1 INTRODUCTION
Neutrophilic inflammation is the trademark of cystic fibrosis (CF) lung
disease (Tirouvanziam, 2006). Excessive and persistent accumulation of
neutrophils in the airways, associated with impaired bacterial clearance
and tissue damage, are early events in the life of patients with CF
(Davis and Ferkol, 2013; Sly et al., 2013). In CF airways, neutrophils
release proteases, mainly elastase that induces inflammatory responses,
impairs ciliary function in epithelial cells, disables CXCL8-induced
bacterial killing and causes bronchomalacia and bronchiectasis (Hartl et
al., 2007; Davis and Ferkol, 2013).
In addition, recruited neutrophils produce the so-called
neutrophil extracellular traps (NETs), consisting of a nuclear DNA
backbone decorated by granular enzymes that help to capture and kill,
extracellularly, invading bacteria (Brinkmann et al., 2004). However,
recent observations in preclinical and clinical CF models indicate that
the excessive accumulation of NETs in the airways plays a key
pathogenetic role in lung disease (Cheng and Palaniyar, 2013). Abundant
NETs can be found in the airways of people with CF and significantly
correlate with impaired lung function, suggesting that excessive
NETosis, in CF, may act as a double edge sword between host-defence and
auto-inflammation (Marcos et al., 2015). Indeed, more recent
observations show that Pseudomonas aeruginosa isolated from
patients with CF is resistant to the bactericidal activity of NETs
(Young et al., 2011). Moreover, excessive NETs formation may play a
pathogenetic role in vasculitis (Kessenbrock et al., 2009) and provide a
scaffold for platelet adhesion and thrombus formation (Fuchs et al.,
2010), thus mediating micro- and macrovascular occlusion.
The basic mechanisms of vital NETosis have been recently uncovered. Upon
appropriate stimulation of neutrophils, the nuclear envelop
disintegrates and allows mixing of chromatin with granular enzymes, such
as myeloperoxidase and elastase, which together with type IV
peptidyl-arginine deiminase (PAD), promote chromatin de-condensation
before extracellular release of large filament of DNA-enzymes complexes,
as NETs (Neeli et al., 2008; Wang et al., 2009; Papayannopoulos et al.,
2010; Thiam et al., 2020). Although the discovery of the process of
NETosis and of the underlying mechanisms is relatively recent, its
pharmacological modulation remains largely unknown. While PAD4
inhibitors, such as GSK484 (Mondal and Thompson, 2019), BMS-P5 (Li et
al., 2020) or the more historic Cl-amidine, are described in the
literature they remain in a non-clinical stage so far. Thus, current
therapies for CF lack of specific approaches to tackle excessive
NETosis. Type 4 phosphodiesterases (PDE4), the major isoform of PDEs
expressed by myeloid cells, control a variety of inflammatory mechanisms
in immune cells. In neutrophils, PDE4 are key mediators of cAMP
degradation and, as a downstream effect, of neutrophil adhesion and
migration, cytokine and chemokine release, synthesis of lipid mediators
and of reactive oxygen species (Sanz et al., 2005). In several animal
models, genetic deficiency of PDE4 reduces neutrophilic inflammation
(Jin and Conti, 2002; Ariga et al., 2004; Jin et al., 2005). In
agreement with genetic ablation, pharmacological blockade of PDE4
reduces leukocyte recruitment at the site of inflammation (Sanz et al.,
2002, 2007). Moreover, it was reported that PDE4 blockade
promotes neutrophil apoptosis thus driving resolution of inflammation
(Sousa et al., 2010). From a mechanistic point of view, we have recently
discovered that selective blockade of PDE4 in human neutrophils
down-regulates Src family kinase activities (SFK), through
protein kinase A (PKA)-mediated activation of COOH-terminal Src Kinase
(CSK), a major endogenous regulator of SFK (Totani et al., 2014).
Through these mechanisms, roflumilast, an oral selective PDE4 inhibitor
approved for clinical use in patients with severe chronic obstructive
pulmonary disease, prevents the release of NETs from neutrophils
adherent on fibrinogen and challenged with bacterial endotoxin (Totani
et al., 2016). Here, we tested the hypothesis that PDE4 inhibitors may
control NETosis in CF. The efficacy of PDE4 blockade was exploredin vitro, using CF neutrophils, and in vivo, in a mouse
model of Pseudomonas aeruginosa chronic lung infection.
2 METHODS