Direct injection of plasmid DNA into the skin induces dermatitis by activation of monocytes through toll-like receptor 9.
BACKGROUND
Direct injection of naked DNA into skin can be efficiently used to transfer genes into keratinocytes in vivo. However, bacterial DNA is known to be a potent stimulus for vertebrate immune cells and immune systems. Towards the clinical use of this method, this study examined whether the application of plasmid DNA by direct injection induces any adverse skin effects.


METHODS
Several plasmid preparations were prepared and directly injected into rat and human skin. Migration, IL-6 production, and reactive oxygen production assays were performed to determine the type of the primary cells responsible for the reaction. Involvement of toll-like receptor (TLR) 9 was examined by experiments using TLR9-knockout mice.


RESULTS
Injection of several plasmid preparations into rat and human skin resulted in an inflammatory reaction at the treated site. Contamination by endotoxin in the plasmid preparation was shown to worsen this skin inflammation reaction. Immunohistochemical analysis showed that the infiltrating cells in the skin lesions were predominantly monocytes and neutrophils. Further experiments examining migration, IL-6 production, and reactive oxygen production indicated that the primary responsible cells were monocytes rather than neutrophils. Since it was recently shown that cytosine-guanosine dinucleotide (CpG) motif is critical for immune reaction induction in bacterial DNA and cellular responses were mediated by TLR9, we injected plasmids into the ear skin of TLR9-knockout mice. A decrease in ear swelling was noted in the knockout mice, compared to controls, suggesting that plasmid-DNA-induced dermatitis was mediated mostly by TLR9.


CONCLUSIONS
This study demonstrates that injection of plasmid DNA induces skin inflammation initiated by monocyte activation via TRL9. We should therefore attempt to counteract this dermatitis during the clinical use of the naked DNA injection method in skin.
Sawamura
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D
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. smartdai@med.hokudai.ac.jp
Abe
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R
Goto
M
M
Akiyama
M
M
Hemmi
H
H
Akira
S
S
Shimizu
H
H
D000818:Animals; D002465:Cell Movement; D004247:DNA / Q000008:administration & dosage*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003872:Dermatitis / Q000209:etiology* / Q000276:immunology; D004427:Ear Diseases / Q000209:etiology; D004731:Endotoxins / Q000494:pharmacology; D006801:Humans; D007279:Injections, Subcutaneous; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009000:Monocytes / Q000276:immunology*; D009504:Neutrophils / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000276:immunology; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D011922:Rats, Mutant Strains; D017382:Reactive Oxygen Species / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012867:Skin / Q000187:drug effects*; D051217:Toll-Like Receptor 9
C408982:CPG-oligonucleotide; D004268:DNA-Binding Proteins; D004731:Endotoxins; D015850:Interleukin-6; D009838:Oligodeoxyribonucleotides; D017382:Reactive Oxygen Species; D011956:Receptors, Cell Surface; C495371:TLR9 protein, human; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D004247:DNA