Fabry disease peripheral blood immune cells release inflammatory cytokines: Role of globotriaosylceramide Pablo N. De Francesco, Juan M. Mucci, Romina Ceci, Carlos A. Fossati, Paula A. Rozenfeld ⁎ LISIN, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata (1900), Argentina abstract article info Article history: Received 4 February 2013 Accepted 5 February 2013 Available online 13 February 2013 Keywords: Fabry disease Proinflammatory cytokine Globotriaosylceramide Macrophage Dendritic cell TLR4 Fabry disease is an X-linked lysosomal disorder (LD) due to deficiency of the enzyme α-galactosidase A (αGal), which leads to the accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3). Several mechanisms contribute to the diverse physiopathological alterations observed in this disease, and it has been suggested that an underlying proinflammatory state could play a significant role. The aim of this study is to investigate the presence of a proinflammatory state in the different subsets of peripheral blood mononuclear cells (PBMC) and to understand the mechanisms that contribute to its onset and perpet- uation. We have shown that cultured PBMC from Fabry patients present a higher proinflammatory cytokine expression and production. Moreover, we determined that among PBMC, dendritic cells and monocytes present a basal proinflammatory cytokine production profile, which is further exacerbated with an inflamma- tory stimulus. Finally we established that normal, monocyte-derived dendritic cells and macrophages display the same proinflammatory profile when cultured in the presence of Gb3 and an inhibitor of αGal. Further- more, this effect can be abolished using a TLR4 blocking antibody, indicating that TLR4 is necessary in the pro- cess. In summary, our results demonstrate the presence of a proinflammatory state involving two key subsets of innate immunity, and provide direct evidence of Gb3 having a proinflammatory role, likely mediated by TLR4, a finding that could help in the understanding of the underlying causes of the inflammatory pathogen- esis of Fabry disease. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Fabry disease is an X-linked lysosomal disorder (LD) due to deficiency of the enzyme α-galactosidase A (αGal, EC 3.2.1.22, codified by the gene GLA) [1,2]. Absent or reduced enzyme activity results in impaired catab- olism of neutral glycosphingolipids, particularly globotriaosylceramide (Gb3), which in turn leads to intracellular deposition of such lipids [3]. Although tissue and organ dysfunctions can be partly attributed to direct buildup of Gb3, the exact molecular mechanisms that link this accumula- tion to the eventual cell and tissue damage have not yet been clearly established. In a number of different LDs, other concurrent pathological mechanisms are elicited, which together contribute to the phenotypic expression of each disease [4]. Growing evidence shows immune system irregularities associated to lysosomal disorders [5]. In several different LDs, immune cells from affected patients display a constitutive proinflammatory pattern of cytokine expression [6–8]. For Fabry disease in particular, a number of immune alterations have been described. Leucocytes and endothelial cells have been shown to exhibit an activated profile. Deposition of globotriaosylceramide occurs in numerous cell types including vascular endothelial and smooth muscle cells. Fabry disease is associated to vas- cular injury and a prothrombotic state, that could be related to the find- ing of higher surface expression of integrins involved in cell adhesion [9]. The evidence for endothelium activation was confirmed lately by Shen et al. [10]. These authors showed that accumulated Gb3 induces oxidative stress and up-regulates adhesion molecules. The mechanisms of endothelial compromise may be associated to the decreased nitric oxide bioavailability detected in a Gla null mouse model, which could account for aberrant vascular phenotypes [11]. A pro-oxidative and proinflammatory state that correlates with urinary Gb3 levels has been recently described [12]. Our group has previously reported alter- ations in the number of leukocyte subpopulations, as well as in their surface marker levels [13]. Later on, we have described a higher apopto- tic rate in peripheral blood mononuclear cells (PBMC), which could be directly attributed to the effects of elevated Gb3 levels [14]. In this work, we investigate the existence of an underlying proinflammatory state in the PBMC of Fabry patients, in particular Molecular Genetics and Metabolism 109 (2013) 93–99 Abbreviations: LD, lysosomal disorder; PBMC, peripheral blood mononuclear cells; DC, dendritic cells; MΦ, macrophage; NK, natural killer; Gb3, globotriaosylceramide; lysoGb3, globotriaosylsphingosine; αGal, α-galactosidase A; GLA (Gla), human (murine) α-galactosidase A gene; ERT, enzyme replacement therapy; DGJ, 1-deoxygalactonojirimycin; TLR4, Toll-like receptor 4; LPS, lipopolysaccharide; DMSO, dimethyl sulfoxide. ⁎ Corresponding author at: LISIN, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115, La Plata (1900), Argentina. Fax: +54 221 422 6947. E-mail addresses: paurozen@biol.unlp.edu.ar, paularozenfeld@gmail.com (P.A. Rozenfeld). 1096-7192/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymgme.2013.02.003 Contents lists available at SciVerse ScienceDirect Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme