3.3 Effect of PDE4 inhibition on lung inflammation and infection
in mice
First, the efficacy of oral administration of roflumilast was tested in
a murine model of Pseudomonas aeruginosa infection to mimic a
chronic lung infection similar to the one typically established in the
airways of people with CF. C57Bl/6NCrlBR mice were challenged with 1 x
106 MDR-RP73 embedded in agar beads by intratracheal
administration to induce chronic infection. Mice were treated with
roflumilast (5 mg/Kg) or vehicle (4,4% DMSO in saline) by gavage once a
day for five days, starting 2 hours before infection. Mice body weight
and health status were monitored daily. After 5 days of infection (2
hours after the last treatment), mice were sacrificed, BALF was
collected and analysed for total and differential cell count, protein
content, as markers of vascular permeability, and interleukin (IL)-1β,
tumour necrosis factor (TNF)-α and KC, the analogue of IL-8 in the
mouse, levels as indices of inflammation. The amount of free-DNA in the
BALF was analysed as an indirect measurement of NETs. In addition, since
treatments that impair neutrophil activities may potentially reduce
immune responses to bacterial infection, CFU were counted in BAL and in
homogenized lung tissue. The effects of oral administration of
roflumilast on bacterial load and inflammatory (cells and cytokines)
markers in BALF, are summarized in Table 2. No difference was observed
in lung CFU after 5 days of infection between roflumilast and
vehicle-treated animals. We observed a clear trend towards a reduction
of the inflammatory response in the roflumilast-treated group compared
to vehicle. Statistically significant differences were detected in total
cells and in the number of neutrophils, but no differences were observed
in the number of macrophages. Roflumilast treatment induced a
statistically significant reduction of TNF-α, while the reduction of KC
and IL-1β levels was not significant. Free-DNA and total protein content
appeared also reduced in the BALF of treated mice, but the differences
did not reach statistical significance. Based on these results, we
reasoned that, compared with the oral route, intratracheal
administration could yield higher drug concentration in the airways and
more effectively control neutrophil recruitment and activation. Three
doses of roflumilast (0.5, 1 or 5 mg/kg/day) were tested for toxicity in
non-infected animals. No evidence of side effects was recorded for all
doses (data not shown). Therefore, we explored the efficacy of aerosol
administration of roflumilast (5 mg/Kg/day for 5 days) in C57Bl/6NCrlBR
mice infected with Pseudomonas aeruginosa MDR-RP73 embedded in
agar beads. To exclude possible effects of the drug during the
initial phase of bacterial infection, the first treatment was started 4
hours post infection. For each group of treatment, half animals were
sacrificed 28 hours after the infection, 2 hours after the second
treatment, to analyse the effect of the drug in the acute phase, and the
remaining were sacrificed 5 days post infection, 2 hours after the last
treatment. At these time points, BALF and lung were collected for
analyses. We observed that the number of total cells, neutrophils and
macrophages, increased at 5 days respect to 28 hours, in the
vehicle-treated-group (Figures 6a, b and c), indicating persistence of
cell recruitment. Treatment with roflumilast, which did not modify
inflammatory cell in BALF at 28 hours of infection, significantly
reduced total cells and the number of neutrophils at 5 days post
infection compared to vehicle-treated animals (Figures 6a and b). On the
contrary, the macrophage count was not affected by roflumilast (Figure
6c). Moreover, roflumilast-treated mice showed significantly lower
free-DNA in BALF at 5 days, compared with vehicle-treated animals
(Figure 7a). To unequivocally confirm that the reduction of free-DNA was
a consequence of NETosis inhibition, we analysed the presence of
citrullinated Histone H3 in supernatants and lysates from neutrophils
recovered in the BALF. To this purpose, pools of supernatants and cell
lysates from all BALF of each group were subjected to Western blot
analysis of citrullinated Histone H3. As shown in Figures 7b-c,
citrullinated Histone H3, which was undetectable in samples collected
after 28 hours infection, increased, both in supernatants and inside the
cells in samples collected 5 days post infection. At this time point,
BALF supernatants and cells collected from animals treated with
roflumilast displayed reduced citrullinated Histone H3 compared to
vehicle-treated mice, conclusively demonstrating that PDE4 inhibition
controls DNA release in inflamed airways by blocking biochemical events
necessary for NETs formation. Measurements of body weight, as an index
of the general health status of infected animals, indicated a rapid
decrease in body weight in both groups of treatment. The group of mice
treated with roflumilast recovered weight more rapidly than the
vehicle-treated group. The improvement in recovery was statistically
significant 5 days after infection (Figure 8). The bacterial load in
BALF and lung at 28 hours and 5 days was not affected by roflumilast
treatment and decreased by approximately an order of magnitude 5 days
post infection, compared to the 28 hours time point, in all groups of
treatment (Supplemental Figure 1). Likewise, the amounts of KC, TNF-α
and MIP2 decreased at 5 days and were not significantly affected by
roflumilast (Supplemental Figure 2).
Correlation analyses between inflammation markers and the body weight
loss at 5 days post infection, including all animals treated with
roflumilast or vehicle of both experimental protocols (per os orper aerosol ) (Supplemental Figure 3), showed that the number of
neutrophils ( a) as well as the amount of free-DNA (b), KC (d)
and total proteins (f) in the BALF, positively correlated with weight
loss. In contrast, no or very weak correlations were found between the
number of macrophages (e), the amount of IL-1β (g), TNF-α (c) and weight
loss. Unexpectedly, bacterial load (CFU in homogenates of lung tissue),
did not correlate with weight loss (h).