3.1 PDE4 blockade prevents NETs release
To analyse NETs release in vitro , we stimulated neutrophils, adherent on fibrinogen/coated surfaces, with bacterial endotoxin fromEscherichia coli at 37°C, 5%CO2. NETs were visualized by confocal microscopy after DNA staining. After 4 hours of stimulation with endotoxin, most of the neutrophils showed de-condensation of their nuclei and DNA appeared widespread into the cytoplasm and outside the cell (Figure 1a) . Few extracellular DNA filaments were visible at this early time point. After 18 hours, macroscopic NETs structures (Figure 1a) were diffused among cells. In order to explore the potential role of CFTR in NETosis, we evaluated, in parallel, NETs formation in neutrophils untreated or exposed to CFTRinh-172, a selective CFTR inhibitor. As shown in Figure 1a, NETs formation was not apparently influenced by CFTR inhibition. For a more quantitative analysis, extracellular free-DNA was measured in supernatants. Released DNA was barely detectable after 4 hours in unstimulated cells and it was not significantly enhanced by the classical chemo-attractants fMLP and C5a. A substantial increment was instead observed in the presence of endotoxin or PMA (Figure 1b). A similar pattern was observed after 18 hours of incubation (Figure 1b). Free-DNA quantitation confirmed that CFTR inhibition did not significantly influence NETs release (Figure 1b).
Next, we examined the impact of PDE4 blockade on neutrophil morphology and DNA release using confocal microscopy and free-DNA quantitation. NETs structures were visualized by DNA and myeloperoxidase staining with DRAQ-5 and an FITC-conjugated anti-myeloperoxidase antibody, respectively. After 18 hours of incubation, DNA filaments decorated by myeloperoxidase were diffused among cells. Notably, rolipram (10 µM) preserved morphological integrity, nuclear DNA and cytoplasmic myeloperoxidase localization (Figure 2). Consistent with this, RNO (the active metabolite of roflumilast) concentration-dependently (0.1-1 µMol/L) reduced DNA release by neutrophils from healthy volunteers, being this effect more evident in the presence of CFTRinh-172 (Figure 3a). Similar measurements were carried out with neutrophils isolated from 7 patients with CF. As shown in Figure 3b, RNO, concentration-dependently reduced free-DNA release.
NETs formation requires the activation of Peptidylarginine deiminase 4 (PAD4) that converts arginine to citrulline on nuclear histones and promotes chromatin de-condensation. Thus, Histone H3 citrullination, may be considered a specific NETosis marker (Wang et al., 2009; Papayannopoulos et al., 2010; Thiam et al., 2020). To unequivocally confirm that reduction of free-DNA was a consequence of inhibition of NETosis, we assessed the presence of citrullinated Histone H3 in cell lysates from neutrophils isolated from healthy volunteers and people with CF, by Western blot analysis. Figures 3 shows that RNO reduced citrullination of Histone H3, demonstrating that PDE4 blockade controls biochemical events necessary for NETs formation, in healthy (Figure 3c) and CF (Figure 3d) neutrophils.