**1. Cystic Fibrosis**

Cystic fibrosis (CF) is due to the mutation of the gene coding for the CFTR Chloride channel. Among more than 2000 mutations of the *cftr* gene, the most common (F508del) results in a protein folding defect, its retention in the endoplasmic reticulum (ER), and degradation by the proteasome [1]. CF affects many organs where the CFTR protein is normally expressed, however, the progressive lung destruction is the main cause of morbidity and mortality. In the healthy lung, mucociliary clearance, which requires optimal hydration of the airway surface liquid (ASL), propels inhaled particles, micro-organisms, and allergens towards the pharynx so that they can be expectorated and removed from the lungs, thus protecting from infection and inflammation. Mutations of *cftr* result in defective Cl<sup>−</sup> secretion and Na<sup>+</sup> hyperabsorption in airway epithelium. This ion transport dysfunction contributes to a reduction of the periciliary fluid volume and impairs mucociliary clearance, as seen in Figure 1, thus favoring bacterial colonization and sustained inflammation.

The focus on the function of CFTR in regulating epithelial ion transport and airway surface hydration provided a convincing explanation of the pathogenesis of airway disease in CF. The discovery in the CF epithelium of reduced ASL height and poor mucociliary clearance [2] was a significant step towards understanding this process in greater detail, as shown in Figure 1. However, the studies of altered ion and water transport alone failed to fully elucidate the mechanism by which the *cftr* gene mutation leads to pathogenesis in the CF lung. Furthermore, evidence began to emerge in the mid-1990s that airway inflammation begins much earlier than had previously been appreciated, even in children who are asymptomatic at the time of testing [3,4]. Overall, despite decades of research, the precise pathogenesis of the lung disease in CF remains not well understood.

**Figure 1.** Cystic fibrosis (CF) and non-CF airway epithelial transport. (**a**) In non-CF airway epithelium, the airway surface liquid (ASL light blue) layer height is finely regulated by transepithelial chloride secretion and sodium absorption that involve the activities of the CFTR chloride channel (green arrow through CFTR) and the epithelial sodium channel (grey arrow through ENaC), respectively. In this tissue, the CFTR protein is normally expressed and trafficked (ER: endoplasmic reticulum, G: Golgi) toward the apical membrane of epithelial cells to play a role as a chloride channel and to down regulate ENaC activity. (**b**) In the case of the most common CF mutation, the CFTR protein is retained in the ER and degraded by the proteasome (P). This results in altered chloride secretion trough CFTR and regulation of ENaC (red cross) and leads to an ASL height decrease that favors bacterial (green rectangle) and neutrophil (N) accumulation in the airway. (**c**) Classical hypothesis for CF airway pathogenesis showing the central role of dehydration/ altered mucociliary clearance (red) that results from ion transport abnormality.

#### **2. Persistent Inflammation in Cystic Fibrosis**

The acute inflammatory response is a normal host-protective response to contain foreign invaders. In CF, the inflammatory reaction is characterized by a predominant neutrophilic component and increased concentrations of pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-8, IL-17, IL-33, GM-CSF, G-CSF, and HMGB-1 [5]. An excessive airway inflammation is developed in early life of patients with CF and persists even in absence of infection [3,6,7].

Two main factors lead to sustained airway inflammation: an increased neutrophil recruitment and their defective elimination (for recent reviews Chmiel et al. [5,8]). Recurrent infections result in neutrophil over-recruitment in CF patients' airways. In addition, it has been proposed that the excess of pro-inflammatory mediators' production by airway epithelial cells, which triggers neutrophil recruitment, is related to a CFTR-dependent activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, as well as a decreased interferon-γ production [9–11]. Increased neutrophil count is also related to the defective mechanisms of elimination related to the altered airway mucociliary clearance resulting from CFTR loss of function and Na<sup>+</sup> hyper-absorption, as seen in Figure 1. Furthermore, neutrophils from CF patients are less sensitive to apoptosis and CF macrophages show a decreased phagocytic activity [12–15].

Neutrophils also present functional defects. The loss of bacteria-killing capacity of CF neutrophils contributes to the maintenance of infection and persistence of inflammation [16,17]. The altered efferocytosis and mucociliary clearance result in the necrosis of neutrophils in the airways. This results in the liberation of toxic and pro-inflammatory compounds and to the recruitment of new neutrophils, leading to the persistence of inflammation [18,19].

Overall, it does not appear that the excessive inflammatory response in CF results from altered triggering mechanisms but is mainly due to its inefficiency to contain infection and to abnormality in its resolution phase [5,8].

#### **3. Resolution of Inflammation**

The acute inflammatory response is normally self-limited with an active resolution phase designed to restore tissue homeostasis. The resolution of inflammation was initially thought to be a passive process and that inflammatory mediators from the initiation of the acute response (e.g., chemoattractants, complement components, prostaglandins, chemokines and cytokines) would simply dilute and dissipate to stop the infiltration of leukocytes into the tissues. However, studies performed by Serhan and colleagues on mouse inflammatory exudates revealed that acute inflammatory responses involve an active resolution phase carried out by specialized pro-resolving eicosanoid mediators (SPM) such as Lipoxins (LX) [20], resolvins (Rv) [21], protectins (PD) [22], and maresins (Mar) [23] that are locally produced from polyunsaturated fatty acids.

The temporal evolution of acute inflammation and its active resolution involves the sequential biosynthesis and activity of characteristic classes of an eicosanoid mediator in a process termed "class switching" [24]. Leukotriene B4 (LTB4) plays its role in amplification and propagation of inflammation [24] acting in concert with the peptide Interleukin 8 (IL8) as a potent neutrophil chemo-attractant [25,26]. Both LTB4 and IL8 are negatively correlated with pulmonary function in CF. Lipoxin A4 (LXA4) is the first eicosanoid mediator to be expressed in the active resolution phase and down-regulates neutrophil effector functions: limiting neutrophil chemotaxis, adherence and transmigration, and inhibiting superoxide anion and peroxynitrite generation [27]. Leukotrienes and lipoxins are closely related metabolites of arachidonic acid and can be synthesised from a common unstable intermediate [25]. They exhibit counter-regulatory effector functions and LXA4 inhibits LTB4 induced neutrophil transmigration in a dose dependent manner [28]. Along with lipoxins, other endogenous lipid mediators, including omega-3-derived resolvins, protectins, and maresins, stimulate and promote resolution of inflammation, clearance of microbes, tissue regeneration, and pain reduction, but do not evoke unwanted immunosuppression. SPMs are also involved in the regulation of adaptive immunity of B and T lymphocytes and can lower the amount of antibiotics needed to clear infections and reduce the potential for antibiotic resistance [29]. The abnormal process of resolution of inflammation is now considered a pathophysiologic basis associated with widely occurring diseases such as cardiovascular disease, neurodegenerative diseases, asthma, diabetes, and obesity, as well as arthritis, periodontal diseases, and cystic fibrosis.

#### **4. Abnormalities of Lipid Levels in Cystic Fibrosis**

Before the gene defect responsible for CF was identified, it was suggested that fatty acid metabolism abnormalities were responsible for the clinical symptoms of the CF disease. In 1962 Kuo et al. proposed that the disease was characterized by low linoleic acid (LA) concentrations in plasma and tissues [30]. Several groups also reported an important unbalance of unsaturated fatty acid with increased release of arachidonic acid (AA) and decreased levels of docosahexaenoic acid (DHA) in CF [31–33]. These lipid abnormalities were mainly explained by the fat malabsorption caused by the pancreatic insufficiency present in 85% of the patients [34,35]. In addition, CFTR dysfunction has been related to increased expression and activity of phospholipase A2 (PLA2) [36–41]. Since the release of AA by phospholipases is rate-limiting for the eicosanoid synthesis, an abnormal activity of PLA2 is a possible mechanism involved in increased AA levels in CF [42]. Increased oxidative stress, which is well documented in CF, has been also suggested as a cause of the fatty acid abnormalities seen in CF [43,44]. However, several studies were performed with supplementation of DHA (that should correct the unbalance between AA and DHA) to patients with CF, and failed to show significant clinical improvement [45–47].

A few groups have studied the levels of lipoxin, a metabolite of AA that could play a role in the pathophysiology of CF airway disease [48,49]. The mean content in LXA4, either absolute or adjusted to neutrophil count, did not significantly change in bronchoalveolar lavage (BAL) samples from patients with CF when compared to BAL samples from individuals without CF [48,50]. However, a significant decrease in LXA4 levels adjusted to neutrophil count was found in BAL from patients with CF when compared to BAL from patients with an airway inflammation but without CF [49]. Furthermore, a significant decrease in LXA4 levels adjusted to the pro-inflammatory cytokine IL8 (LXA4/IL8 ratio) was found in BAL samples of CF patients compared to control subjects [48,49]. In BAL and sputum samples from patients with CF, inter-individual changes in LXA4 content did not correlate with IL8 content [48]. The abnormality of eicosanoid class switching has been further investigated by analyzing LXA4/LTB4 ratios in BAL samples. In individuals without CF, LXA4/LTB4 ratio was related to infection, and was significantly higher in sterile samples than that measured in BAL samples from which pathogens were cultured. In contrast, in BAL samples from patients with CF, LXA4/LTB4 ratio did not vary with infection status and was uniformly depressed when compared with BAL from control children [48]. Consistent with reduced levels of DHA, RvD1/Il8 was also found to be decrease in sputa from CF patients, even in the absence of an acute infection or exacerbation [51]. Taken together, these data demonstrated that abnormalities in lipid metabolism, including SPM biosynthesis, characterize the CF airway disease.
