The histone methyltransferase Smyd2 is a negative regulator of macrophage activation by suppressing interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) production

Abstract

SET and MYND domain-containing 2 (Smyd2), a histone 3 lysine 4- and histone 3 lysine 36 (H3K36)-specific methyltransferase, plays critical roles in cardiac development and tumorigenesis. However, the role of Smyd2 in immunity and inflammation remains poorly understood. In this study, we report that Smyd2 is a novel negative regulator for macrophage activation and M1 polarization. Elevated Smyd2 expression suppresses the production of proinflammatory cytokines, including IL-6 and TNF, and inhibits the expression of important cell surface molecules, including major MHC-II and costimulatory molecules. Furthermore, macrophages with high Smyd2 expression inhibit Th-17 cell differentiation but promote regulatory T cell differentiation as a result of increased TGF-β production and decreased IL-6 secretion. In macrophages, Smyd2 specifically facilitates H3K36 dimethylation at Tnf and Il6 promoters to suppress their transcription and inhibits NF-κB and ERK signaling. Therefore, our data demonstrate that epigenetic modification by Smyd2-mediated H3K36 dimethylation at Tnf and Il6 promoters plays an important role in the regulation of macrophage activation during inflammation.

Keywords: Autoimmunity; ChiP; Immunology; Macrophage; Signaling.

FIGURE 1.

Expression of Smyd2 during macrophage differentiation and activation. Mouse bone marrow (BM) cells were differentiated into M0 macrophages with MCSF for 6 days. A, M0 macrophages were stimulated with 100 ng/ml LPS or PBS for 6 h and collected later for detection of Smyd family mRNA expression by Q-PCR. B, the mRNA levels of Smyd2 in bone marrow cells and M0 cells detected by Q-PCR. C and D, the quantities of mRNA and protein of Smyd2 in bone marrow-derived macrophages stimulated with LPS for 0, 2, 4, 8, 16, or 24 h were measured by Q-PCR and Western blot analysis. E, luciferase (luc) assay of Raw 264.7 cells transfected with IL-6 or the TNF promoter reporter plasmid with the Smyd2 plasmid or its catalytic mutant control. *, p < 0.05; **, p < 0.01; NS, no significance. Data are mean ± S.D. of technical replicates from one of three independent experiments. In A–C and E, controls were normalized to one. In Q-PCR experiments, β-actin served as an internal control. The experiments were performed at least three times with similar results obtained.

FIGURE 2.

Smyd2 inhibits the production of proinflammatory cytokines and the expression of costimulatory molecules. A, the quantities of mRNA (top panel) and protein (bottom panel) of Smyd2 in MCSF-driven bone marrow-derived macrophages 24 h after transfection with the Smyd2 mutant (Mutant) or Smyd2 plasmid (Smyd2) were measured by Q-PCR and Western blot analysis. B, the expression of IL-6 and TNF mRNA (top panel) and protein (bottom panel) in macrophages transfected as described in A and subsequently stimulated with 100 ng/ml LPS for 4 and 24 h, respectively, were determined by Q-PCR and ELISA. C, the quantities of mRNA (top panel) and protein (bottom panel) of Smyd2 in MCSF-driven bone marrow-derived macrophages 24 h after transfection with control siRNA (si-Ctrl) or two kinds of Smyd2 siRNA (si-RNA1 and si-RNA2) were measured by Q-PCR and Western blot analysis. D, expression of IL-6 and TNF mRNA (top panel) and protein (bottom panel) in macrophages transfected as described in C and then stimulated with 100 ng/ml LPS for 4 and 24 h, respectively, were determined by Q-PCR and ELISA. E, macrophages transfected as described in A and subsequently stimulated for 24 h with 100 ng/ml LPS were analyzed for the expressions of the indicated surface markers by flow cytometry. The shaded area and solid line refer to the isotype control and the indicated markers, respectively. MFI, mean fluorescence intensity. *, p < 0.05; **, p < 0.01; NS, no significance. Data are mean ± S.D. of technical replicates from one of three independent experiments. In Q-PCR experiments, controls were normalized to one, and β-actin served as an internal control. The experiments were performed at least three times with similar results obtained.

FIGURE 3.

Smyd2 suppresses TLR-mediated activation of the NF-κB and MAPK signaling pathways. A and B, MCSF-driven bone marrow-derived macrophages were transfected with the Smyd2 mutant (Mutant) control or the Smyd2 plasmid (Smyd2) for 24 h. After stimulation for 0–4 h with LPS, the phosphorylated (p-) or total protein in lysates of mutant control and macrophages overexpressing Smyd2 were analyzed by Western blot analysis. Total protein and β-actin served as loading controls. Graphed data were derived from the Western blot analysis by ImageJ software (National Institutes of Health). *, p < 0.05. Western blot data are representative of one of three independent experiments. Data are mean ± S.D. of technical replicates. The experiments were performed at least three times with similar results obtained.

FIGURE 4.

Suppression of IL-6 and TNF-α production by Smyd2 through its H3K36 methyltransferase activity. A, the dimethylation of histone 3 lysine 4 (H3K4me2), trimethylation of histone 3 lysine 4 (H3K4me3), trimethylation of histone 3 lysine 27 (H3K27me3), dimethylation of histone 3 lysine 36 (H3K36me2), and RelA at the Il6 and Tnf promoter regions in MCSF-driven bone marrow-derived macrophages stimulated for 2 h with LPS or PBS were analyzed by ChIP assay. The promoter sequences were detected by quantitative PCR. B, the promoter sequences were detected in mutant control- or Smyd2 plasmid-transfected macrophages as in A. Macrophages were transfected for 24 h before stimulation with 100 ng/ml LPS for another 2 h. C, the recruitment of Smyd2 to the Il6, Tnf, Tafaip3, Il12b, or Jmjd3 promoter locus with Smyd2 antibody in MCSF-driven bone marrow-derived macrophages without stimulation were analyzed by ChIP assay. Promoter sequences in input DNA and DNA recovered from antibody-bound chromatin segments were detected by quantitative PCR. IgG served as a ChIP control and input as a normalized control. *, p < 0.05; **, p < 0.01. Data are mean ± S.D. of technical replicates from one of three independent experiments. The experiments were performed at least three times with similar results obtained.

FIGURE 5.

Macrophages overexpressing Smyd2 suppress Th-17 but promote Treg cell differentiation. MCSF-driven bone marrow-derived macrophages were transfected with mutant control (Mut) or Smyd2 plasmid (Smyd2) and then stimulated for 4 h with LPS. A, the macrophages were cocultured with purified CD4⁺CD25⁻ T cells (1:1) under suboptimal Th-17 differentiation conditions. After 4 days of culture, Th-17 cell differentiation was evaluated by flow cytometry gated on CD4⁺ T cells (left panel) or ELISA detection of IL-17 (right panel). B, the macrophages were cocultured under suboptimal Treg differentiation conditions. Three days later, iTreg cell differentiation was evaluated by flow cytometry detection of Foxp3 (left panel). The expression of TGF-β1 mRNA in cocultured macrophages transfected with the mutant or Smyd2 plasmid (right panel) was determined by Q-PCR. *, p < 0.05. Data are mean ± S.D. of technical replicates from one of three independent experiments. The experiments were performed at least three times with similar results obtained.