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.