The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice

Zhilong Jiang 0 Melane L Fehrenbach 0 Giulia Ravaioli 0 Blerina Kokalari 0 Imre G Redai 0 Steven A Sheardown 3 Stephen Wilson 2 Colin Macphee 4 Angela Haczku 0 1 0 Pulmonary, Allergy and Critical Care Division, University of Pennsylvania , Philadelphia, PA , USA 1 Pulmonary, Allergy and Critical Care Division, Translational Research Laboratories , 125 South 31st Street, Philadelphia, PA 19104-3403 , USA 2 GSK Laboratory Animal Sciences , GlaxoSmithKline, Stevenage , UK 3 Takeda Cambridge Limited , 418 Cambridge Science Park, Cambridge , UK 4 Department of Vascular Biology and Thrombosis, GlaxoSmithKline , King of Prussia, PA , USA Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models. Methods: Lp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. - hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice. Results: PAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released -hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice. (Continued on next page) - (Continued from previous page) Conclusions: We conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge. Introduction Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a 45-kDa protein of 441 amino acids encoded by the pla2g7 gene in humans. In the blood it travels mainly with low density lipoprotein (LDL) and less than 20% is associated with high density lipoprotein (HDL). This enzyme is produced by myeloid derived cells and it functions to hydrolyze oxidized/polar phospholipids. Whether Lp-PLA2 is a pro- or anti-inflammatory mediator is the subject of intense debate and numerous studies involving clinical trials and animal models [1]. Lp-PLA2 is implicated in the development of atherosclerosis [2]. A meta-analysis on a total of 79,036 participants in 32 prospective studies found that serum Lp-PLA2 positively correlated with an increased risk of coronary heart disease and stroke [3]. In atherosclerotic lesions the main sources of Lp-PLA2 include LDL from the circulation, and de novo synthesis by the inflammatory cells found in the plaque (macrophages, platelets, mast cells) [4]. Products of Lp-PLA2 can upregulate expression of adhesion molecules, activate leucocytes and recruit macrophages and monocytes into inflammatory areas [5-7]. Inhibition of Lp-PLA2 by the highly potent and selective inhibitor darapladib effectively ameliorated the clinical severity of atherosclerosis and deceased inflammation in the plaque area in a swine model [8]. Therefore, targeting of Lp-PLA2 has become an attractive strategy for the treatment of atherosclerosis. Lp-PLA2 is also called platelet-activating factor acetylhydrolase (PAF-AH), as it can cleave platelet-activating factor (PAF) in vitro by hydrolysis of the acetyl group at the sn-2 position, producing lyso-PAF and acetate [9,10]. PAF plays a prominent role in the pathogenesis of IgE mediated allergic inflammation and anaphylaxis (reviewed in [11-15]). Therapeutic targeting of PAF however did not affect asthma symptoms [16]. Nonetheless because of its PAF catalyzing activity [17-22], inhibition of PAF-AH/Lp-PLA2 raised the concern of an increased predisposition to allergic inflammation or anaphylaxis. Although the published direct evidence to support this concern is limited, there were clinical associations reported between low PAF-AH/Lp-PLA2, high plasma PAF and increased incidents and severity of asthma [23-26] and anaphylaxis [19]. A single nucleotide polymorphism of Val-279-Phe in the PAF-AH/Lp-PLA2 gene with functional deficiency was shown to be highly prevalent in Japan (about 4% of the general Japanese population) [27]. According to a 1999 study by Stafforini et al. PAF-AH/Lp-PLA2 deficiency was increased in asthmatics in comparison with healthy subjects in Japan with the greatest asthma severity found in homozygous PAFAH/Lp-PLA2 deficient subjects [25]. In animal models of lung injury and sepsis elevated PAF-AH/Lp-PLA2 levels were reported to be associated with inhibitory effects during the acute inflammatory process [28,29]. Exogenous administration of PAF-AH/Lp-PLA2 reduced mortality [18] and over-expression of PAF-AH/Lp-PLA2 attenuated inflammation in mouse models of sepsis [17,18,30] suggesting that this enzyme may have protective effects against inflammatory mechanisms involving PAF. This suggestion was contested in a clinical study conducted very similarly to that of Stafforinis. In this work Satoh and colleagues found no difference in the allele frequency between asthmatic patients and healthy controls and the V279F mutant allele prevalence was consistent regardless of asthma type or severity of disease [31]. In a more recent study Japanese patients underwent a bronchoprovocation test with PAF showed no difference in airway responsiveness whethe (...truncated)