Dimethyl fumarate inhibits dendritic cell maturation via nuclear factor κB (NF-κB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 (MSK1) signaling

Abstract

Dimethyl fumarate (DMF) is an effective novel treatment for multiple sclerosis in clinical trials. A reduction of IFN-γ-producing CD4(+) T cells is observed in DMF-treated patients and may contribute to its clinical efficacy. However, the cellular and molecular mechanisms behind this clinical observation are unclear. In this study, we investigated the effects of DMF on dendritic cell (DC) maturation and subsequent DC-mediated T cell responses. We show that DMF inhibits DC maturation by reducing inflammatory cytokine production (IL-12 and IL-6) and the expression of MHC class II, CD80, and CD86. Importantly, this immature DC phenotype generated fewer activated T cells that were characterized by decreased IFN-γ and IL-17 production. Further molecular studies demonstrated that DMF impaired nuclear factor κB (NF-κB) signaling via reduced p65 nuclear translocalization and phosphorylation. NF-κB signaling was further decreased by DMF-mediated suppression of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its downstream kinase mitogen stress-activated kinase 1 (MSK1). MSK1 suppression resulted in decreased p65 phosphorylation at serine 276 and reduced histone phosphorylation at serine 10. As a consequence, DMF appears to reduce p65 transcriptional activity both directly and indirectly by promoting a silent chromatin environment. Finally, treatment of DCs with the MSK1 inhibitor H89 partially mimicked the effects of DMF on the DC signaling pathway and impaired DC maturation. Taken together, these studies indicate that by suppression of both NF-κB and ERK1/2-MSK1 signaling, DMF inhibits maturation of DCs and subsequently Th1 and Th17 cell differentiation.

FIGURE 1.

DMF inhibits dendritic cell maturation. A, BMDCs were stimulated by LPS (100 ng/ml) in the presence or absence of different dosages of DMF (17, 35, and 70 μm). Cytokine secretion was determined by ELISA at 6 and 24 h. Error bars indicate S.E. B, DCs were generated in vivo, purified, and stimulated by LPS (100 ng/ml) in the presence or absence of DMF (70 μm) for 24 h. MHC class II, CD80, and CD86 were then stained for flow cytometric analysis. Numbers indicate the percentages of MHC class II^high, CD80-positive, or CD86^high. *, p < 0.01. Data are derived from one experiment representative of three independent experiments.

FIGURE 2.

LPS/DMF-DCs generate less T cell activation and proliferation characterized by decreased IFN-γ and IL-17 production. Purified DCs were stimulated by LPS (100 ng/ml) in the presence or absence of DMF (70 μm) for 24 h, supernatant was then removed, and DCs were washed twice by warm PBS. MOG_35–55-specific T cells were then added to the conditioned DC culture in the presence of MOG_35–55 peptides (2 μg/ml). A, T cell activation was determined by flow cytometric analysis of CD44, an effector T cell marker, at both 48 h and 72 h after co-culture was set up. The number above the gate indicates the percentage of activated CD4⁺CD44⁺ T cells. B, cytokine production including IFN-γ and IL-17 in the co-culture system supernatant was evaluated by ELISA. C, proliferation was assessed by [³H]thymidine incorporation. Cells were pulsed at 48 h with [³H]thymidine and harvested 18 h later. [³H]Thymidine incorporation was measured using a TopCount NXT (PerkinElmer Life Sciences). *, p < 0.01. Data are derived from one experiment, which is representative of at least three independent experiments. Error bars indicate S.E.

FIGURE 3.

DMF inhibits p65 phosphorylation and nuclear translocation. A and B, BMDCs were stimulated by LPS (100 ng/ml) in the presence or absence of DMF (70 μm). A, cells were fixed by 2% paraformaldehyde at 5 and 60 min after LPS stimulation and then labeled with p65 and DAPI, revealing increased cytoplasmic retention of p65 in DMF conditions. The immunohistochemical results are quantified in B, which shows the ratio of nuclear p65 over whole cell p65 in LPS (black bar) and LPS+DMF (white bar) conditions. Data are derived from one experiment representative of two independent experiments. *, p < 0.01. Error bars indicate S.E. C, time course of phospho-p65 and IκBα expression in LPS and LPS+DMF conditions. BMDCs were stimulated by LPS (500 ng/ml) in the presence or absence of DMF (70 μm). Whole cell protein was then extracted at 0, 5, 20, 30, 40, and 60 min, and phospho-p65, p65, IκBα, and β-actin levels were determined by Western blot and quantified in D as the ratio of phospho-p65/p65 or IκBα/β-actin. Data are derived from one experiment representative of three independent experiments.

FIGURE 4.

DMF inhibits ERK1/2-MSK1 signaling. Serum-starved-BMDCs were treated with DMF (70 μm) or ERK1/2 inhibitor U0126 (10 μm) 1 h before the addition of LPS (100 ng/ml). Whole cell protein was then extracted at 5, 10, 20, and 30 min, and p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, p-MSK1, and β-actin were determined by Western blot. A, time course of expression of p-ERK1/2 and p-MSK1. B, quantification of Western blot images. Values are expressed as the ratio of p-ERK1/2/ERK1/2, and p-MSK1/β-actin. C, time course of expression of p-JNK, JNK, p-p38, and p38 in each condition. Data are derived from one experiment representative of three independent experiments.

FIGURE 5.

DMF-mediated-MSK1 reduction interferes with p65 signaling. A, serum-starved-BMDCs were treated with DMF (70 μm) or MSK1 inhibitor H89 (10 μm) 1 h before the addition of LPS (100 or 500 ng/ml). Whole cell protein was then extracted at 5, 20, 30, and 40 min, and p^Ser10-histone-3, histone-3, p^Ser276-p65, p65, IκBα, and β-actin were determined by Western blot. B, quantification of Western blot images. Values are expressed as the ratio of p^Ser276-p65/p65, p^Ser10-histone-3/histone-3, and IκBα/β-actin. Data are derived from one experiment representative of three independent experiments.

FIGURE 6.

DMF-mediated MSK1 reduction is essential for the inhibitory effect of DMF on DC maturation. Purified DCs were stimulated by LPS (100 ng/ml), LPS+H89 (10 μm), or LPS+DMF (70 μm) for 24 h. Untreated condition was included as control. A, MHC class II^high, CD80, and CD86^high expression was determined by flow cytometry. B, cells were first gated on MHC class II^high and then gated on CD80 and CD86^high. C, cytokine secretion in each condition was evaluated by ELISA. *, p < 0.01. Data are derived from one experiment representative of two independent experiments. Error bars indicate S.E.

FIGURE 7.

TLR signaling and proposed model for DMF-mediated impairment of DC maturation. All TLR signaling (except TLR3) events recruit the adaptor MyD88, followed by the interleukin-1 receptor-associated kinase (IRAK) family of protein kinases, leading to TNF receptor-associated factor-6 (TRAF6) activation followed by NF-κB activation. In addition to MyD88-dependent pathway, MyD88-independent (TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent) pathway is utilized in TLR3/4 signaling. TLR3/4 recruit the adaptor TRIF, which can subsequently lead to activation of interferon regulatory factor 3 (IRF3). IRF3 then translocates into the nucleus and transcribes target genes, e.g. TNF-α. TNF-α binds to the TNF receptor and induces delayed NF-κB activation. In our study, we show that DMF suppresses p65 phosphorylation and nuclear localization (Point 1). p65 activation is further reduced by DMF-mediated MSK1 reduction (Point 2), which results in decreased phosphorylation of p65 at serine 276 (Point 3) and histone-3 at serine 10 (Point 4). IKKϵ, IκB kinase ϵ. TAK1, TGF-β activated kinase-1, TBK1, TANK-binding kinase-1.