**5. Cellular Mechanisms Involved in the Defective Production of SPM in Cystic Fibrosis**

To date, approximately 30 kinds of SPMs have been found in inflammatory exudates: lipoxins, D-series and E-series resolvins, protectins, and maresins. They are produced at inflammatory sites from essential fatty acids (AA, DHA and eicosapentaenoic acid (EPA)) by the interaction of lipoxygenase (LO) activities within several cell types including leukocytes, platelets and epithelium, as seen in Figure 2 [25,52]. The level of expression of 15LO in airway epithelial cells and macrophages and the levels of 12LO in platelets play a central role in the eicosanoid class-switching from leukotrienes B4 (LTB4), a pro-inflammatory lipid mediator, toward SPMs. Indeed, the activity of 15LO favors LXA4 (pro-resolving) synthesis at the expense of LTB4 (pro-inflammatory) synthesis that involves leukotriene A4 hydrolase (LTA4H) activity (Figure 2) [25]. Cellular location of 5LO also plays a crucial role in LTB4/SPM equilibrium. Inhibition of the CaM kinases by RvD1 favors extra-nuclear location of 5LO, and LXA4 biosynthesis at the expense of LTB4 [53]. A reduced expression of 12/15LO in the airway of a CF mouse model as well as a reduced 15LO2 level in the BAL of patients with F508del mutation was described [48,49]. A defective 12LO activity has been reported in platelets during CFTR inhibition [54]. The 15LO expression was shown to be decreased by 50% in nasal epithelium of patients with CF compared to non-CF [55]. The 5LO was initially thought to be restricted to leucocytes, but is also expressed in human bronchial epithelial cells [56]. The cellular mechanism by which CFTR could affect 5LO, 12LO, 15LO, 15LO2 and/or LTA4H level of expression, cellular localization and activities in CF airways remains unclear. However, several data suggested that the abnormal SPM production in CF is related to abnormal LO expression and/or activity in cells that fail to normally express CFTR, including CF airway epithelium and platelets [48,49,54,55].

**Figure 2.** Schematic view of specialized pro-resolving eicosanoid mediators (SPM) biosynthesis pathways. Lipoxin and resolvin result from lipoxygenases interactions. Transcellular biosynthesis can occur via the interactions of two or more cell types expressing different enzymes. Epithelial cells and macrophages express the 15-lipoxygenase (15LO), that metabolizes docosahexaenoic acid (DHA) and arachidonic acid (AA) to produce 17(S)-HpDHA and 15-Hydroxyeicosatetraenoic acid (15(S)-HETE), respectively. These products of 15LO activity are the substrates for the 5LO expressed in neutrophils, to synthetize resolvin and lipoxin (pro-resolving mediators). The 5LO can also metabolize AA into leukotriene A4 (LTA4), which can be substrate of both, the 15LO to produce lipoxin or the LTA4H to produce leukotriene B4 (pro-inflammatory mediator). The pro-resolving pathways are illustrated in green, the pro-inflammatory pathway in red, and enzyme/product/substrate involved in both pathways in brown. The precise mechanism by which CFTR could affect LO activity is not known (green dotted arrow).

#### **6. SPM Impact on CF Airway Epithelial Transport and ASL Height**

In CF, the decreased levels in ASL height has been reported to be mainly due to a defect in trafficking and function of CFTR as a Cl− channel as well as its impact on ENaC regulation leading to an increase in Na+ absorption (Figure 2).

The calcium-activated chloride channel TMEM16A (Transmembrane member 16A) and the SLC26A9 (Solute Carrier Family 26 Member 9) are two other anion channels that provide the critical regulation of mucus hydration of airway epithelium [57]. Both TMEM16A and SLC26A9 activities which attenuate airway inflammation and prevent mucus obstruction during airway inflammation have been considered as alternative therapeutic targets to bypass CFTR dysfunction in the airway epithelia of CF patients However, strategies for increasing intracellular calcium concentration to stimulate calcium-activated chloride secretion have been plagued by the amplification of the calcium dependent pro-inflammatory response [58,59]. Therefore, research for pharmacological long-lasting stimulation of TMEM16A independent of intracellular Ca2+ has been intensified [60]. The SLC26A9, a member of the solute carrier 26 family of anion transporters was shown to act as a modifier of CF lung disease severity and the response to CFTR modulator therapy [61,62]. An alternative strategy to deliver artificial non-toxic Cl− transporters to CF epithelia have emerged [60].

On another side, consistent with their effect in resolution of inflammation and stimulation of innate immunity, LXA4 and RvD1 treatments have been shown to target epithelium and to regulate airway epithelial ion transport [63]. LXA4 stimulates CFTR-independent chloride secretion and inhibit amiloride-sensitive Na+ absorption resulting in an ASL height increase in human CF and non-CF airway epithelium [64–67]. This effect involves the apical ATP (adenosine triphosphate) secretion through pannexin channels induced by LXA4, leading to purino-receptor activation and further triggering of an intracellular calcium signal [63,65]. RvD1 also increases the CF ASL height of human bronchial epithelium in a calcium-dependent manner by decreasing the amiloride-sensitive Na+ absorption and stimulating CFTR-independent Cl− secretion. In vivo studies reflect these in vitro data showing that LXA4 and RvD1 restored the nasal transepithelial potential difference in CF mice [67]. These observations, shown in Figure 3, support the concept that the abnormal production of SPM in CF contributes to the dehydration of the airway surface and the defective airway clearance.

**Figure 3.** Novel hypothetical model for cystic fibrosis (CF) airway pathogenesis. In CF and non-CF airway bacterial infection triggers inflammation. In non-CF cells (top), self-limited inflammation involves the biosynthesis of specialized pro-resolving eicosanoid mediators (SPM) that enhance mucociliary clearance, bacterial killing, resolution of inflammation, and tissue repair, resulting in tissue returning to homeostasis (green). In CF cells (bottom), after initiation of inflammation, the eicosanoid class switching is altered and SPM are abnormally produced. This results into a defective mucociliary clearance, sustained inflammation, inefficient bacterial killing and tissue repair that lead to respiratory failure (red). This model enhances the role of SPM in the resolution phase of inflammation compared to the impact of dehydration and altered mucociliary clearance.
