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Isolation, Transfection, and Culture of Primary Human Monocytes
Published on: December 16, 2019
Plasmid DNA uptake and subsequent cellular activation characteristics in human monocyte-derived cells in primary
Yuga Fukuhara1, Tomoyuki Naoi, Yoshiyuki Ogawa
1Department of Biopharmaceutics and Drug Metabolism, Graduated School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Abstract:
Plasmid DNA (pDNA) uptake and subsequent cellular activation characteristics were studied in three types of human monocyte-derived cells, that is, human monocytes, macrophages, and dendritic cells (DCs) in primary culture. Naked pDNA was bound to and taken up by the macrophages and DCs while only significant binding occurred in the monocytes. pDNA binding to these monocyte-derived cells was significantly inhibited by polyinosinic acid (poly[I]), dextran sulfate, maleylated bovine serum albumin (Mal-BSA) and to a lesser extent by polycytidylic acid (poly[C]), but not by dextran or galactosylated BSA (Gal-BSA), mannosylated BSA (Man-BSA), suggesting that a specific mechanism for polyanions is involved in the pDNA binding. In cellular activation studies, naked pDNA could not induce TNF-alpha production from any monocyte-derived cells, regardless of the abundant presence of CpG motifs in the pDNA. However, when complexed with cationic liposomes, pDNA produced a significant amount of TNF-alpha from the human macrophages. TNF-alpha induction was not observed in the monocytes or DCs. Moreover, calf thymus DNA (CT DNA) complexed with cationic liposomes also induced TNF-alpha production to a similar extent in the human macrophages. These results indicate that, among human monocyte-derived cells, macrophages are activated by DNA when complexed with cationic liposomes in a CpG motif-independent manner.
Insights
Human macrophages, but not monocytes or dendritic cells, are activated by plasmid DNA (pDNA) when complexed with cationic liposomes. This DNA activation occurs independently of CpG motifs, highlighting a specific cellular response mechanism.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Plasmid DNA (pDNA) uptake and cellular activation are critical for gene therapy and immunotherapy.
- Human monocyte-derived cells, including monocytes, macrophages, and dendritic cells (DCs), play key roles in immune responses.
- The interaction of pDNA with these cells is complex and can be influenced by various factors.
Purpose of the Study:
- To investigate the uptake and cellular activation of plasmid DNA (pDNA) in human monocytes, macrophages, and dendritic cells (DCs).
- To determine the mechanism of pDNA binding and the conditions under which pDNA induces cellular activation, specifically TNF-alpha production.
Main Methods:
- Primary cultures of human monocytes, macrophages, and DCs were used.
- pDNA binding assays were performed with and without inhibitory agents (polyanions like polyinosinic acid, dextran sulfate).
- Cellular activation was assessed by measuring TNF-alpha production following pDNA exposure, both naked and complexed with cationic liposomes.
Main Results:
- Macrophages and DCs efficiently bound and took up naked pDNA, while monocytes showed significant binding only.
- pDNA binding was inhibited by specific polyanions, suggesting a receptor-mediated mechanism.
- Naked pDNA did not induce TNF-alpha production. However, pDNA complexed with cationic liposomes significantly induced TNF-alpha in macrophages, but not in monocytes or DCs. Calf thymus DNA complexed with liposomes also activated macrophages.
Conclusions:
- Human macrophages are activated by DNA when complexed with cationic liposomes, independent of CpG motifs.
- This activation pathway is specific to macrophages among the studied monocyte-derived cells.
- Cationic liposomes play a crucial role in mediating DNA-induced cellular activation in macrophages.
