Dengue virus type 2 infection inhibits the expression of toll-like receptors 4 and 7 in murine bone marrow-derived dendritic cells

Abstract

Objective To analyze the changes in the expression of Toll-like receptors 4 (TLR4) and 7 on the surface of murine bone marrow-derived dendritic cells following dengue virus type 2 (DEN2) infection. Methods DEN2 NGC strain was infected into BALB/c suckling mice through intracranial injection and injected into C6/36 cells to induce the in vivo and in vitro proliferation of DEN2, respectively. RT-PCR was performed to identify DEN2 RNA. Reed-Muench method was used to determine the 50% tissue culture infective dose (TCID50) of DEN2. Dendritic cells (DCs) were prepared by stimulating bone marrow cells isolated form C57BL/6 mice with IL-4 and GM-CSF and then identified by flow cytometry. The prepared murine bone marrow-derived DCs were infected with DEN2 and observed with direct immunofluorescence assay. Dynamic changes in the expression of CD11c, CD86 and I-A/I-E molecules on DCs after DEN2 infection were detected by flow cytometry. Levels of DEN2 RNA and the expression of TLR4 and TLR7 at mRNA level were dynamically detected by real-time quantitative PCR. Results The TCID50 of DEN2 to C6/36 cells was 10-5.8. Murine bone marrow-derived DCs were acquired with a purity of 70% and could be infected with DEN2 in vitro. The percentages of CD86 and I-A/I-E molecules on the surface of DCs infected with 1×105 TCID50 of DEN2 were statistically different from those of the negative control group. Neither of the two groups showed a significant difference in the percentages of membrane molecules over time. However, the percentages of membrane molecules on DCs increased with increasing viral load. Compared with the negative control group, the levels of DEN2 RNA in infection groups were increased with increasing virus load, while the expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level was decreased with increasing viral load. Conclusion DEN2 infection promotes the maturation of DCs. Expression of TLR4 and TLR7 on DEN2 infected-DCs at mRNA level decreases with increasing viral load, which suggests that TLR4 and TLR7 are closely related to viral infection and play a certain role in the pathogenesis of DEN infection. Key words: Dengue virus type 2; DC; TLR4; TLR7