ORIGINAL ARTICLE CYSTIC FIBROSIS Reduced 15-lipoxygenase 2 and lipoxin A4/leukotriene B4 ratio in children with cystic fibrosis Fiona C. Ringholz1, Paul J. Buchanan1, Donna T. Clarke1, Roisin G. Millar1, Michael McDermott2, Barry Linnane1,3,4, Brian J. Harvey5, Paul McNally1,2 and Valerie Urbach1,6 Affiliations: 1National Children’s Research Centre, Crumlin, Dublin, Ireland. 2Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland. 3Midwestern Regional Hospital, Limerick, Ireland. 4Centre for Interventions in Infection, Inflammation and Immunity (4i), Graduate Entry Medical School, University of Limerick, Limerick, Ireland. 5Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin, Ireland. 6Institut National de la Sante´ et de la Recherche Me´dicale, U845, Faculte´ de Me´decine Paris Descartes, Paris, France. Correspondence: Valerie Urbach, National Children’s Research Centre, Crumlin, Dublin 12, Ireland. E-mail: [email protected] ABSTRACT Airway disease in cystic fibrosis (CF) is characterised by impaired mucociliary clearance, persistent bacterial infection and neutrophilic inflammation. Lipoxin A4 (LXA4) initiates the active resolution of inflammation and promotes airway surface hydration in CF models. 15-Lipoxygenase (LO) plays a central role in the ‘‘class switch’’ of eicosanoid mediator biosynthesis from leukotrienes to lipoxins, initiating the active resolution of inflammation. We hypothesised that defective eicosanoid mediator class switching contributes to the failure to resolve inflammation in CF lung disease. Using bronchoalveolar lavage (BAL) samples from 46 children with CF and 19 paediatric controls we demonstrate that the ratio of LXA4 to leukotriene B4 (LTB4) is depressed in CF BAL (p,0.01), even in the absence of infection (p,0.001). Furthermore, 15-LO2 transcripts were significantly less abundant in CF BAL samples (p,0.05). In control BAL, there were positive relationships between 15-LO2 transcript abundance and LXA4/LTB4 ratio (p50.01, r50.66) and with percentage macrophage composition of the BAL fluid (p,0.001, r50.82), which were absent in CF. Impoverished 15-LO2 expression and depression of the LXA4/LTB4 ratio are observed in paediatric CF BAL. These observations provide mechanistic insights into the failure to resolve inflammation in the CF lung. @ERSpublications Reduced 15-LO2 expression in the lower airways of children with CF, associated with a depressed LXA4/LTB4 ratio http://ow.ly/tzZWa This article has supplementary material available from erj.ersjournals.com Received: June 21 2013 | Accepted after revision: Feb 03 2014 | First published online: April 2 2014 Support statement: This work was supported by grants from the National Children’s Research Centre, Ireland (E10/08), the Health Research Board of Ireland (1403 HRA_POR/2011/21) and Institut National de la Sante´ et de la Recherche Me´dicale, France. Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com Copyright ßERS 2014 394 Eur Respir J 2014; 44: 394–404 | DOI: 10.1183/09031936.00106013 CYSTIC FIBROSIS | F.C. RINGHOLZ ET AL. Introduction Cystic fibrosis (CF) is caused by mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and characterised by impaired mucociliary clearance, persistent bacterial infection and neutrophil mediated inflammation. Sequential biosynthesis of eicosanoid classes in inflammatory exudates (termed ‘‘class switching’’) directs the temporal progression of acute inflammation from initiation to propagation and finally to active resolution [1]. Prostaglandins initiate the inflammatory response. Leukotrienes follow and are typified by leukotriene B4 (LTB4) which amplifies and propagates inflammation [1]. LTB4 and interleukin (IL)-8 are both potent neutrophil chemoattractants [2, 3] and negatively correlate with pulmonary function in CF [4, 5]. Lipoxin A4 (LXA4) biosynthesis initiates the active resolution phase of inflammation [6], followed by biosynthesis of the resolvins and protectins. LXA4 inhibits neutrophil effector functions [7] and counter-regulates the effector functions of LTB4, most notably inhibiting LTB4-induced neutrophil transmigration [8–10]. LXA4 also suppresses production of IL-8 by leukocytes and bronchial epithelial cells [11–13]. Mice treated with analogues of LXA4 and subsequently challenged with Pseudomonas aeruginosa contained the bacterial challenge more effectively [14]. LXA4 augments airway epithelial innate defence by stimulating tight junction formation [15], enhancing calcium-activated chloride secretion [16], and by restoration of airway surface liquid height in CF airway epithelial cells [17]. LXA4 concentration in CF bronchoalveolar lavage (BAL) has been variously reported as significantly suppressed or not significantly different from controls [14, 18]. LXA4 is biosynthesised in the respiratory tract by transcellular cooperation of neutrophils [19], eosinophils [20], alveolar macrophages [21] or airway epithelial cells [22], each expressing different lipoxygenase (LO) enzymes (fig. 1)[2, 23]. Upregulation of 15-LO activity favours LXA4 biosynthesis at the expense of leukotriene biosynthesis [1, 24]. This occurs both as a result of 15-LO product, 15-hydroxyeicosatetraenoic acid (HETE), competing for catalytic sites at the 5-LO enzyme and thereby reducing the formation of 5(S) HETE, and by competition for the common biosynthetic intermediate leukotriene A4 [1, 22–24]. We hypothesised that ‘‘eicosanoid mediator class switching’’ is defective in CF and may play a mechanistic role in the failure to resolve inflammation in CF lung disease. We measured eicosanoids associated with the ‘‘class switch’’ [1] from propagation (leukotrienes) to resolution of inflammation (lipoxins), and the relative expression of mRNA transcripts for eicosanoid synthetic enzymes (5-LO, 12-LO and 15-LO1/2 and leukotriene A4 hydrolase (LTA4H)) in BAL from children with CF and compared this with paediatric a) Neutrophil AA 5-LO LTA4H LTA4 5-LO FIGURE 1 An illustration of lipoxin A4 (LXA4) biosynthesis pathways involving transcellular cooperation in LTB4 LXA4 the airways. a) The neutrophil donates the leukotriene A4 (LTA4) intermediate 15S H(p)ETE formed by the action of 5-lipoxygenase b) (LO) on arachidonic acid (AA) to the acceptor airway epithelial cell or alveolar macrophage, whereby 15-LO1/2 (15- 15-LO1/2 15-LO1/2 LO isoform 1 or 2) catalyses LXA4 formation. Leukotriene B4 (LTB4)is also generated from the LTA4 inter- AA mediate by the action of leukotriene A4 hydrolase (LTA4H). b) Airway epithelial Airway cell or alveolar macrophage 15-LO1/2 epithelial cell activity catalyses the conversion of AA OR Macrophage to 15(S)-hydroperoxyeicosatetraenoic acid 15(S) H(p)ETE, which is donated to the acceptor neutrophil and converted to LXA4 by 5-LO catalysis. DOI: 10.1183/09031936.00106013 395 CYSTIC FIBROSIS | F.C. RINGHOLZ ET AL. controls. In addition, we conducted subgroup analysis based on the presence or absence of microbial pathogens (.104 CFU?mL-1) detected in the BAL fluid. Methods Study population Children with CF were recruited through SHIELD CF (Study of Host Immunity and Early Lung Disease in Children with CF), a longitudinal study established around the preschool surveillance bronchoscopy programmes at Our Lady’s Children Hospital, Crumlin (OLCHC), the National Children’s Hospital, Tallaght and University Hospital Limerick, Limerick, Ireland. Children without CF undergoing bronchoscopy for clinical reasons were recruited as paediatric controls (see the online supplementary material for indications and treatment characteristics). Assessments were undertaken when the children were in a stable clinical condition. Children were excluded from the analysis if they were taking leukotriene receptor antagonists or treatment doses of antibiotics in the 2 weeks prior to bronchoscopy. Ethical approval for this study was granted by the research ethics committee at each hospital and written informed consent was obtained from the parents of all participating subjects. Bronchoscopy, BAL collection and processing to obtain a supernatant and cell pellet were performed as previously described (online supplementary material) [25]. Measurements An aliquot of BAL was sent to the microbiology lab at OLCHC where testing, including conventional bacterial and viral studies, was performed according to Clinical Pathology Accreditation guidelines (online supplementary material). Samples were considered to have positive microbial culture if one or more pathogen was detected on viral studies or cultured at a concentration of .104 CFU?mL-1. LXA4 and LTB4 concentrations were measured by ELISA (Oxford Biomedical Research, Rochester Hills, MI, USA and Cayman Chemical, Ann Arbor, MI, USA) (for specificities of antisera see online supplementary table E1). IL-8 was measured by ELISA using mouse anti-IL8 capture antibody and biotinylated goat anti- IL8 detection antibody (R&D Systems, Abingdon, UK). Neutrophil elastase (NE) activity was measured as described previously (online supplementary material) [25]. Total and differential cell counts were obtained by the trypan blue exclusion method and light microscopy (for further detail see the online supplementary material). Total RNA was extracted from the BAL cell pellets using RNAqueous kit (Ambion Inc., Austin, TX, USA). RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Dun Laoghaire, Ireland).