Chlorella sorokiniana-Induced Activation and Maturation of Human Monocyte-Derived Dendritic Cells through NF-κB and PI3K/MAPK Pathways

Abstract

Chlorella sorokiniana (CS) is a unicellular green alga. The extracts of Chlorella have been used as treatments for relieving hypertension and modulating immune response. The detailed mechanisms are not clear yet. In this study, we sought to study the molecular mechanisms for the polysaccharide fraction of CS-induced immune response. We pulsed dendritic cells (DCs) with CS and found that CS could maturate DCs. CS-maturated DC could activate naïve T cells and stimulate T-cell proliferation and IFN-γ secretion. Furthermore, CS activated PI3K and MAPKs signaling pathways in DCs by interacting with TLR4 receptor. These CS-activated signaling pathways could further activate NF-κB and induce IL-12 production in DCs. This study provides molecular mechanisms for CS-induced DCs activation and immune response.

Figure 1

The effects of CS on DC maturation. (a) Human DCs were treated with CS (30 μg/mL), LPS (1 μg/mL), or medium alone for 48 h, and surface markers were analyzed by flow cytometry (dotted line, isotype control; solid line: specific mAb). The values shown were the percentage of gated cells (Gated %). (b) DCs were treated with CS at different concentrations, and the expressions of CD83 and CD86 were analyzed by flow cytometry (black bar: CD83; gray bar: CD86). LPS was used as positive control. (c) Human DCs were cultured in the presence of 1 μg/mL LPS or various concentrations of CS for 48 h. IL-12 secretion was analyzed by ELISA after incubation. (d) The time-dependent effect of CS (30 μg/mL) treatments on IL-12 secretion in DCs. IL-12 secretion was analyzed by ELISA. *P < 0.05 compared to control. N.D: nondetectable. ((e) and (f)) QRT-PCR analysis of IL-12 p35 and IL-12 p40. DCs were incubated in the presence of CS (30 μg/mL) for 3, 6, 18, 24, and 48 h. Representative images of three independent experiments were shown here. Lane M: marker. (g) The effect of CS on DC endocytosis. Human PBMCs were cultured for 6 days, and monocytes were induced to differentiated DCs (refer to Section 2). Immature DCs were stimulated with medium alone, LPS (1 μg/mL), or CS (30 μg/mL) for 48 h, and then incubated with FITC-dextran (0.5 mg/mL) for 1 h at 4°C (dotted line) or 37°C (solid line). (h) Human monocyte-derived DC were incubated with CS (30 μg/mL) for the indicated period of time. NF-κB assay was described in Section 2. The binding activity of NF-κB was shown as relative OD₄₅₀ levels. LPS (1 μg/mL) treatment for 2 h was used as positive control. *P < 0.05 compared to control.

Figure 2

Allogeneic T-cell responses induced by CS-treated DCs. Immature DCs were stimulated with CS (30 μg/mL) or LPS (1 μg/mL) for 48 h. Allogeneic T cells (1 × 10⁵) were cocultured in 96-well U-bottom microplates with CS-treated DCs as described. (a) T-cell proliferation was determined by using an Alamar Blue assay 5 days post coculturing. Experimental groups were compared to corresponding control groups. (b) After 2 days of coculturing, the production of IFN-γ by T cells was analyzed by ELISA. Data were expressed as means ± SEM of triplicates from three independent experiments. *P < 0.05 compared to control. N.D: nondetectable.

Figure 3

CS induces IL-12 secretion through PI3K/AKT and MAPKs pathways in DCs. ((a) and (b)) Time course of pAKT, p44/42 ERK, p38 MAPK, and p46/54 JNK phosphorylation in CS-stimulated DCs. Human DCs were treated with CS (30 μg/mL), and cell lysates were collected at different time points. The level of MAPK phosphorylations was analyzed by Western blotting (N = 3). (c) Effect of PI3K inhibitor LY294002 on MAPKs in CS-stimulated DCs. Human DCs were pretreated with various concentration of LY294002 (5, 25, 50, and 100 μM) for 1 h prior to CS (30 μg/mL) for 15 min. Cell lysates were collected, and MAPK phosphorylations were analyzed (N = 3). (d) The PI3K inhibitor LY294002 repressed CS-induced NF-κB p65 binding to DNA. Human DCs were pretreated with various concentration of LY294002 (5, 25, 50, and 100 μM) for 1 h prior to 2 h stimulation by CS (30 μg/mL). *P < 0.05 compared to CS alone. ((e) and (f)) Inhibitors against PI3K, NF-κB, and MAPKs blocked CS-induced IL-12 secretion (e) and CD83, CD86 upregulation (f) (black bar: CD83; gray bar: CD86) in DC. Immature DCs were preincubated for 1 h with one of the following compounds: LY294002 (25 μM), Helenalin (2.5 μM), SB203580 (20 μM), PD98059 (50 μM), or JNK inhibitor II (20 μM) and followed by CS (30 μg/mL) stimulation for an additional 48 h. *P < 0.05 compared to CS alone. N.D: nondetectable.

Figure 4

CS induces IL-12 expression through a TLR4-dependent ROS-regulated signaling pathway. (a) Anti-TLR-4 neutralizing antibody blocks CS-induced IL-12 secretion in DCs. DCs were preincubated with 20 μg/mL anti-TLR-2, anti-TLR-4, or control IgG for 1 h prior to LPS (1 μg/mL), LTA (1 μg/mL), or CS (30 μg/mL) treatments for 24 h. Conditioned media were collected for IL-12 detection. (b) NF-κB binding activity was examined in DC treated with CS (30 μg/mL) for 2 h with or without the pretreatments of neutralizing antibody against TLRs or control antibody for 1 h. The effects of neutralizing antibodies treatments were compared to corresponding control IgG treatments. *P < 0.05 compared to control antibodies. N.D: nondetectable.

Figure 5

Model of CS-mediated signaling pathways in regulating IL-12 p70 expression in DC.