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.