Based on the concentrations used for determining the epithelial membrane permeability (Fig. 1), 64 μM (7 μg/mouse, 0.35 mg/kg) of both Esc(1–21) and LL-37 stimulated the host immune system, causing a considerable increase in the number of neutrophils and macrophages in the bronchoalveolar lavage (BAL) compared to the amount found in the control animals receiving the vehicle phosphate buffered saline (PBS) (Fig. 2a, red bars). However, the lung inflammatory reaction was negligible when the peptides were used at 20 μM (2 μg/mouse, 0.1 mg/kg), as demonstrated by the invariant number of total immune cells in the BAL compared to PBS-treated mice (Fig. 2b). In parallel, no observable effects on inflammation-related genes expression (such as those encoding for the cytokines IL-6, IL-10 or the tumor necrosis factor-α TNFα, and NF-kB) were observed in the lungs of mice, at 24 hours after peptide treatment at a concentration of 0.1 mg/kg (Fig. 3a).Figure 2Effect of Esc(1–21), Esc(1–21)-1c and LL37 on the number of lung inflammatory cells in mice. The number of macrophages, neutrophils and total inflammatory cells in the BAL of mice was counted at 24 hours after peptide administration at 7 μg/mouse (0.35 mg/kg) (panel a) or 2 μg/mouse (0.1 mg/kg) (panel b). Results are mean ± SEM from three independent experiments; n = 4–6 mice for each group in each experiment. Following one-way analysis of variance (ANOVA), posthoc comparisons were made using the Dunnett’s multiple comparison test when the P-value was significant (p < 0.05). *p < 0.05, **p < 0.01, for peptide-treated mice versus PBS-receiving animals. Figure 3Comparison among Esc(1–21), Esc(1–21)-1c and LL-37 on the in vivo gene expression in the lungs of mice. Effects of peptides on the expression of different inflammatory genes (panel a) or mucociliary-associated genes (panel b) in the lungs of mice after 24 hours from peptide administration at 0.1 mg/kg. AMPs treatments at 0.1 mg/kg do not noticeably affect the expression of inflammation and mucociliary clearance-related genes. Results are mean ± SEM from two independent experiments; n = 4–6 mice for each group in each experiment.