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. 2015 Mar 20;10(3):e0121574.
doi: 10.1371/journal.pone.0121574. eCollection 2015.

Sulforaphane epigenetically regulates innate immune responses of porcine monocyte-derived dendritic cells induced with lipopolysaccharide

Xueqi Qu  1 , Maren Pröll  1 , Christiane Neuhoff  1 , Rui Zhang  1 , Mehmet Ulas Cinar  2 , Md Munir Hossain  3 , Dawit Tesfaye  1 , Christine Große-Brinkhaus  1 , Dessie Salilew-Wondim  1 , Ernst Tholen  1 , Christian Looft  1 , Michael Hölker  1 , Karl Schellander  1 , Muhammad Jasim Uddin  1
Affiliations

Sulforaphane epigenetically regulates innate immune responses of porcine monocyte-derived dendritic cells induced with lipopolysaccharide

Xueqi Qu et al. PLoS One. .

Abstract

Histone acetylation, regulated by histone deacetylases (HDACs) is a key epigenetic mechanism controlling gene expressions. Although dendritic cells (DCs) are playing pivotal roles in host immune responses, the effect of epigenetic modulation of DCs immune responses remains unknown. Sulforaphane (SFN) as a HDAC inhibitor has anti-inflammatory properties, which is used to investigate the epigenetic regulation of LPS-induced immune gene and HDAC family gene expressions in porcine monocyte-derived dendritic cells (moDCs). SFN was found to inhibit the lipopolysaccharide LPS induced HDAC6, HDAC10 and DNA methyltransferase (DNMT3a) gene expression, whereas up-regulated the expression of DNMT1 gene. Additionally, SFN was observed to inhibit the global HDAC activity, and suppressed moDCs differentiation from immature to mature DCs through down-regulating the CD40, CD80 and CD86 expression and led further to enhanced phagocytosis of moDCs. The SFN pre-treated of moDCs directly altered the LPS-induced TLR4 and MD2 gene expression and dynamically regulated the TLR4-induced activity of transcription factor NF-κB and TBP. SFN showed a protective role in LPS induced cell apoptosis through suppressing the IRF6 and TGF-ß1 production. SFN impaired the pro-inflammatory cytokine TNF-α and IL-1ß secretion into the cell culture supernatants that were induced in moDCs by LPS stimulation, whereas SFN increased the cellular-resident TNF-α accumulation. This study demonstrates that through the epigenetic mechanism the HDAC inhibitor SFN could modulate the LPS induced innate immune responses of porcine moDCs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression of class I, class II, and class IV HDAC genes in porcine moDCs stimulated with LPS.
The expression of HDAC family genes in moDCs were influenced with LPS (1μg/ml) stimulation for 24 h. moDCs were generated from adherent monocytes at day 7 in vitro, which were treated with or without LPS. The class I (A), class II (B), and class IV (C) of HDACs mRNA were quantified by qRT-PCR and normalized with the housekeeping gene HRPT1. The results were combined from three independent experiments and each experiment performed in triplicate. The data were represented as the mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01).
Fig 2
Fig 2. The effects of LPS on DNMT gene expression.
Expression of genes that encode the enzymes responsible for methylating CpG sites of DNA were quantified by qRT-PCR including the maintenance methyltransferase DNMT1 and the de novo methyltransferase DNMT3a in moDCs in response to LPS exposure (24 h) compared with control. The results were combined from three independent experiments and each experiment was performed in triplicate. The data were represented as the mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01).
Fig 3
Fig 3. SFN pre-incubation inhibited LPS induce cell death in a time denpendent manner.
moDCs at day 7 were used for cell viability assay by WST-1 kit. A SFN dose-dependent assay was used to confirm cell viability of moDCs after stimulating with different concentration of SFN (Control, 5 μM, 10 μM, 15 μM, 20 μM, and 50 μM) for 24h (A). For the effects of SFN on LPS induced cell death, moDCs were pre-incubated 24 h with or without SFN (10 μM) before exposed to LPS (1 μg/ml) for 1, 3, 6, 12, and 24 h (B). The results were combined from three independent experiments and each experiment was performed in triplicate. The data were represented as the mean ± standard deviations (SD) (* P < 0.05; **P<0,01; ***P<0,001).
Fig 4
Fig 4. SFN inhibits HDAC activity and regulates genes which encode epigenetic enzymes.
moDCs at day 7 in cell culture were used for this experiment. Relative HDAC activity assay was measured using the Color-de-Lys HDAC colorimetric activity assay kit. To confirm the global HDAC deacetylation of moDCs, cells were stimulated with different concentration of SFN (Control, 5 μM, 10 μM, 15 μM, and 20 μM) for 24 h (A). Equal amounts of isolated nuclear protein were subjected to HDAC activity analysis. The effects of SFN (10 μM) on gene expression of epigenetic encoding enzymes in porcine moDCs stimulated with LPS were examined. The HDAC6 (B), HDAC10 (C), DNMT3a (D) and DNMT1 (E) mRNA expression was quantified using qRT-PCR. The moDCs were pre-treated for 24 h with or without SFN before stimulating with LPS (1 μg/ml) for additional 24 h. The results (A, B, C, D, and E) were represented as the mean ± standard deviation (SD) of three independent experiments and each experiment was performed in duplicate (*p
Fig 5
Fig 5. SFN inhibits LPS induced moDC maturation and enhances the phagocytic activity.
moDCs at day 7 in culture were used for cell phagocytosis and cell differentiation status analysis. moDCs were pre-incubated for 1 h with or without SFN (10 μM) before stimulation for 24 h LPS (1.0 μg/ml) or to the indicated concentrations. CD40, CD80, and CD86 cellular surface markers expression were analyzed by flow cytometry (A). The flow cytometry results shown were from one experiment of two independent experiments. CD40, CD80 and CD86 mean fluorescence intensity (MFI) determined by flow cytometry (B). The flow cytometry results were combined from two independent experiments and each experiment was performed from triplications. Data are mean ± standard deviations (SD) (the letters a and b P<0.01). The phagocytic activity of moDCs was examined after stimulating with different concentration of LPS (0,5 μg/ml, 1,0 μg/ml, and 2,0 μg/ml) with or without 24 h pre-treatment with SFN (C). The mRNA expression of DCs surface markers CD40, CD80 and CD86 were quantified using qRT-PCR (D). The mRNA expression and phagocytosis results were combined from three independent experiments and each experiment was performed in four replications. The data represented as the mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01; *** P < 0.001).
Fig 6
Fig 6. The effects of SFN on LPS induced up-regulation of TLR4 and MD2 gene expression.
moDCs were pre-incubated for 1 h with or without SFN (10 μM) before stimulation for 0, 1, 3, 6, 12, 24 h with LPS (1.0 μg/ml). The TLR4 and MD2 mRNA expression were quantified by qRT-PCR. The results were combined from three independent experiments and each experiment was performed in four replications. The data represented as the mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01; *** P < 0.001).
Fig 7
Fig 7. The effects of SFN on LPS induced NF-κB and TBP expression.
moDCs were pre-incubated for 1 h with or without SFN (10 μM) before exposure to LPS (1.0 μg/ml) for 0, 1, 3, 6, 12, 24 h or to the indicated time. The transcription factor NF-κB and TBP mRNA expression were quantified by qRT-PCR (A and C). Data are mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01; *** P < 0.001) of triplication samples from three independent experiments. NF-κB and TBP protein expression were examined by western blotting. The results were determined from one experiment representative of two experiments. The p50 and p65 protein of NF-κB family were analyzed by western blotting by the selected time points (0, 3, 12, and 24 h) (B). The western blotting result was from one experiment of three independent experiments.
Fig 8
Fig 8. SFN affects gene expressions and protein productions of cytokine.
moDCs were pre-incubated for 24 h with or without SFN (10 μM) before stimulation with LPS (1 μg/ml) for additional 24 h. The cell lysate proteins of IRF6 and TNF-α were analyzed using western blotting (A). The effects of SNF on gene expression of pro-inflammatory cytokines TNF-α, IL-1ß, IL-8, and IFN-γ were quantified by qRT-PCR (B). The pro-inflammatory cytokines TNF-α (C) and IL-1ß (D) secreted in cell culture supernatant were determined by ELISA. The results were combined from three independent experiments and each experiment was performed in triplicates. The data were represented as the mean ± standard deviations (SD) (* P < 0.05; ** P < 0.01; *** P < 0.001).
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