Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance

Abstract

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

Figure 1. ABLs carrying selected bioactive lipids facilitate phagolysosome maturation.

dTHP1 cells were uninfected (a) or infected (b) with BCG at the MOI of 5, treated with ABLs carrying the indicated bioactive lipid for 18 hours and then stained with Lysosensor green DND189 for pH determination. Results are shown as mean ± standard deviation of the values obtained from the triplicate of each condition and are representative of three separate experiments. *p < 0.001 in comparison with untreated control by one sided Student’s t Test. (c) BCG was labeled with 100 μg/ml of the pH sensitive dye NHS and used to infect dTHP1 cells (MOI 5). Infected cells were then treated with ABLs carrying the indicated bioactive lipid. The fluorescence expressed in relative fluorescence units is directly proportional to intraphagosomal pH. Results are shown as mean ± standard deviation of the values obtained from the triplicate of each condition and are representative of three separate experiments. *p < 0.001 in comparison with untreated control by one sided Student’s t test.

Figure 2. ABLs carrying selected bioactive lipids promote ROS production.

Uninfected (a–c) or BCG infected (MOI 5) dTHP1 cells (d–f) were labeled with DCF at a concentration of 10 μM and subsequently treated with ABLs carrying the indicated bioactive lipid for 20 (a,d), 40 minutes (b,e) or 18 hours (c,f). The intensity of the fluorescence of DCF is proportional to the amount of ROS produced. The results are shown as mean ± standard deviation of relative fluorescence units performed in triplicates and are representative of 3 separate experiments. *p < 0.0001 in comparison with untreated control (a), or with BCG infected untreated control (f) by one sided Student’s t test.

Figure 3. ABLs promote intracellular mycobacterial killing by a ROS dependent mechanism.

dTHP1 cells were infected with BCG at the MOI of 5 and then treated for 3 days with ABLs carrying PA, PI3P, or PI5P in the presence or absence of 100 U/ml PEG-Cat. Results are expressed as mean ± standard deviation of CFU values performed in triplicate and are representative of two independent experiments. *p < 0.01 and **p < 0.0001 by one sided Student’s t test.

Figure 4. ABLs carrying PA or PI5P promote intracellular bacterial killing in primary macrophages expressing a pharmacologically inhibited CFTR.

Monocyte derived macrophages (MDM) isolated from healthy subjects (n = 8) were infected with P. aeruginosa, as described in materials and methods, and then treated for 2 hours with the ABLs carrying PA, PI3P or PI5P in the absence (a–c) or in the presence (d–f) or of the CFTR inhibitor INH172. Bacterial growth was assessed by CFU assay. The results shown are obtained from triplicate for each condition. Statistical analysis was performed by using two sided Wilcoxon matched-pairs signed rank test and p value is indicated in the single panels.

Figure 5. ABLs carrying PA or PI5P induce ROS dependent intracellular killing of P. aeruginosa.

Primary monocyte derived macrophages (10⁶ cells/ml) were exposed to the CFTR inhibitor INH172 at a concentration of 10 μM and infected with P. aeruginosa, as described in materials and methods, and then treated for further 2 hours with the indicated ABLs in the presence or absence of catalase (PEG-Cat), used at the concentration of 100 U/ml. Bacterial growth was assessed by CFU assay. The results are shown as mean ± standard deviation of the values obtained from the triplicate of each condition and are representative of two separate experiments. *p < 0.0001 by one sided Student’s t test.

Figure 6. ABL carrying PA or PI5P promote intracellular bacterial killing in CF macrophages.

Monocyte derived macrophages (10⁶ cells/ml), isolated from CF patients (n = 8), were infected with P. aeruginosa, as described in materials and methods, and then treated for further 2 hours with ABL/PA (a), ABL/PI3P (b), or ABL/PI5P (c). Bacterial growth was assessed by CFU assay. Survival is expressed as a percentage of bacterial survival and was calculated as the ratio between the CFU obtained after two hours of infection in the presence or absence of the indicated ABLs and the CFU obtained at time 0, before the addition of the ABLs. Statistical analysis was performed by using two sided Wilcoxon matched-pairs signed rank test and p value is indicated in the single panels.

Figure 7. The microbicidal function of BAL cells from bacterial pneumonia patients is enhanced by ABLs.

Bronchoalveolar lavage cells, from 6 patients with bacterial pneumonia, were treated for 18 hours with the indicated ABLs. Intracellular bacterial growth was evaluated by CFU. The number above each column indicates the percentage of killing is indicated above each column and was calculated by using the following formula: (1- CFU sample treated with ABLs/CFU untreated control) x100. The results are shown as mean ± standard deviation of the values obtained from the triplicate of each condition. ^○p < 0.05, ^●p < 0.001, and *p < 0.0001 in comparison with untreated cells by one sided Student’s t test.