Antimicrobial Peptide Reduces Cytotoxicity and Inflammation in Canine Epidermal Keratinocyte Progenitor Cells Induced by Pseudomonas aeruginosa Infection

Abstract

The direct effects and antimicrobial activity of synthetic antimicrobial peptides (AMPs) obtained from dogs, including cBD, cBD103, and cCath, against P. aeruginosa wild-type strain PAO1 and canine keratinocytes were analyzed. Antibacterial effects on planktonic bacteria were assessed by determining the minimum bactericidal concentrations (MBCs) of AMPs and by a time-kill assay. Antibiofilm effects were assessed using the microtiter plate assay. We also evaluated the effects of AMPs on cell cytotoxicity and host immune response induced by stimulating canine epidermal keratinocyte progenitor (CPEK) cells with PAO1 and its LPS. cBD, cBD103, and cCath all exhibited dose-dependent antimicrobial and antibiofilm effects. In particular, 25 μg/mL cBD103 showed rapid bactericidal activity within 60 min and inhibited biofilm formation. In addition, pretreatment with cBD103 (25 µg/mL) and cCath (50 µg/mL) 1 h before stimulation significantly reduced the cytotoxicity of the CPEK cells by PAO1 and LPS-induced IL-6 and TNF-a expressions. cBD had little effect on the response to PAO1 and LPS in the cells. These results indicate the therapeutic potential of AMPs in P. aeruginosa skin infections. However, further studies on the mechanism of action of AMPs in keratinocytes and clinical trials are needed.

Keywords: antibacterial activity; antibiofilm effect; antimicrobial peptides; dogs; keratinocytes; lipopolysaccharides; pseudomonas aeruginosa.

Figure 1

(A) Antimicrobial activity of cBD, cBD103, and cCath on PAO1. (B) Time-kill assay against Pseudomonas aeruginosa.

Figure 2

Inhibitory activity against pseudomonal biofilm formation of (A) cBD, (B) cBD103, and (C) cCath. Biofilm formation was evaluated by crystal violet staining. The absorbance was measured at 595 nm using a microplate absorbance reader. All experiments were performed independently in triplicate and analyzed using one-way analysis of variance with Tukey’s multiple comparisons. Results are expressed as mean ± standard deviation. *** p < 0.001.

Figure 3

Effects of (A) cBD, (B) cBD103, and (C) cCath on the viability of CPEK cells. Cell viability and proliferation were evaluated using an EZ-Cytox cell viability kit. Values are expressed as the mean ± standard deviation and were analyzed using one-way analysis of variance and Tukey’s multiple comparisons test in three independent experiments. *** p < 0.001.

Figure 4

Pseudomonas aeruginosa-induced cytotoxicity of CPEK cells. (A) Infectivity of P. aeruginosa in CPEK cells was established by inoculating PAO1 at multiples of infection of 0.1, 1, 10, or 100 for a designated time (2, 4, or 6 h). (B) The effect of antimicrobial peptides on P. aeruginosa-induced cytotoxicity on CPEK cells. Cell cytotoxicity was assessed using an EZ-LDH cell cytotoxicity assay kit. Results are expressed as mean ± standard deviation and were analyzed using one-way analysis of variance and Tukey’s multiple comparisons test in three independent experiments. ** p < 0.01.

Figure 5

Cytokine release following stimulation with lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa. The releases of (A) IL-6 and (B) TNF-α were quantified in the cell supernatants using an enzyme-linked immunosorbent assay. Antimicrobial peptides were pretreated 1 h before the LPS stimulation. Data are expressed as the mean ± standard deviation. Data were analyzed in three independent experiments conducted in triplicate using one-way analysis of variance with Tukey’s multiple comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001.