TLR7 Expression And Immune Response Mediated By TLR7 In Mice Dendritic Cells

Dendritic cells(DCs) have been considered to be the most potent professional antigen-presenting cells(APC), which are involved in innate immune response and specific immune response by Toll like receptors(TLRs) recognizing microbial organisms. Recently, people have explored a special kind of TLRs, TLR7, which express in many immune cells, particularly, in DC. It has been proved that TLR7 can be the pattern recognition receptor upon some anti-viral compounds and virus single strand RNA (ssRNA) to induce immune response in DC. Up to now, there have been many doubts about TLR7, such as the subcellular localization of TLR7 and the immune response after TLR7 recognition of ligand. So, our research about TLR7 will reveal the anti-infection molecular mechanism of body.For exploring the immune response induced by TLR7 in DC, we took advantage of murine immortalized dendritic cell lines DC2.4 as cells model in the subject, to detect TLR7 and TLR4 expression in DC2.4 and the immune response induced by TLR7 after recognizing ligand. The following have been done:1. Detection of TLR7 gene and protein in DC2.4According to TLR7 andβ-actin gene completed sequences in Gene bank, designed respectively the corresponding upstream primer and downstream primer, reverse transcription PCR detected TLR7 mRNA expression in DC2.4, Western blot detected TLR7 protein expression in DC2.4. The results showed that there was TLR7 mRNA expression, but no TLR7 protein expression in DC2.4.2. Detection of TLR4 protein in DC2.4Culture DC2.4 on glass coverslips in 6–well plates with RPMI 1640 medium supplemented with 10% FBS, on the logarithmic growth phase, indirect immunofluorescence assay was adopted to detect TLR4 protein expression. The results showed that there was TLR4 protein expression in DC2.4.3. LPS---the ligand of TLR4, induced TLR7 protein expression in DC2.4 DC2.4 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, on the logarithmic growth phase, which were added with LPS (1mg/L). DC2.4 cells were collected at 12 h, 24 h, 48 h and 72 h, and at the same time, TLR7 protein was extracted. Negative control and positive control were no-LPS cells and placenta respectively. Western blot was employed to analyze the expression of TLR7 protein. The results showed that TLR7 protein started to express in DC2.4 after 12 hours by LPS stimulation, but it was not detected after 72 hours. There was TLR7 protein expression in positive control but no in negative control.4. TLR7-mediated DC immune responses by recognition of ImiquimodDC2.4 cells were cultured with 1×108 /L in 96–well plates, the cells were divided into four groups, L0 group was negative control, L group was added with LPS(1mg/L), I group was added with Imiquimod(10mol/L), LI group was added with Imiquimod(10mol/L)when 12h after being added with LPS(1mg/L) first . Cell supernatants were collected 12h post culturing, respectively. ELISA tested the secretion levels of IL-12 and IFN-α. The operation was strictly in accordance with the Manual for kit. The data was treated statistically with SPSS11.0 software. The results showed that IL-12 secretion increased but IFN-αsecretion had no change after Imiquimod stimulating DC2.4 expressed TLR7 protein. Conclusion: 1. TLR4 protein and TLR7 mRNA are expressed in DC2.4, but TLR7 protein can not be detected in DC2.4.2. LPS, the ligand of TLR4, can stimulate immature DC to express TLR7 protein,but the protein can not be detected after 72h.3. In the immature DC, the induced TLR7 protein can recognize Imiquimod, activate signal transduction pathway and mediate DC secreting cytokines, the secretion level of IL-12 is increased significantly, but IFN-αhas no change.