Immunomodulatory Effects of (R)-Sulforaphane on LPS-Activated Murine Immune Cells: Molecular Signaling Pathways and Epigenetic Changes in Histone Markers

Abstract

The aim of this study was to explore the immunomodulatory effects of the natural enantiomer (R)-Sulforaphane (SFN) and the possible signaling pathways involved in an ex vivo model of LPS-stimulated murine peritoneal macrophages. Furthermore, we studied the epigenetic changes induced by (R)-SFN as well as the post-translational modifications of histone H3 (H3K9me3 and H3K18ac) in relation to the production of cytokines in murine splenocytes after LPS stimulation. (R)-SFN was able to modulate the inflammatory response and oxidative stress induced by LPS stimulation in murine peritoneal macrophages through the inhibition of reactive oxygen species (ROS), nitric oxide (NO) and cytokine (IL-1β, IL-6, IL-17, IL-18 and TNF-α) production by down-regulating the expression of pro-inflammatory enzymes (iNOS, COX-2 and mPGES-1). We also found that activation of the Nrf-2/HO-1 axis and inhibition of the JAK2/STAT-3, MAPK, canonical and non-canonical inflammasome signaling pathways could have been responsible for the immunomodulatory effects of (R)-SFN. Furthermore, (R)-SFN modulated epigenetic modifications through histone methylation (H3K9me3) and deacetylation (H3K18ac) in LPS-activated spleen cells. Collectively, our results suggest that (R)-SFN could be a promising epinutraceutical compound for the management of immunoinflammatory diseases.

Keywords: (R)-sulforaphane; antioxidant; epigenetic; histone; inflammation; macrophages; spleen cells.

Figure 1

Enantioselective synthesis of (R)-sulforaphane by the DAG methodology.

Figure 2

Effect of (R)-SFN on cell viability. Cells were treated with (R)-SFN (200–1.6 µM) for 18 h. Cell survival was represented as the percentage of viability with respect to 100% of untreated control cells.

Figure 3

Effects of (R)-SFN on NO (A) and ROS (B) production and iNOS protein expression (C). Cells were treated with (R)-SFN (12.5 and 6.25 µM) for 30 min (min) and then exposed to 5 µg/mL of LPS for 18 h. The β-actin housekeeping gene was used as a control to normalize the densitometry performed. Data are expressed as means ± standard errors (SEMs) (n = 6). (+++) p < 0.001 vs. control cells (unstimulated); (***) p < 0.001 vs. LPS-DMSO-treated cells.

Figure 3

Effects of (R)-SFN on NO (A) and ROS (B) production and iNOS protein expression (C). Cells were treated with (R)-SFN (12.5 and 6.25 µM) for 30 min (min) and then exposed to 5 µg/mL of LPS for 18 h. The β-actin housekeeping gene was used as a control to normalize the densitometry performed. Data are expressed as means ± standard errors (SEMs) (n = 6). (+++) p < 0.001 vs. control cells (unstimulated); (***) p < 0.001 vs. LPS-DMSO-treated cells.

Figure 4

Treatment with (R)-SFN decreased the production of TNF-α (A), IL-17 (B), IL-6 (C) and IL-1β (D) levels. Murine peritoneal macrophages were treated with (R)-SFN (12.5 or 6.25 µM) 30 min before stimulation with LPS (5 µg/mL) for 18 h, then cytokine secretion was analyzed by enzyme-linked immunosorbent assay (ELISA) in cell supernatants. Data are expressed as means ± SEMs (n = 8). (++) p < 0.01; (+++) p < 0.001 vs. control cells (unstimulated); (***) p < 0.001 vs. LPS-DMSO-treated cells; (#) p < 0.05 vs. cells treated with 6.25 µM (R)-SFN.

Figure 5

(R)-SFN decreased COX-2 and mPGES-1 expression in murine peritoneal macrophages. Cells were treated with (R)-SFN (12.5 and 6.25 µM) for 30 min and then exposed to 5 µg/mL of LPS for 18 h. Histograms show the densitometric analysis, normalized to the β-actin housekeeping gene of COX-2 or mPGES-1 proteins. Data are represented as means ± SEMs (n = 6). (+++) p < 0.001 vs. un-stimulated control cells; (***) p < 0.001 vs. LPS-DMSO-treated cells; (#) p < 0.05 vs. cells treated with 6.25 µM (R)-SFN.

Figure 6

(R)-SFN increased the expression of Nrf-2 and HO-1 in murine peritoneal macrophages activated by LPS. Murine immune cells were treated with (R)-SFN for 30 min, followed by stimulation with LPS for 18 h. Histograms show the densitometric analysis of Nrf-2 and HO-1 proteins normalized to the β-actin housekeeping gene. Data are expressed as means ± SEMs (n = 6). (**) p < 0.01; (***) p < 0.001 vs. LPS-DMSO-treated cells.

Figure 7

(R)-SFN decreased the phosphorylation of p38, JNK and ERK in LPS-activated peritoneal macrophages. Cells were pretreated with (R)-SFN (12.5 and 6.25 µM) for 30 min followed by LPS stimulation for 18 h. As controls, cells were also treated with DMSO (solvent control) and LPS or untreated in the absence of LPS. Histograms show the densitometric analysis of pp38, pJNK and pERK proteins normalized to the p38, JNK and ERK housekeeping gene, respectively. Data are expressed as means ± SEMs (n = 6). (+++) p < 0.001 vs. unstimulated control cells; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 vs. LPS-DMSO-treated cells.

Figure 8

(R)-SFN reduced the phosphorylation of the JAK2/STAT-3 pathway in LPS-activated peritoneal macrophages. Murine peritoneal macrophages were treated with ®-SFN (12.5 and 6.25 µM) for 30 min, followed by LPS stimulation for 18 h. Histograms show the densitometric analysis of pJAK2 and pSTAT-3 proteins normalized to the β-actin housekeeping gene. Data are expressed as means ± SEMs (n = 6). (+++) p < 0.001 vs. unstimulated control cells; (***) p < 0.001 vs. LPS-DMSO-treated cells.

Figure 9

(R)-SFN down-regulated the inflammasome signaling pathway in murine peritoneal macrophages. Murine peritoneal macrophages were treated with (R)-SFN (12.5 and 6.25 µM) for 30 min, followed by LPS stimulation for 18 h. Histograms show the densitometric analysis of NLRP3 (A), ASC (B), caspase 1 (C), caspase 11 (D) and IL-18 (E) proteins normalized to the β-actin housekeeping gene. Data are expressed as means ± SEMs (n = 6). (+) p < 0.05; (++) p < 0.01; (+++) p < 0.001 vs. unstimulated control cells; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 vs. LPS-DMSO-treated cells; (##) p < 0.01; (###) p < 0.001 vs. cells treated with 6.25 µM (R)-SFN.

Figure 10

Effects of (R)-SFN on the production of IL-6 (A), IL-17 (B), IL-1β (C) and on the modification of the expression of H3 histones: H3K18ac (D), H3K27me3 (E) and H3K9me3 (F). Splenocytes were treated with (R)-SFN (12.5 and 6.25 μM) and stimulated with LPS for 24 h. Histones were isolated from spleen cells with acid extraction and were evaluated by Western blotting. The H3 housekeeping gene was used as a control to normalize the densitometry performed. Cytokine secretion was analyzed by ELISA in spleen cell supernatants. Results are presented as means ± SEMs (n = 6). (++) p < 0.01; (+++) p < 0.001 vs. unstimulated control cells; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 vs. LPS-DMSO-treated cells; (#) p < 0.05; (###) p < 0.001 vs. cells treated with (R)-SFN.