Introduction: Chronic lung infections sustained by Pseudomonas aeruginosa are commonly associated with a persistent inflammatory state, which represents the major process leading to substantial airway damage and reduced life expectancy. Antimicrobial peptides (AMPs) have been shown to profoundly modulate the host immune response, thus gaining increasing attention as novel therapeutic solutions to tackle uncontrolled inflammation during chronic infections. In the present study, we examined the anti-inflammatory potential of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K, which have previously demonstrated a significant antibacterial and antibiofilm activity in in vivo-like models of P. aeruginosa lung infections. Materials and methods: Human lung epithelial cells (A549) were exposed to P. aeruginosa lipopolysaccharide (LPS; 800 ng/mL) to induce an inflammatory response. The anti-inflammatory activity of lin-SB056-1 and (lin-SB056-1)2-K was evaluated by monitoring the production of pro-inflammatory cytokines (IL-8 and IL-6) through a bead-based flow cytometric assay. Lactate dehydrogenase assay and propidium iodide exclusion assay was performed to determine peptide cytotoxicity. LPS-neutralizing activity of the peptides was analysed by the Limulus amebocyte lysate (LAL) assay. Galleria mellonella was exploited to assess the in vivo protective effect of the peptides against P. aeruginosa infection. Results: Exposure of LPS-stimulated A549 cells to non-toxic concentrations of both lin-SB056-1 and (lin-SB056-1)2-K resulted in a dose-dependent decrease in cytokine release. The dendrimeric derivative demonstrated a stronger anti-inflammatory activity than its monomeric counterpart, reducing the levels of IL-8 by more than 3 times at 9.6 μM. Interestingly, the enhanced anti-inflammatory effect of (lin-SB056-1)2-K correlated with its higher ability to neutralize endotoxin as compared to lin-SB056-1. In particular, it showed almost 90% LPS-binding ability at the concentration of 9.6 μM. Although the dendrimeric peptide exerted a considerable antimicrobial and anti-inflammatory activity in vitro, it resulted to be poorly effective in protecting G. mellonella larvae from a lethal challenge with P. aeruginosa. Discussion and conclusions: In addition to the remarkable antibiofilm properties stated in previous studies, the anti-inflammatory potential of (lin-SB056-1)2-K might have a critical role in improving the clinical outcome of P. aeruginosa chronic lung infection. Despite these promising results, further studies aimed at enhancing peptide stability and bioavailability are warranted to solve the lack of protective effect observed in the G. mellonella systemic infection model.

Anti-inflammatory strategies to tackle Pseudomonas aeruginosa lung infections: exploring the potential of the antimicrobial peptide lin-SB056-1 and its dendrimeric derivative

Arianna Pompilio;Giovanni Di Bonaventura;
2019

Abstract

Introduction: Chronic lung infections sustained by Pseudomonas aeruginosa are commonly associated with a persistent inflammatory state, which represents the major process leading to substantial airway damage and reduced life expectancy. Antimicrobial peptides (AMPs) have been shown to profoundly modulate the host immune response, thus gaining increasing attention as novel therapeutic solutions to tackle uncontrolled inflammation during chronic infections. In the present study, we examined the anti-inflammatory potential of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K, which have previously demonstrated a significant antibacterial and antibiofilm activity in in vivo-like models of P. aeruginosa lung infections. Materials and methods: Human lung epithelial cells (A549) were exposed to P. aeruginosa lipopolysaccharide (LPS; 800 ng/mL) to induce an inflammatory response. The anti-inflammatory activity of lin-SB056-1 and (lin-SB056-1)2-K was evaluated by monitoring the production of pro-inflammatory cytokines (IL-8 and IL-6) through a bead-based flow cytometric assay. Lactate dehydrogenase assay and propidium iodide exclusion assay was performed to determine peptide cytotoxicity. LPS-neutralizing activity of the peptides was analysed by the Limulus amebocyte lysate (LAL) assay. Galleria mellonella was exploited to assess the in vivo protective effect of the peptides against P. aeruginosa infection. Results: Exposure of LPS-stimulated A549 cells to non-toxic concentrations of both lin-SB056-1 and (lin-SB056-1)2-K resulted in a dose-dependent decrease in cytokine release. The dendrimeric derivative demonstrated a stronger anti-inflammatory activity than its monomeric counterpart, reducing the levels of IL-8 by more than 3 times at 9.6 μM. Interestingly, the enhanced anti-inflammatory effect of (lin-SB056-1)2-K correlated with its higher ability to neutralize endotoxin as compared to lin-SB056-1. In particular, it showed almost 90% LPS-binding ability at the concentration of 9.6 μM. Although the dendrimeric peptide exerted a considerable antimicrobial and anti-inflammatory activity in vitro, it resulted to be poorly effective in protecting G. mellonella larvae from a lethal challenge with P. aeruginosa. Discussion and conclusions: In addition to the remarkable antibiofilm properties stated in previous studies, the anti-inflammatory potential of (lin-SB056-1)2-K might have a critical role in improving the clinical outcome of P. aeruginosa chronic lung infection. Despite these promising results, further studies aimed at enhancing peptide stability and bioavailability are warranted to solve the lack of protective effect observed in the G. mellonella systemic infection model.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11564/721806
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