TLR4 mediated the maturation of DCs induced by EV71 VLPs.

<p>(A) Human DCs were pre-incubated with 20<i> </i>µg/mL of anti-TLR2, anti-TLR4, or IgG2a (as an isotype control) separately for 1<i> </i>h and were then challenged with EV71 VLPs (10<i> </i>µg/mL) for 16<i> </i>h. The control was not treated with EV71 VLPs. Supernatants from the cell cultures were collected to determine levels of IL-12 p70, IL-12 p40, and IL-10. Significant differences in the levels of cytokines produced by DCs treated with Abs and those treated with IgG2a are indicated by asterisks (*<i>p</i><0.05). (B) Human DCs were pre-incubated with 20<i> </i>µg/mL of anti-TLR4 mAb or IgG2a (as an isotype control) separately for 1<i> </i>h and were then treated with 10<i> </i>µg/mL EV71 VLPs for 30<i> </i>min on ice to allow binding to the cell surface. Surface-bound EV71 VLPs were measured with flow cytometry through immunostaining with anti-EV71 monoclonal antibodies. The control cells (no VLPs; dotted line) were stained with the same primary antibody followed by a secondary antibody. The bar diagram shows the MFI with statistics for the anti-TLR4 mAb effect on binding of EV71 VLPs to DCs. (C) HEK293 cells were transfected with control plasmids, TLR4, or TLR4+MD2. The transfected cells were stimulated with medium alone, LPS (50<i> </i>ng/mL), or EV71 VLP (10<i> </i>µg/mL) for 24<i> </i>h, and IL-8 protein in the supernatants was measured by ELISA. This experiment was repeated three times with similar results. (D) Proteinase K-treated EV71 VLPs abrogated the production of IL-12 p70, IL-12 p40, and IL-10 by DCs. Human DCs were incubated with medium alone (Control), proteinase K (10<i> </i>ng/mL), EV71 VLPs (10<i> </i>µg/mL), proteinase K-treated EV71 VLPs, LPS (100<i> </i>ng/mL), or proteinase K-treated LPS for 24<i> </i>h. At the end of the incubation time, the production of IL-12 p70, IL-12 p40, and IL-10 was analyzed by ELISA. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).