Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways.

Previously, we showed that bacterial DNA and vertebrate DNA/cationic liposome complexes stimulate potent inflammatory responses in cultured mouse macrophages. In the present study, we examined whether endocytosis and subsequent acidification are associated with these responses. The endocytosis inhibitor, cytochalasin B, reduced tumor necrosis factor alpha (TNF-alpha) production by a plasmid DNA (pDNA)/cationic liposome complex. The endosomal acidification inhibitor, monensin, inhibited cytokine production by pDNA or a calf thymus DNA/liposome complex. These results suggest, similarly to CpG motif-dependent responses, that endocytosis and subsequent endosomal acidification are also required for these inflammatory responses. It is intriguing that another inhibitor of endosomal acidification, bafilomycin A, stimulated the production of TNF-alpha mRNA and its protein after removal of the pDNA/liposome complex and inhibitors, although it inhibited the release of interleukin-6. Similar phenomena were observed in the activation of macrophages by CpG oligodeoxynucleotide, calf thymus DNA, and Escherichia coli DNA complexed with liposomes. Moreover, bafilomycin A also induced a high degree of TNF-alpha release after stimulation with naked pDNA. These results suggest that bafilomycin A increases TNF-alpha production induced by DNA at the transcriptional level via an as-yet unknown mechanism. Furthermore, we investigated the contribution of Toll-like receptor 9 (TLR9), the receptor of CpG motifs, to the cell activation by the DNA/cationic liposome complex using the macrophages from TLR9-/- mice. We observed a reduced inflammatory cytokine release from macrophages of TLR9-/- mice compared with wild-type mice. However, the cytokine production was not completely abolished, suggesting that the DNA/cationic liposome complex can induce macrophage activation via TLR9-dependent and -independent pathways.

Cellular uptake and activation characteristics of naked plasmid DNA and its cationic liposome complex in human macrophages.

Plasmid DNA (pDNA) is an important macromolecular therapeutic agent suitable for DNA-based therapies, such as non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs abundant in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits transgene expression, while modulating immunological consequences following vaccination. We studied cellular uptake and activation characteristics of naked pDNA and its cationic liposome complex in human macrophage-like cells. The present study has demonstrated that naked pDNA was recognized by human macrophage-like cells via specific mechanisms for polyanions. Moreover, it has shown that pDNA complexed with cationic liposomes activates human macrophage-like cells to induce the production of tumor necrosis factor-alpha (TNF-alpha) in a CpG motif-independent manner, while any types of naked DNA could not induce TNF-alpha production from these cells, regardless of the presence of CpG motifs in pDNA or oligonucleotide (ODN). These findings form an important basis for DNA-based therapies including gene therapy and DNA vaccination.

Restricted cytokine production from mouse peritoneal macrophages in culture in spite of extensive uptake of plasmid DNA.

The production of inflammatory cytokines from macrophages (Mphi), upon stimulation with plasmid DNA (pDNA) containing CpG motifs, is a critical process for DNA-based therapies such as DNA vaccination and gene therapy. We compared Mphi activation, following stimulation with naked pDNA, based on the production of cytokines from cell lines (RAW264.7 and J774A1) and peritoneal Mphis in primary culture. The Mphi cell lines RAW264.7 and J774A1 produced a significant amount of tumour necrosis factor-alpha (TNF-alpha) upon stimulation with naked pDNA and this response required endosomal acidification. On the other hand, peritoneal Mphis (both resident and elicited) in primary culture did not secrete TNF-alpha or interleukin-6, although they contain the mRNA of toll-like receptor-9 (TLR-9) and are able to respond to CpG oligodeoxynucleotides. This unresponsiveness was not a result of impaired cellular uptake of pDNA because the primary cultured Mphis showed a higher uptake of pDNA than the RAW264.7 and J774A1 cell lines. These findings have important implications for Mphi activation by naked pDNA as it has been generally assumed that pDNA that contains CpG motifs is a potent agent for inducing inflammatory cytokines in vivo, based on evidence from in vitro studies using Mphi cell lines.