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, monensm, 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, bafilonivcin 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/catioinic 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.