DC-SIGN Aptamer Inhibits Atherosclerotic Inflammatory Reaction Mediated By Dendritic Cells

Part 1Homocysteine Enhanced Binding of Dendritic Cells to Endothelial Cells mediated by DC-SIGNObjective Elevated plasma homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS), which is recognized as inflammation and immune reactions. And Hcy may enhance dendritic cells (DCs)-endothelial Cells (ECs) interaction, a pivotal early event in atherogenesis, by upregulating expression of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) in cultured DCs. The purpose of this study is to approach the mechanism of the generation and progress of AS by investigating the effect of Hcy on the expression of DC-SIGN on DCs and the interaction between DCs and ECs or T lymphocytes.Methods Immunophenotype, such as HLA-DR, CD80, CD86 and CD83, of Hcy-treated DCs was monitored by flow cytometry. The endocytic activity of Hcy-treated DCs was measured by flow cytometry using a FITC-dextran uptake assay. Hcy-treated DCs were coincubated with cultured human umbilical vein endothelial cells (HUVECs), and adhesion of DCs to ECs and migration of DCs through endothelial monolayer growing on the insert of transwell plate were detected by confocal microscope and multi-detection microplate reader. Allogeneic T cell activation by Hcy-treated DCs was tested by mixed lymphocyte reaction (MLR). The expression of DC-SIGN on Hcy-treated DCs was detected by Western Blot and immunofluorescence stain.Results The majority of immature DCs generated in control cultures with GM-CSFand IL-4 expressed high level of HLA-DR, moderate levels of CD80 and CD86, and low level of CD83, while the mature DCs exposed to LPS expressed high levels of HLA-DR, CD86 and CD83, moderate level of CD80. The presence of Hcy did not change the phenotype of immature and mature DCs and not modulate the endocytic activity of mature DCs yet. Mature DCs in the presence of Hcy with high concentration were more effective than control in stimulating T-cell proliferation. Hcy promoted adhesion and migration of DCs to ECs in a concentration-dependent fashion. And this effect could be inhibited by anti-DC-SIGN monoclonal antibody. The expression of DC-SIGN on DCs was significantly upregulated by Hcy in a concentration-dependent manner as assessed by Western blot and immunofluorescence stain.Conclusions Although Hcy is unable to mature DCs or enhance the endocytic activity of DCs, it may potentialize the interaction of DCs-ECs and DC-T cells by upregulating the expression of DC-SIGN on DCs, and anti-DC-SIGN monoclonal antibody could inhibit the interaction, which indicates a novel pathophysiological mechanism for Hcy to promote atherogenesis.Part 2Selection of DNA aptamers against DC-SIGN proteinObjective Systematic evolution of ligands by exponential enrichment (SELEX) procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. Dendritic cells (DCs) are professional antigen presenting cells (APCs) which make an essential function in immune reactions. DC-SIGN is a novel DC-specific adhesion receptor that takes part in the interaction between DCs and T cells or vascular endothelial cells (ECs). In this study, we aimed at generating the high affinityaptamers against DC-SIGN to develop potential therapeutics for infection, tumour and atherosclerosis.Methods The DNA oligonucleotide library contains a 35-base central random sequence and two primers, one of them is biotin-labeled, were synthesized. DNA aptamers with a high affinity to recombinant human DC-SIGN/CD209/Fc Chimera were selected repeatedly by microwell plate and streptavidin magnesphere paramagnetic particles and then amplified by PCR. The PCR products were sequenced after being purified and cloned. At last, the affinity of the aptamers to DC-SIGN was measured by lipid scintillation counter and K_d was calculated by nonlinear regression analysis.Results 11 rounds of selection have been performed, as DC-SIGN protein is the target. The binding ratio of ssDNA and DC-SIGN protein was increased along with the rounds of selection, which indicated that ssDNA sequence were enriched significantly. After the PCR products were purified and cloned, 26 clones were picked in random to be sequenced. The affinity of synthesized DNA to DC-SIGN was measured and K_d was calculated by nonlinear regression analysis. As a result, No.16 apmater has the highest affinity to DC-SIGN, and its K_d value is 21.73 nmol/L.Conclusions We selected an aptamer which has high affinity to DC-SIGN by SELEX technology. After being purified, cloned, sequenced and measurement of the affinity to DC-SIGN, the aptamer is finally determinated as 5'-GGCGAAAATTTGTGGAT ATAGAGGGTTACTCGGAT-3'Part 3Effect of DC-SIGN aptamer on the interaction of dendritic cells and endothelial cells or T lymphocytesObjective Dendritic cells (DCs) are known to be the most powerful professional antigen-presenting cells (APCs) and be appreciated as critical controllers of the immune response. DC-SIGN is a novel DC-specific adhesion receptor that takes part in the interaction between DC and T cells or vascular endothelial cells. We have selected high affinity DNA aptamer against DC-SIGN by systematic evolution of ligands by exponential enrichment (SELEX) procedure. In this study, we aimed at observing the effect of the aptamer on the interaction between DCs and endothelial cells (ECs) or T lymphocytes, in order to demonstrate the potential therapeutics for infection, tumour and atherosclerosis.Methods DCs, human umbilical vein endothelial cells (HUVECs) and T cells were separated and cultured. Hcy-stimulated DCs or DCs in control were pretreated by anti-DC-SIGN monoclonal antibody or DC-SIGN aptamer before they were coincubated with ECs. Adhesion of DCs to ECs and migration of DCs through endothelial monolayer in transwell plate were detected by confocal microscope and multi-detection microplate reader. Allogeneic T cell activation by Hcy-stimulated mature DCs was tested by mixed lymphocyte reaction (MLR), and the effect of antibody and aptamer in MLR was investigated and compared.Results Adhesion and migration of DCs to ECs were inhibited by anti-DC-SIGN monoclonal antibody and DC-SIGN aptamer, both of which also reduced the promotion of adhesion and migration of DCs to ECs by the treatment of Hcy. In addition, both of antibody and aptamer inhibited the T cell proliferation stimulated by mature DCs even if DCs were activated by Hcy. And there is no significant difference between antibody and aptamer in their effects on the interaction of DCs and ECs or T cells.Conclusions Both anti-DC-SIGN monoclonal antibody and DC-SIGN aptamer could inhibit the interaction between Hcy-stimulated DCs and ECs or T cells, as well as DCs in control. Furthermore, the ability of aptamer is not inferior to antibody.