4 DISCUSSION
The mechanisms of neutrophilic inflammation in CF lung disease remain to
be fully understood and no established pharmacological treatments are
currently available to control this process. However, recent studies
strongly suggest that NETs are main determinants of lung inflammation
and damage in CF. Therefore, identifying therapies that preserve the
positive effects of neutrophils, while reducing the detrimental effects
of NETs and cytotoxic components, are essential for achieving innovative
therapeutic advances (Khan et al., 2019). Here, we provide evidence that
PDE4 inhibition prevents NETosis in, in vitro and in vivo,CF relevant models.
In this work, we tested the selective PDE4 inhibitor roflumilast, mainly
because this drug has been tested in phase II and III clinical trials
(Rabe, 2011) and approved as adjuvant to reduce the risk of exacerbation
in patients with severe COPD (Schudt et al., 2011). Like CF, COPD is, in
fact, characterized by excessive neutrophilic lung infiltration as well
as by an imbalance in the oxidant/antioxidant and protease/antiprotease
equilibrium, two neutrophil-mediated processes, regarded as major
determinants of the progressive lung damage in both diseases. PDE4
blockade in lung immune cells prevents the progression of inflammation
in COPD (Rabe, 2011; Baye, 2012). Moreover, roflumilast reduced
neutrophil and eosinophil accumulation in BAL of healthy volunteers
subjected to segmental administration of endotoxin (Hohlfeld et al.,
2008). Further indication for the clinical use of PDE4 inhibitors in CF
derives from the evidence that PDE4 inhibition rescues CFTR activity in
varying experimental settings (Blanchard et al., 2014).
RNO reduced NETs release by neutrophils in vitro (Figures 1-3).
This was clearly demonstrated by combining confocal microscopy with
measurements of free- DNA and citrullinated Histone H3. These analyses
were conducted on neutrophils from healthy donors, exposed or not to
CFTRinh-172 to mimic a CF status, and on neutrophils from donors with CF
with different CFTR mutations and disease severity (Table1). The overall
picture emerging from these experiments shows that while CFTR blockade
did not modify the entity of NETs release, RNO was significantly more
potent at inhibiting NETosis in CFTRinh-172-treated normal neutrophils
as well as in CF neutrophils, compared to untreated normal neutrophils.
While confirming previous data from our and other’s laboratory
(Shishikura et al., 2016; Totani et al., 2016), the present results
provide the novel observation that PDE4 blockade is more efficient at
reducing NETs under CF conditions, suggesting that impairment of CFTR
function induces signalling events that favour the activity of PDE4
inhibitor.
In addition to reducing NETs, RNO preserved neutrophil integrity as well
as the stability of MPO-containing intracellular granules (Figure 4).
These are key events in neutrophil-driven inflammation, since the
release in the airways of the content of proteolytic enzymes, stored in
neutrophil granules, sustains CF inflammation and lung damage. Along
these lines, the observation that RNO preserves both neutrophil
integrity and the apoptotic process, further supports the hypothesis
that PDE4 inhibition may sustain the resolution program of inflammation,
which requires a discrete neutrophil apoptosis to be preserved. A recent
work links NETosis with a delayed neutrophil apoptosis in CF and
suggests that promotion of apoptosis may allow more appropriate
neutrophil disposal, decreasing NETs formation and thus inflammation
(Gray et al., 2018). The existence of a functional link between the two
processes, NETosis and apoptosis, is also supported by the results of
Remijsen et al. (Remijsen et al., 2011) showing that inhibition of
either autophagy or NADPH oxidase activity, which are essential for NETs
formation, results in cell death characterized by hallmarks of
apoptosis, suggesting that switching-on apoptosis program might function
as a stop signal for NETosis and vice versa .QQD In keeping with
this, our results in in vitro models demonstrate that PDE4
blockade reduces NETosis while preserves apoptosis and this is
particularly relevant in CF neutrophils. Biochemical pathways mediated
by cAMP-activated PKA and regulating kinases belonging to the src family
as well as PI3K/Akt pathway appears to play a relevant role in this
effect (Sousa et al., 2010; Totani et al., 2014, 2016).
In vitro data were confirmed in a preclinical mouse model of respiratory
infection by Pseudomonas aeruginosa , which often colonizes the
airways of patients with CF. We used two different routes of roflumilast
administration, by gavage or intratracheal, and monitored markers of
neutrophilic inflammation at short (28 hours) and extended time (5
days). The results showed that while these markers were barely affected
by the drug given per os , the aerosol administration was
effective in reducing the accumulation of neutrophils at 5 days but not
after 28 hours of infection. Accordingly, roflumilast-treated mice
showed a significant reduction in the accumulation of free-DNA in BALF
at 5 days, as well as reduced citrullination of Histone H3, both in BALF
supernatants and cells, thus conclusively demonstrating that PDE4
inhibition controls key biochemical steps necessary for NETs formationin vivo . Of note, roflumilast did not modify DNA release after 28
hours of infection and at this early time point we did not observe
measurable amounts of citrullinated Histone H3. Although we are unable
to provide a clear explanation for this finding, we may hypothesize that
at this early time point: i) DNA release is not a result of NETosis; ii)
PAD4-independent mechanisms may be responsible for NETosis. Further
studies are necessary to clarify this point.
In this model, roflumilast effects on neutrophil functions correlated
with a more rapid weight recovery, which reached statistical
significance at 5 days of infection (Figure 8a). The hypothesis that
roflumilast improves the animal wellbeing by modulating neutrophil
recruitment and function was supported by the observation of a direct
correlation between neutrophil number and free-DNA with body weight
(Supplemental Figure 3). On the contrary, the bacterial load, which was
not appreciably modified by roflumilast, was not influent.
Consistent with this scenario, free-DNA accumulated in BALF ofPseudomonas aeruginosa -infected mice, positively correlated with
neutrophil counts. In this condition, free-DNA may be largely
represented by NETs, as indicated by Histone H3 data. Thus, NETs release
appears to represent a neutrophil function mainly involved in the
pathogenesis of lung inflammation during Pseudomonas aeruginosainfection. This interpretation is consistent with recent evidence that
free-DNA, abundant in CF sputum, shows NETs characteristics (Dwyer et
al., 2014). Moreover, in the airways of people with CF, NETs components,
such as elastase and others granule proteins, perpetuate lung damage and
inflammation (Marcos et al., 2015; Dittrich et al., 2018), while
decondensed chromatin, the main structure of NETs, increases the
viscosity of endobronchial secretions, further hampering muco-ciliary
clearance. Clinical observations also confirm the pathogenetic role of
NETs in CF, by revealing a positive correlation between the impairment
of respiratory function and the level of free-DNA or elastase in CF
airways, or the level of MPO and antibodies to PAD4 in circulating blood
(Marcos et al., 2015; Dittrich et al., 2018; Yadav et al., 2019).
In summary our study describes a series of pharmacological activities
associated with PDE4 blockade in neutrophils, which could be beneficial
in a CF clinical setting. Our data, showing that PDE4 inhibitors may
sustain a local signal driving neutrophilic inflammation towards
physiological resolution, indicate that PDE4 may be a potential novel
target to promote a ”correction” of neutrophilic inflammation, rather
than a complete suppression, which could be detrimental. As PDE4
inhibitors have been recently approved for clinical use in COPD
(Rabe, 2011; Wedzicha et al.,
2016) and psoriasis (Rich et al., 2016), our present results encourage
further research to validate the use of these drugs in patients with CF.