6B). The serum concentration of self-DNA

in patients with DNA-related autoimmune diseases is higher than that in healthy subjects 7–9. In addition, circulating CpG DNA has been reported to be a pathogenic factor of SLE 23, 24. However, it is unclear whether the augmented self-DNA in serum affects the immune response to CpG DNA in autoimmune diseases. In the present study, we clearly demonstrated that DNase I-treated DNA, but not intact DNA, increases the CpG motif- and TLR9-dependent cytokine production in murine macrophages. As shown in Fig. 3B, it was found that only the DNase I-treated DNA, but not DNase II-treated one, has an ability to increase CpG DNA-induced cytokine production. Both DNase I and DNase II are endonucleases and cleave the PO bond in DNA, which yields polynucleotides with a 5′-mono-phosphate and 3′-mono-phosphate, respectively 13, 25. Taking into HTS assay consideration these results, an oligonucleotide

with a phosphate group selleck chemicals at the 5′-terminal is required for increased cytokine production from macrophages. In addition, the results showing that dNMPs and dNTPs but not deoxynucleosides increased the TNF-α release by CpG DNA at the comparable level (Fig. 3A), support the importance of the presence of a phosphate at the 5′-end of DNA. Moreover, the results in Fig. 3A indicate that this activity of DNA with 5′-phosphate to increase CpG DNA-induced cytokine production is dependent on the type of base, because TMP and TTP were much less effective than other dNMP or dNTP. It is still unknown how short the DNA is when DNA is fully cleaved by DNase I. However, the present study has demonstrated that mononucleotides are sufficient to increase the CpG DNA-dependent cytokine release from macrophages. TNF-α production in RAW264.7 cells was not proportional to the concentration of ODN1668; a 3-fold increase in the concentration of ODN1668 resulted in a 18-fold increase in TNF-α

check production (Fig. 1A). ODN1668 at a concentration of 1 μM or lower was hardly effective for cytokine production (data not shown). In addition, CpG DNA was often used at the equal concentration to this study in multiple reports of immune responses to DNA 26, 27. Therefore, the concentrations of ODN1668 used in the present study (1 and 3 μM for RAW264.7 cells and splenic macrophages, respectively) were similar levels to those used in literatures 26, 27, even though they were higher than the concentration of DNA in the serum of active SLE patients (about 3.2±1.1 μg/mL) 28. It was excluded that increased cellular uptake or stabilization is involved in the DNase I-treated DNA-mediated increase in cytokine production (Fig. 5). Zwiorek et al.