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Invariant natural killer T cells are phenotypically and functionally altered in Fabry disease
Molecular Genetics and Metabolism 108 (2013) 241–248
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Invariant natural killer T cells are phenotypically and functionally altered in Fabry disease Catia S. Pereira a, Olga Azevedo b, M. Luz Maia a, Ana F. Dias a, Clara Sa-Miranda a, M. Fatima Macedo a, c, d,⁎ a
Lysosome and Peroxisome Biology Unit (UniLiPe), IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre nº 823, 4150-180, Porto, Portugal Serviço de Cardiologia, Centro Hospitalar do Alto Ave, Portugal ESS Jean Piaget, Vila Nova de Gaia, Portugal d SACS, Universidade de Aveiro, Portugal b c
a r t i c l e
i n f o
Article history: Received 24 December 2012 Received in revised form 27 January 2013 Accepted 27 January 2013 Available online 1 February 2013 Keywords: Lysosomal storage disorders Fabry disease Enzyme replacement therapy iNKT cells Cytokines
a b s t r a c t Fabry disease is a lysosomal storage disease belonging to the group of sphingolipidoses. In Fabry disease there is accumulation of mainly globotriaosylceramide due to deficiency of the lysosomal enzyme α-galactosidase A. The lysosome is an important compartment for the activity of invariant natural killer T (iNKT) cells. iNKT cells are lipid-specific T cells that were shown to be important in infection, autoimmunity and tumor surveillance. In several mouse models of lysosomal storage disorders there is a decrease in iNKT cell numbers. Furthermore, alterations on iNKT cell subsets have been recently described in the Fabry disease mouse model. Herein, we analyzed iNKT cells and their subsets in Fabry disease patients. Although there were no differences in the percentage of iNKT cells between Fabry disease patients and control subjects, Fabry disease patients presented a reduction in the iNKT CD4 + cells accompanied by an increase in the iNKT DN cells. Since iNKT cell subsets produce different quantities of pro-inflammatory and antiinflammatory cytokines, we analyzed IFN-γ and IL-4 production by iNKT cells of Fabry disease patients and mice. We found a significant reduction in the production of IL-4 by mice splenic iNKT cells and human iNKT cell subsets, but no significant alterations in the production of IFN-γ. Altogether, our results suggest a bias towards a pro-inflammatory phenotype in Fabry disease iNKT cells. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Lysosomal storage disorders are a group of inherited metabolic defects that arise mainly due to abnormalities in lysosomal hydrolases being characterized by the accumulation of different types of undegraded substrates in the lysosome. They can be classified according to the type of material that is accumulated. Fabry disease is a sphingolipidosis caused by mutations in the GLA gene, which encodes the lysosomal hydrolase α-galactosidase A. The decrease in the activity of this enzyme leads to the progressive lysosomal accumulation of mainly globotriaosylceramide (Gb3). However, secondary accumulation of other lipids such as lyso-Gb3
Abbreviations: iNKT, invariant natural killer T; α-GalCer, α-galactosylceramide; Gb3, globotriaosylceramide; ERT, enzyme replacement therapy; PMA, phorbol 12-myristate 13-acetate; TCR, T cell receptor. ⁎ Corresponding author at: Lysosome and Peroxisome Biology Unit (UniLiPe), Institute for Molecular and Cell Biology (IBMC), Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Fax: +351 226099157. E-mail addresses: [email protected] (C.S. Pereira), [email protected] (O. Azevedo), [email protected] (M.L. Maia), [email protected] (A.F. Dias), [email protected] (C. Sa-Miranda), [email protected] (M.F. Macedo). 1096-7192/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymgme.2013.01.018
is also detected [1]. Lipid accumulation is more pronounced in capillary endothelial, renal, cardiac and nerve cells [2]. Consequently, the clinical manifestations of Fabry disease include left ventricular hypertrophy and dysfunction, dysrhythmias and cardiac conduction abnormalities; proteinuria and renal failure; and acroparesthesias, neuropathic pain, stroke and white matter lesions [3]. Enzyme replacement therapy (ERT) is available for Fabry disease patients since 2001. This treatment was shown to efficiently promote de clearance of the endothelial deposits of Gb3 in the skin, heart and kidney [4]. ERT was also demonstrated to reduce myocardial mass, improving left ventricular function, to reduce or halt the decline of glomerular filtration rate and to improve pain and quality of life [5]. The lysosome, whose function is impaired in Fabry disease, is an important compartment for the immune response and especially for the lipid-specific T lymphocytes activity. Invariant natural killer T (iNKT) cells are lipid-specific T lymphocytes that are characterized by the expression of a semi-invariant T cell receptor (TCR) constituted by and invariant TCR Vα chain (Vα24Jα18 in humans and Vα14Jα18 in mice) paired with a limited repertoire of TCR Vβ chains [6]. iNKT cells recognize lipid antigens when they are associated with MHC class I-like CD1d molecules. These cells have a low frequency in peripheral blood and are more abundant in liver and fat tissue [7,8].
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Since peripheral blood is the main biological sample available from humans, iNKT cell studies are often challenging. Several studies described an important role of iNKT cells in anti-tumor, autoimmune and anti-microbial responses. Intrinsic iNKT cell numerical and functional abnormalities have been reported in several human diseases, such as type I diabetes, multiple sclerosis, cancers and infections [7]. iNKT cells are also important targets of cancer immunotherapy [9,10]. Currently, clinical trials involving iNKT cells are being conducted for melanoma and multiple myeloma [10]. Human iNKT cells can be divided in three subsets according to the CD4 and CD8 expression: positive only for CD4 (iNKT CD4 +), only for CD8 (iNKT CD8 +), or negative for both molecules (iNKT DN). These subsets have different cytokine production profiles. When stimulated ex-vivo, iNKT CD4 + cells produce both Th1 and Th2 cytokines while iNKT CD4 − cells produce mainly Th1 cytokines [11,12]. When iNKT cells are expanded in vitro and then stimulated, iNKT CD4 + cells produce more Th2 cytokines and iNKT CD4 − cells produce more Th1 cytokines [13]. The most potent known antigen for both human and murine iNKT cells is α-galactosylceramide (α-GalCer). However, this compound is not present in mammals. In the past years, a few mammalian lipids have been identified as iNKT cell antigens, including sphingolipids (β-glucosylceramide) and phospholipids (etherbonded lysophosphatidylethanolamine) [14,15]. These antigens stimulate human iNKT cells at different degrees and might be involved in the process of thymic selection and peripheral activation. iNKT cells were studied in several mouse models of lysosomal storage disorders. A reduction of iNKT cell percentage was found in Sandhoff disease [16–18], Tay–Sachs disease [17], Fabry disease [17,19,20], NPC1 deficiency [17,21], NPC2 deficiency [22,23], GM1 gangliosidosis [17,22] and multiple sulfatase deficiency [18]. iNKT CD4+ and CD4− cell subsets were also analyzed in GM1 gangliosidosis, NPC2 deficiency and Fabry disease mouse models. GM1 gangliosidosis mice presented no alterations in iNKT cell subsets, while Fabry disease mice exhibited a decrease in splenic iNKT CD4+ cells and NPC2 deficient mice displayed a reduction in the hepatic iNKT CD4+ subset [22,24]. Studies in patients with lysosomal storage disorders are scarce. iNKT cell percentage was analyzed in patients with Gaucher, Fabry and Niemann–Pick C diseases, but no reduction was found in these diseases [19,25–27]. Here, we analyzed Fabry disease patients, regarding the iNKT cell phenotype and function. In addition, iNKT cell function was assessed in Fabry disease mice. We also analyzed the effect of ERT overtime on iNKT cells and subsets of Fabry disease patients.
2. Methods 2.1. Population studied This study included 15 Fabry disease patients, previously diagnosed in our laboratory and regularly followed up and treated at the Cardiology departments of Centro Hospitalar do Alto Ave, Guimarães, Portugal (n = 14) and Hospital de São Teotónio, Viseu, Portugal (n = 1). They were all adult Caucasian patients (mean age of 48 ±14) and gave informed consent to participate in the study. At the beginning of this study, 4 patients had begun ERT 4 months before with agalsidase alfa — Replagal© (Shire Human Genetic Therapies). The patients that were under treatment were analyzed longitudinally, every 4 months, over a total period of 28 months. Patient 1 died in an accident and was only analyzed during 16 months. Unless stated, all the results presented in the results section (Section 3) correspond to the first determination obtained for each patient. Twenty-seven blood donors from the Instituto Português do Sangue or from the Immuno-haemotherapy department of Hospital de São João, Porto, Portugal were studied as controls. The mean age of controls was 41 ± 12.
2.2. Mononuclear cells isolation from human peripheral blood, iNKT cell expansion and analysis of their cytokine production profile Peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque-1077® (Sigma) density centrifugation, following the manufacturer's instructions. iNKT cells were expanded by culturing PBMCs in RPMI 10% iFBS medium, at a concentration of 0.5× 106 cells/mL, with 100 ng/mL of α-GalCer (Alexis-Coger SA) in a 24 well plate. At day 1 of culture, 100 U/mL of recombinant human IL-2 (kindly provided by the National Cancer Institute) was added. After 11–14 days, cells were collected, counted and stimulated with 25 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma), 1 μg/mL ionomycin (Sigma) and 10 μg/mL brefeldin A (Sigma) for 5 h before staining with cytokinespecific monoclonal antibodies. 2.3. Analyzes of cytokine production by mouse iNKT cells Fabry disease mice (GLA−/−) with C57BL/6 background were provided by the National Institutes of Health (Bethesda, MD, USA) and maintained at the IBMC-INEB animal facility (IBMC, Porto, Portugal). C57BL/6J mice were used as wild type controls. All procedures were performed in conformity with the institutional and national guidelines. The animals were analyzed at 12–15 weeks of age. The control and the Fabry disease mice were injected with 2 μg of α-GalCer, intraperitoneal (i.p.), diluted at 10μg/mL in saline solution or with saline solution alone. Two hours after injection, the mice were sacrificed, the spleen was removed and the liver was perfused and harvested as previously described [24]. Single-cell suspensions of hepatic mononuclear cells and splenocytes were prepared as previously described [24]. After cell suspension preparation, cells were counted and resuspended in RPMI supplemented with 10% iFBS and 10 μg/mL of Brefeldin A (Sigma), at a concentration of 5 ×106 cells/mL. Cells were then plated in 96-round bottomed well plates (1×106 cells/well) and incubated for 2 h, at 37 °C, 5% CO2. After the end of the incubation period, cells were stained for determination of cytokine production by flow cytometry. 2.4. Flow cytometry Human PBMCs were stained with antibody/CD1d tetramer cocktails diluted in PBS/2%FBS/0.01%NaN3 (flow cytometry solution) for 20 min, at 4 °C, in the dark. For PBMCs, the staining mixture was composed by anti-human CD3 (SK7), anti-human CD4 (RPA-T4), anti-human CD8 (RPA-T8) and anti-human CD161 (HP-3G10) antibodies (all from eBioscience) and the human CD1d tetramer loaded with PBS57 (from the National Institute of Health tetramer core facility). After staining, cells were washed with flow cytometry solution and then fixed with PBS 1% formaldehyde. For cytokine detection studies, human in vitro expanded iNKT cells or mice splenocytes and hepatic mononuclear cells were first stained with antibody/tetramer cocktail diluted in flow cytometry solution. For splenocytes and hepatic mononuclear cells the following antibodies were used together with the mouse CD1d tetramer loaded with PBS57: anti-mouse CD3 (17A2, Biolegend); anti-mouse CD4 (RM4-5, BD Biosciences). After extracellular staining, cells were washed, and then fixed by incubating with PBS 2% formaldehyde for 10min, at room temperature, in the dark. Cells were permeabilized by incubating with 0.5% saponin (Sigma) diluted in flow cytometry solution, for 5 min and washed again. Cells were then stained for 30 min, at room temperature. For human cells the following antibodies were used: anti-human IFN-γ (4S.B3) and anti-human IL-4 (8D4-8) antibodies or the corresponding isotype controls (all from eBioscience). For mice cells, anti-mouse IFN-γ (XMG1.2) and anti-mouse IL-4 (11B11) antibodies (both from Biolegend) were used. After incubation, cells were washed and resuspended in PBS. Samples were acquired on a 3-laser BD FACS Canto™ II flow cytometer using BD FACSDiva™
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software (BD Biosciences). All flow cytometry analyses were performed using the FlowJo software (Tree Star).
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3. Results 3.1. iNKT CD4 + cells are reduced in Fabry disease patients
2.5. Statistical analysis All statistical analyses were performed using GraphPad Prism 5 software. Shapiro–Wilk test was applied to determine the normal distribution of the variables. Student's t-test was used for normally distributed variables. For variables that did not follow a normal distribution, Mann–Whitney U test was used. Values of p b 0.05 were considered statistically significant.
We analyzed the percentage of iNKT cells in Fabry disease patients by flow cytometry using an anti-CD3 antibody and the PBS57 loaded CD1d tetramer (Fig. 1A). In accordance with the previous studies, which analyzed the percentage of Vα24 + cells, we found no differences between control subjects and patients regarding the percentage of iNKT cells over the total of CD3 + cells (Fig. 1B). We further characterized iNKT cells using the cell surface markers CD4, CD8 and CD161. Interestingly, Fabry disease patients presented a decrease
Fig. 1. iNKT cell percentage and phenotype in control subjects and Fabry disease patients. A — iNKT cell gating strategy. B — iNKT cell percentage in T cells. C — CD4+ cell percentage in iNKT cells. D — CD8+ cell percentage in iNKT cells. E — DN cell percentage in iNKT cells. F — CD161+ cells percentage in iNKT cells. Filled squares (■) represent patients receiving ERT. *p b 0.05.
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in the percentage of iNKT CD4 + cells and an increase in the percentage of iNKT DN cells when compared to control subjects (Figs. 1C and E). iNKT CD8+ cells presented no significant alterations (Fig. 1D). The 15 Fabry disease patients under study included 4 patients submitted to ERT for 4 months. When the patients receiving ERT were compared to patients not receiving ERT, no significant differences were found (Fig. 1). To determine if the reduction observed in the iNKT CD4 + cell subset was specific for iNKT cells, we analyzed the percentage of CD4 + and CD8 + T cells in total lymphocytes. Fabry disease patients presented no alterations in the percentage of CD4+ or CD8+ T cells (Supplemental Figs. S1A and B). Then, we determined the percentage of iNKT cells expressing the natural killer cell marker CD161. No differences were found in this subset, when comparing patients with control subjects or patients under ERT with untreated patients (Fig. 1F). We performed a longitudinal evaluation of the iNKT cell percentage and phenotype overtime for each of the 4 patients under ERT. We analyzed these 4 Fabry disease patients every 4 months, starting 4 months after the beginning of ERT and over a total period of 24 months. As shown in Fig. 2, the percentages of iNKT cells and iNKT CD4 + and DN cells do not present major variations within the
individuals, overtime. It is only the iNKT CD8 + subset that seems slightly decreased during the first 8 months of ERT. The percentage of iNKT CD161 + cells remained stable, over a year, in the tested individuals (Fig. 2E). 3.2. Fabry disease patients present an imbalance in iNKT cell cytokine production The imbalance in iNKT cell subsets found in Fabry disease patients suggested a functional alteration in the iNKT cell pool, since iNKT cell subsets have distinct cytokine production profiles. Therefore, we hypothesized that iNKT cell cytokine production might be altered in Fabry disease patients. To test this hypothesis, the expanded iNKT cells were stimulated using PMA and ionomycin for 5 h. During this time, cells were cultured with brefeldin A to avoid cytokine secretion, allowing the cytokine production determination by flow cytometry. An isotype control was used to define flow cytometry gates (Fig. 3A). We analyzed 11 control subjects and 10 Fabry disease patients (not under ERT). In Fabry disease patients, the production of IFN-γ by iNKT cells was not significantly altered (Fig. 3B). However, there was a tendency towards a reduction in the percentage of iNKT cells producing IL-4 (Fig. 3B). Interestingly, the analysis of IL-4
Fig. 2. Longitudinal study of iNKT cells in Fabry disease patients. The percentage of iNKT cells and iNKT cell subsets was studied in Fabry disease patients 1 to 4, over a period of 28 months of enzyme replacement therapy. A — iNKT cell percentage. B — iNKT CD4+ cell percentage. C — iNKT CD8+ cell percentage. D — iNKT DN cell percentage. E — iNKT CD161+ cell percentage.
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production by the iNKT cell subsets revealed a significant decrease in the percentage of IL-4 producing cells in both the CD4 + and CD4 − iNKT cell subsets, but no alterations in IFN-γ production (Figs. 3C and D). When we analyzed the co-production of IFN-γ and IL-4 in the iNKT cell subsets, we observed that the reduction in total IL-4 production is mainly due to a decrease of cells producing both IFN-γ and IL-4, with an increase in cells producing only IFN-γ (Fig. S2).
3.3. The imbalances in cytokine production by the iNKT cells are also present in the Fabry disease mouse model Due to the low percentage of iNKT cells in human peripheral blood, ex vivo analysis of cytokine production is difficult. Therefore, the analysis of cytokine production by patients' iNKT cells was based in cultured cells that might acquire different characteristics from those present in vivo. Furthermore, the higher variability existent between human subjects often hides the presence of small differences. We had previously described a reduction in the splenic iNKT CD4 + cell subset in the Fabry disease mouse model [24], which is consistent with the results presented here for Fabry disease patients peripheral blood. To test in vivo the capacity of cytokine production of iNKT cells, we injected Fabry and control mice with the lipid antigen α-GalCer or the corresponding volume of saline solution. After 2 h, mice were sacrificed and the liver and spleen were collected for analysis. iNKT cell cytokine production was analyzed by flow cytometry (Fig. 4A). Mice injected with saline displayed no IL-4 or IFN-γ production by iNKT cells, confirming that these cells were responding to α-GalCer stimuli (Fig. 4A). We found a significant reduction in the percentage of IL-4 producing iNKT cells in the spleen of Fabry disease mice when compared to control mice (Fig. 4B). Interestingly, when we analyzed cytokine production by the splenic iNKT cell subsets, we found that the reduction in IL-4 production was present in both iNKT cell subsets, but was more pronounced in the iNKT CD4 − subset, which is in accordance with the results observed in Fabry disease patients (Figs. 3C, D, 4D and E). In the liver, no significant differences were found regarding IL-4 production (Fig. 4). For IFN-γ no significant differences were found in both organs tested; however, there
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was a tendency towards a lower production of this cytokine by both splenic and hepatic iNKT cells (Fig. 4). 4. Discussion In the present study, we analyzed iNKT cells, their subsets and respective cytokine production in Fabry disease patients. To identify iNKT cells, we used the CD1d tetramer loaded with PBS57. This is a more specific approach than that used in previous studies where an antibody against Vα24 was used, since there are T cells expressing Vα24 that are not iNKT cells [7]. Although we found no differences in the total percentage of iNKT cells, we found a significant decrease in the iNKT CD4 + subset accompanied by an increase in the iNKT DN subset. This alteration is in accordance with our recent findings in the Fabry disease mouse model [24]. In humans, iNKT cell subsets are known to have distinct origins and proliferation and survival requirements. iNKT CD4 + cell peripheral population is sustained mainly by thymic output, whereas iNKT CD4 − cells proliferate essentially at the periphery [28]. Furthermore, while iNKT CD4 + cells respond better to IL-7, iNKT CD4 − cells proliferate more efficiently in the presence of IL-15 [28]. Therefore, an alteration in the thymus or in the availability of the IL-15 or IL-7 in Fabry disease patients might explain the differences observed in this study. Enzyme deficiency and consequent lipid accumulation in Fabry disease may also have a direct effect on iNKT cell development, proliferation and survival. It was shown in mice that the deficiency of α-galactosidase A leads to the activation of iNKT cells, both in vitro and in vivo [29]. Furthermore, iNKT cells from Fabry disease mice display an activated phenotype and an increased apoptosis, consistent with chronic exposure to self-antigens [29]. This indicates that the accumulated material in Fabry disease might truly stimulate iNKT cells. On the other hand, it was recently shown that glycolipid accumulation in splenic dendritic cells from Fabry disease mice was responsible for a reduction in their antigen presentation capacity, resulting in a lower stimulation of iNKT cells [30]. In both studies, the effects of the enzyme deficiency on iNKT cell subsets were not analyzed. This would be important because it is possible that the enzyme deficiency
Fig. 3. IL-4 and IFN-γ production by expanded iNKT cells from control subjects and Fabry disease patients. Expanded iNKT cells were stimulated with PMA/ionomycin and cytokine production was determined by flow cytometry. A — Representative example of cytokine versus isotype control staining. B — IL-4 and IFN-γ expression by total iNKT cells. C — IL-4 and IFN-γ expression by iNKT CD4+ cells. D — IL-4 and IFN-γ expression by iNKT CD4− cells. *pb 0.05; **p b 0.01.
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Fig. 4. IL-4 and IFN-γ production by splenic and hepatic iNKT cells in Fabry and control mice. Control or Fabry disease mice were injected with 2μg of α-GalCer or the equivalent volume of saline solution. A — Representative example of cytokine production in mice injected with α-GalCer versus saline solution. B — IL-4 expression by splenic and hepatic iNKT cells. C — IFN-γ expression by splenic and hepatic iNKT cells. D — IL-4 expression by the iNKT CD4+ cell subset. E — IFN-γ expression by iNKT CD4+ cell subset. F — IL-4 expression by the iNKT CD4− cell subset. G — IFN-γ expression by the iNKT CD4− cell subset. n (control) = 5; n (Fabry) = 5. *p b 0.05; **p b 0.01; ***p b 0.001.
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and consequent lipid accumulation leads to a preferential stimulation of one of the iNKT cell subsets. This could explain the alterations in the iNKT cell subsets in Fabry disease patients that we found in our study. A reduction in the iNKT CD4 + cells was also described in the mouse model of NPC2 deficiency, but not of GM1 gangliosidosis [22]. This indicates that the iNKT CD4 + reduction occurs due to a specific defect and not due to a general lysosomal impairment. We also analyzed the effect of ERT on iNKT cells and iNKT cell subsets using two different approaches. We compared the percentage of iNKT cell and iNKT cell subsets between patients under ERT (n = 4) and not under ERT (n = 11) and found no significant differences. However, an effect of ERT on iNKT cells cannot be excluded, because the number of patients under treatment is small and there is a high variability between subjects' iNKT cell populations. Then, we analyzed the percentage of iNKT cells and iNKT cell subsets in the 4 Fabry disease patients over 28 months of ERT. We found no significant variations on iNKT cell populations, except for a decrease in the iNKT CD8 + population in the first months after the beginning of the treatment. The study of Fabry disease patients before starting ERT would be important to confirm this reduction. We recently showed in the Fabry disease mouse model that ERT administered weekly from 4 to 12 weeks of age prevents the decrease in the iNKT CD4 + subset but does not correct the reduction already present at the start of the treatment in the 4 week-old mice. [24]. This might explain why we found no significant variations in the longitudinal study of these Fabry disease patients: ERT may be able to prevent a further decrease of iNKT CD4 + cells, but it seems unable to recover the lost population. The human iNKT cell subsets produce different amounts of proinflammatory and anti-inflammatory cytokines: iNKT CD4 + cells present a Th2 bias, while iNKT CD4 − cells present a shift towards a Th1 phenotype [11–13]. Here, we demonstrated that iNKT cell cytokine production is altered in both Fabry disease patients and mice. In Fabry disease patients we found a reduction in the percentage of cells producing IL-4 in both the iNKT CD4 − and iNKT CD4 + subsets, when compared to control subjects. However, this reduction did not reach statistical significance on total iNKT cells. This could be explained by the increased variation between subjects caused by the different percentages of iNKT CD4 + and CD4 − cells. In Fabry disease mice, injection of α-GalCer resulted in a lower percentage of splenic iNKT CD4 − and iNKT CD4 + cells expressing IL-4, when compared to control mice. Interestingly, in both humans and mice, the reduction was more pronounced in the iNKT CD4 − subset. The reduction in IL-4 production and simultaneously in the iNKT subset responsible for most of its production (iNKT CD4 +) strongly suggests a bias towards a Th1 phenotype in Fabry disease iNKT cells, which could result in a pro-inflammatory environment. Inflammation has been reported in several lysosomal storage disorders and its impact on disease pathology has been discussed [31,32]. Fabry disease patients present an overexpression of MHC class II in monocytes [19,26], which is in accordance with a pro-inflammatory state. Furthermore, a recent study analyzing Fabry disease patients under ERT, showed that the plasma level of the pro-inflammatory cytokine TNF-α is increased in patients compared to control subjects [33]. 5. Conclusion Fabry disease patients present a decrease in the percentage of iNKT CD4+ cells accompanied by an increase in iNKT DN cells. Moreover, these patients present a reduction in the production of the antiinflammatory cytokine IL-4 by both the iNKT CD4+ and iNKT CD4− cells. Consistently, Fabry disease mice also present a decrease in IL-4 production by splenic iNKT cells. These alterations suggest a proinflammatory status in Fabry disease, which can contribute to disease pathology. In this cohort, where 4 out of 15 patients were under ERT, no significant differences were observed between treated and untreated
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patients. However, the longitudinal study of patients under ERT suggests that the first months of ERT seem to promote a reduction of iNKT CD8+ cells. This reduction can have a positive effect on disease pathology since iNKT CD8+ cells are more pro-inflammatory than iNKT CD4+ cells. Our results show that iNKT cell population is altered in percentage and function in both Fabry disease mice and patient's. These alterations might contribute to Fabry disease pathology through the induction of inflammation. On the other hand, the imbalance of iNKT cell subsets suggests that the enzyme deficiency and consequent lipid accumulation in Fabry disease play a role in iNKT cell development and survival. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ymgme.2013.01.018. Role of the funding source The funding sources had no influence in the study design, data collection, analysis, or interpretation, manuscript writing or in the decision to submit the paper for publication. Acknowledgments This work is funded by FEDER Funds through the Operational Competitiveness Programme — COMPETE and by National Funds through FCT — Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-015955 [PTDC/SAU-ORG/110112/2009]. Financial support was also given by Shire Human Genetics, USA, the Piaget Institute, Portugal, and the Portuguese Society of Metabolic diseases through a Genzyme research fellowship. The authors would like to thank the National Institutes of Health (NIH), USA, for providing the CD1d-PBS57 tetramer, the National Cancer Institute (NCI), USA for providing recombinant human IL-2 and the Instituto Português do Sangue and the Immuno-Haemotherapy department of Hospital de São João, Porto, for providing blood samples of control subjects. We also thank Dr. Emanuel Correia for Fabry disease patients' recruitment, Professor R.O. Brady for kindly providing the Fabry knockout mice and Dr. Lorena G Rodrigues for management of mice colonies. References [1] J.M. Aerts, J.E. Groener, S. Kuiper, W.E. Donker-Koopman, A. Strijland, R. Ottenhoff, C. van Roomen, M. Mirzaian, F.A. Wijburg, G.E. Linthorst, A.C. Vedder, S.M. Rombach, J. Cox-Brinkman, P. Somerharju, R.G. Boot, C.E. Hollak, R.O. Brady, B.J. Poorthuis, Elevated globotriaosylsphingosine is a hallmark of Fabry disease, Proc. Natl. Acad. Sci. 105 (2008) 2812–2817. [2] R.J. Desnick, Y.A. Ioannou, C.M. Eng, Alpha-galactosidase A deficiency: Fabry disease, The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, pp. 3733–3774. [3] D.P. Germain, Fabry disease, Orphanet J. Rare Dis. 5 (2010) 30. [4] C.M. Eng, N. Guffon, W.R. Wilcox, D.P. Germain, P. Lee, S. Waldek, L. Caplan, G.E. Linthorst, R.J. Desnick, Safety and efficacy of recombinant human α-galactosidase A replacement therapy in Fabry's disease, N Engl J. Med. 345 (2001) 9–16. [5] A. Pisani, B. Visciano, G.D. Roux, M. Sabbatini, C. Porto, G. Parenti, M. Imbriaco, Enzyme replacement therapy in patients with Fabry disease: state of the art and review of the literature, Mol. Genet. Metab. 107 (2012) 267–275. [6] D.I. Godfrey, S. Stankovic, A.G. Baxter, Raising the NKT cell family, Nat. Immunol. 11 (2010) 197–206. [7] S.P. Berzins, M.J. Smyth, A.G. Baxter, Presumed guilty: natural killer T cell defects and human disease, Nat. Rev. Immunol. 11 (2011) 131–142. [8] L. Lynch, M. Nowak, B. Varghese, J. Clark, Andrew E. Hogan, V. Toxavidis, Steven P. Balk, D. O'Shea, C. O'Farrelly, Mark A. Exley, Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production, Immunity 37 (2012) 574–587. [9] E. Vivier, S. Ugolini, D. Blaise, C. Chabannon, L. Brossay, Targeting natural killer cells and natural killer T cells in cancer, Nat. Rev. Immunol. 12 (2012) 239–252. [10] M.A. Exley, L. Lynch, B. Varghese, M. Nowak, N. Alatrakchi, S.P. Balk, Developing understanding of the roles of CD1d-restricted T cell subsets in cancer: reversing tumor-induced defects, Clin. Immunol. 140 (2011) 184–195. [11] J.E. Gumperz, S. Miyake, T. Yamamura, M.B. Brenner, Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining, J. Exp. Med. 195 (2002) 625–636.
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Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Invariant natural killer T cells are phenotypically and functionally altered in Fabry disease Catia S. Pereira a, Olga Azevedo b, M. Luz Maia a, Ana F. Dias a, Clara Sa-Miranda a, M. Fatima Macedo a, c, d,⁎ a
Lysosome and Peroxisome Biology Unit (UniLiPe), IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre nº 823, 4150-180, Porto, Portugal Serviço de Cardiologia, Centro Hospitalar do Alto Ave, Portugal ESS Jean Piaget, Vila Nova de Gaia, Portugal d SACS, Universidade de Aveiro, Portugal b c
a r t i c l e
i n f o
Article history: Received 24 December 2012 Received in revised form 27 January 2013 Accepted 27 January 2013 Available online 1 February 2013 Keywords: Lysosomal storage disorders Fabry disease Enzyme replacement therapy iNKT cells Cytokines
a b s t r a c t Fabry disease is a lysosomal storage disease belonging to the group of sphingolipidoses. In Fabry disease there is accumulation of mainly globotriaosylceramide due to deficiency of the lysosomal enzyme α-galactosidase A. The lysosome is an important compartment for the activity of invariant natural killer T (iNKT) cells. iNKT cells are lipid-specific T cells that were shown to be important in infection, autoimmunity and tumor surveillance. In several mouse models of lysosomal storage disorders there is a decrease in iNKT cell numbers. Furthermore, alterations on iNKT cell subsets have been recently described in the Fabry disease mouse model. Herein, we analyzed iNKT cells and their subsets in Fabry disease patients. Although there were no differences in the percentage of iNKT cells between Fabry disease patients and control subjects, Fabry disease patients presented a reduction in the iNKT CD4 + cells accompanied by an increase in the iNKT DN cells. Since iNKT cell subsets produce different quantities of pro-inflammatory and antiinflammatory cytokines, we analyzed IFN-γ and IL-4 production by iNKT cells of Fabry disease patients and mice. We found a significant reduction in the production of IL-4 by mice splenic iNKT cells and human iNKT cell subsets, but no significant alterations in the production of IFN-γ. Altogether, our results suggest a bias towards a pro-inflammatory phenotype in Fabry disease iNKT cells. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Lysosomal storage disorders are a group of inherited metabolic defects that arise mainly due to abnormalities in lysosomal hydrolases being characterized by the accumulation of different types of undegraded substrates in the lysosome. They can be classified according to the type of material that is accumulated. Fabry disease is a sphingolipidosis caused by mutations in the GLA gene, which encodes the lysosomal hydrolase α-galactosidase A. The decrease in the activity of this enzyme leads to the progressive lysosomal accumulation of mainly globotriaosylceramide (Gb3). However, secondary accumulation of other lipids such as lyso-Gb3
Abbreviations: iNKT, invariant natural killer T; α-GalCer, α-galactosylceramide; Gb3, globotriaosylceramide; ERT, enzyme replacement therapy; PMA, phorbol 12-myristate 13-acetate; TCR, T cell receptor. ⁎ Corresponding author at: Lysosome and Peroxisome Biology Unit (UniLiPe), Institute for Molecular and Cell Biology (IBMC), Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Fax: +351 226099157. E-mail addresses: [email protected] (C.S. Pereira), [email protected] (O. Azevedo), [email protected] (M.L. Maia), [email protected] (A.F. Dias), [email protected] (C. Sa-Miranda), [email protected] (M.F. Macedo). 1096-7192/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymgme.2013.01.018
is also detected [1]. Lipid accumulation is more pronounced in capillary endothelial, renal, cardiac and nerve cells [2]. Consequently, the clinical manifestations of Fabry disease include left ventricular hypertrophy and dysfunction, dysrhythmias and cardiac conduction abnormalities; proteinuria and renal failure; and acroparesthesias, neuropathic pain, stroke and white matter lesions [3]. Enzyme replacement therapy (ERT) is available for Fabry disease patients since 2001. This treatment was shown to efficiently promote de clearance of the endothelial deposits of Gb3 in the skin, heart and kidney [4]. ERT was also demonstrated to reduce myocardial mass, improving left ventricular function, to reduce or halt the decline of glomerular filtration rate and to improve pain and quality of life [5]. The lysosome, whose function is impaired in Fabry disease, is an important compartment for the immune response and especially for the lipid-specific T lymphocytes activity. Invariant natural killer T (iNKT) cells are lipid-specific T lymphocytes that are characterized by the expression of a semi-invariant T cell receptor (TCR) constituted by and invariant TCR Vα chain (Vα24Jα18 in humans and Vα14Jα18 in mice) paired with a limited repertoire of TCR Vβ chains [6]. iNKT cells recognize lipid antigens when they are associated with MHC class I-like CD1d molecules. These cells have a low frequency in peripheral blood and are more abundant in liver and fat tissue [7,8].
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Since peripheral blood is the main biological sample available from humans, iNKT cell studies are often challenging. Several studies described an important role of iNKT cells in anti-tumor, autoimmune and anti-microbial responses. Intrinsic iNKT cell numerical and functional abnormalities have been reported in several human diseases, such as type I diabetes, multiple sclerosis, cancers and infections [7]. iNKT cells are also important targets of cancer immunotherapy [9,10]. Currently, clinical trials involving iNKT cells are being conducted for melanoma and multiple myeloma [10]. Human iNKT cells can be divided in three subsets according to the CD4 and CD8 expression: positive only for CD4 (iNKT CD4 +), only for CD8 (iNKT CD8 +), or negative for both molecules (iNKT DN). These subsets have different cytokine production profiles. When stimulated ex-vivo, iNKT CD4 + cells produce both Th1 and Th2 cytokines while iNKT CD4 − cells produce mainly Th1 cytokines [11,12]. When iNKT cells are expanded in vitro and then stimulated, iNKT CD4 + cells produce more Th2 cytokines and iNKT CD4 − cells produce more Th1 cytokines [13]. The most potent known antigen for both human and murine iNKT cells is α-galactosylceramide (α-GalCer). However, this compound is not present in mammals. In the past years, a few mammalian lipids have been identified as iNKT cell antigens, including sphingolipids (β-glucosylceramide) and phospholipids (etherbonded lysophosphatidylethanolamine) [14,15]. These antigens stimulate human iNKT cells at different degrees and might be involved in the process of thymic selection and peripheral activation. iNKT cells were studied in several mouse models of lysosomal storage disorders. A reduction of iNKT cell percentage was found in Sandhoff disease [16–18], Tay–Sachs disease [17], Fabry disease [17,19,20], NPC1 deficiency [17,21], NPC2 deficiency [22,23], GM1 gangliosidosis [17,22] and multiple sulfatase deficiency [18]. iNKT CD4+ and CD4− cell subsets were also analyzed in GM1 gangliosidosis, NPC2 deficiency and Fabry disease mouse models. GM1 gangliosidosis mice presented no alterations in iNKT cell subsets, while Fabry disease mice exhibited a decrease in splenic iNKT CD4+ cells and NPC2 deficient mice displayed a reduction in the hepatic iNKT CD4+ subset [22,24]. Studies in patients with lysosomal storage disorders are scarce. iNKT cell percentage was analyzed in patients with Gaucher, Fabry and Niemann–Pick C diseases, but no reduction was found in these diseases [19,25–27]. Here, we analyzed Fabry disease patients, regarding the iNKT cell phenotype and function. In addition, iNKT cell function was assessed in Fabry disease mice. We also analyzed the effect of ERT overtime on iNKT cells and subsets of Fabry disease patients.
2. Methods 2.1. Population studied This study included 15 Fabry disease patients, previously diagnosed in our laboratory and regularly followed up and treated at the Cardiology departments of Centro Hospitalar do Alto Ave, Guimarães, Portugal (n = 14) and Hospital de São Teotónio, Viseu, Portugal (n = 1). They were all adult Caucasian patients (mean age of 48 ±14) and gave informed consent to participate in the study. At the beginning of this study, 4 patients had begun ERT 4 months before with agalsidase alfa — Replagal© (Shire Human Genetic Therapies). The patients that were under treatment were analyzed longitudinally, every 4 months, over a total period of 28 months. Patient 1 died in an accident and was only analyzed during 16 months. Unless stated, all the results presented in the results section (Section 3) correspond to the first determination obtained for each patient. Twenty-seven blood donors from the Instituto Português do Sangue or from the Immuno-haemotherapy department of Hospital de São João, Porto, Portugal were studied as controls. The mean age of controls was 41 ± 12.
2.2. Mononuclear cells isolation from human peripheral blood, iNKT cell expansion and analysis of their cytokine production profile Peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque-1077® (Sigma) density centrifugation, following the manufacturer's instructions. iNKT cells were expanded by culturing PBMCs in RPMI 10% iFBS medium, at a concentration of 0.5× 106 cells/mL, with 100 ng/mL of α-GalCer (Alexis-Coger SA) in a 24 well plate. At day 1 of culture, 100 U/mL of recombinant human IL-2 (kindly provided by the National Cancer Institute) was added. After 11–14 days, cells were collected, counted and stimulated with 25 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma), 1 μg/mL ionomycin (Sigma) and 10 μg/mL brefeldin A (Sigma) for 5 h before staining with cytokinespecific monoclonal antibodies. 2.3. Analyzes of cytokine production by mouse iNKT cells Fabry disease mice (GLA−/−) with C57BL/6 background were provided by the National Institutes of Health (Bethesda, MD, USA) and maintained at the IBMC-INEB animal facility (IBMC, Porto, Portugal). C57BL/6J mice were used as wild type controls. All procedures were performed in conformity with the institutional and national guidelines. The animals were analyzed at 12–15 weeks of age. The control and the Fabry disease mice were injected with 2 μg of α-GalCer, intraperitoneal (i.p.), diluted at 10μg/mL in saline solution or with saline solution alone. Two hours after injection, the mice were sacrificed, the spleen was removed and the liver was perfused and harvested as previously described [24]. Single-cell suspensions of hepatic mononuclear cells and splenocytes were prepared as previously described [24]. After cell suspension preparation, cells were counted and resuspended in RPMI supplemented with 10% iFBS and 10 μg/mL of Brefeldin A (Sigma), at a concentration of 5 ×106 cells/mL. Cells were then plated in 96-round bottomed well plates (1×106 cells/well) and incubated for 2 h, at 37 °C, 5% CO2. After the end of the incubation period, cells were stained for determination of cytokine production by flow cytometry. 2.4. Flow cytometry Human PBMCs were stained with antibody/CD1d tetramer cocktails diluted in PBS/2%FBS/0.01%NaN3 (flow cytometry solution) for 20 min, at 4 °C, in the dark. For PBMCs, the staining mixture was composed by anti-human CD3 (SK7), anti-human CD4 (RPA-T4), anti-human CD8 (RPA-T8) and anti-human CD161 (HP-3G10) antibodies (all from eBioscience) and the human CD1d tetramer loaded with PBS57 (from the National Institute of Health tetramer core facility). After staining, cells were washed with flow cytometry solution and then fixed with PBS 1% formaldehyde. For cytokine detection studies, human in vitro expanded iNKT cells or mice splenocytes and hepatic mononuclear cells were first stained with antibody/tetramer cocktail diluted in flow cytometry solution. For splenocytes and hepatic mononuclear cells the following antibodies were used together with the mouse CD1d tetramer loaded with PBS57: anti-mouse CD3 (17A2, Biolegend); anti-mouse CD4 (RM4-5, BD Biosciences). After extracellular staining, cells were washed, and then fixed by incubating with PBS 2% formaldehyde for 10min, at room temperature, in the dark. Cells were permeabilized by incubating with 0.5% saponin (Sigma) diluted in flow cytometry solution, for 5 min and washed again. Cells were then stained for 30 min, at room temperature. For human cells the following antibodies were used: anti-human IFN-γ (4S.B3) and anti-human IL-4 (8D4-8) antibodies or the corresponding isotype controls (all from eBioscience). For mice cells, anti-mouse IFN-γ (XMG1.2) and anti-mouse IL-4 (11B11) antibodies (both from Biolegend) were used. After incubation, cells were washed and resuspended in PBS. Samples were acquired on a 3-laser BD FACS Canto™ II flow cytometer using BD FACSDiva™
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software (BD Biosciences). All flow cytometry analyses were performed using the FlowJo software (Tree Star).
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3. Results 3.1. iNKT CD4 + cells are reduced in Fabry disease patients
2.5. Statistical analysis All statistical analyses were performed using GraphPad Prism 5 software. Shapiro–Wilk test was applied to determine the normal distribution of the variables. Student's t-test was used for normally distributed variables. For variables that did not follow a normal distribution, Mann–Whitney U test was used. Values of p b 0.05 were considered statistically significant.
We analyzed the percentage of iNKT cells in Fabry disease patients by flow cytometry using an anti-CD3 antibody and the PBS57 loaded CD1d tetramer (Fig. 1A). In accordance with the previous studies, which analyzed the percentage of Vα24 + cells, we found no differences between control subjects and patients regarding the percentage of iNKT cells over the total of CD3 + cells (Fig. 1B). We further characterized iNKT cells using the cell surface markers CD4, CD8 and CD161. Interestingly, Fabry disease patients presented a decrease
Fig. 1. iNKT cell percentage and phenotype in control subjects and Fabry disease patients. A — iNKT cell gating strategy. B — iNKT cell percentage in T cells. C — CD4+ cell percentage in iNKT cells. D — CD8+ cell percentage in iNKT cells. E — DN cell percentage in iNKT cells. F — CD161+ cells percentage in iNKT cells. Filled squares (■) represent patients receiving ERT. *p b 0.05.
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in the percentage of iNKT CD4 + cells and an increase in the percentage of iNKT DN cells when compared to control subjects (Figs. 1C and E). iNKT CD8+ cells presented no significant alterations (Fig. 1D). The 15 Fabry disease patients under study included 4 patients submitted to ERT for 4 months. When the patients receiving ERT were compared to patients not receiving ERT, no significant differences were found (Fig. 1). To determine if the reduction observed in the iNKT CD4 + cell subset was specific for iNKT cells, we analyzed the percentage of CD4 + and CD8 + T cells in total lymphocytes. Fabry disease patients presented no alterations in the percentage of CD4+ or CD8+ T cells (Supplemental Figs. S1A and B). Then, we determined the percentage of iNKT cells expressing the natural killer cell marker CD161. No differences were found in this subset, when comparing patients with control subjects or patients under ERT with untreated patients (Fig. 1F). We performed a longitudinal evaluation of the iNKT cell percentage and phenotype overtime for each of the 4 patients under ERT. We analyzed these 4 Fabry disease patients every 4 months, starting 4 months after the beginning of ERT and over a total period of 24 months. As shown in Fig. 2, the percentages of iNKT cells and iNKT CD4 + and DN cells do not present major variations within the
individuals, overtime. It is only the iNKT CD8 + subset that seems slightly decreased during the first 8 months of ERT. The percentage of iNKT CD161 + cells remained stable, over a year, in the tested individuals (Fig. 2E). 3.2. Fabry disease patients present an imbalance in iNKT cell cytokine production The imbalance in iNKT cell subsets found in Fabry disease patients suggested a functional alteration in the iNKT cell pool, since iNKT cell subsets have distinct cytokine production profiles. Therefore, we hypothesized that iNKT cell cytokine production might be altered in Fabry disease patients. To test this hypothesis, the expanded iNKT cells were stimulated using PMA and ionomycin for 5 h. During this time, cells were cultured with brefeldin A to avoid cytokine secretion, allowing the cytokine production determination by flow cytometry. An isotype control was used to define flow cytometry gates (Fig. 3A). We analyzed 11 control subjects and 10 Fabry disease patients (not under ERT). In Fabry disease patients, the production of IFN-γ by iNKT cells was not significantly altered (Fig. 3B). However, there was a tendency towards a reduction in the percentage of iNKT cells producing IL-4 (Fig. 3B). Interestingly, the analysis of IL-4
Fig. 2. Longitudinal study of iNKT cells in Fabry disease patients. The percentage of iNKT cells and iNKT cell subsets was studied in Fabry disease patients 1 to 4, over a period of 28 months of enzyme replacement therapy. A — iNKT cell percentage. B — iNKT CD4+ cell percentage. C — iNKT CD8+ cell percentage. D — iNKT DN cell percentage. E — iNKT CD161+ cell percentage.
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production by the iNKT cell subsets revealed a significant decrease in the percentage of IL-4 producing cells in both the CD4 + and CD4 − iNKT cell subsets, but no alterations in IFN-γ production (Figs. 3C and D). When we analyzed the co-production of IFN-γ and IL-4 in the iNKT cell subsets, we observed that the reduction in total IL-4 production is mainly due to a decrease of cells producing both IFN-γ and IL-4, with an increase in cells producing only IFN-γ (Fig. S2).
3.3. The imbalances in cytokine production by the iNKT cells are also present in the Fabry disease mouse model Due to the low percentage of iNKT cells in human peripheral blood, ex vivo analysis of cytokine production is difficult. Therefore, the analysis of cytokine production by patients' iNKT cells was based in cultured cells that might acquire different characteristics from those present in vivo. Furthermore, the higher variability existent between human subjects often hides the presence of small differences. We had previously described a reduction in the splenic iNKT CD4 + cell subset in the Fabry disease mouse model [24], which is consistent with the results presented here for Fabry disease patients peripheral blood. To test in vivo the capacity of cytokine production of iNKT cells, we injected Fabry and control mice with the lipid antigen α-GalCer or the corresponding volume of saline solution. After 2 h, mice were sacrificed and the liver and spleen were collected for analysis. iNKT cell cytokine production was analyzed by flow cytometry (Fig. 4A). Mice injected with saline displayed no IL-4 or IFN-γ production by iNKT cells, confirming that these cells were responding to α-GalCer stimuli (Fig. 4A). We found a significant reduction in the percentage of IL-4 producing iNKT cells in the spleen of Fabry disease mice when compared to control mice (Fig. 4B). Interestingly, when we analyzed cytokine production by the splenic iNKT cell subsets, we found that the reduction in IL-4 production was present in both iNKT cell subsets, but was more pronounced in the iNKT CD4 − subset, which is in accordance with the results observed in Fabry disease patients (Figs. 3C, D, 4D and E). In the liver, no significant differences were found regarding IL-4 production (Fig. 4). For IFN-γ no significant differences were found in both organs tested; however, there
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was a tendency towards a lower production of this cytokine by both splenic and hepatic iNKT cells (Fig. 4). 4. Discussion In the present study, we analyzed iNKT cells, their subsets and respective cytokine production in Fabry disease patients. To identify iNKT cells, we used the CD1d tetramer loaded with PBS57. This is a more specific approach than that used in previous studies where an antibody against Vα24 was used, since there are T cells expressing Vα24 that are not iNKT cells [7]. Although we found no differences in the total percentage of iNKT cells, we found a significant decrease in the iNKT CD4 + subset accompanied by an increase in the iNKT DN subset. This alteration is in accordance with our recent findings in the Fabry disease mouse model [24]. In humans, iNKT cell subsets are known to have distinct origins and proliferation and survival requirements. iNKT CD4 + cell peripheral population is sustained mainly by thymic output, whereas iNKT CD4 − cells proliferate essentially at the periphery [28]. Furthermore, while iNKT CD4 + cells respond better to IL-7, iNKT CD4 − cells proliferate more efficiently in the presence of IL-15 [28]. Therefore, an alteration in the thymus or in the availability of the IL-15 or IL-7 in Fabry disease patients might explain the differences observed in this study. Enzyme deficiency and consequent lipid accumulation in Fabry disease may also have a direct effect on iNKT cell development, proliferation and survival. It was shown in mice that the deficiency of α-galactosidase A leads to the activation of iNKT cells, both in vitro and in vivo [29]. Furthermore, iNKT cells from Fabry disease mice display an activated phenotype and an increased apoptosis, consistent with chronic exposure to self-antigens [29]. This indicates that the accumulated material in Fabry disease might truly stimulate iNKT cells. On the other hand, it was recently shown that glycolipid accumulation in splenic dendritic cells from Fabry disease mice was responsible for a reduction in their antigen presentation capacity, resulting in a lower stimulation of iNKT cells [30]. In both studies, the effects of the enzyme deficiency on iNKT cell subsets were not analyzed. This would be important because it is possible that the enzyme deficiency
Fig. 3. IL-4 and IFN-γ production by expanded iNKT cells from control subjects and Fabry disease patients. Expanded iNKT cells were stimulated with PMA/ionomycin and cytokine production was determined by flow cytometry. A — Representative example of cytokine versus isotype control staining. B — IL-4 and IFN-γ expression by total iNKT cells. C — IL-4 and IFN-γ expression by iNKT CD4+ cells. D — IL-4 and IFN-γ expression by iNKT CD4− cells. *pb 0.05; **p b 0.01.
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Fig. 4. IL-4 and IFN-γ production by splenic and hepatic iNKT cells in Fabry and control mice. Control or Fabry disease mice were injected with 2μg of α-GalCer or the equivalent volume of saline solution. A — Representative example of cytokine production in mice injected with α-GalCer versus saline solution. B — IL-4 expression by splenic and hepatic iNKT cells. C — IFN-γ expression by splenic and hepatic iNKT cells. D — IL-4 expression by the iNKT CD4+ cell subset. E — IFN-γ expression by iNKT CD4+ cell subset. F — IL-4 expression by the iNKT CD4− cell subset. G — IFN-γ expression by the iNKT CD4− cell subset. n (control) = 5; n (Fabry) = 5. *p b 0.05; **p b 0.01; ***p b 0.001.
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and consequent lipid accumulation leads to a preferential stimulation of one of the iNKT cell subsets. This could explain the alterations in the iNKT cell subsets in Fabry disease patients that we found in our study. A reduction in the iNKT CD4 + cells was also described in the mouse model of NPC2 deficiency, but not of GM1 gangliosidosis [22]. This indicates that the iNKT CD4 + reduction occurs due to a specific defect and not due to a general lysosomal impairment. We also analyzed the effect of ERT on iNKT cells and iNKT cell subsets using two different approaches. We compared the percentage of iNKT cell and iNKT cell subsets between patients under ERT (n = 4) and not under ERT (n = 11) and found no significant differences. However, an effect of ERT on iNKT cells cannot be excluded, because the number of patients under treatment is small and there is a high variability between subjects' iNKT cell populations. Then, we analyzed the percentage of iNKT cells and iNKT cell subsets in the 4 Fabry disease patients over 28 months of ERT. We found no significant variations on iNKT cell populations, except for a decrease in the iNKT CD8 + population in the first months after the beginning of the treatment. The study of Fabry disease patients before starting ERT would be important to confirm this reduction. We recently showed in the Fabry disease mouse model that ERT administered weekly from 4 to 12 weeks of age prevents the decrease in the iNKT CD4 + subset but does not correct the reduction already present at the start of the treatment in the 4 week-old mice. [24]. This might explain why we found no significant variations in the longitudinal study of these Fabry disease patients: ERT may be able to prevent a further decrease of iNKT CD4 + cells, but it seems unable to recover the lost population. The human iNKT cell subsets produce different amounts of proinflammatory and anti-inflammatory cytokines: iNKT CD4 + cells present a Th2 bias, while iNKT CD4 − cells present a shift towards a Th1 phenotype [11–13]. Here, we demonstrated that iNKT cell cytokine production is altered in both Fabry disease patients and mice. In Fabry disease patients we found a reduction in the percentage of cells producing IL-4 in both the iNKT CD4 − and iNKT CD4 + subsets, when compared to control subjects. However, this reduction did not reach statistical significance on total iNKT cells. This could be explained by the increased variation between subjects caused by the different percentages of iNKT CD4 + and CD4 − cells. In Fabry disease mice, injection of α-GalCer resulted in a lower percentage of splenic iNKT CD4 − and iNKT CD4 + cells expressing IL-4, when compared to control mice. Interestingly, in both humans and mice, the reduction was more pronounced in the iNKT CD4 − subset. The reduction in IL-4 production and simultaneously in the iNKT subset responsible for most of its production (iNKT CD4 +) strongly suggests a bias towards a Th1 phenotype in Fabry disease iNKT cells, which could result in a pro-inflammatory environment. Inflammation has been reported in several lysosomal storage disorders and its impact on disease pathology has been discussed [31,32]. Fabry disease patients present an overexpression of MHC class II in monocytes [19,26], which is in accordance with a pro-inflammatory state. Furthermore, a recent study analyzing Fabry disease patients under ERT, showed that the plasma level of the pro-inflammatory cytokine TNF-α is increased in patients compared to control subjects [33]. 5. Conclusion Fabry disease patients present a decrease in the percentage of iNKT CD4+ cells accompanied by an increase in iNKT DN cells. Moreover, these patients present a reduction in the production of the antiinflammatory cytokine IL-4 by both the iNKT CD4+ and iNKT CD4− cells. Consistently, Fabry disease mice also present a decrease in IL-4 production by splenic iNKT cells. These alterations suggest a proinflammatory status in Fabry disease, which can contribute to disease pathology. In this cohort, where 4 out of 15 patients were under ERT, no significant differences were observed between treated and untreated
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patients. However, the longitudinal study of patients under ERT suggests that the first months of ERT seem to promote a reduction of iNKT CD8+ cells. This reduction can have a positive effect on disease pathology since iNKT CD8+ cells are more pro-inflammatory than iNKT CD4+ cells. Our results show that iNKT cell population is altered in percentage and function in both Fabry disease mice and patient's. These alterations might contribute to Fabry disease pathology through the induction of inflammation. On the other hand, the imbalance of iNKT cell subsets suggests that the enzyme deficiency and consequent lipid accumulation in Fabry disease play a role in iNKT cell development and survival. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ymgme.2013.01.018. Role of the funding source The funding sources had no influence in the study design, data collection, analysis, or interpretation, manuscript writing or in the decision to submit the paper for publication. Acknowledgments This work is funded by FEDER Funds through the Operational Competitiveness Programme — COMPETE and by National Funds through FCT — Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-015955 [PTDC/SAU-ORG/110112/2009]. Financial support was also given by Shire Human Genetics, USA, the Piaget Institute, Portugal, and the Portuguese Society of Metabolic diseases through a Genzyme research fellowship. The authors would like to thank the National Institutes of Health (NIH), USA, for providing the CD1d-PBS57 tetramer, the National Cancer Institute (NCI), USA for providing recombinant human IL-2 and the Instituto Português do Sangue and the Immuno-Haemotherapy department of Hospital de São João, Porto, for providing blood samples of control subjects. We also thank Dr. Emanuel Correia for Fabry disease patients' recruitment, Professor R.O. Brady for kindly providing the Fabry knockout mice and Dr. Lorena G Rodrigues for management of mice colonies. References [1] J.M. Aerts, J.E. Groener, S. Kuiper, W.E. Donker-Koopman, A. Strijland, R. Ottenhoff, C. van Roomen, M. Mirzaian, F.A. Wijburg, G.E. Linthorst, A.C. Vedder, S.M. Rombach, J. Cox-Brinkman, P. Somerharju, R.G. Boot, C.E. Hollak, R.O. Brady, B.J. Poorthuis, Elevated globotriaosylsphingosine is a hallmark of Fabry disease, Proc. Natl. Acad. Sci. 105 (2008) 2812–2817. [2] R.J. Desnick, Y.A. Ioannou, C.M. Eng, Alpha-galactosidase A deficiency: Fabry disease, The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, pp. 3733–3774. [3] D.P. Germain, Fabry disease, Orphanet J. Rare Dis. 5 (2010) 30. [4] C.M. Eng, N. Guffon, W.R. Wilcox, D.P. Germain, P. Lee, S. Waldek, L. Caplan, G.E. Linthorst, R.J. Desnick, Safety and efficacy of recombinant human α-galactosidase A replacement therapy in Fabry's disease, N Engl J. Med. 345 (2001) 9–16. [5] A. Pisani, B. Visciano, G.D. Roux, M. Sabbatini, C. Porto, G. Parenti, M. Imbriaco, Enzyme replacement therapy in patients with Fabry disease: state of the art and review of the literature, Mol. Genet. Metab. 107 (2012) 267–275. [6] D.I. Godfrey, S. Stankovic, A.G. Baxter, Raising the NKT cell family, Nat. Immunol. 11 (2010) 197–206. [7] S.P. Berzins, M.J. Smyth, A.G. Baxter, Presumed guilty: natural killer T cell defects and human disease, Nat. Rev. Immunol. 11 (2011) 131–142. [8] L. Lynch, M. Nowak, B. Varghese, J. Clark, Andrew E. Hogan, V. Toxavidis, Steven P. Balk, D. O'Shea, C. O'Farrelly, Mark A. Exley, Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production, Immunity 37 (2012) 574–587. [9] E. Vivier, S. Ugolini, D. Blaise, C. Chabannon, L. Brossay, Targeting natural killer cells and natural killer T cells in cancer, Nat. Rev. Immunol. 12 (2012) 239–252. [10] M.A. Exley, L. Lynch, B. Varghese, M. Nowak, N. Alatrakchi, S.P. Balk, Developing understanding of the roles of CD1d-restricted T cell subsets in cancer: reversing tumor-induced defects, Clin. Immunol. 140 (2011) 184–195. [11] J.E. Gumperz, S. Miyake, T. Yamamura, M.B. Brenner, Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining, J. Exp. Med. 195 (2002) 625–636.
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