Experimental studies of lipid mediator modulation of airway responsiveness

Abstract: Lipid mediators play an important role in responsiveness of the airways. The roles of prostanoids and leukotrienes in inducing airway inflammation and contraction are reasonably well established. The functions of specialised pro-resolving lipid mediators (SPMs), which are thought to mediate pro-resolving and anti-inflammatory effects, are less well-studied. The current knowledge on SPM functions, specifically in airway inflammation and contractility is limited. Furthermore, mast cells are important innate immune effectors cells found in the lung, known to release a host of pro-inflammatory and contractile cytokines and lipids in allergic airway inflammation and airway hyperreactivity. However, if the presence of mast cells in airways is necessary for antigen-induced airway contractions and induction of airway hyperreactivity remains to be clarified. In addition, though it is known that unselective cyclo-oxygenase (COX)-inhibition results in increased contractions in airways upon mast cell activation by antigen, the exact mechanism behind this is unknown. The aim of this thesis was thus to investigate if selected SPMs have anti-hyperreactive properties, how COX-inhibition results in increased airway constriction and if mast cells are necessary for antigen-induced contraction and airway hyperreactivity. To this end, mouse and guinea pig models of airway hyperreactivity and allergic inflammation were used. Tracheae were dissected free from surrounding tissue and divided in segments of equal size. The isometric contractions of these isolated tracheal preparations were studied in myographs, either immediately after dissection from mice that received intranasal administration of HDM and SPMs beforehand, or after four days of incubation of the segments with cytokines and SPMs. Alongside this, concentrations of released mast cell mediators were determined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). First, it was found that four-day intranasal administration of the SPMs lipoxin A4 (LXA4) and resolvin D1 (RvD1) could reduce house dust mite (HDM)-induced airway hyperreactivity. The reducing effect of LXA4 was replicated when added during four days of incubation of tracheal segments with HDM and TNFα and was also present for lipoxin B4 (LXB4). A potential involvement of the ALX/FPR2 receptor was found, though this should be further backed up by future investigations. Second, IL-13 induced a steroid-resistant airway hyperreactivity in vitro that could be reduced by cysteinyl maresins. This effect could be blocked by three different cysteinyl leukotriene (CysLT) receptor 1 antagonists. However, LTD4 could not reproduce the anti-hyperreactive effect nor did it interfere with cysteinyl maresin signalling. The exact receptor signalling remains to be clarified. Third, prostaglandin D2 (PGD2) was found to be produced by the COX-1 enzyme in mast cells present in tracheal segments from ovalbumin (OVA)-sensitised guinea pigs, when exposed to OVA in vitro. Further release of contractile mediators like histamine and cysteinyl leukotrienes from mast cells was inhibited by PGD2. This was done via the DP1 receptor, therefore PGD2 and the DP1 receptor likely function as an inhibitory, autocrine signalling axis for mast cells. Fourth, the presence of mast cells in lung tissue was necessary for HDM-induced airway contractions and mast cell absence led to reduced airway hyperresponsiveness in mouse models. Repeated HDM-exposure via intranasal instillation led to airway hyperreactivity mediated by carbachol and serotonin (5-HT) in isolated tracheal segments. The data suggest that mast cell activation occurred as an interplay between nerve-endings and mast cells, as mouse mast cells expressed the M3-receptor and activation led to release of 5-HT. To conclude, lipid mediators and mast cells play an essential role in the modulation of airway responsiveness. They do this by either inducing contractions after antigen-exposure (mast cells), reducing cytokine and antigen-induced airway hyperreactivity (SPMs) or inhibiting release of pro-contractile mediators and ultimately airway contractions (PGD2). This makes SPMs and their receptors, as wells as mast cells promising future drugs or drug targets for the treatment of airway hyperreactivity as seen in for example asthma.