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).