The role of Lipoxin A4 in Cystic Fibrosis Lung Disease

In Cystic Fibrosis (CF), mutations of the CFTR gene result in defective Cl− secretion and Na+ hyperabsorption by epithelia which leads to airway lumen dehydration and mucus plugging and favours chronic bacterial colonization, persistent inflammation and progressive lung destruction. Beyond this general description, the pathogenesis of CF lung disease remains obscure due to an incomplete understanding of normal innate airway defense. This mini-review aims to highlight the role of the pro-resolution lipid mediator, Lipoxin A4, which is inadequately produced in CF, on several aspects of innate immunity that are altered in CF airway disease.

Whilst the field continues to celebrate the success, for a minority of people with CF, in achieving therapeutic benefits via CFTR modulation strategies, reduction of lung inflammation and restoration of airway hydration / muco-ciliary clearance remain core goals of CF therapy for the majority. Several anti-inflammatory approaches have been examined in CF, however, the ideal anti-inflammatory drug is not yet available [9]. A recent systematic review of the risks and benefits of Inhaled corticosteroids (ICS) in CF, examining evidence from 3 trials, concluded that there is insufficient evidence to establish whether ICS are beneficial in CF, but withdrawal in those already taking them has been shown to be safe [ 0]. It is established that ICS use can have adverse effects on growth. A systematic review of the efficacy of non-steroidal anti-inflammatory drugs in CF concluded that treatment with high-dose ibuprofen was associated with a significantly lower annual rate of decline in lung function (especially in children), however, the adoption of ibuprofen into therapy has not been universally accepted [ , 2]. Redressing the imbalance in fatty acid metabolism described in CF, by supplementation of Docosahexaenoic Acid may be helpful, and efforts are ongoing to evaluate the potential therapeutic benefit [ 3].

The Specialized Pro-resolving Mediators
New perspectives have emerged in inflammation research with the discovery of new classes of biologically active lipid mediators playing specialised roles in the active resolution of inflammation -the "specialized pro-resolving mediators" (SPM) [ 4]. Furthermore, the acute inflammatory response is a protective mechanism that evolved to eliminate invading organisms and yet be self-limited with an active resolution phase designed to restore tissue homeostasis. The resolution phase is carried out by the actions of SPM which are non- The temporal evolution of acute inflammation toward its active resolution is directed by the sequential expression and activity of characteristic classes of eicosanoid mediator in a process termed "class switching" [ 5]. Prostaglandins are biosynthesised early, initiating the inflammatory response. Leukotrienes follow, typified by Leukotriene B4 (LTB4) which plays its role in amplification and propagation of inflammation [ 5] acting in concert with the cytokine Interleukin 8 (IL-8) as a potent CSBJ Abstract: In Cystic Fibrosis (CF), mutations of the CFTR gene result in defective Clsecretion and Na + hyperabsorption by epithelia which leads to airway lumen dehydration and mucus plugging and favours chronic bacterial colonization, persistent inflammation and progressive lung destruction. Beyond this general description, the pathogenesis of CF lung disease remains obscure due to an incomplete understanding of normal innate airway defense. This mini-review aims to highlight the role of the pro-resolution lipid mediator, Lipoxin A4, which is inadequately produced in CF, on several aspects of innate immunity that are altered in CF airway disease.

The role of Lipoxin A4 in Cystic Fibrosis Lung Disease
Valérie Urbach a,b,* , Gerard Higgins a , Paul Buchanan a , Fiona Ringholz a  [ 6,7]. Lipoxin A4 (LXA4) is the first eicosanoid of the SPM family to be expressed in the active resolution phase of inflammation. LXA4 production is followed by the biosynthesis of Resolvins and Protectins at the inflammatory site.

Anti-inflammatory properties of Lipoxin A4
The anti-inflammatory properties of LXA4 have been reported in a wide variety of tissues. LXA4 inhibits nuclear factor-kappaB activation, which results in inhibition of pro-inflammatory cytokine release and inhibition of inflammatory responses in microglial cells, astrocytoma cells, macrophages, peripheral blood mononuclear cells (PBMC), polymorphonuclear leukocytes (PMN) and intestinal epithelial cells [8][9][10][11][12][13][14][15][16][17][18][19][20] . This is a critical point since delayed neutrophil apoptosis appears to be a component of the pathophysiology in patients with inflammatory diseases, including cystic fibrosis [27,28] and frequently correlates with disease severity and outcome. In the airways, in vitro and in vivo studies also report that LXA4 displays diverse and potent anti-inflammatory actions [ 9,29,30]. In human airways, LXA4 suppresses IL-8 production by leukocytes and bronchial epithelial cells [30,3 ]. LXA4 was shown to arrest neutrophilic inflammation and decrease infection in a mouse model of chronic airway inflammation and infection [32]. LXA4 has been proposed as a novel regulators of adaptive immunity and may have therapeutic potential in chronic immune disorders [33]. The pro-resolution properties of LXA4 are mediated by the ALX/FPR2 receptor. The ALX/FPR2 is a G protein-coupled receptor of seven-transmembrane domains that is expressed mainly by mammalian phagocytic leukocytes. The known effect of ALX/FPR2 receptor in the in host defense and inflammation is not only triggered by LXA4 since other proresolution mediators such as resolving D also mediate their effects through this receptor  [ 5,4 ,42]. The levels of LXA4 have been reported to be decreased in chronic airway inflammatory disease such as asthma, chronic obstructive pulmonary disease and CF [29,32,43,44]. A decreased proportion of pro-resolving compounds (LXA4) compared to pro-inflammatory (LTB4) is associated with decrease of lung function [45]. The absolute content of LXA4 concentration in CF BAL fluid from patients with CF is not significantly different from controls [46]. However, a significant suppression in LXA4/neutrophil ratios in BAL fluid of patients with CF compared with pulmonary inflammatory controls was reported [32,46]. More specifically, in vitro studies support a role for CFTR in LXA4 production. CFTR inhibition reduced LXA4 synthesis by 50% during platelets/ PMN co-incubation by inhibiting the lipoxin synthase activity of platelets 2-LO. Platelets from patients with CF generated 40% less LXA4 compared to healthy subject [47]. The decreased LXA4 production in CF provides a mechanistic explanation of the failure to actively resolve acute airway inflammation seen in these patients.
Lipoxin A4 regulates ion transport and the airway surface liquid height LXA4 stimulates a rapid and transient intracellular Ca 2+ increase and induces Clsecretion through human bronchial epithelial cells by Ca 2+ -activated Clchannel and not CFTR [48]. Furthermore, LXA4 effects on ion transport lead into an increase of the airway surface liquid (ASL) layer height in models of fully differentiated bronchial epithelia derived from primary culture of bronchial brushings from patients with CF (Figure ). LXA4 exerts this effect on the ASL dynamics via the ALX/FPR2 receptor which is expressed in the apical membrane of airway epithelial cells. The sustained increase in ASL height induced by LXA4 in non-CF and CF bronchial epithelial results from stimulation of an intracellular calcium signal and Ca 2+activated Clsecretion via NPPB sensitive Clchannels [49]. LXA4 thus restores Clsecretion and adequate ASL height which are affected in CF airways, highlighting a role for LXA4 in the control of innate immune defence. The inadequate endogenous LXA4 biosynthesis in CF contributes to the reduce ASL volume and impaired mucociliary clearance in addition to alter resolution of inflammation in the airway, thus amplifying the vicious circle of airway dehydration, chronic infection and inflammation.

Lipoxin A4 regulates airway epithelial integrity
Epithelial repair is a key process required to maintain epithelial barrier integrity and respiratory function, however in CF, repeated Figure 1. LXA4 restores the Airway Surface Liquid (ASL) layer in CF bronchial epithelium. Live cell imaging using confocal microscopy of bronchial epithelium in primary culture from a child with CF before and after treatment with LXA4. The bronchial epithelial cells are stained in green using calcein green and the ASL in red using dextran coupled to texas-red.
infections and inflammatory insults result in damage to the airways, triggering the repair process [50]. Epithelial repair initially involves cell spreading and migration, followed by proliferation to repopulate the denuded area that has been created by injury [5 -53]. This is then followed by differentiation of the epithelium [54]. Recent research suggests that epithelial repair and differentiation of the CF airway epithelium is down-regulated or delayed [55][56][57][58][59]. More specifically, cell migration and proliferation appear to both be reduced during repair in CF bronchial epithelial cells compared to non-CF [60]. This delay in repair of the CF epithelium renders the lung more susceptible to ongoing bacterial infection and thus may lead to more epithelial damage [6 ]. It was recently reported that LXA4 can trigger epithelial cell migration and proliferation and thus play a role in repair of corneal and bronchial epithelia [62-64] [65-67]. LXA4 triggers an increase in migration, proliferation, and wound repair of non-CF and CF bronchial epithelia. These responses to LXA4 are mediated by the ALX/FPR2 receptor via the downstream activation of KATP channels and ERK MAP kinase phosphorylation [60]. This effect of LXA4 both ion transport and repair are consistent with the role of ion channels in two key processes of repair, migration and proliferation [68]. In particular potassium channels have been shown in numerous cell types to be involved in cell migration and proliferation [57,58,[69][70][71][72]. Furthermore, LXA4 enhances airway epithelial tight junction formation. LXA4 stimulates ZO-, claudin-and occludin expression and trafficking at the apical membrane resulting in enhanced transepithelial electrical resistance in human airway epithelia [73]. Therefore the reduced levels of LXA4 in the CF airways [74] may account for the reduced capacity for epithelial repair in the CF epithelium.
Therapeutic potential for LXA4 in cystic fibrosis treatment Clearance of airway secretions has been a first line therapy for patients with CF and a variety of airway clearance therapies have been developed [75,76]. One of the greatest challenges into reversing the CF defect in the airways is to design strategies to overcome the absence of functional CFTR by stimulating chloride secretion via alternative pathways, thus restoring airway hydration and mucociliary clearance. This can be achieved via the activity of calcium-activated chloride channels stimulated by agents that raise the intracellular calcium concentration. This strategy has been plagued by the attendant side effects of the amplification of the calcium dependant pro-inflammatory response resulting in the undesirable activation of NFĸB [26]. Thus identification of agents, particularly natural endogenous biologicals, that stimulate alternative non-CFTR Clsecretory pathways and promote ASL hydration and optimal ASL height recovery are likely to be of therapeutic benefit in improving mucociliary clearance in patients with CF. The effects of LXA4 inhalation has been evaluated in a pilot study of eight asthmatic and healthy adult subjects. The challenge was tolerated, had no adverse effect on pulse or blood pressure and demonstrated favourable effects on specific airway conductance [77].

Conclusion and future prospect
Taken together, the discovery of the multiple LXA4 functions in restoring bronchial epithelium ion transport in enhancing ASL height, in restoring epithelial barrier function and in reducing inflammation might provide significant advance in improving quality of life and longevity for CF patients (Figure 2).