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