Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: Dysfunctional Pseudomonas aeruginosa uptake in macrophages with pharmacologically inhibited or naturally mutated cystic fibrosis transmembrane conductance regulator channel (CFTR) and its enhancement by apoptotic body-like liposome/phosphatidylinositol 5-phosphate (ABL/PI5P) stimulation. (A) Monocyte-derived macrophages (MDMs) from healthy donors, treated or not with INH172, or from cystic fibrosis (CF) patients were infected with multidrug-resistant (MDR) P. aeruginosa (BAA-2113 strain) at an MOI of 30. (B, C) CF MDMs were stimulated or not with ABL/PI5P for 30 min before infection (B) or before and during infection (C) . Cells were then infected with MDR P. aeruginosa (BAA-2113 strain) at an MOI of 30. The bacterial uptake was quantified by colony-forming unit (CFU) assay and indicated as phagocytosis index, calculated as the ratio between the CFUs obtained immediately after the infection and the inoculum. (A) Statistical analysis was performed by using the two-sided Mann–Whitney test and *p < 0.05; **p < 0.01 in comparison with control cells (healthy donors, n = 6; CF patients, n = 6). (B, C) Statistical analysis was performed by using the two-sided Wilcoxon rank sum test ( B , n = 6) p = 0.03 and ( C , n = 9) p = 0.004.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Derivative Assay, Infection, Colony-forming Unit Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: Treatment with ABL/PI5P rescues impaired phagosome acidification and ROS production in MDM with pharmacologically inhibited CFTR. Primary MDMs, treated or not with INH172, were exposed for 1 h to 1-µm microbeads (Mb). Phagosomal pH (A) and ROS (B) were assessed, by staining with Lysosensor green DND 189 or DCF, after 90 min of stimulation with ABL/PI5P, respectively. Results are shown as mean + standard deviation of the values obtained from triplicate cultures and are representative of experiments with cells by two different donors. *p < 0.05; **p < 0.01, in comparison with INH172-unstimulated cells and in comparison with untreated cells by one-sided Student’s t test.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Staining, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: ABL/PI5P promotes both ROS and phagolysosome acidification-dependent intracellular P. aeruginosa killing in MDM with pharmacologically inhibited CFTR. (A) Primary MDMs were exposed to the CFTR inhibitor INH172 at a concentration of 10 µM, infected with MDR P. aeruginosa (BAA-2113 strain) and then stimulated for further 2 h with ABL/PI5P. (B) Primary MDMs were exposed to the CFTR inhibitor INH172 at a concentration of 10 µM, infected with MDR P. aeruginosa (BAA-2113 strain), and then stimulated for a further 2 h with ABL/PI5P in the presence or absence of catalase (PEG-Cat) or Concanamycin A (Conc A), at a concentration of 100 U/ml or 10 nM, respectively. Bacterial growth was assessed by CFU assay, and replication index was calculated as the ratio between the CFU obtained after 2 h of infection, in the presence or absence of ABL/PI5P, and the CFU was obtained before the addition of liposomes. The results are shown as mean + standard deviation of the values obtained from triplicate of each condition. **p < 0.01; ****p < 0.0001 by two-sided Student’s test.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Concentration Assay, Infection, Colony-forming Unit Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: ABL/PI5P enhances intracellular bacterial killing in CF macrophages characterized by impaired antimicrobial activity. MDM isolated from CF patients (n = 12) were infected with MDR P. aeruginosa (BAA-2113 strain) and then stimulated for another 2 h with ABL/PI5P. Bacterial growth was assessed by CFU assay, and replication index was calculated as the ratio between the CFU obtained after 2 h of infection in the presence or absence of ABL/PI5P and the CFU obtained before the addition of liposomes. (A) CF patients have been defined as “functional” or “controller” on the basis of bacterial replication index, less or higher than 1, respectively. Bacterial replication index is shown in “controller” ( B , n = 6) and “impaired” ( C , n = 6) macrophages from CF patients following ABL/PI5P stimulation. Statistical analysis was performed by using the two-sided Mann–Whitney test (A) and two-sided Wilcoxon matched-pairs signed rank test (B, C) . (A) p = 0.0022; (B) p = not significant; (C) p = 0.0313.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Activity Assay, Isolation, Infection, Colony-forming Unit Assay, Functional Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: ABL/PI5P stimulation modulates NF-κB and cytokine production in MDM with pharmacologically inhibited CFTR. MDMs were treated or not with INH172, infected or not with MDR P. aeruginosa (Pa, BAA-2113 strain), and then stimulated or not with ABL/PI5P for 2 h. Thereafter, cells were lysed (A) or supernatants were collected (B–F) , and both were stored at −20°C until analysis. (A) Cell lysates were analyzed by NF-κB p65 (Total/Phospho) Human InstantOne™ ELISA kit, and results are shown as the ratio between phosphorylated and total NF-κB p65. The production of TNF-α (B) , IL-1β (C) , IL-6 (D) , IL-10 (E) , and TGF-β (F) was analyzed by ELISA. The results are shown as mean + standard deviation of the values obtained from triplicate of each conditions and are representative of experiments with cells from at least three different donors. *p < 0.05; **p < 0.01; ***p < 0.001 one-sided t test.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: ABL/PI5P treatment modulates KC and MIP-2 production in a murine model of MDR P. aeruginosa chronic lung infection. C57Bl/6NCrlBR mice were infected with MDR P. aeruginosa (RP73 strain) and then treated with ABL/PI5P (n = 16) or vehicle (n = 16), as described in the Material and Methods section. At day 6 post-infection, mice were sacrificed. Levels of KC (A) , MIP-2 (B) , TNF-α (C) , and JE (D) upon treatment with ABL/PI5P or vehicle in the supernatants of lung homogenates were measured by ELISA. Data are shown as mean values + standard error. The data are pooled from two independent experiments. Statistical analysis was performed by using the two-sided Mann–Whitney test. Statistical significance is indicated: *p < 0.05. Outlier data, identified by Grubbs’ test, were excluded by the analysis.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

doi: 10.3389/fimmu.2020.532225

Figure Lengend Snippet: ABL/PI5P treatment reduces neutrophilic recruitment in a murine model of MDR P. aeruginosa chronic lung infection. C57Bl/6NCrlBR mice were infected with MDR P. aeruginosa (RP73 strain) and then treated with ABL/PI5P (n = 16) or vehicle (n = 16), as described in the Materials and Methods section. At day 6 postinfection, mice were sacrificed, BALF was collected, and lungs were excised, homogenized, and plated onto TSA to determine bacterial burden. Counts of total number of cells (A) , neutrophils (B) , and macrophages (C) were performed in BALF. (D) The bacterial burden and was assessed by CFU assay. Data are shown as mean values + standard error. The data are pooled from two independent experiments. Statistical analysis was performed by using two-sided Mann–Whitney test. Statistical significance is indicated: *p < 0.05. Outlier data, identified by Grubbs’ test, were excluded by the analysis.

Article Snippet: Thus, we tested the efficacy of ABL/PI5P administrated by Penn-Century MicroSprayer ® Aerosoliser in mice, 5 min after infection with MDR P. aeruginosa embedded in agar beads.

Techniques: Infection, Colony-forming Unit Assay, MANN-WHITNEY