Fabry disease peripheral blood immune cells release inflammatory cytokines: Role of globotriaosylceramide

https://doi.org/10.1016/j.ymgme.2013.02.003Get rights and content

Abstract

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 perpetuation. 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 inflammatory 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. Furthermore, this effect can be abolished using a TLR4 blocking antibody, indicating that TLR4 is necessary in the process. 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 pathogenesis of Fabry disease.

Highlights

► PBMC from Fabry patients present a higher proinflammatory cytokine profile. ► Dendritic cells and monocytes from Fabry patients secrete proinflammatory cytokines. ► Gb3 is the responsible of this proinflammatory profile, likely mediated by TLR4.

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 catabolism 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 accumulation 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], [7], [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 vascular injury and a prothrombotic state, that could be related to the finding 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 alterations in the number of leukocyte subpopulations, as well as in their surface marker levels [13]. Later on, we have described a higher apoptotic 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 in the subsets related to the innate immune system, and the possible effect of excessive Gb3 levels on the initiation and maintenance of this state.

Section snippets

Patient's and normal control's samples

Blood samples from 29 Fabry patients, 15 males and 14 females (mean age: 30.2 ± 18.4 years old; range: 3–74 years old) were taken for this study. Diagnosis of Fabry disease was established by clinical examination reduced enzymatic activity and genetic test. Clinical manifestations of the patients included angiokeratoma, acroparesthesia, cornea verticillata, abdominal pain, proteinuria and hypohidrosis; however none of the patients suffered from renal insufficiency, heart failure or cerebrovascular

Expression and production of proinflammatory cytokines in Fabry PBMC

To assess the existence of a proinflammatory state in PBMC from Fabry patients, we first analyzed the expression and production of proinflammatory cytokines in PBMC from patients and normal controls.

Unstimulated, freshly isolated PBMC from Fabry patients showed a basal increased expression of the proinflammatory cytokines IL-1β and TNF-α compared to normal controls (p = 0.0098 and p = 0.0496, respectively), whereas the expression levels of IL-1α, IL-6, and IFN-γ were unaltered (Fig. 1).

When the

Discussion

The idea of a chronic stimulation of the immune system in LDs has been introduced a long time ago [17], through findings of increase cytokine levels in sera from affected patients. However, the mechanism that explained these observations was far from being elucidated. In the last few years, much work has been done in an attempt to clarify this key aspect. Production of proinflammatory cytokines such as IL-1β and TNF-α seems to be a common phenomenon among a wide range of LDs [5]. Particularly,

Conclusions

In summary, PBMC from Fabry patients display a proinflammatory cytokine expression and production profile. In particular, dendritic cells and monocytes, two innate immune subsets, could be the main source of this basal proinflammatory state. Additionally, Gb3 can induce the same cytokine profile on normal monocyte-derived macrophages and dendritic cells, indicating that it could be responsible for the generation of this proinflammatory profile. Finally, Gb3 seems to depend on TLR4 to achieve

Role of the funding source

The funding source had no involvement neither in the study design; in the collection, analysis, interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication.

The following are the supplementary data related to this article.

. Expression of cytokines in PBMC from normal controls (white bars), untreated Fabry patients (light gray bars) and Fabry patients undergoing ERT (dark gray bars). Values shown correspond to average expression normalized to the

Acknowledgments

We wish to thank the Banco de Sangre, Instituto de Hemoterapia de la Provincia de Buenos Aires, La Plata for kindly providing the buffy coats used in this study.

This work was supported by Shire HGT (USA) [research grant 2007/2008 to PR]; and Agencia Nacional de Promoción Científica y Tecnológica (Argentina) [PICT 2007 No. 01070 to PR].

PR acts as a consultant and has received research grants from Shire HGT.

References (31)

  • R.J. Desnick et al.

    α-Galactosidase A deficiency: Fabry disease

  • J.A. Castaneda et al.

    Immune system irregularities in lysosomal storage disorders

    Acta Neuropathol.

    (2008)
  • V. Barak et al.

    Cytokines in Gaucher's disease

    Eur. Cytokine Netw.

    (1999)
  • P. Formichi et al.

    Psychosine-induced apoptosis and cytokine activation in immune peripheral cells of Krabbe patients

    J. Cell. Physiol.

    (2007)
  • T. DeGraba et al.

    Profile of endothelial and leukocyte activation in Fabry patients

    Ann. Neurol.

    (2000)
  • Cited by (0)

    View full text