Acetylcorynoline Impairs the Maturation of Mouse Bone Marrow-Derived Dendritic Cells via Suppression of IκB Kinase and Mitogen-Activated Protein Kinase Activities

<div><p>Background</p><p>Dendritic cells (DCs) are major modulators in the immune system. One active field of research is the manipulation of DCs as pharmacological targets to screen novel biological modifiers for the treatment of inflammatory and autoimmune disorders. Acetylcorynoline is the major alkaloid component derived from <i>Corydalis bungeana</i> herbs. We assessed the capability of acetylcorynoline to regulate lipopolysaccharide (LPS)-stimulated activation of mouse bone marrow-derived DCs.</p> <p>Methodology/Principal Findings</p><p>Our experimental data showed that treatment with up to 20 µM acetylcorynoline does not cause cytotoxicity in cells. Acetylcorynoline significantly inhibited the secretion of tumor necrosis factor-α, interleukin-6, and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility complex class II, CD40, and CD86 on DCs was also decreased by acetylcorynoline, and the endocytic capacity of LPS-stimulated DCs was restored by acetylcorynoline. In addition, LPS-stimulated DC-elicited allogeneic T-cell proliferation was blocked by acetylcorynoline, and the migratory ability of LPS-stimulated DCs was reduced by acetylcorynoline. Moreover, acetylcorynoline significantly inhibits LPS-induced activation of IκB kinase and mitogen-activated protein kinase. Importantly, administration of acetylcorynoline significantly attenuates 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity.</p> <p>Conclusions/Significance</p><p>Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of DC maturation and function.</p> </div

Inhibitory effect of acetylcorynoline on the contact hypersensitivity response in mice.

<p>Contact hypersensitivity response was showed by hematoxylin and eosin staining (A), and thickness of the challenged ear was calculated (original magnification times 40) (B). Mice that were not sensitized but were challenged with DNFB provided as negative controls. One representative result from three independent experiments is shown. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between unsensitized and sensitized mice (<i>p</i><0.001); an asterisk (*) indicates significant differences between the DNFB-challenged control samples and DNFB plus acetylcorynoline-challenged samples (^*<i>p</i><0.05).</p

Effects of acetylcorynoline on the viability of mBM-DCs.

<p>mBM-DCs were treated with serially diluted acetylcorynoline for 24 h. The viability of cells was determined by propidium iodide staining/flow cytometry assay (A). The apoptosis of cells was determined by annexin V-fluorescein staining/flow cytometry assay (B), as described in the text. The data represent the mean ± SD (n = 3). An asterisk (*) indicates significant differences between acetylcorynoline untreated control samples and acetylcorynoline treated samples (^**<i>p</i><0.01, ^***<i>p</i><0.001).</p

Inhibitory effects of acetylcorynoline on TNF-α, IL-6, and IL-12p70 secretion in LPS-stimulated mBM-DCs.

<p>mBM-DCs were pretreated with 10 or 20 µM acetylcorynoline. After 1 h of incubation, the cells were washed, followed by stimulation with 100 ng/ml LPS for 24 h (6 h for TNF-α). Media were collected and assayed for TNF-α, IL-6 and IL-12p70 levels by using as ELISA kit. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between LPS-stimulated and unstimulated cells (<i>p</i><0.001); an asterisk (*) indicates significant differences between the LPS-stimulated control samples and acetylcorynoline-pretreated, LPS-stimulated samples (^*<i>p</i><0.05, ^**<i>p</i><0.01).</p

Inhibitory effects of acetylcorynoline on the proliferation of naïve allogeneic T lymphocytes by LPS-stimulated mBM-DCs.

<p>mBM-DCs were pretreated with 10 or 20 µM acetylcorynoline. After 1 h of incubation, the cells were washed, followed by stimulation with 100 ng/ml LPS for 16 h. DCs were then washed and incubated with 25 µg/ml mitomycin C for 30 min at 37°C. Finally, the cells were washed and diluted with the prepared splenocytes in a ratio of 1∶50 and 1∶100 in culture plates for 3 days. The proliferation of T cells was assessed by the MTT assay. DCs were sufficiently fixed with mitomycin C according to a DC-only control (data not shown). The values of unstimulated dendritic cells served as control values in the calculation of percentage of proliferation. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between LPS-stimulated and unstimulated cells (<i>p</i><0.001); an asterisk (*) indicates significant differences between the LPS-stimulated control samples and acetylcorynoline-pretreated, LPS-stimulated samples (^*<i>p</i><0.05).</p

Inhibitory effects of acetylcorynoline on MHC class II, CD40, and CD86 expression in LPS-stimulated mBM-DCs.

<p>mBM-DCs were pretreated with 10 or 20 µM acetylcorynoline. After 1 h of incubation, the cells were washed, followed by stimulation with 100 ng/ml LPS for 16 h. The expression of MHC class II, CD40 and CD86 on CD11c⁺ cells was determined by flow cytometry. The data are represented as the mean fluorescent intensity ± SD (n = 3). A hash (#) indicates significant differences between LPS-stimulated and unstimulated cells (<i>p</i><0.001); an asterisk (*) indicates significant differences between the LPS-stimulated control samples and acetylcorynoline-pretreated, LPS-stimulated samples (^*<i>p</i><0.05, ^**<i>p</i><0.01).</p

Inhibitory effect of acetylcorynoline on the translocation of NF-κB p65 in LPS-stimulated mBM-DCs.

<p>mBM-DCs were pretreated with 10 or 20 µM acetylcorynoline. After 1 h of incubation, the cells were washed, followed by stimulation with 100 ng/ml LPS for 1 h. Cells were lysed and the nuclear fraction determined for relative binding activity of NF-κB p65 by using the Universal EZ-TFA Transcription Factor Assay Colorimetric kit. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between LPS-stimulated and unstimulated cells (<i>p</i><0.001); an asterisk (*) indicates significant differences between the LPS-stimulated control samples and acetylcorynoline-pretreated, LPS-stimulated samples (^*<i>p</i><0.05, ^**<i>p</i><0.01).</p

Chemical structure of acetylcorynoline.

<p>Chemical structure of acetylcorynoline.</p

Inhibitory effects of acetylcorynoline on the migratory capacity of LPS-stimulated mBM-DCs.

<p>mBM-DCs were pretreated with 10 or 20 µM acetylcorynoline. After 1 h of incubation, the cells were washed, followed by stimulation with 100 ng/ml LPS for 16 h. The <i>in vitro</i> migration of DCs in response to 100 ng/ml SLC/CCL21 was determined by chemotaxix assay and flow cytometry. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between LPS-stimulated and unstimulated cells (<i>p</i><0.001); an asterisk (*) indicates significant differences between the LPS-stimulated control samples and acetylcorynoline-pretreated, LPS-stimulated samples (^*<i>p</i><0.05, ^**<i>p</i><0.01).</p

Inhibitory effects of acetylcorynoline on antigen uptake in LPS-stimulated mBM-DCs.

<p>mBM-DCs were pretreated with 10 or 20 µM acetylcorynoline. After 1 h of incubation, the cells were washed, followed by stimulation with 100 ng/ml LPS for 16 h. Cell were then incubated with 1 mg/ml FITC-dextran at 37°C for 1 h and analysed by flow cytometry. Control experiments were performed at 4°C for 1 h. The data are represented as the mean fluorescent intensity the mean ± SD (n = 3). A hash (#) indicates significant differences between LPS-stimulated and unstimulated cells (<i>p</i><0.001); an asterisk (*) indicates significant differences between the LPS-stimulated control samples and acetylcorynoline-pretreated, LPS-stimulated samples (^*<i>p</i><0.05).</p