A surface antigen of Orientia tsutsugamushi activates human monocyte-derived dendritic cells via nuclear factor-kB & p38 mitogen-activated protein kinase pathways

Figures

• 

  Fig. 1. Expression and purification of recombinant Sat56 protein in Escherichia coli. (A) The protein was separated by SDS-PAGE and stained with Coomassie blue. (B) Sta56 was separated by SDS-PAGE and detected with antibodies by immunoblotting. Lane M, protein molecular weight marker.

• 

  Fig. 2. Sta56 induced interleukin-12 p40, IL-12 p70 and IL-10 production in human dendritic cells in vitro. Human dendritic cells were cultured in the presence or absence of Sta56 0.1, 0.2 and 1 μg/ml for 48 h. The supernatants were the collected for cytokine analysis including such as IL-12 p40, IL-12 p70 and IL-10. Each data point represents the mean ± SE of three determinations. *P<0.05 unstimulated vs stimulated dendritic cells.

• 

  Fig. 3. Effect of Sta56 on (A) the expression of human dendritic cells. Human dendritic cells were treated in the presence or absence of Sta56 (1 mg/ml) or lipopolysaccharide (LPS, 100 ng/ml) for 48 h. The cells were harvested on day 7 of culture, stained with different mAbs and analyzed using flow cytometry (dotted line, isotype control; solid line, specific mAbs). (B) Phagocytic capacity of monocyte derived-dendritic cells. Immature monocyte derived-dendritic cells (1×106 cells/ml/well) were incubated for 24 h with or without Sta56 (0.1 or 1 μg/ml). The dendritic cells stimulated with lipopolysaccharide (100 ng/ml) served as the positive maturation controls. The cells were incubated with fluorescein isothiocyanate-dextran for one hour at 4°C (dotted lines) or 37°C (solid lines). The values shown in the flow cytometry profiles are the mean fluorescence intensity indexes indices. These results are representative of three independent experiments with similar results. CD, cluster of differentiation; HLA, human leukocyte antigen DR.

• 

  Fig. 4. Sta56 enhanced T-cell responses. (A) Immature dendritic cells were stimulated with Sta56 (0.1 μg, 0.2 μg, and 1 μg/ml) for 48 h. Autologous T-cell proliferation was measured after five days of co-culture with the dendritic cells. Data are presented as means ± SEM of three independent experiments. The supernatants were analyzed for (B) interferon-g, interleukin-10 and interleukin-5 produced by the activated T-cells after two days of culture. *P<0.05 compared to control. CPM, counts per minute.

• 

  Fig. 5. Neutralization with anti-toll-like receptor-4 mAbs inhibits the synthesis of interleukin (IL)-12 p40, IL-12 p70 and IL-10. Human dendritic cells were pre-incubated with 20 mg/ml anti-toll-like receptor (TLR)-1, TLR-2, TLR-3 and TLR-4 mAbs separately for one hour. The dendritic cells were challenged with Sta56 (1 μg/ml) for 20 h. The cell culture supernatants were collected for IL-12 p40, IL-12 p70 and IL-10 analysis. Data are presented as mean ± standard error of three independent experiments. *P<0.05 between the dendritic cells treated with and without Abs.

• 

  Fig. 6. The effect of NF-kB, p38 MAPK, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase inhibition on cytokine release and expression of surface markers in Sta56-treated dendritic cells. Cells were pre-treated with 0.1 per cent dimethyl sulphoxide (DMSO), 10 mM helenalin, 20 mM SB203580, 50 mM PD98059 or 20 mM of SP600125 for 1 h and incubated with 1 mg/ml Sta56 for 24 h. (A) IL-12 p40, IL-12 p70 and IL-10 levels in cell supernatants analyzed by ELISA. The data are presented as the mean ± standard error of three independent experiments. *P<0.05 versus inhibitor untreated Sta56-induced dendritic cells. (B) The Sta56-induced upregulation of CD40, CD80, CD86, CD83 and HLA-DR in human monocyte-derived DC. Day 6 immature DC was pre-treated with as above. The cell surface expression of CD40, CD80, CD86, CD83 and HLA-DR was measured using the flow cytometry (dotted line, isotype control; solid line, specific mAbs). The values shown in the flow cytometry profiles are the mean fluorescence intensity indexes. These results are representative of three independent experiments with similar results.

• 

  Fig. 7. Human monocyte derived-dendritic cells were treated with Sta56 (1 μg/ml) for the indicated time periods. Cytosolic fractions were prepared and analyzed for (A) the phosphorylation level of IkBa by Western blotting. The lower panel shows the blot probed for a-tubulin to demonstrate equal loading of the samples. (B) The phosphorylation level of mitogen-activated protein kinase by Western blotting. The lower panel shows the blot probed for anti-total p38 polyclonal antibody to demonstrate equal loading of the samples. This experiment was repeated three times with similar results.

• 

  Fig. 8. ELISA of nuclear p50 and p65 nuclear factor-kB subunits of the dendritic cells treated with Sta56 for two hours. Each data point represents the mean ± standard error of three determinations. *P<0.05 compared to control.