DNA containing unmethylated cytosine-guanine motifs (CpG DNA) initiates innate immune responses, including the secretion of cytokines from macrophages. Some antimicrobial peptides modulate the responses to CpG DNA, although the molecular mechanisms of this process remain unclear. This study examined the effects of four alpha-helical antimicrobial peptides on the immune responses induced by CpG DNA. The antimicrobial peptide FIKRIARLLRKIF, known as Kn2-7, increased the CpG DNA-dependent secretion of interleukin-10 (IL-10) and tumor necrosis factor-alpha from mouse macrophage-like RAW264.7 cells. Kn2-7 enhanced the cellular uptake of CpG DNA; this effect was decreased by the substitution of arginine residues with alanine residues, and increased by the substitution of lysine residues with arginine residues. The degree to which these peptides enhanced the cellular uptake of CpG DNA correlated well with their ability to increase CpG DNA-dependent IL-10 secretion. In contrast, Kn2-7 synthesized with D-amino acids did not increase CpG DNA-dependent IL-10 secretion, although the ability of the D-form of Kn2-7 to enhance the cellular uptake of CpG DNA was not diminished relative to that of Kn2-7. These results indicate that enhanced cellular uptake of CpG DNA is necessary but insufficient to augment CpG DNA-dependent immune responses. (C) 2020 Elsevier Inc. All rights reserved.