Abstract
We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing 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.
Highlights
TLR9 is the only TLR that recognizes DNA
The band was not detected in the lysates of the untreated or mock-transfected HEK-Blue human Toll-like receptor 9 (hTLR9) cells, indicating that FLAG-tagged human macrophage scavenger receptor-1 (hMSR1) was expressed in the HEK-Blue hTLR9/hMSR1 cells
We demonstrated that HEK-Blue hTLR9 cells efficiently responded to PS CpG DNA, which has high binding affinity for cell membranes, whereas they hardly responded to natural, PO CpG DNA, in spite of the fact that the cells expressed human TLR9
Summary
TLR9 is the only TLR that recognizes DNA. Its ligand is a DNA molecule containing an unmethylated cytosine–phosphate–guanine (CpG) motif, i.e., CpG DNA. Msr1-mediated DNA uptake could be involved in TLR-mediated immune stimulation by nucleic acid drug candidates. We hypothesized that low cellular uptake of CpG DNA by HEK-Blue hTLR9 cells might explain the weak or absent response to PO CpG DNA. Such cells would be useful for the screening of nucleic acid drug candidates with diverse physicochemical properties To this end, we transduced HEK-Blue hTLR9 cells with human MSR1 to obtain HEK-Blue hTLR9/hMSR1 cells in the hope that the transfection of the MSR1 gene to HEK-Blue hTLR9 cells would increase the uptake of PO DNA. We determined 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 containing the ssCpG (tetraCpG) as model TLR9 ligands. HEK-Blue hTLR9 cells and HEK-Blue hTLR7 cells were used for the analysis of cellular responses to CpG DNA
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
