Image_3_Evaluation of phages and liposomes as combination therapy to counteract Pseudomonas aeruginosa infection in wild-type and CFTR-null models.tif

Multi drug resistant (MDR) bacteria are insensitive to the most common antibiotics currently in use. The spread of antibiotic-resistant bacteria, if not contained, will represent the main cause of death for humanity in 2050. The situation is even more worrying when considering patients with chronic bacterial infections, such as those with Cystic Fibrosis (CF). The development of alternative approaches is essential and novel therapies that combine exogenous and host-mediated antimicrobial action are promising. In this work, we demonstrate that asymmetric phosphatidylserine/phosphatidic acid (PS/PA) liposomes administrated both in prophylactic and therapeutic treatments, induced a reduction in the bacterial burden both in wild-type and cftr-loss-of-function (cftr-LOF) zebrafish embryos infected with Pseudomonas aeruginosa (Pa) PAO1 strain (PAO1). These effects are elicited through the enhancement of phagocytic activity of macrophages. Moreover, the combined use of liposomes and a phage-cocktail (CKΦ), already validated as a PAO1 “eater”, improves the antimicrobial effects of single treatments, and it is effective also against CKΦ-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CKΦ and PS/PA liposomes administrations in in vitro model of human bronchial epithelial cells, carrying the homozygous F508del-CFTR mutation, and in THP-1 cells differentiated into a macrophage-like phenotype with pharmacologically inhibited CFTR. Our results open the way to the development of novel pharmacological formulations composed of both phages and liposomes to counteract more efficiently the infections caused by Pa or other bacteria, especially in patients with chronic infections such those with CF.</p

Image_1_Evaluation of phages and liposomes as combination therapy to counteract Pseudomonas aeruginosa infection in wild-type and CFTR-null models.TIF

Multi drug resistant (MDR) bacteria are insensitive to the most common antibiotics currently in use. The spread of antibiotic-resistant bacteria, if not contained, will represent the main cause of death for humanity in 2050. The situation is even more worrying when considering patients with chronic bacterial infections, such as those with Cystic Fibrosis (CF). The development of alternative approaches is essential and novel therapies that combine exogenous and host-mediated antimicrobial action are promising. In this work, we demonstrate that asymmetric phosphatidylserine/phosphatidic acid (PS/PA) liposomes administrated both in prophylactic and therapeutic treatments, induced a reduction in the bacterial burden both in wild-type and cftr-loss-of-function (cftr-LOF) zebrafish embryos infected with Pseudomonas aeruginosa (Pa) PAO1 strain (PAO1). These effects are elicited through the enhancement of phagocytic activity of macrophages. Moreover, the combined use of liposomes and a phage-cocktail (CKΦ), already validated as a PAO1 “eater”, improves the antimicrobial effects of single treatments, and it is effective also against CKΦ-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CKΦ and PS/PA liposomes administrations in in vitro model of human bronchial epithelial cells, carrying the homozygous F508del-CFTR mutation, and in THP-1 cells differentiated into a macrophage-like phenotype with pharmacologically inhibited CFTR. Our results open the way to the development of novel pharmacological formulations composed of both phages and liposomes to counteract more efficiently the infections caused by Pa or other bacteria, especially in patients with chronic infections such those with CF.</p

Image_4_Evaluation of phages and liposomes as combination therapy to counteract Pseudomonas aeruginosa infection in wild-type and CFTR-null models.TIF

Multi drug resistant (MDR) bacteria are insensitive to the most common antibiotics currently in use. The spread of antibiotic-resistant bacteria, if not contained, will represent the main cause of death for humanity in 2050. The situation is even more worrying when considering patients with chronic bacterial infections, such as those with Cystic Fibrosis (CF). The development of alternative approaches is essential and novel therapies that combine exogenous and host-mediated antimicrobial action are promising. In this work, we demonstrate that asymmetric phosphatidylserine/phosphatidic acid (PS/PA) liposomes administrated both in prophylactic and therapeutic treatments, induced a reduction in the bacterial burden both in wild-type and cftr-loss-of-function (cftr-LOF) zebrafish embryos infected with Pseudomonas aeruginosa (Pa) PAO1 strain (PAO1). These effects are elicited through the enhancement of phagocytic activity of macrophages. Moreover, the combined use of liposomes and a phage-cocktail (CKΦ), already validated as a PAO1 “eater”, improves the antimicrobial effects of single treatments, and it is effective also against CKΦ-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CKΦ and PS/PA liposomes administrations in in vitro model of human bronchial epithelial cells, carrying the homozygous F508del-CFTR mutation, and in THP-1 cells differentiated into a macrophage-like phenotype with pharmacologically inhibited CFTR. Our results open the way to the development of novel pharmacological formulations composed of both phages and liposomes to counteract more efficiently the infections caused by Pa or other bacteria, especially in patients with chronic infections such those with CF.</p

Image_2_Evaluation of phages and liposomes as combination therapy to counteract Pseudomonas aeruginosa infection in wild-type and CFTR-null models.tif

Multi drug resistant (MDR) bacteria are insensitive to the most common antibiotics currently in use. The spread of antibiotic-resistant bacteria, if not contained, will represent the main cause of death for humanity in 2050. The situation is even more worrying when considering patients with chronic bacterial infections, such as those with Cystic Fibrosis (CF). The development of alternative approaches is essential and novel therapies that combine exogenous and host-mediated antimicrobial action are promising. In this work, we demonstrate that asymmetric phosphatidylserine/phosphatidic acid (PS/PA) liposomes administrated both in prophylactic and therapeutic treatments, induced a reduction in the bacterial burden both in wild-type and cftr-loss-of-function (cftr-LOF) zebrafish embryos infected with Pseudomonas aeruginosa (Pa) PAO1 strain (PAO1). These effects are elicited through the enhancement of phagocytic activity of macrophages. Moreover, the combined use of liposomes and a phage-cocktail (CKΦ), already validated as a PAO1 “eater”, improves the antimicrobial effects of single treatments, and it is effective also against CKΦ-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CKΦ and PS/PA liposomes administrations in in vitro model of human bronchial epithelial cells, carrying the homozygous F508del-CFTR mutation, and in THP-1 cells differentiated into a macrophage-like phenotype with pharmacologically inhibited CFTR. Our results open the way to the development of novel pharmacological formulations composed of both phages and liposomes to counteract more efficiently the infections caused by Pa or other bacteria, especially in patients with chronic infections such those with CF.</p

Reduction of IL-8 is associated with a relevant decrease of GBA2 expression in CF bronchial cells.

<p>IB3-1 (A), CuFi-1 (B) or CF primary bronchial cells (C) were transfected with GBA2 siRNA or scrambled oligonucleotides for 24 h and then infected with PAO1 (10–50 CFU/cell). IL-8 mRNA expression was measured as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g001" target="_blank">figure 1</a>. The data reported on the y-axis are relative to scrambled-treated cells (A, B and C) or scrambled-treated uninfected cells (D, E and F) and represent the mean ± SE of five (IB3-1, panels A and D), eight (CuFi-1, panels B and E) and four (CF primary bronchial, panels C and F) independent experiments performed in duplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

Miglustat and Genz-529648 inhibit GBA2 activity in IB3-1 and CuFi-1 cells infected by <i>P. aeruginosa</i>.

<p>IB3-1 and CuFi-1 cells were treated with [2 µM] miglustat, [10 nM] Genz-529648 or solvent alone for 1 hour prior to infection with heat-killed PAO1 for 4 hours. Total β–glucosidase (A), GBA1 (B) and GBA2 (C) activities were measured as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g002" target="_blank">figure 2</a>. The data reported are the mean ± standard error of the mean of 3 (IB3-1) or 2 (CuFi-1) independent experiments in triplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

GBA2 silencing reduces the IL-8 protein release in CuFi-1 cells.

<p>CuFi-1 cells were transfected with GBA2 siRNA or scrambled oligonucleotides and then infected with PAO1 as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104763#pone-0104763-g005" target="_blank">figure 5</a>. The supernatants were collected at the end of infection, and IL-8 protein release was measured as detailed in the “Methods” section. The data reported are the mean ± SE of eight independent experiments performed in duplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

GBA2-Encoded β-Glucosidase Activity Is Involved in the Inflammatory Response to <i>Pseudomonas aeruginosa</i>

<div><p>Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by <i>P. aeruginosa</i> and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by <i>P. aeruginosa</i>. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely <i>N</i>-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by <i>P. aeruginosa</i>. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2.</p></div

Infection with PAO1 increases β-glucosidase activity in IB3-1 and CuFi-1 cells.

<p>IB3-1 and CuFi-1 cells were infected with heat-killed PAO1 for 4 hours. The cells were then scraped and centrifuged; the cellular pellets were resuspended in water containing protease inhibitors and sonicated. Similar amounts of cellular proteins were used to perform the enzymatic assays to detect the activities of total β-glucosidase (A), GBA1 (B) and GBA2 (C), as reported in the Methods section. The data reported are the mean ± standard error of the mean of 4 (IB3-1) or 3 (CuFi-1) independent experiments in triplicate. Comparisons between groups were made by using Student’s <i>t</i> tests.</p

Inhibition of <i>P. aeruginosa</i> stimulated IL-8 mRNA expression by alkylated iminosugars in IB3-1 and CuFi-1 cells.

<p>Inhibition of <i>P. aeruginosa</i> stimulated IL-8 mRNA expression by alkylated iminosugars in IB3-1 and CuFi-1 cells.</p