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Updated: Jun 5, 2026
Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo
Published on: September 18, 2014
5'-Phosphate oligodeoxynucleotides enhance the phosphodiester-CpG DNA-induced inflammatory response in macrophages
Hiroyuki Yoshida1, Makiya Nishikawa, Tsuyoshi Kiyota
1Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Abstract:
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-α production irrespective of treatment with DNase I; however, DNase I-treated, but not untreated, non-CpG DNA increased the PO-CpG DNA-mediated TNF-α production. This increase was not observed with phosphorothioate-CpG DNA or ligands for TLR3, TLR4 or TLR7. Deoxynucleotides with a 5'-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'-phosphate; such molecules could therefore be considered as exacerbating factors for CpG motif-related inflammation.
Insights
DNA fragments from dying cells can worsen autoimmune diseases. DNA with a 5'-phosphate group, like that from DNase I treatment, significantly boosts immune responses to CpG DNA motifs, potentially exacerbating inflammation.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Dying cells release genomic DNA, which is degraded into smaller fragments.
- Unmethylated CpG motifs in DNA are characteristic of bacteria but also present in mammalian DNA.
- CpG motifs have been implicated in exacerbating DNA-associated autoimmune diseases.
Purpose of the Study:
- To investigate if DNA fragments and their degradation products influence immune responses to phosphodiester (PO)-CpG DNA.
- To determine the role of DNA structure, specifically the 5'-phosphate group, in modulating CpG DNA-mediated immune activation.
Main Methods:
- Treatment of murine macrophage-like RAW264.7 cells with various DNA fragments and degradation products.
- Measurement of Tumor Necrosis Factor-alpha (TNF-α) production as an indicator of immune response.
- In vivo studies involving subcutaneous injection of DNA fragments and PO-CpG DNA into mouse footpads to assess inflammation.
Main Results:
- DNase I-treated non-CpG DNA, but not untreated DNA, significantly increased PO-CpG DNA-mediated TNF-α production in macrophages.
- Deoxynucleotides with a 5'-phosphate group mimicked the effect of DNase I-treated DNA, enhancing TNF-α production.
- Co-injection of DNase I-treated DNA with PO-CpG DNA in mice led to significantly increased paw swelling compared to PO-CpG DNA alone.
Conclusions:
- DNA molecules possessing a 5'-phosphate group can potentiate inflammatory responses triggered by PO-CpG DNA.
- These findings suggest that DNA fragments with 5'-phosphate groups may act as exacerbating factors in CpG motif-related inflammatory conditions and autoimmune diseases.
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