This study demonstrates the utility of polypoid-like structured DNA (polypodna) preparations as potent immunostimulators in a murine model.

Dying cells release genomic DNA into the surroundings where the DNA is first degraded to oligodeoxynucleotides, then to nucleotides, nucleosides and so on. Given that the unmethylated CpG dinucleotide (CpG motif), which is characteristic of bacterial DNA, is also contained in mammalian DNA and has been reported to be involved in the exacerbation of DNA-associated autoimmune diseases, we investigated whether nucleotides and nucleosides affect immune responses to phosphodiester (PO)-CpG DNA. Addition of non-CpG DNA to RAW264.7, murine macrophage-like cells, induced no significant TNF-a production irrespective of treatment with DNase I; however, DNase I-treated, but not untreated, non-CpG DNA increased the PO-CpG DNA-mediated TNF-a production. This increase was not observed with phosphorothioate-CpG DNA or ligands for TLR3, TLR4 or TLR7. Deoxynucleotides with a 5 0 -phosphate showed similar effects to those of DNase I-treated non-CpG DNA, but DNase II-treated DNA or deoxynucleosides did not. Subcutaneous injection of PO-CpG DNA into the mouse footpad induced little swelling of the paw; however, significant swelling was observed when DNase I-treated DNA was co-injected with PO-CpG DNA. These results imply that PO-CpG DNA-dependent inflammatory responses are increased by DNA molecules with a 5 0 -phosphate; such molecules could therefore be considered as exacerbating factors for CpG motif-related inflammation.Keywords: 5 0 -Phosphate . CpG motif . Cytokines . DNase I . Macrophages Supporting Information available onlineIntroduction DNA is one of the fundamental components of many types of organisms. A unique property of DNA is the species difference in the frequency of unmethylated CpG dinucleotides (CpG motifs) in genomic DNA; the motifs are abundant in bacterial or viral DNA but few in mammalian genomic DNA [1]. This difference would have evolved so that the mammalian innate immune systems can recognize the CpG motif as a danger signal using Toll-like receptor-9 (TLR9) expressed in DC, B cells and macrophages, which is followed by the release of inflammatory cytokines and type I interferons (IFNs) [2]. TLR9 as well as other TLRs for nucleic acids, such as TLR3 and TLR7, exists in the intracellular compartment, and the binding of CpG motif to TLR9 Eur. J. Immunol. 2011. 41: 425-436 DOI 10.1002 Innate immunity 425 occurs in the late-endosome/lysosome compartments after the internalization of DNA [3,4]. The reason for the intracellular localization of TLR9 is considered to be aimed at avoiding an unnecessary immune response to self-DNA [5], which also contains some CpG motifs as genomic and mitochondrial DNA. Extracellular and intracellular deoxyribonucleases (DNases) would also reduce the unexpected immune activation by self-DNA. Although these multiple mechanisms prevent unwanted recognition of self-DNA as danger signals, self-DNA is shown to be an activator of DC in systemic autoimmune diseases and to induce cytokine production via the TLR9 pathway [6]. Several previous experiments have provided evidence about ...