The cells transfected with vacant pcDNA3.1 vector were used as mock controls. Western Blotting of hMSR1 in HEK-Blue hTLR Cells The cells were lysed in a lysis buffer (PicaGene Dual Sea Pansy Luminescence Kit, Toyo Ink, Tokyo, Japan), and the cell lysates were reduced by the addition of dithiothreitol to 100?mM. (tetrapodna) made up of ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties. Introduction Various classes of nucleic acid drugs have been marketed or are being developed. Attention must be paid to toxicity issues during Rilapladib the development of nucleic acid drugs1. Nucleic acid drug candidates have several toxicity issues, including off-target effects, immune stimulation, hematoxicity, hepatotoxicity, and nephrotoxicity. Immune stimulation occurs when toll-like receptors (TLRs) recognize DNA or RNA. Several reports have suggested that certain small interfering RNA molecules cause immune stimulation Ywhaz via TLRs2C8. Therefore, it is clearly important to evaluate unintentional TLR-mediated immune stimulation in the development of nucleic acid drug candidates. TLR9 is the only TLR that recognizes DNA. Its ligand is usually a DNA molecule made up of an unmethylated cytosineCphosphateCguanine (CpG) motif, to obtain HEK-Blue hTLR9/hMSR1 cells in the hope that this transfection of the gene to HEK-Blue hTLR9 cells would increase the uptake of PO DNA. We first evaluated the effect of transfection of the gene around the cellular uptake of DNA. We then Rilapladib decided whether HEK-Blue hTLR9/hMSR1 cells respond to both PS and PO CpG DNAs. We selected phosphorothioate CpG2006 (PS CpG2006), a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA made up of the ssCpG (tetraCpG) as model TLR9 ligands. HEK-Blue hTLR9 cells and HEK-Blue hTLR7 cells were also used for the analysis of cellular responses to CpG DNA. Results Establishment of HEK-Blue hTLR9/hMSR-1 Cells Physique?1A shows the results of western blotting analysis of the cell lysates using anti-hMSR1 antibody. The lysate of HEK-Blue hTLR9/hMSR1 cells revealed a band of approximately 75?kDa, which corresponded to the FLAG-tagged hMSR1. The band was not detected in the lysates of the untreated or mock-transfected HEK-Blue hTLR9 cells, indicating that FLAG-tagged hMSR1 was expressed in the HEK-Blue hTLR9/hMSR1 cells. We examined the localization of hMSR1 in the HEK-Blue hTLR9 cells using confocal microscopy. Physique?1B presents confocal microscopy images of untreated, mock-transfected, and cDNA used in the present study contained hMSR1 signal-anchor sequences, so it is reasonable to assume that hMSR1 is appropriately sorted to the correct destination (the cell membrane). It has been reported that ligation to MSR1 induces clathrin-mediated endocytosis, and that the ligands are then sorted to endosomes31. The mechanistic details of the uptake of DNA by HEK-Blue hTLR9/hMSR1 cells were not investigated in this study, but the efficient response to PO CpG DNA strongly suggests that the cells take up DNA in a similar manner to that adopted by other types of cells that express MSR1, such as dendritic cells. Several reports suggest that hMSR1 is usually involved in the cellular uptake of PS CpG DNA32. However, the present study exhibited that hMSR1 expression had no significant effect on Rilapladib the cellular uptake of Alexa Fluor 488-PS CpG DNA (data not shown) or on SEAP release (Fig.?4). PS CpG DNA binds strongly to cell surfaces33, and would mask any hMSR1-mediated cellular uptake of PS CpG DNA, even if it occurred. Although most TLR9 is found in around the endosomes, TLR9 is also detected on the surface of cells in some cell types34,35. Some reports discussed that this cell surface TLR9 promoted the cellular uptake of CpG DNA as well as CpG DNA-coupled siRNA36C38. In these studies, PS CpG DNA and the antisense strand of siRNA were conjugated and, therefore, a strong binding of PS CpG DNA to the cell surface could lead to efficient uptake of the conjugate. Zhang fragment amplified by polymerase chain reaction (PCR) from a cDNA clone of human MSR1 (GE Healthcare UK Ltd., Buckinghamshire, England) into the multi-cloning site of pcDNA3.1. Transfection of hMSR1-expressing Plasmid DNA in HEK-Blue Cells HEK-Blue hTLR7 and HEK-Blue.
