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 ).