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_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_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

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

MSU crystals enhance antimycobacterial activity.

<p>(<b>A</b>) Differentiated THP-1 (dTHP1) cells were infected with BCG at the MOI of 1 and then stimulated or not with 0.5, 5, 50 μg/ml of MSU for 3 and 5 days. The results are expressed as means ± Standard Deviation (SD) of CFU values performed in triplicate and are representative of three independent experiments. * p ≤ 0.001 in comparison with non-stimulated control cells. (<b>B</b>) Stimulation of human macrophages with MSU enhances phagocytosis of BCG. Differentiated THP-1 cells were exposed to BCG at the MOI of 1 for 3 hour in the presence or not of 0.05, 0.5, 5 μg/ml MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.05 in comparison with non-stimulated control cells.</p

Co-administration of BCG with MSU crystals enhances the clearance and efficacy of BCG vaccination.

<p>Five mice per group were vaccinated with i) 10^<b>6</b> CFU BCG, ii) 10^<b>6</b> CFU BCG+MSU [200 μg], or iii) PBS alone. (<b>A</b>) Mice were sacrificed after 15 days from immunization and BCG colonies enumerated by the draining axillary lymph nodes. Immunized and control mice were infected 10 weeks post immunization with <i>M</i>. <i>tuberculosis</i> Erdman (≈ 100 CFU/mouse) by the aerogenic route. Twenty-eight days later, mice were sacrificed and bacterial loads were determined by CFU counting in the lungs (<b>B</b>) and spleens (<b>C</b>). * p < 0.05 in comparison with BCG vaccinated mice.</p

MSU crystals promote maturation of phagosomes containing BCG.

<p>(<b>A</b>) dTHP-1 cells were infected with NHS labelled BCG at the MOI of 1 and then stimulated overnight with 5 μg/ml of MSU, in the presence or absence 10 μM Chloroquine. Results are expressed in terms of mean ± SD of arbitrary fluorescence units of triplicate values and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells, ° p < 0.001 in comparison with MSU stimulated cells. (<b>B</b>) dTHP-1 cells were infected with BCG, stimulated overnight with 5 μg/ml of MSU, in the presence or absence of 10 μM Chloroquine (Cq), and then labelled with 1 μM Lysosensor green DND 189. Results are expressed as mean ± SD of pH values from cultures performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells, ° p < 0.001 in comparison with BCG infected MSU stimulated cells (<b>C</b>) Protease activity of BCG infected dTHP-1 cells stimulated overnight with 5 μg/ml of MSU was analysed by loading of 10 μg/ml DQ red BSA for 2h at 37°C. Results are expressed as mean ± SD of triplicate values and are representative of three independent experiments. * p ≤ 0.01 in comparison with non-stimulated control cells. (<b>D</b>) Confocal microscopy representative images out of 10 per condition showing the increase of Auramine-stained BCG (green) residing in LAMP-3 positive vacuoles (red) after stimulation with 5 μg/ml of MSU. One representative experiment out of three is shown. (<b>E</b>) Summary of the mean percentage ± standard deviation (SD) of BCG co-localizing in LAMP-3-positive vacuoles determined by acquiring at least 10 images per condition and by counting ≥ 50 dTHP-1 cells per sample, after stimulation or not with MSU. Three different experiments were assessed. * p < 0.05 in comparison with BCG-infected cells (data were analyzed using the unpaired Student’s <i>t</i>-test).</p

MSU crystals induce trained anti-mycobacterial innate immunity.

<p>(<b>A</b>) Diagram showing the course of <i>in vitro</i> preincubation experiment. THP-1 cells were cultured with 5 or 50 μg/ml MSU for 3 days. Thereafter, the medium was changed to remove MSU stimulus and cells cultured for further 4 days. Finally, cells were exposed to BCG at the MOI of 10 for 3 hours (T0), washed and cultured for further 3 days (T3). (<b>B</b>) Intracellular mycobacterial growth was monitored in BCG infected THP-1 cells prestimulated or not with 5 or 50 μg/ml MSU. Results are shown as mean ± SD of CFU values performed in triplicate and are representative of three independent experiments. ° p < 0.05 and * p < 0.001 in comparison with same time non pre-stimulated control cells. (<b>C</b>) Intracellular mycobacterial growth was monitored in THP-1 cells, pre-stimulated or not with 50 μg/ml MSU, and exposed or not to 10 μM chloroquine after BCG infection for 3 days. Results are shown as mean ° SD of CFU values performed in triplicate. * p < 0.05 and ° p < 0.01 in comparison with same time non pre-stimulated control cells. (<b>D</b>) IL-1β production in the supernatant of BCG infected THP-1 cells pre-stimulated or not with either 5 or 50 μg/ml MSU. Results are shown as mean ± SD of values performed in triplicate and are representative of three independent experiments. * p < 0.05 in comparison with same time non pre-stimulated control cells.</p

MSU crystals reduce intracellular BCG viability in a phagolysosome maturation dependent and ROS mediated manner.

<p>BCG infected dTHP-1 cells were stimulated or not with 5 μg/ml MSU and CFU assays were performed at day 3 post-infection. (<b>A</b>) In order to ascertain whether phagolysosome maturation was responsible for intracellular mycobacterial killing, 10 μM chloroquine (Cq) or 20 mM NH₄Cl was added to BCG-infected cells together with MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells. (<b>B</b>) In order to ascertain the role of ROS in intracellular mycobacterial killing, 100 U/ml PEG-catalase was added to BCG infected cells together with MSU. Results are expressed as mean ± SD of CFU values performed in triplicate and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells.</p

MSU crystals induce phagolysosome dependent ROS generation.

<p>dTHP-1 cells were infected or not with BCG at the MOI of 5 and then labelled with 10 μM DCF for 60 min. Thereafter, cells were washed twice, stimulated or not overnight with 5 μg/ml MSU in the presence or absence of 10 μM Chloroquine (Cq). Results are expressed as means ± SD of arbitrary fluorescence units of triplicate values and are representative of two independent experiments. * p < 0.001 in comparison with non-stimulated control cells. ° p < 0.001 in comparison with MSU stimulated cells or with MSU-stimulated BCG-infected cells.</p