| Toll-like receptors, which serve as pattern recognition receptors (PRRs), most express on immune cells, such as dendritic cell, B cell and macrophage. TLRs can activate specific signaling pathways through recognizing different microbial components, then initiate and modulate innate and adaptive immune responses. To date, there are13TLRs had been found in mammals. There are10TLRs in people and13TLRs in mouse.TLR9, which is responsible for sensing unmethylated CpG-DNA, plays an important role in many immune diseases such as autoimmune diseases, cancer, etc. Therefore, TLR9can be used as a potential therapeutic target for treatment of diseases. Dendritic Cells, as the most potent professional antigen presenting cells (APCs), plays a crucial role at the cross-talk of innate and adaptive immune systems. CpG oligonucleotides (CpG-ODN) can activate TLR9pathway and affect the immune function of BMDCs. The dedicated clinical drugs is very limited, thus, looking for new angent to TLR9signaling pathways of BMDCs is expected to offer more theapeutic potentials against these diseases.Our previous research has shown that D181can inhibit the activation of the TLR4signaling pathway and regulate the generation of inflammatory factors. What’s more, the TLR4signaling pathways related disease can be improved by D181. In this study, mouse bone marrow-drived dendritic cells were used to research the effects of D181on the activation of TLR9signaling. First we used CCK-8assay to evaluate the effect of D181on viability of BMDCs and found that D181(10～200μM) didn’t affect BMDCs’viability. At the same time, we analyzed the effect of D181on the cell apoptosis by Annexin V/PI staining, the result showed that D181didn’t induce the apoptosis of DCs during the concentration of0～200μM. D181could inhibit the expression of many flammatory cytokines (IL-12, IL-6, CXCL-10and TNF-a) induced by CpG, and CXCL-10is the most significant one. CXCL10(interferon-γ inducible protein10, IP-10), which can affect the interaction between DCs and T cells, is produced by fibroblasts, endothelial cells and DCs cells when induced by IFN-y. CXCL10plays an important role in variety of diseases such as the occurrence and development of SLE, AR, tumors, etc. In order to investigate the inhibitory mechanism of D181on TLR9signaling pathway, we detected the expression of surface markers (CD80, CD86, CD40and MHC II) on BMDCs by flow cytometry and found that D181markedly inhibited the maturation of the DCs with a low level of expression of surface molecules. We carried out phagocytosis assay to revel how D181influences DCs endocytic capacity of FITC-dextran, and found that D181not affect the BMDCs endocytosis.To further explore the impact of D181on CXCL-10and its mechanism, we pretreated BMDCs with different concentrations of D181before CpG stimulation, then detected the expression of CXCL-10on mRNA and protein levels and found that D181can dose-dependent inhibit CXCL-10expression on both situations. To study the role of CXCL-10changes on BMDCs, Transwell assay were used. We founded that D181can down-regulate BMDCs chemotactic ability, compared with the separate CpG stimulation. To study the mechanism of D181on CXCL-10, we added four different pathway inhibitors for ERK, JNK, p38and STAT1into the medium, then explored CXCL-10expression, results showed that ERK, JNK, p38and STAT1signaling pathway inhibitors can significantly reduce CXCL-10expression. Then, western blot analysis suggested that D181can inhibit the phosphorylation of ERK, JNK, p38and STAT1. In summary, D181can inhibit the maturation and function of CpG-induced BMDCs and exhibit an immunosuppressive effect on the TLR9signaling of mouse bone marrow-derived DCs, this suggests that the potential application of D181in the treatment of autoimmune inflammatory diseases. All these results suggest that D181could be a promising therapeutic agent for CXCL-10associated diseases. |
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