Topographical and Biological Evidence Revealed FTY720-Mediated Anergy-Polarization of Mouse Bone Marrow-Derived Dendritic Cells In Vitro

Abnormal inflammations are central therapeutic targets in numerous infectious and autoimmune diseases. Dendritic cells (DCs) are involved in these inflammations, serving as both antigen presenters and proinflammatory cytokine providers. As an immuno-suppressor applied to the therapies of multiple sclerosis and allograft transplantation, fingolimod (FTY720) was shown to affect DC migration and its crosstalk with T cells. We posit FTY720 can induce an anergy-polarized phenotype switch on DCs in vitro, especially upon endotoxic activation. A lipopolysaccharide (LPS)-induced mouse bone marrow-derived dendritic cell (BMDC) activation model was employed to test FTY720-induced phenotypic changes on immature and mature DCs. Specifically, methods for morphology, nanostructure, cytokine production, phagocytosis, endocytosis and specific antigen presentation studies were used. FTY720 induced significant alterations of surface markers, as well as decline of shape indices, cell volume, surface roughness in LPS-activated mature BMDCs. These phenotypic, morphological and topographical changes were accompanied by FTY720-mediated down-regulation of proinflammatory cytokines, including IL-6, TNF-α, IL-12 and MCP-1. Together with suppressed nitric oxide (NO) production and CCR7 transcription in FTY720-treated BMDCs with or without LPS activation, an inhibitory mechanism of NO and cytokine reciprocal activation was suggested. This implication was supported by the impaired phagocytotic, endocytotic and specific antigen presentation abilities observed in the FTY720-treated BMDCs. In conclusion, we demonstrated FTY720 can induce anergy-polarization in both immature and LPS-activated mature BMDCs. A possible mechanism is FTY720-mediated reciprocal suppression on the intrinsic activation pathway and cytokine production with endpoint exhibitions on phagocytosis, endocytosis, antigen presentation as well as cellular morphology and topography

Nanocrystalline Co_0.85Se Anchored on Graphene Nanosheets as a Highly Efficient and Stable Electrocatalyst for Hydrogen Evolution Reaction

For the first time, a porous and conductive Co_0.85Se/graphene network (CSGN), constructed by Co_0.85Se nanocrystals being tightly connected with each other and homogeneously anchored on few-layered graphene nanosheets, has been synthesized by a facile one-pot solvothermal method. Compared to unhybridized Co_0.85Se, CSGN exhibits much faster kinetics and better electrocatalytic behavior for hydrogen evolution reaction (HER). The HER mechanism of CSGN is improved to Volmer–Tafel combination, instead of Volmer–Heyrovsky combination, for Co_0.85Se. CSGN has a very low Tafel slope of 34.4 mV/dec, which is much lower than that of unhybridized Co_0.85Se (41.8 mV/dec) and is the lowest ever reported for Co_0.85Se-based electrocatalysts. CSGN delivers a current density of 55 mA/cm² at 250 mV overpotential, much larger than that of Co_0.85Se (33 mA/cm²). Furthermore, CSGN shows superior electrocatalytic stability even after 1500 cycles. The excellent HER performance of CSGN is attributed to the unique porous and conductive network, which can not only guarantee interconnected conductive paths in the whole electrode but also provide abundant catalytic active sites, thereby facilitating charge transportation between the electrocatalyst and electrolyte. This work provides insight into rational design and low-cost synthesis of nonprecious transition-metal chalcogenide-based electrocatalysts with high efficiency and excellent stability for HER

Topographical and morphological changes of BMDCs upon LPS-activation are reversible by FTY720.

<p>(<b>A</b>) Morphologies of LPS- and/or FTY720- treated BMDCs. Day 6 BMDCs were exposed to LPS (1 µg/ml) and/or FTY720 (500 nM) for additional 48 h, followed by microscopic observations. Scale bar  = 20 µm. (<b>B</b>) Statistical results on the shape indices of each group. *<i>P</i><0.05, n = 20 (20 cells randomly selected from high power fields from 3 separate experiments). Atomic force microscope observations on the untreated (<b>C</b>), FTY720 alone (<b>D</b>), LPS alone (<b>E</b>) and LPS+FTY720- (<b>F</b>) treated groups were shown as representative images. Enlarged areas were indicated in squares in dark and green, respectively. The statistical results of the cell volume (<b>G</b>), RMS roughness (<b>H</b>) and peak to valley distance (<b>I</b>) are analyzed and shown in histograms. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 10 (10 cells randomly selected from 3 separate experiments).</p

Scanning electron microscopic observation.

<p>Day 6 BMDCs were treated with LPS (1 µg/mL) and/or FTY720 (500 nM) for additional 48 h before SEM observation. Scale bar = 5 µm.</p

Cytometric Bead Array analysis.

<p>CBA assays on IL-10 (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>), IL-12 (<b>D</b>), IFN-γ (<b>E</b>) and MCP-1 (<b>F</b>) were shown in their respective panels. For these assays, Day 6 BMDCs were treated with LPS (1 µg/mL) and/or FTY720 (500 nM) for 24 h. The culture supernatants of each group were subjected to CBA analysis by flow cytometry. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 3. The cytokine concentrations were calculated from the standard curve (<i>R</i>²>0.95) via data analysis by a four parameter linear fitting program provided by the manufacturer.</p

FTY720 alters the surface phenotype of mouse bone marrow-derived dendritic cells upon LPS activation.

<p>Expression of CD11c (<b>A</b>), MHC II molecule I-A^d (<b>B</b>), CD80 (<b>C</b>), CD86 (<b>D</b>) and CD40 (<b>E</b>) was shown in their respective panels. For these assays, mouse bone marrow cells were differentiated for 6 d to prepare BMDCs that were exposed to LPS (1 µg/mL) and/or FTY720 (500 nM) for additional 24 h. Representative results out of three independent experiments were shown. The mean fluorescence intensities (MFI) of each marker were analyzed for statistical difference. *<i>P</i><0.05, n = 3.</p

Cell viability determination.

<p>(<b>A</b>) MTT assays on the bone marrow cells. Mouse bone marrow cells were isolated and exposed to FTY720 with different concentrations for 48 h. Cytotoxicity was indicated by the ratio of OD_FTY720 to OD_control. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 3. (<b>B</b>) MTT assays on BMDCs. Day 6 BMDCs were treated with FTY720 at concentrations of 0, 0.1, 0.5 and 1.0 µM for 48 h. Data are shown as mean ± SEM. *<i>P</i><0.05, n = 3. (<b>C</b>) Apoptosis assays on BMDCs. The cell death rates of BMDCs was determined by Annexin V-FITC and PI staining flow cytometry. Cells were treated with FTY720 for 48 h at concentrations ranged from 0.5 to 10 µM.</p

CCR7 transcription, NO production as well as phagocytosis and endocytosis of BMDCs.

<p>(<b>A</b>) CCR7 mRNA production analysis. Day 6 BMDCs were treated with LPS (1 µg/mL) and/or FTY720 (500 nM) for 24 h. Total mRNA were extracted, reverse transcripted and quantified by the real-time PCR. *<i>P</i><0.05, n = 3. (<b>B</b>) Nitric oxide production assays. Supernatants of cells with similar treatments were obtained and analyzed by diazotization reaction and colorimetry. *<i>P</i><0.05, n = 3. (<b>C</b>) Phagocytosis assay of BMDCs. Day 6 BMDCs were treated with LPS (1 µg/ml) alone or FTY720 (500 nM) alone for 24 h and subjected to fluorescence-conjugated beads uptake assays. Representative of results on the percentage of phagocytes were shown in each flow chart. Statistical analysis on the percentage of phagocytotic cells were presented as mean ± SEM. *<i>P</i><0.05, n = 3. (<b>D</b>) Endocytosis assay of BMDCs. Similar experimental design to (<b>C</b>) were employed. Cells were treated with FITC-conjugated dextran and analyzed by FACS. Representative results on the percentage of endocytotic cells were shown. Statistical data were presented as mean ± SEM. *<i>P</i><0.05, n = 3.</p