Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37

Abstract

Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.

FIG. 1.

Production of nitrate/nitrite in segments of rat aortas during 24 h of incubation, as measured with Griess reagent. LPS alone (filled bar) increased the amount of nitrate/nitrite production compared to the control (Ctrl). Compared to that induced by LPS alone, the production of nitrate/nitrite was lower in segments coincubated with the peptides at 2 μM (*). However, fragment 106 was less effective at inhibiting nitrate/nitrate production than LL-37 at 2 μM (#). The data were analyzed by one-way repeated-measurement ANOVA followed by post hoc testing by the Holm-Sidak method (P < 0.05). Values are means + SEM (n = 7).

FIG. 2.

Concentration-response curves of the hemolytic activities of the peptides towards human erythrocytes. The 18-mer LLKKK (▴) and BMAP-27 (▵) induced significantly more (*) hemolysis, while fragments 106 (▪) and 110 (□) induced significantly less hemolysis, than LL-37 (•). Fragment 106 induced significantly less hemolysis than fragment 110 (#). The data were analyzed by two-way repeated-measurement ANOVA for the factors of different peptides and peptide concentrations followed by post hoc testing by the Holm-Sidak method (P < 0.05). Values are means ± SEM (n = 7). Note the log scales on both the x and y axes.

FIG. 3.

DNA fragmentation in cultured human vascular smooth muscle cells after 16 h of incubation with LL-37 (•) or fragment 106 (▪) or 110 (□). LL-37 at 6 and 20 μM as well as fragment 106 at 6 μM induced significant DNA fragmentation compared to the control (*). The data were analyzed by two-way repeated-measurement ANOVA for the factors of different peptides and peptide concentrations followed by post hoc testing by the Holm-Sidak method (P < 0.05). DNA fragmentation is expressed as the fold increase in absorbance over that of the control. Values are means ± SEM (n = 8).

FIG. 4.

Chemotaxis of human neutrophils in response to LL-37, fragments 106 and 110, the 18-mer LLKKK, and the classical chemoattractant formyl peptide, fMLP. fMLP (0.1 μM) displayed a statistically significant chemoattractant activity (filled bar, §) compared to the control (open bar) (Student's paired t test; P < 0.05). The concentration-response curve for LL-37 was biphasic, with statistically significant chemoattractant activities at 0.1 and 1 μM but not at 10 μM (•,*). The concentration-response curves for fragments 106 (▪) and 110 (□) as well as that for the 18-mer LLKKK (▴) were similar to that of LL-37, except that at 10 μM, the 18-mer LLKKK induced a larger chemotactic response than that induced by LL-37 (#). The data were analyzed by two-way repeated-measurement ANOVA for the factors of different peptides and peptide concentrations followed by post hoc testing by the Holm-Sidak method (P < 0.05). Values are means ± SEM (n = 4).

FIG. 5.

Inhibition of antibacterial activity by serum, as assessed by a radial diffusion assay using E. coli. In the absence of serum (open bars), LL-37 and fragments 106 and 110 (all at 20 μM) inhibited bacterial growth to the same extent. Although serum alone was weakly antibacterial (*), it markedly decreased the antibacterial activity of LL-37 and, to a smaller extent, the activity of fragment 110 (*). The activity of fragment 106 was not significantly affected. The data were analyzed by two-way repeated-measurement ANOVA for the factors of different peptides and serum concentrations followed by post hoc testing by the Holm-Sidak method (P < 0.05). *, statistically significantly different from the value in the absence of serum; #, statistically significantly different from the value for LL-37 in the presence of serum at the same concentration. Values are means + SEM (n = 5).