Figures
- FIGURE 1
Phagocytosis and antifungal activity for phagocytes taken from healthy and CF donors. For phagocytosis (a) and fungal germination (b), polymorphonuclear cells (PMN) and monocytes (MNC) were co-incubated with FLuorescent Aspergillus REporter (FLARE) conidia in a 1:3 ratio and imaged directly with confocal microscopy. The proportion of cells containing conidia was assessed 4 h post-inoculation. Error bars indicate sem for three healthy and three CF donors. For fungicidal activity (c), PMN (5×105 per mL), peripheral blood mononuclear cells (PBMC) and MNC (2.5×106 per mL) were stimulated with Aspergillus fumigatus conidia (2.5×106 per mL) for 18 h and fungal metabolic activity was assessed with an XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay. Error bars indicate sem for eight healthy and eight CF donors for PMN and PBMC and for four healthy and four CF donors for MNC. *: p<0.05 (Mann–Whitney U-test).
- FIGURE 2
Production of reactive oxygen species (ROS) by phagocytes from healthy and CF donors. Polymorphonuclear cells (PMN) (a), peripheral blood mononuclear cells (PBMC) (b) and monocytes (MNC) (c) (2.5×106 per mL) were stimulated with Aspergillus fumigatus conidia (5×107 per mL) and chemiluminescence was measured directly (as relative light units (RLUs)·s–1 over a 2-h period). The total ROS production (d) was calculated as the area under the curve (AUC). Error bars indicate sem for four-to-six healthy and four-to-eight CF donors. *: p<0.05 (Mann–Whitney U-test).
- FIGURE 3
Effect of inhibition of phagocytosis on the production of reactive oxygen species (ROS) against Aspergillus fumigatus. ROS production by polymorphonuclear cells (PMN) (a) and peripheral blood mononuclear cells (PBMC) (b) (2.5×106 per mL) is shown after pre-incubation with cytochalasin-B (Cyto-B) (5 µg·mL–1) and stimulation with Aspergillus fumigatus conidia (5×107 per mL). Error bars indicate sem for four healthy and four CF donors. RLU: relative light unit.
- FIGURE 4
Pre-treatment of phagocytes from healthy and CF donors with diphenyleneiodonium chloride (DPI). The effect of DPI pre-incubation on the fungicidal activity of peripheral blood mononuclear cells (PBMC) (a) (2.5×106 per mL) and polymorphonuclear cells (PMN) (b) (5×105 per mL) against Aspergillus fumigatus (2.5×106 per mL) was assessed by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) colorimetric assay. Error bars indicate sem for five healthy and five CF donors. *: p<0.05 (Mann–Whitney U-test).
- FIGURE 5
Specificity of production of reactive oxygen species (ROS) against Aspergillus fumigatus conidia by phagocytes from healthy and CF donors. Polymorphonuclear cells (PMN) (a) (2.5×106 per mL) and peripheral blood mononuclear cells (PBMC) (b) (2.5×106 per mL) were stimulated with A. fumigatus conidia (5×107 per mL), 2 µm latex beads (5×107 per mL), depleted zymosan (200 µg·mL–1) and lipopolysaccharide (LPS) (50 ng·mL–1). Total ROS production is shown as the area under the curve (AUC) over 2 h (mean±sem) for five healthy and three CF donors. *: p<0.05 (Mann–Whitney U-test).
- FIGURE 6
Effect of pre-treatment with lipopolysaccharide (LPS) or cystic fibrosis (CF) serum on the production of reactive oxygen species (ROS) by phagocytes from healthy and CF donors. ROS production by LPS pre-treated healthy and CF polymorphonuclear cells (PMN) (a) (2.5×106 per mL) and peripheral blood mononuclear cells (PBMC) (b) (2.5×106 per mL) was measured upon stimulation with Aspergillus fumigatus conidia (5×107 per mL), depleted zymosan (200 µg·mL–1) or 2 µm latex beads (5×107 per mL). Total ROS production, as calculated by the area under the curve (AUC) of relative light units (RLUs) by chemiluminescence over 1 h, is shown compared to non-primed controls. Error bars indicate ±sem for five healthy and three CF donors. Total ROS production by healthy PMN (c) (2.5×106 per mL) and PBMC (d) (2.5×106 per mL) incubated with 20% healthy or CF serum and stimulated with A. fumigatus conidia (5×107 per mL) or 2 µm latex beads (5×107 per mL) was also determined. Error bars indicate ±sem for five healthy donors.
- FIGURE 7
Effect on production of reactive oxygen species (ROS) by polymorphonuclear cells (PMN) from healthy and CF donors on treatment with different sizes of bead and Pseudomonas aeruginosa. PMN (2.5×106 per mL) were stimulated with beads of different sizes (0.5–6 µm; 5×107 per mL), Aspergillus fumigatus conidia and P. aeruginosa. Chemiluminescence was measured directly for 2 h and total ROS production was calculated as the area under the curve (AUC). Error bars indicate ±sem for nine healthy and 11 CF donors. RLU: relative light unit. *: p<0.05 (Mann–Whitney U-test).
- FIGURE 8
Correlation of excessive reactive oxygen species (ROS) production to clinical disease severity. ROS production against Aspergillus fumigatus by polymorphonuclear cells (PMN) from 13 cystic fibrosis (CF) patients (compared to PMN from healthy patients) was correlated with the number of exacerbations in the previous year (a). Analysis was performed using Spearman's rank correlation coefficient (Spearman's rho). In addition, the 13 CF patients were divided into two groups based on percentage predicted forced expiratory volume in 1 s (FEV1) scores and their ROS production was compared. *: p<0.05 (Mann–Whitney U-test).
Supplementary Materials
Supplementary Material
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Supplementary material 00068-2017_Supplementary
