A novel antagonist of TLR9 blocking all classes of immunostimulatory CpG-ODNs

The recognition of microbial CpG-DNA by toll-like receptor 9 (TLR9) might promote excessive inflammatory response or inflammatory disorder. To prevent possible clinical pathological injury following the TLR9 activation, here we have investigated a series of CpG-DNA sequences from conventional microbes using a bioinformatics tool of pattern search, and successfully identified CpG-ODN c41 from Pseudomonas aeruginosa genome, which contains a novel motif, '3 × N-CGCG'. Using ELISA and MTT assays, we found that CpG-ODN c41 was non-stimulatory and non-cytotoxic and was able to inhibit the immunostimulatory activity caused by all classes of optimal stimulatory CpG-DNAs in murine 264.7 cells and human monocytes. Laser confocal microscopy demonstrated that CpG-ODN c41 competitively blocked the optimal stimulatory CpG-DNAs from binding to TLR9 in a dose-dependent fashion, thereby preventing TLR9 from triggering the inflammatory response. Moreover, CpG-ODN c41-mediated protection could take up a lethal challenge by stimulatory CpG-ODN in vivo. This study suggests that CpG-ODN c41 is a strong TLR9 antagonist that could be used as a therapeutic agent for CpG-ODN-mediated over-inflammatory responses, may also be used to treat autoimmune diseases.