IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation

Abstract

Background: Overexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. Since both IFN-γ and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity.

Methods: Monocytes (Human monocytic THP-1 cells) were stimulated with IFN-γ and LPS. CCL2 gene expression was determined by real-time RT-PCR. CCL2 protein was determined by ELISA. Signaling pathways were identified by using epigenetic inhibitors and STAT1 siRNA. Acetylation of H3K27 was analyzed by Western blotting. The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR.

Results: Our results show that the co-incubation of THP-1 monocytes with IFN-γ and LPS significantly enhanced the expression of CCL2, compared to treatment with IFN-γ or LPS alone. Similar results were obtained using primary monocytes and macrophages. Interestingly, IFN-γ priming was found to be more effective than LPS priming in inducing synergistic expression of CCL2. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. Mechanistically, we showed that IFN-γ priming induced acetylation of lysine 27 on histone 3 (H3K27ac) in THP-1 cells. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN-γ in this synergy.

Conclusion: Our findings suggest that IFN-γ, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

Keywords: CCL2; H3K27 ac; IFN-γ; LPS; inflammation; monocytes/macrophages.

Figure 1

IFN-γ and LPS cooperatively induce CCL2 in monocytic cells/macrophages. THP-1 monocytic cells, primary monocytes, and THP-1-derived macrophages were treated for 24h with IFN-γ (10 ng/mL) alone or in combination with LPS (10 ng/mL). Total RNA was extracted and Ccl2 mRNA expression was quantified by real-time RT-PCR. CCL2 protein was measured in cell supernatants using commercial ELISA kit. All data are expressed as mean ± SEM (n ≥ 3). Group means between two data sets were compared using Student’s t-test and those of more than two data sets were compared using one-way ANOVA with post-hoc Tukey’s test. All p-values < 0.05 were considered significant (ns, non-significant, *p< 0.05, **p< 0.01, and ***p< 0.001). Elevated CCL2 gene (A) and secreted protein (B) expression is shown in THP-1 monocytic cells co-stimulated with IFN-γ and LPS compared to cells treated with IFN-γ and LPS alone. Primary monocytes (C) also show increased CCL2 secreted protein expression after co-stimulation with IFN-γ and LPS compared to stimulation with either IFN-γ or LPS. In addition to monocytic cells and primary monocytes, THP-1-derived macrophages co-stimulated with IFN-γ and LPS also display elevated expression of CCL2 transcripts (D) and secreted protein (E). (F and G) THP-1 cells were primed with IFN-γ and LPS, separately, followed by LPS and IFN-γ treatment, respectively, for 24h. Ccl2 mRNA and protein expression was quantified and statistically analyzed as described above. The data show that only the IFN-γ priming followed by LPS stimulation led to elevated CCL2 mRNA (F) and secreted protein (G) expression in monocytic cells compared to controls stimulated with IFN-γ or LPS alone.

Figure 2

Synergistic expression of CCL2 is dependent on STAT1. (A) THP-1 monocytic cells were transfected with control/scrambled siRNA or STAT1 siRNA and incubated for 40h. Total RNA was extracted and real-time RT-PCR was performed to measure STAT1 mRNA expression. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2^−ΔΔCT method. Relative STAT1 mRNA expression was expressed as fold expression over average of control (scrambled siRNA) gene expression. All data are expressed as mean ± SEM (n ≥ 3) and group means between two data sets were compared using Student’s t-test. All p-values < 0.05 were considered significant. The data show significant suppression of STAT1 mRNA expression in cells transfected with STAT1 siRNA compared to control siRNA transfected cells (**p< 0.01). (B and C) STAT1-deficient cells were treated for 24h with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) and Ccl2 mRNA (B) and protein (C) expression was determined using real-time RT-PCR and ELISA, respectively. Target mRNA levels were normalized against GAPDH mRNA and gene expression relative to control was calculated using 2^−ΔΔCT method. Relative CCL2 mRNA expression was expressed as fold change over average of control (vehicle treatment) gene expression. All data are expressed as mean ± SEM (n ≥ 3) and group means between two data sets were compared using Student’s t-test. The data show significant suppression of (B) CCL2 mRNA (***p< 0.001) and (C) CCL2 secreted protein (**p< 0.01) in cells co-stimulated with IFN-γ and LPS as compared to those stimulated with IFN-γ or LPS alone. (D) Western blot showing phosphorylation of STAT1 after IFN-γ (10 ng/mL) treatment over time indicates the optimal STAT1 phosphorylation at 120 min.

Figure 3

H3K27 acetylation levels at different sites of CCL2 promotor region. (A) THP-1 monocytic cells were treated with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) for 4 h and the treatment with vehicle alone served as control. Cell lysates were used for determination of H3K27 acetylation by Western blotting. Briefly, cell lysates were resolved using 12% SDS-PAGE and blots were probed with rabbit anti-human H3K27 antibody (1:1000 dilution) at 4°C overnight. Blots were washed, incubated for 2h with HRP-conjugated secondary antibody (1:2500 dilution), and immunoreactive bands were developed and visualized using ChemiDoc™ MP Imaging Systems. (B) Western blot band densities were quantified and data were expressed as mean ± SEM (n = 3) values which were compared for various treatments using one-way ANOVA and post-hoc Tukey’s test. All p-values < 0.05 were considered significant (ns, non-significant, *p< 0.05, **p< 0.01, and ***p< 0.001). The data show increased H3K27 acetylation (H3K27 ac) in the cells stimulated with IFN-γ or IFN-γ+LPS compared to cells stimulated with LPS only (***p< 0.001). (C) The schematic diagram of CCL2 gene promotor region is shown. (D–I) THP1 cells were stimulated with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) by incubation for 24h at 37°C. Chromatin immunoprecipitation was done on cell lysate as described in methodology using antibodies specific to H3K27, Histone H3, and normal rabbit IgG for overnight at 4°C. H3K27 acetylation levels induced by treatments compared to control (vehicle) were detected by qPCR using primers specific to the closest regions of transcription start site at the CCL2 promotor. Data (mean ± SEM, n = 3) were expressed as fold enrichment levels and were compared for different treatments against control using one-way ANOVA with post-hoc Tukey’s test. All p-values < 0.05 were considered significant (*p< 0.05 and **p< 0.01).

Figure 4

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate synergy between IFN-γ and LPS for the CCL2 production. THP-1 monocytic cells were treated with anacardic acid (HATs inhibitor; 50 μM) overnight or with TSA (HDACs inhibitor; 25 nM) for 6 h, followed by treatments with IFN-γ (10 ng/mL) and/or LPS (10 ng/mL) for 24h, and the treatment with vehicle alone served as control. Ccl2 mRNA expression was determined by real-time RT-PCR and target gene expression were normalized to GAPDH expression. Relative changes in Ccl2 gene expression were calculated using 2^−ΔΔCT method and expressed as fold change over its expression in control (vehicle treatment). CCL2 secreted protein expression was detected by ELISA as described in materials and methods. All data were expressed as mean ± SEM values (n = 3) and group means between two data sets were compared using Student’s t-test. All p-values < 0.05 were considered significant (**p<0.01, ***p<0.001). The data show the reduced expression of CCL2 (A) mRNA (***p<0.001) and (B) secreted protein (**p<0.01) in the cells that were treated with HAT-inhibitor anacardic acid before co-stimulation with IFN-γ and LPS as compared to similarly stimulated cells that were not pre-treated with anacardic acid. Interestingly, increased expression of CCL2 (C) mRNA (**p<0.01) and (D) secreted protein (***p<0.001) were observed in the cells that were treated with HDAC-inhibitor TSA before stimulation with LPS only as compared to similarly stimulated cells that were not pre-treated with TSA, suggesting that TSA priming could mimic effect of and substitute for IFN-γ in cooperativity with LPS.