IL-4 or IFN-γ/LPS in THP-1-derived macrophages. (A) Left: temporal dynamics of levels of IL-4Rα protein and phosphorylated IL-4Rα (S387, T476, and T487) in response to IL-4. Right: correlation matrix for IL-4-treated groups. (B) Left: graph shows temporally distinct and successive peaks (arrows) of activating phosphorylating events for JAK1 and STAT6 at 10 min after the addition of IL-4, for AKT1 at 1 h, for ERK2 at 4–8 h (and diminished activity at 24 h), and for MYC at ~4 h, whereas PPARγ (S112) peaks at 24 h. Right: correlation matrix for IL-4-treated groups shows that JAK1 and STAT6 activations strongly correlate temporally (high r value), whereas the delayed activation of ERK2, MYC, or the S112 phosphorylation of PPARγ is reflected in a low r value or an adverse correlation in the comparison with JAK1 or STAT6. (C) Left: a strong increase in downstream targets of PPARγ is observed at 24 h, including FABP4, LPL, and rate-limiting enzymes of RA signaling (RDH10 and ALDH1A2). TGM2 and CD209 are markedly increased at 24 h. Right: correlation matrix for all treatment groups shows a high correlation for all these proteins. (D) Proposed model of temporal sequence of activation events during IL-4-induced M2-type macrophage polarization based on time-course proteomic and phosphoproteomic data and functional studies. Inhibitors of pan-Raf, MEK, HDACs, and PPARγ inhibit M2-type polarization. BRAF-inhibitor-mediated activation of MEK/ERK signaling, PPARγ agonist treatment, or direct stimulation of RA signaling with the RAR agonist AM580 or with ATRA promotes M2-type polarization. “P” indicates phosphorylation. Time when phosphorylation peaks is shown. TF: transcription factor. For correlation matrices: Pearson correlation coefficients (r) shown in heatmap with p values in parenthesis. See also Figures S2 and S12; Table S2.

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

(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).

Figure 7.. Panobinostat and trametinib block M2-type macrophage polarization in vivo and inhibit CNV

(A) MEK inhibitors and HDAC inhibitors attenuate VEGF-A expression in IL-4-treated THP-1-derived macrophages. Normalized to housekeeping gene 36B4 (semiquantitative RT-PCR). Cytokine treatment for 4 days. N = 3/group (each in triplicate). (B) Semiquantitative RT-PCR of choroid/RPE lysates of mice treated with either panobinostat, trametinib, or DMSO (control) for 3 days after laser-induced CNV induction. Graphs show Arg1 (M2), COX2 (M1), and CXCL9 (M1) expression normalized to CD68. N = 8–10 mice/group (each in triplicate). (C) Confocal microscopy images of CNV lesions 3 days after laser-induced injury and treatment with either DMSO, trametinib, or panobinostat. Arg1⁺ macrophages (white; yellow arrows), SMA⁺ cells (red), and CD31⁺ neovessels (green) in DMSO-treated mice, but no Arg1 or CD31 and only reduced SMA staining is observed in trametinib or panobinostat treated mice (top row). Panobinostat or trametinib treatment does not prevent macrophage infiltration (F4/80⁺ cells [white]; bottom row). Scale bars, 100 μm. (D) In skin wounds infiltrating F4/80⁺ (white) macrophages are strongly Arg1⁺ (green). Panobinostat-treated mice show infiltration of F4/80⁺ macrophages, which do not stain for Arg1. Scale bars, 20 μm. (E) Semiquantitative RT-PCR shows inhibition of Arg1 expression in skin wounds after trametinib or panobinostat treatment (normalized to 36b4). N = 8–10 mice/group (each in triplicate). Graphs represent data as means ± SEM. p values are shown (t test). See also Table S3.