Emodin Bidirectionally Modulates Macrophage Polarization and Epigenetically Regulates Macrophage Memory

Abstract

Macrophages are pleiotropic cells capable of performing a broad spectrum of functions. Macrophage phenotypes are classified along a continuum between the extremes of proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. The seemingly opposing functions of M1 and M2 macrophages must be tightly regulated for an effective and proper response to foreign molecules or damaged tissue. Excessive activation of either M1 or M2 macrophages contributes to the pathology of many diseases. Emodin is a Chinese herb-derived compound and has shown potential to inhibit inflammation in various settings. In this study, we tested the ability of emodin to modulate the macrophage response to both M1 and M2 stimuli. Primary mouse macrophages were stimulated with LPS/IFNγ or IL4 with or without emodin, and the effects of emodin on gene transcription, cell signaling pathways, and histone modifications were examined by a variety of approaches, including microarray, quantitative real-time PCR, Western blotting, chromatin immunoprecipitation, and functional assays. We found that emodin bidirectionally tunes the induction of LPS/IFNγ- and IL4-responsive genes through inhibiting NFκB/IRF5/STAT1 signaling and IRF4/STAT6 signaling, respectively. Thereby, emodin modulates macrophage phagocytosis, migration, and NO production. Furthermore, emodin inhibited the removal of H3K27 trimethylation (H3K27m3) marks and the addition of H3K27 acetylation (H3K27ac) marks on genes required for M1 or M2 polarization of macrophages. In conclusion, our data suggest that emodin is uniquely able to suppress the excessive response of macrophages to both M1 and M2 stimuli and therefore has the potential to restore macrophage homeostasis in various pathologies.

Keywords: cell signaling; emodin; histone modification; inflammation; innate immunity; macrophage; macrophage memory; polarization.

FIGURE 1.

Emodin inhibits LPS/IFNγ- and IL4-induced transcriptional changes in macrophages. A and B, mouse peritoneal macrophages were stimulated with LPS (100 ng/ml) and IFNγ (20 ng/ml) for 24 h (A) or IL4 (10 ng/ml) for 6 h (B) with or without emodin (Em, 50 μm). Gene expression was then detected using a whole-genome microarray. Genes significantly increased (top panel) or decreased (bottom panel) by LPS/IFNγ or IL4, respectively. The y axes correspond to normalized intensity values for gene expression and the x axes to treatments. Each line represents one gene, and the red and green colors mark high and low expression of genes, respectively, in the LPS/IFNγ or IL4 treatment groups. Con, control. C, Venn diagrams showing genes significantly changed by LPS/IFNγ or IL4 and by emodin under LPS/IFNγ or IL4 stimulation. D, principle component analysis of genes significantly changed in one of the treatment groups.

FIGURE 2.

Emodin inhibits the induction of signaling pathways associated with macrophage polarization and function. Shown are the most significantly affected pathways relevant for macrophage activation as determined by Ingenuity IPA canonical pathway analyses.

FIGURE 3.

Effects of emodin on the expression of genes that regulate macrophage activation. A and B, heat maps showing the expression of genes associated with the most significantly affected macrophage canonical signaling pathways. Right panels, the -fold changes of genes caused by LPS/IFNγ or IL4 treatment compared with control (Con) and emodin (Em) treatment compared with LPS/IFN or IL4 for select macrophage activation genes.

FIGURE 4.

Emodin inhibits the expression of M1 and M2 genes. A and B, qPCR was performed to verify the microarray results for select M1 and M2 genes. Error bars represent the mean ± S.E. For each treatment, n = 4. Con, control; Em, emodin. C, macrophages were stimulated with IL4 with or without emodin (0–50 μm) for 6 h. The cells were lysed, and IRF4 protein levels were detected by Western blotting. Results are shown as the mean ± S.E. for two independent experiments (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Emodin modulates the functions of activated macrophages. Mouse peritoneal macrophages were stimulated with LPS (100 ng/ml) and IFNγ (20 ng/ml) or IL4 (10 ng/ml) with or without emodin (50 μm) for 24 h. A, macrophages were washed, and the cells were incubated with FITC-labeled E. coli bioparticles for 4–6 h. Fluorescence was detected with a microplate reader as an indicator of phagocytosis. Results are shown as the mean ± S.E. (n = 4). B, macrophages were seeded into the top chamber of a transwell insert in DMEM, and DMEM with MCP1 (20 ng/ml) was placed in the bottom of the well. After 4 h, cells were fixed, stained with DAPI, and imaged with five fields of view at ×200 magnification per membrane. Results are shown as the mean ± S.E. for two independent experiments (n = 3). C, macrophages were incubated with LPS/IFNγ with emodin (Em) at various concentrations. After 24 h, the medium was collected, and the NO content was detected. Results are shown as the mean ± S.E. (n = 4).*, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Emodin inhibits LPS/IFNγ- and IL4-induced activation of signaling pathways. A and B, macrophages were stimulated with (A) LPS (100 ng/ml) and IFNγ (20 ng/ml) or (B) IL4 (10 ng/ml) with or without emodin (Em, 50 μm) for 24 h. Cells were lysed, and cytoplasmic and nuclear fractions were collected. Transcription factors were then detected in both fractions using Western blotting. Bottom panels, quantifications of blots of nuclear fractions normalized to the loading control (Con) TATA-binding protein (TBP). Results are shown as the mean ± S.E. for two independent experiments (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Emodin inhibits LPS/IFNγ- and IL4-induced histone modifications in macrophages. Macrophages were stimulated with LPS (100 ng/ml) and IFNγ (20 ng/ml) or IL4 (10 ng/ml) with or without emodin (50 μm) for 24 h. A, global histone modification levels were detected using Western blotting. The experiment was performed in triplicate. Con, control; Em, emodin. B and C, ChIP-PCR was used to detect gene-specific changes in histone modifications. Results are shown as the mean ± S.E. (n = 3). *, p ≤ 0.05; **, p ≤ 0.01.

FIGURE 8.

Emodin inhibits macrophage memory. A, macrophage treatments for testing the effects of emodin on macrophage memory. Macrophages were incubated with IFNγ (20 ng/ml) or IL4 (10 ng/ml) with or without emodin (50 μm) for 24 h. The cells were washed and incubated for 2 or 5 days (washout periods) and then stimulated with either IL4 or LPS for 6 h. B and C, gene expression was analyzed with qPCR after the first treatment with IFNγ or IL4 for 24 h and after the 2- or 5-day (d) washout period and compared with the baseline control (Con, set as 1). Em, emodin. D and E, gene expression was analyzed after the cells were restimulated with LPS or IL-4 for 6 h following the 2- or 5-day washout period. F and G, in crossover experiments, macrophages were first treated with IL4 or IFNγ for 24 h. After the 2- or 5-day washout period, the cells were further treated with LPS or IL4 for 6 h, respectively, and gene expression was analyzed. B–G, results are shown as the mean ± S.E. for two independent experiments (n = 3). H and I, macrophages were lysed after the 5-day rest period, and cytoplasmic and nuclear protein fractions were collected and analyzed via Western blotting. Results are shown as the mean ± S.E. for two independent experiments (n = 4). *, p ≤ 0.05; **, p ≤ 0.01; ***, p < 0.001.

FIGURE 9.

The effects of emodin on naive macrophages. A, macrophages were incubated with various concentrations of emodin (0–50 μm) for 24 h, and gene expression was analyzed using qPCR. Con, control. B, macrophages were incubated with or without emodin (50 μm) for 24 h. Then the cells were washed and incubated for 2 days and further stimulated with either IL4 or LPS for 6 h, and gene expression was analyzed with qPCR. Results are shown as the mean ± S.E. for two independent experiments (n = 3). *, p ≤ 0.05; **, p ≤ 0.01.