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Updated: Jun 23, 2026
Intralymphatic Immunotherapy and Vaccination in Mice
Published on: February 2, 2014
TLR9-dependent systemic interferon-beta production by intravenous injection of plasmid DNA/cationic liposome complex
Hiroyuki Yoshida1, Makiya Nishikawa, Sachiyo Yasuda
1Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Insights
CpG lipoplex triggers a strong immune response in vivo, mediated by Toll-like receptor 9 (TLR9). Interferon-beta (IFN-beta) is produced by non-splenic phagocytic cells, while interleukin-6 (IL-6) originates from splenic macrophages.
Area of Science:
- Immunology
- Molecular Biology
- Pharmacology
Background:
- The in vivo type I interferon (IFN) response to DNA/cationic liposome complexes (lipoplex) is poorly understood.
- Pro-inflammatory cytokine responses to lipoplex are known to involve unmethylated CpG motifs and Toll-like receptor (TLR)-9.
Purpose of the Study:
- To investigate the in vivo immune response to CpG-containing lipoplex.
- To elucidate the roles of TLR9, phagocytic cells, and the spleen in lipoplex-induced cytokine production.
Main Methods:
- Intravenous injection of CpG- or non-CpG lipoplex into mice.
- Measurement of IFN-beta and IL-6 in serum and organs via ELISA.
- Evaluation of TLR9, phagocytic cells, and spleen involvement using knockout, clodronate liposome-treated, and splenectomized mice.
Main Results:
- CpG lipoplex significantly increased IFN-beta and IL-6 levels in serum and organs; non-CpG lipoplex had minimal effect.
- Responses were dependent on TLR9, phagocytic cells, and spleen for IL-6 but not IFN-beta.
- Monocyte accumulation was observed in lung capillaries following lipoplex administration.
Conclusions:
- IFN-beta production following CpG lipoplex injection occurs via a TLR9-dependent pathway involving phagocytic cells outside the spleen.
- IL-6 production is primarily mediated by splenic macrophages, contrasting with IFN-beta production.
Background:
The type I interferon (IFN) response to DNA/cationic liposome complex, or lipoplex, has been reported in cultured cells, but little is known about the response in vivo. Studies of the pro-inflammatory cytokine response to lipoplex have shown the importance of the unmethylated CpG dinucleotide (CpG motif) and its receptor, Toll-like receptor (TLR)-9.
Methods:
CpG- and non-CpG lipoplex consisting of CpG- or non-CpG plasmid DNA, respectively, and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol liposomes were intravenously injected into mice. IFN-beta and interleukin (IL)-6 in the serum and organs were measured by the enzyme-linked immunosorbent assay. The involvement of TLR9, phagocytic cells and the spleen in the responses was evaluated using TLR9(-/-), clodronate liposome-treated-, and splenectomized mice, respectively. Accumulation of blood cells in the lung was evaluated histologically.
Results:
CpG lipoplex induced a large increase in the levels of IFN-beta and IL-6 in the serum, liver, spleen, lung and kidney, whereas non-CpG lipoplex scarcely had any effect. Neither formulation led to significant cytokine production in TLR9(-/-) mice. Clodronate liposome-treated mice showed a large reduction in both IFN-beta and IL-6 levels. Splenectomized mice receiving CpG lipoplex also showed a significantly low production of IL-6 but a similar level of IFN-beta production to that of unsplenectomized mice. A large number of monocytes were found in the capillary vessels around the pulmonary alveoli of mice receiving lipoplex.
Conclusions:
These findings indicate that, in contrast to the production of IL-6 from splenic macrophages, IFN-beta is produced from phagocytic cells other than splenic macrophages after the injection of CpG lipoplex through the TLR9-dependent pathway.
