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Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Published on: November 9, 2019
Cellular activation by plasmid DNA in various macrophages in primary culture
Hiroyuki Yoshida1, Makiya Nishikawa, Sachiyo Yasuda
1Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Abstract:
Macrophages are an important group of cells responsible for the inflammatory response to unmethylated CpG dinucleotide (CpG motif) in plasmid DNA (pDNA) via Toll-like receptor 9 (TLR9). This finding is primarily based on in vitro studies. Previous in vivo studies also have suggested that tissue macrophages are involved in inflammatory cytokine release in the circulation following intravenous administration of pDNA to mice. However, the relationship between the in vitro and in vivo studies has not been sufficiently clarified. To gain insight into which types of cells are responsible for the production of cytokines upon interaction with pDNA, peritoneal macrophages, splenic macrophages, hepatic nonparenchymal cells (NPCs) including Kupffer cells and mesangial cells were isolated from mice. All types of primary cultured cells, except for mesangial cells, express TLR9 at varying levels. Splenic macrophages and hepatic NPCs were activated to produce tumor necrosis factor-alpha (TNF-alpha) by naked pDNA, whereas peritoneal macrophages and mesangial cells were not. pDNA complexed with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride/cholesterol liposome induced TNF-alpha in the splenic macrophages but not in the other cell types. These results indicate that splenic macrophages and hepatic NPCs are closely involved in TNF-alpha production in response to pDNA.
Insights
Splenic macrophages and hepatic nonparenchymal cells (NPCs) are key players in the inflammatory response to plasmid DNA (pDNA). These cells, but not others, produce tumor necrosis factor-alpha (TNF-alpha) when exposed to pDNA.
Area of Science:
- Immunology
- Cell Biology
Background:
- Macrophages mediate inflammatory responses to unmethylated CpG motifs in plasmid DNA (pDNA) via Toll-like receptor 9 (TLR9).
- In vitro and in vivo studies suggest tissue macrophages contribute to cytokine release after pDNA administration, but the precise cell types involved require clarification.
Purpose of the Study:
- To identify specific cell types responsible for cytokine production upon interaction with plasmid DNA (pDNA) in vivo.
- To elucidate the role of different macrophage populations and hepatic nonparenchymal cells (NPCs) in the inflammatory response to pDNA.
Main Methods:
- Isolation of primary peritoneal macrophages, splenic macrophages, and hepatic NPCs (including Kupffer cells) from mice.
- Assessment of Toll-like receptor 9 (TLR9) expression in isolated cell types.
- Stimulation of cells with naked pDNA and liposome-complexed pDNA to measure tumor necrosis factor-alpha (TNF-alpha) production.
Main Results:
- Splenic macrophages and hepatic NPCs express TLR9 and produce TNF-alpha in response to naked pDNA.
- Peritoneal macrophages and mesangial cells did not produce TNF-alpha in response to naked pDNA.
- Liposome-complexed pDNA induced TNF-alpha production specifically in splenic macrophages, not other tested cell types.
Conclusions:
- Splenic macrophages and hepatic NPCs are critical for TNF-alpha production following pDNA exposure.
- The cellular response to pDNA, particularly TNF-alpha production, is dependent on both the cell type and the form of pDNA (naked vs. liposome-complexed).
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