The Role of Lipoprotein-Associated Phospholipase A₂ in a Murine Model of Experimental Autoimmune Uveoretinitis

April The Role of Lipoprotein-Associated Phospholipase A in a Murine Model of Experimental Autoimmune Uveoretinitis G. L. Crawford 0 1 J. Boldison 0 1 D. A. Copland 0 1 P. Adamson 0 1 D. Gale 0 1 M. Brandt 0 1 L. B. Nicholson 0 1 A. D. Dick 0 1 0 1 Academic unit of Ophthalmology, School of Clinical Sciences, University of Bristol , Bristol , United Kingdom , 2 School of Cellular and Molecular Medicine, University of Bristol , Bristol , United Kingdom , 3 Ophthiris Discovery Performance Unit , GlaxoSmithKline, Stevenage , United Kingdom , 4 Ophthiris Discovery Performance Unit, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America, 5 Platform Technology Sciences, King of Prussia , Pennsylvania , United States of America 1 Academic Editor: Andrew W Taylor, Boston University School of Medicine, UNITED STATES Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of LpPLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in LpPLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity. - Funding: http://www.bbsrc.ac.uk/home/home.aspx, Biotechnology and biological sciences research council: BB/J500197/1, received by GLC (nee Gemma Beers) and http://www.gsk.com/ GlaxoSmithKline: STU100020289, received by GLC (nee Gemma Beers). The funder GlaxoSmithKline provided support in the form of salaries for authors PA, DG & MB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section. Non-anterior uveitis (posterior, pan and intermediate uveitis) is a collective term used to describe a range of intraocular inflammatory disorders affecting the uvea and retina, and whilst more rare than anterior uveitis, is significantly more sight threatening [1]. Uveitis accounts for 10% of the 285 million visually impaired patients worldwide [2] with considerable financial and social implications, as it predominantly effects the working age population [3]. Uveitis is presumed autoimmune or immune mediated when infectious aetiology has been excluded [1, 46]. In mice, experimental autoimmune uveoretinitis (EAU) is an antigen-specific Th1/Th17 CD4+ T cell-directed murine model, employed to mimic human non-infectious, non-anterior uveitis, which has been utilised extensively to develop a detailed understanding of the immuno-pathogenesis of vascular inflammation, retinal leukocyte infiltration and mechanisms of tissue damage [68]. In this model, tissue destruction is dependent upon activation and infiltration of mononuclear cell infiltration [6, 912]. Tissue inflammation is held in check and homeostasis maintained, in part through microglia (resident retinal myeloid cell population) but following breakdown of blood ocular barrier [13] circulating macrophages infiltrate the retina early in the course of EAU [7, 14]. The migration of CD4+ T cells to the retina causes activation and further accumulation of CD11b+ macrophages. Macrophages display broad heterogeneity and their phenotype and behaviour is regulated by a plethora of stimuli found in the local microenvironment. For example, macrophages can inhibit T-cell proliferation in the eye but also produce tissue-damaging superoxides [7, 15, 16]. Notwithstanding, regulating macrophage activation will reduce inflammation [17, 18] and as such, an ability to manipulate macrophage phenotype and/or migration has the potential to abrogate EAU onset. One means of repressing macrophage activation is through prevention of hydrolysis of oxidised low density lipoproteins (oxLDL), catalysed by the enzyme Lipoprotein Associated Phospholipase A2 (Lp-PLA2). This reaction produces lysophosphatidylcholine (LPC) and oxidised non-esterified fatty acids (ox-NEFA) two potent pro-inflammatory mediators, which up-regulate expression of chemokines and adhesion molecules, induce macrophage migration and promote pro-inflammatory cytokine release [19, 20]. The phospholipase A2 (PLA2) family are a group of enzymes involved in catalysing the hydrolysis of fatty acyl moieties from the sn-2 position of oxidized phospholipid molecules [19]. Lp-PLA2 is a 45kDa secreted enzyme, expressed by monocytes, macrophages, T cells and mast cells. This enzyme contains a catalytic serine residue [21] and is active in basal physiological conditions. Unlike other PLA2 family members, Lp-PLA2 functions independently of calcium and is highly specific for substrates with a short chain fatty acyl residue at the sn-2 position, meaning it does not hydrolyse membrane phospholipids [22]. Lp-PLA2 suppression has been used to modulate disease in both atherosclerosis and diabetic macular oedema models [23], which in common with uveitis, present with macrophage mediated tissue damage and underlying vascular pathology. It was first noted that Lp-PLA2 expression was high in atherosclerotic plaques, particularly those which were necrotic and prone to rupture and a meta-analysis of nearly 80,000 people demonstrated a continuous association between the mass and activity of Lp-PLA2 and risk of coronary heart disease [24]. A diabetic and hypercholesterolemic swine model was used to test the efficacy of Lp-PLA2 depletion in atherosclerosis. In short, treatment of atherosclerotic pigs with Darapladib, a selective, reversible inhibitor of Lp-PLA2 [25] induced a significant reduction in lesion development and necrotic core area [26]. Similar results (...truncated)