FIGURE LEGENDS
Figure 1 . (a) Neutrophils isolated from healthy subject were allowed to adhere on fibrinogen-coated slides in the absence or presence of endotoxin (10 µg/ml) and cultured for 4 or 18 hours. At the end of the incubation, samples were fixed and stained for DNA (blue staining, DRAQ-5). (b) Neutrophils from healthy subjects pre-treated with CFTRInh-172 (10 µMoles/L) or vehicle were allowed to adhere on fibrinogen-coated surfaces and stimulated with different agonists for 4 or 18 hours. At the end of the incubation, free-DNA was quantitated. Briefly 2.5 µl of endonuclease (Nuclease micrococcal, from Staphylococcus aureus 50 U/ml) per 500 µl of sample was added and samples incubated at 37°C for 10 min. The reaction was stopped with 5 µl of EDTA (0.5 M). Samples were centrifuged at 10.000 x g for 3 min and supernatants stored at - 20°C until DNA quantification by Quant-iTTM dsDNA high-sensitivity assay kit (Invitrogen). Results are means ± SEM of experiments with cells from 4 different donors performed in duplicate.
Figure 2. Neutrophils isolated from healthy subjects were allowed to adhere on fibrinogen-coated slides in the presence of endotoxin, with or without rolipram (10 µMoles/L) and cultured for 18 hours. At the end of the incubation, samples were fixed and stained for DNA (blue staining, DRAQ-5), myeloperoxidase (green staining, FITC-conjugated anti-myeloperoxidase antibody) and F-actin (red staining, TRIC-phalloidin) and analysed by confocal microscopy. Figure shows images representative of 3 different experiments.
Figure 3 . (a) Neutrophils from healthy subjects, pre-treated for 2 min with increasing concentrations of RNO (0-1000 nMoles/L), were exposed to endotoxin and allowed to adhere on fibrinogen-coated surfaces for 18 hours in the presence or in the absence of CFTRinh-172 (10 µMoles/L). Unstimulated neutrophils, pre-treated with increasing concentrations of RNO (0-1000 nMoles/L), were incubated in parallel. At the end of the incubation, Free-DNA was quantitated.
Results are mean ± SEM of experiments performed with cells from 9-11 different donors in duplicate. *p < 0.05 (ANOVA, Dunnett test), RNO-treated vs untreated samples; ** p < 0.05 (Student T-test), CFTRinh-172-treated vs untreated samples. (b) Neutrophils, from individuals with CF, pre-treated with increasing concentrations of RNO (0-1000 nMoles/L), were incubated with endotoxin and allowed to adhere on fibrinogen-coated surfaces for 18 hours. Unstimulated neutrophils, pre-treated with increasing concentrations of RNO (0-1000 nMoles/L), were incubated in parallel. Results are mean ± SEM of experiments performed with cells from 7 different patients with CF (see Table 1 for patients’ characteristics). *p < 0.05 (ANOVA, Dunnett test) vs untreated samples. The presence of citrullinated Histone H3 in neutrophils from healthy donors (c) or people with CF (d) was analysed after 18 hours of incubation. Samples were then subjected to Western blot analysis using a monoclonal antibody which specifically recognizes citrullinated Histone H3. The figure shows results from one experiment representative of two.
Figure 4. Neutrophils from healthy subjects pre-treated with vehicle (a, c, e, g) or RNO (100 nMoles/L) (b, d, f, h), were exposed to endotoxin and allowed to adhere on fibrinogen coated surfaces for 18 hours in the absence (c, d) or in the presence (g, h) of CFTRinh-172 (10 µMoles/L). Unstimulated neutrophils were incubated in parallel in the absence (a,b) or in the presence (g, h) of CFTRinh-172 (10 µMoles/L). At the end of the incubation, samples were stained for intracellular myeloperoxidase and analysed by flow cytometry. Intact neutrophils were identified on the basis of typical SSC and FSC and analysed for myeloperoxidase content. Results are from one representative experiment.
Figure 5. Neutrophils isolated from healthy subjects (n = 16) (a, c) or from volunteers with CF (n = 7) (b, d) were treated with RNO (100 nMoles/L) (shaded bars) or vehicle (white bars) and allowed to adhere on fibrinogen-coated surfaces in the presence or absence of CFTRinh-172 (10 µMoles/L), for 18 hours with or without endotoxin. At the end of the incubation, the percentage of intact cells (a, b) and Annexin V binding related to intact cells (c, d) were evaluated by flow cytometry (see Figure 4). Intact neutrophils were identified by typical SSC and FSC. *p < 0.05 (Student T test) vs vehicle-treated samples.
Figure 6. C57BL/6 male mice (8 to 10 weeks of age) were infected i.t. with 1 x 106 CFUs of MDR-RP73 embedded in agar beads and per aerosol with roflumilast (5 mg/kg) or placebo (4,4% DMSO in saline) once a day starting from 4 hours post infection. Animals were sacrificed after 28 hours or 5 days of infection and BALF was collected. Total cells (a), neutrophils (b) and macrophages (c) were counted in BALF. Panels show box plots of cell numbers at the time of sacrifice (28 hours and 5 days after infection) of vehicle- (n = 9 per group) or roflumilast-treated mice (n = 9 sacrificed at 28 hours and n = 8 sacrificed at 5 days after infection). The horizontal lines mark the median of values, the edges of each box mark the 25th and 75th percentiles and the vertical lines indicate the highest and lowest values, respectively, which are not outliers (values greater than 1.5 times the length of the box were considered outliers and excluded from the analysis). *p < 0.05 (ANOVA, Dunnett’s test) vs vehicle-treated mice.
Figure 7. C57BL/6 male mice were treated as described in Figure 6 and sacrificed after 28 hours or 5 days of infection. BALF was collected for measurement of free DNA in supernatants and citrullinated Histone H3 in supernatants and in cell lysates. (a) shows box plots of free-DNA values at the time of sacrifice (28 hours and 5 days after infection) of vehicle- (n = 9 per group) or roflumilast-treated mice (n = 9 sacrificed at 28 hours and n = 8 sacrificed at 5 days after infection). *p < 0.05 (ANOVA, Dunnett’s test) vsvehicle-treated mice. The presence of citrullinated Histone H3 was analysed by Western blotting in BALF supernatants (b) as well as in lysates of BALF cells from mice sacrificed at 28 hours and 5 days after infection (c). Pools of BALF supernatants and of cell lysates from all animals of each group were subjected to Western blotting using a monoclonal antibody which recognizes mouse citrullinated Histone H3.
Figure 8. C57BL/6 male mice were treated as described in Figure 6. (a). Mice body weight was monitored daily before treatment to evaluate the health status. * p < 0.05 (ANOVA, Dunnett test)vs vehicle-treated mice. (b) shows box plots of weight loss values at the time of sacrifice (28 hours and 5 days after infection) of vehicle- or roflumilast-treated mice. * p < 0.05 (Student-t test) vs vehicle-treated mice.
Supplemental Figure 1. C57BL/6 male mice were treated as described in Figure 6 and sacrificed after 28 hours or 5 days of infection. BALF was collected and mouse lungs were recovered, homogenized and plated to determine the bacterial load. Box plots are shown.
Supplemental Figure 2. C57BL/6 male mice were treated as described in Figure 6 and sacrificed after 28 hours or 5 days of infection. BALF was collected and inflammatory cytokines (KC, TNF-α and MIP-2) were quantified in BALF supernatant by specific ELISA. Box plots are shown.
Supplemental Figure 3. Correlation analyses between inflammation markers and the body weight loss in mice subjected to chronic infection with 1 x 106 MDR-RP73. Analyses includes all animals treated with roflumilast or vehicle of both experimental protocols (per os and per aerosol ).