3.2 PDE4 blockade preserves neutrophil integrity and apoptosis
NETosis is accompanied by microvesiculation and fragmentation of netting neutrophils (Thiam et al., 2020). Images shown in Figure 2 are suggestive of protective effects by PDE4 inhibitor on neutrophil fragmentation. To obtain a more quantitative readout of this effect, we used flow cytometry. To this end, after 18 hours adhesion, neutrophils from normal volunteers were detached from fibrinogen-coated surfaces by brief exposure to 5 mMol/L EGTA/EDTA, permeabilized and stained with a FITC-conjugated anti-myeloperoxidase antibody. Figure 4 reports representative flow cytometric analyses showing that, compared to unstimulated samples (a), endotoxin stimulation (c),significantly decreased the percentage of intact neutrophils. Moreover, in the absence of endotoxin, 50% of the intact neutrophil population contained large amounts of intracellular myeloperoxidase (a), indicating non-degranulated cells. In contrast, in endotoxin-treated samples, the few neutrophils remaining intact appeared completely degranulated (c). No significant changes were detected in the presence of CFTRinh-172 (Figures 4 e and g, without or with endotoxin, respectively). PDE4 inhibition by RNO (100 nMoles/L) significantly increased the percentage of intact neutrophils as well as of non-degranulated cells in all experimental settings (Figure 4b, d, f and h, and Figure 5a). We also quantitated these effects in neutrophils from 7 donors with CF and consistently observed that RNO (100 nMoles/L) increased the percentage of intact neutrophils in endotoxin-stimulated samples (Figures 5 b). Since neutrophil apoptosis is key for the proper development of the resolution program of the inflammatory response and neutrophils from patients with CF manifest delayed apoptosis (McKeon et al., 2008; Moriceau et al., 2010; Gray et al., 2018), we asked whether preservation of neutrophil integrity by PDE4 inhibition had an impact on neutrophil apoptosis. As shown in Figures 5c and d, flow cytometric analysis of Annexin-V binding, revealed that approximately 80% endotoxin-stimulated normal (± CFTRinh-172) or CF neutrophils, remaining intact after 18 hours, displayed an apoptotic profile. Collectively, these results suggest that PDE4 inhibition controlling NETosis and preserving, at the same time, neutrophil apoptosis, may be useful to mitigate neutrophilic inflammation in CF. To further support this hypothesis, we conducted preclinical studies in a mouse model of bacterial lung inflammation.