Enzyme replacement therapy partially prevents invariant Natural Killer T cell deficiency in the Fabry disease mouse model

Molecular Genetics and Metabolism 106 (2012) 83–91

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Enzyme replacement therapy partially prevents invariant Natural Killer T cell deficiency in the Fabry disease mouse model Maria Fatima Macedo a, b, c,⁎, 1, Rui Quinta a, d, 1, Catia Sofia Pereira a, Maria Clara Sa Miranda a,⁎ 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 ESS Jean Piaget, Escola Superior de Saúde, Alameda Jean Piaget, 4405-678 Gulpilhares, Portugal Health Sciences Department, University of Aveiro, Aveiro, Portugal d School of Health Sciences, University of Minho, Campus de Gualtar, 4710-350, Braga, Portugal b c

a r t i c l e

i n f o

Article history: Received 23 December 2011 Received in revised form 23 February 2012 Accepted 23 February 2012 Available online 1 March 2012 Keywords: iNKT Fabry disease Lysosomal storage diseases Enzyme replacement therapy

a b s t r a c t Fabry disease is a lysosomal storage disease caused by deficient activity of the α-Galactosidase A (α-Gal A) enzyme, which leads to abnormal accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in the lysosome. Glycosphingolipids are known to be invariant Natural Killer T (iNKT) cell antigens. Several animal models of lysosomal storage diseases, including Fabry disease, present a defect in iNKT cell selection by the thymus. We have studied the effect of age and the impact of enzyme replacement therapy on Gb3 accumulation and iNKT cells of Fabry knockout mice. At 4 weeks of age, Fabry knockout mice already showed Gb3 accumulation and a reduction in the percentage of iNKT cells. In older mice (12-week old), we observed an accentuated peripheral iNKT deficiency. 12-week old animals also showed a reduced splenic CD4+/CD4 − iNKT cell ratio due to greater loss in the iNKT CD4 + subset. Treatment of Fabry knockout mice with α-Gal A replacement therapy efficiently reduced Gb3 deposition in the liver and spleen. Moreover, enzyme replacement therapy had a positive effect on the number of iNKT cells in an organ-dependent fashion. Indeed, treatment of Fabry knockout mice with α-Gal A did not alter iNKT cell percentage in the thymus and liver but increased splenic iNKT cell percentage when compared to untreated mice. Study of animals prior to treatment indicates that enzyme replacement therapy stabilized iNKT cell percentage in the spleen. This stabilization is due to a specific effect on the iNKT CD4 + subset, preventing the decrease on the number of these cells that occurs with age in Fabry knockout mice. This study reveals that enzyme replacement therapy has a positive organ and subset-dependent effect in iNKT cells of Fabry knockout mice. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Fabry disease is a rare X-linked lysosomal storage disease (LSD) caused by mutations in the GLA gene, which leads to a deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A). Loss of α-Gal A activity gives rise to a progressive accumulation of glycosphingolipids (GSL), mainly globotriaosylceramide (Gb3), in various tissues throughout the body. Additional accumulating materials include digalactosylceramide [1] and the deacylated form of Gb3 (lyso-Gb3) [2]. Lysosomal storage of Gb3 in endothelial cells is thought to cause a multisystemic vasculopathy with a wide spectrum

Abbreviations: ERT, enzyme replacement therapy; iNKT, invariant Natural Killer T. ⁎ Corresponding authors at: 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. Fax: + 351 226099157. E-mail addresses: [email protected] (M.F. Macedo), [email protected] (R. Quinta), [email protected] (C.S. Pereira), [email protected] (M.C. Sa Miranda). 1 The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 1096-7192/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2012.02.014

of clinical manifestations which often include renal failure, cardiac disease and cerebrovascular disease [3–5]. Enzyme replacement therapy (ERT), with α-Gal A, is the only currently available specific treatment for Fabry disease patients. ERT is effective in the clearance of Gb3 deposits in skin [6,7] and heart [6] microvascular endothelial cells. However, considerable variation in the efficacy of ERT is observed in other cell types [8,9]. Indeed, clearance of Gb3 deposits from kidney podocytes and distal tubular epithelium [8] or cardiac myocytes [9] is not as effective. This is probably due to a restricted access of the enzyme to these cell types, albeit clinical studies indicate that ERT improves cardiac and renal functions and the quality of life of Fabry disease patients [10]. Early therapeutic intervention is important as the best responses to ERT have been described in patients who have started therapy at an early phase of the disease [10–12]. It is well known that the Fabry knockout mouse (α-Gal A knockout mouse) reproduces biochemical abnormalities found in Fabry disease patients [2,13–15]. As a result of impaired catabolism, Gb3 deposition is observed in various tissues, including the skin, heart, liver, kidney, spleen, lung, intestine, brain and sciatic nerve [2,13–15]. In addition to lipid accumulation, these mice develop mild cardiac [16] and renal alterations [17]. The availability of Fabry knockout mice made it possible

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to undertake a series of critical experiments in regard to the evaluation of the efficacy of ERT in Fabry disease [18–25]. Collectively, amongst other findings, these studies have shown that ERT in Fabry knockout mice increases α-Gal A activity [18,19,21,22] and reduces lysosomal lipid accumulation [18,20,21,23–25] in an organ and dose-dependent manner. Uptake of α-Gal A in Fabry knockout mice seems to be more efficient in the liver, followed by the spleen, kidney and ultimately by the heart [18,19,21,22]. The mechanisms involved in the differential uptake of α-Gal A by different organs and cell types seem to be complex and cannot be fully explained by the distribution of the ubiquitous mannose-6-phosphate (M6P) receptor, which interacts with α-Gal A. The M6P receptor is known to be present in cardiac myocytes [26] but injection of α-Gal A into Fabry knockout mice did not show α-Gal A immunostaining in this cell type [22]. However, in the adrenal gland, which is not known to be particularly abundant in M6P receptors [27], administered α-Gal A was found to be present in the adrenal cortical zona fasciculata and in endothelial cells of the cortex and medulla [22]. The late endocytic and lysosomal compartments, where GSL accumulate in Fabry disease, are important compartments for the selection and peripheral function of invariant Natural Killer T (iNKT) cells. iNKT cells are lipid reactive T cells that express markers of Natural Killer (NK) cells and an invariant T cell receptor (TCR) alpha chain (Vα24-Jα18 in humans and Vα14-Jα18 in mice), which have the capacity to recognize foreign as well as self-lipid antigens presented by the MHC class I-like molecule CD1d [28]. iNKT cells, which in mice can be either CD4+ or CD4− CD8− doublenegative, are a thymus dependent population derived from CD4+ CD8+ double-positive thymocytes [29–31]. The most remarkable property of iNKT cells is their capacity to rapidly produce large amounts of various kinds of cytokines with potent immunomodulatory properties in response to their TCR engagement [28]. Human iNKT cell defects have been associated with various disease conditions, including cancer, infection and autoimmunity [32]. iNKT cells have also emerged as potential therapeutic targets in human diseases [33]. A decrease in the number of iNKT cells has been observed in several animal models of LSDs, including Fabry disease [34–37], Multiple sulfatase deficiency [38], GM1 gangliosidosis [34,39], Sandhoff disease [34,38,40], Niemann–Pick type C1 [34,41] and C2 [39,42] and TaySachs [34] disease. However, the alterations in iNKT cell number observed in Fabry knockout mice were not observed in the peripheral blood of Fabry disease patients [36,43]. Due to the limited availability of tissues, by virtue of ethical issues, the analysis of iNKT cells in organs of Fabry disease patients is restricted and has not yet been performed. There are currently two models that try to explain the fact that there is a decrease in the number of iNKT cells in animal models of LSDs: i) the accumulation of undegraded substrates in the lysosome, independently of the nature of the substrate that accumulates, would have a negative effect on the lipid antigen presentation capacity and a consequent reduction in the number of iNKT cells and ii) the specific enzymatic defects present in LSDs, leads to specific alteration in lipid antigen presentation, primarily due to the altered quantity and specificity of lipid antigens. Lipid deposition in adult Fabry knockout mice is known to increase with age [14,15]. In this work, we investigated whether young Fabry knockout mice (4-week old) already accumulate Gb3 or show alterations in iNKT cell number and subsets. In addition, we determined the effect of age on Gb3 accumulation and iNKT cells. Moreover, we evaluated whether in vivo treatment of young Fabry knockout mice with α-Gal A is able to prevent iNKT cell alterations previously observed in adult mice. 2. Material and methods 2.1. Mice and enzyme replacement therapy α-Galactosidase A− males and α-Galactosidase A−/− females (Fabry knockout mice) were provided by the National Institutes of

Health (Bethesda, MD, USA) and a colony was maintained at the Institute for Molecular and Cell Biology (IBMC, Porto, Portugal). Given the genetic background of this mouse colony, C57BL/6J mice were used as wild type controls. Food and water were provided ad libitum. All procedures were performed in conformity with the institution's guidelines and were performed by investigators with training in FELASA category C in laboratory animal science. All work with animals took place within the IBMC/INEB animal facility which is licensed for breeding and experiments with laboratory rodents. 4- and 12-week old male wild type and Fabry knockout mice were studied. ERT was provided to Fabry knockout mice, by weekly intravenous administration of α-Gal A (agalsidase alfa, Replagal©, Shire Human Genetics) at 1.5 mg/kg body weight, via the tail vein, from the fourth to the eleventh week of life. One group of wild type and one group of Fabry knockout mice received weekly infusions of saline solution during the same period. At week twelve, animals were sacrificed and organs were harvested. A minimum of three animals per group was used. 2.2. Gb3 extraction and UPLC-MS/MS quantification in mouse tissues Gb3 quantification was performed as previously described [44]. Briefly, to 100 μL of tissue homogenate, 100 μL of N-Heptadecanoyl ceramide trihexoside (Matreya LLC, PA, USA) was added as an internal standard. Total lipids were extracted with 2 mL of chloroform: methanol 2:1 (v/v) solution and the precipitated protein was removed by centrifugation at 3000 ×g for 10 min. 400 μL of water was added, samples were thoroughly vortexed and the two layers were separated by centrifugation. The lower layer, containing Gb3, was collected and dried under N2. Gb3 was desalted prior to MS by reconstitution in chloroform, followed by solid-phase-extraction on a C-18 column (Chromabond ®, Macherey-nagel, Düren, Germany) and, elution with acetone:methanol 9:1 (v/v). For quantification, the area counts for each isoform were determined and then summed to obtain the total Gb3 area counts. The ratio of the total Gb3 area counts to that of the internal standard was determined and used to calculate the final concentration of Gb3 in each sample. 2.3. Cell preparations Intrahepatic mononuclear cells were separated from mouse livers according to a previously described protocol [45], with minor modifications. In brief, after portal perfusion with DMEM medium (Invitrogen,CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, CA, USA), livers were dissected, homogenized and pressed through strainers. After washing, cells were layered over Percoll (Sigma-Aldrich, St. Louis, MO, USA) gradients and centrifuged for 10 min at 500 ×g. A red blood cell lysis step was performed. Thymus and spleen single cell suspensions were prepared by mechanical dissociation through a nylon cell strainer and washed with PBS. For splenic suspensions an additional red blood cell lysis step was performed. 2.4. Flow cytometry Hepatic, thymic and splenic cell suspensions were stained according to conventional protocols. In brief, 10 6 cells in staining solution (PBS, 0.2% Bovine Serum Albumin, 0.1% NaN3) were incubated for 20 min with combinations of monoclonal antibodies and the CD1dtetramer (loaded or unloaded), followed by three washes in staining solution. Cells were acquired in a FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Data was analyzed using the FlowJo software (TreeStar, Ashland, OR, USA). The following fluorochrome-conjugated monoclonal antibodies were used: rat anti-mouse CD4-FITC (clone RM4.4) and Armenian

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Hamster anti-mouse CD3ε-PE (clone 145-2C11). Antibodies were purchased from eBioscience (San Diego, CA, USA). The Allophycocianinconjugated CD1d-PBS57 tetramer and unloaded CD1d-tetramer were kindly provided by the National Institutes of Health tetramer core facility, Atlanta, GA, USA.

2.5. Immunofluorescent staining of α-galactosidase a and image processing Mouse tissues were collected and fixed in PLP (paraformaldehyde 2%/0.075 M lysine, 0.037 M sodium phosphate, 0.01 M sodium periodate) for 24 h. Tissue blocks were washed with 0.1 M Sorrensons phosphate buffer, embedded in Shandon Cryomatrix (Thermo Scientific, Waltham, MA, USA), snap-frozen, and sectioned with a Leica CM 3050 S Cryostat. Immunofluorescent staining was performed according to standard protocols, using a rabbit polyclonal antibody against α-Gal A (SH-006-CR0020-08; a kind gift of Shire Human Genetics, Cambridge, MA, USA), diluted 1:100, and a secondary antirabbit Alexa Fluor® 488 antibody, diluted 1:1000 (Invitrogen, CA, USA). Z-stack images were acquired using a Leica Scanning Confocal Microscope SP2 AOBS SE (Leica Microsystems, Germany), containing the LCS 2.61 software, a HC PL APO CS 40 xs/1.25 numerical aperture oil-objective, in an oil-based medium. Brightness and contrast adjustments were performed with the public domain ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.6. α-galactosidase A activity assay A fraction of mouse liver and spleen was used to determine α-Gal A activity. Tissue homogenates were obtained using a Heidolph RZR 2021 homogenizer, followed by 10 rounds of sonication at 40% cycle and 10% power. After centrifugation for 15 min at 13000 rpm and 4 °C, protein concentrations were determined in the supernatant of tissue homogenates and adjusted to 1 mg/mL with water. 10 μL of lysates were assayed for α-Gal A activity in the presence of 50 μL of 50 mM citrate; 100 mM-phosphate solution, pH 4.5, containing 2.5 mM fluorogenic α-Gal A substrate 4-methylumbelliferyl-α-Dgalactopyranoside (Glycosynth Ltd, Warrington, UK) and 100 mM N-acetyl-D-galactosamine (Sigma-Aldrich, St. Louis, MO, USA), a specific inhibitor of α-galactosidase B activity. After 1 h at 37 °C, reaction was stopped by adding 1 mL of 0.5 M glycine pH 10.0. Fluorescence intensities were measured using a Hitachi F-2000 spectrofluorometer with an excitation wavelength of 365 nm and an emission wavelength of 445 nm. Enzyme activities are expressed in nmol/h/mg protein.

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2.7. Statistical analysis The results were analyzed using the Student's unpaired t-test and One-way Anova with Tukey's Multiple Comparison test. p b 0.05 was considered significant. Data were statistically analyzed using GraphPad Prism 5 software (Graphpad Software, San Diego, CA, USA). 3. Results 3.1. The deficiency in the number of iNKT cells in untreated Fabry knockout mice aggravates with age and is accompanied by alteration in splenic iNKT cell subsets 3.1.1. Gb3 accumulation is already present in 4-week old untreated Fabry knockout mice and increases with age In order to evaluate the effect of age on lipid deposition of Fabry knockout mice, Gb3 was quantified in the liver and spleen of 4- and 12-week old Fabry knockout and wild type mice by UPLC-MS/MS. At a very young age (4 weeks old), Fabry knockout mice already showed a ≈230-fold elevation in hepatic and ≈50-fold elevation in splenic Gb3 when compared to age-matched wild type mice (Table 1). From the fourth to the twelfth week of life, liver Gb3 of Fabry knockout mice increases approximately four-fold and splenic Gb3 increases ≈1.7 times. 3.1.2. The deficiency in the number of iNKT cells in untreated Fabry knockout mice aggravates with age and is accompanied by alteration in splenic iNKT cell subsets The total number of thymocytes, splenocytes and hepatic leukocytes of wild type and Fabry knockout mice is available as supplemental data (Table S1). No statistically significant differences were observed, with the exception of thymic cells at 12 weeks of age, in which a reduction in thymocytes was found in Fabry knockout mice. The differentiation of thymocytes gives rise to mature T cells that then migrate to the peripheral organs like the spleen and liver. The numbers of splenic and hepatic T cells (identified by the expression of CD3) were similar between wild type and Fabry knockout mice (Table S1). Given the observed increase in Gb3 accumulation from the fourth to twelfth week of life of Fabry knockout mice, we hypothesized that this progressive accumulation of lipids would influence iNKT cells. Towards this aim iNKT cell number and iNKT cell subsets were quantified in the thymus, spleen and liver of 4- and 12-week old Fabry knockout and wild type mice by flow cytometry using CD1d tetramers loaded with the synthetic ligand PBS-57, anti-CD3 and anti-CD4 antibodies.

Table 1 Gb3 quantification by UPLC-MS/MS in liver and spleen of wild type and Fabry knockout mice. Strain

Wild type

Age (weeks)

4

Treatment

Tissue Gb3 (μg/g tissue weight) Liver Spleen

Fabry 12

4

12

12

Baseline

Saline

Baseline

Saline

α-Gal A

n=4

n = 10

n=4

n=4

(1.5 mg/kg) n=3

0.7 ± 0.6 31.4 ± 20.5

3.6 ± 1.3 36.9 ± 25.7

162.0 ± 34.9 1639 ± 330.2a

701.9 ± 292.7b 2817 ± 846.0b

c c

4.3 ± 1.0d 404.2 ± 92.3d

4-week old animals were perfused once a week with saline or α-Gal A (1.5 mg/kg), for a total period of 8 weeks. Tissues were harvested one week after the last injection. Data are expressed as mean ± SD. Statistically significant differences between groups of mice, with p b 0.001, analyzed by One-way Anova with Tukey's Multiple Comparison test are indicated by: a, 4-week Fabry vs 4-week wild type; b, 12-week saline perfused wild type vs 12 weeks saline perfused Fabry; c, 4-week Fabry vs 12 week-saline perfused Fabry; d , 12-week saline perfused Fabry vs 12-week α-Gal A treated Fabry.

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The organ-specific flow cytometry data is summarized in Fig. 1. Fabry knockout mice showed a reduction in the percentage of iNKT cells when compared to wild type mice (Fig. 1). At 4 weeks of age, absence of α-Gal A impaired thymic development of iNKT cells by ≈50%. An analogous reduction was observed in the spleen. The liver showed no alterations in 4-week old mice (Figs. 1 A, B). Similarly to what was observed in younger animals, the 12-week old thymic iNKT cell population was reduced by ≈50% when compared to wild type mice. 12-week old Fabry knockout mice showed a more severe decline in iNKT cell percentage in the periphery than 4-week old mice. The most significant reduction was observed in the spleen,

with ≈75% reduction in iNKT cell percentage when compared to wild type mice. 12-week old Fabry knockout mice also showed reduced frequency of liver iNKT cells when compared to wild type mice (≈39% decrease) (Fig. 1). Similar results were obtained when total iNKT cell numbers per organ were analyzed (Figs. 1C, D). The Fabry mice residual iNKT cell population was further analyzed for CD4 expression. This allows the study of CD4+ and CD4− iNKT cell subsets in Fabry knockout mice (Fig. 2). In 4-week old Fabry knockout mice, no alterations were observed in iNKT CD4+/CD4− ratio in all organs studied (data not shown). Similarly, in both the livers and thymi of 12-week old Fabry knockout mice, we found no

Fig. 1. Fabry knockout mice show a reduction in the percentage and number of iNKT cells that aggravates with age. Splenocytes, thymocytes, and purified liver leukocytes from 4and 12-week old Fabry knockout or wild type (WT) mice were analyzed by flow cytometry for the presence of iNKT cells. iNKT cells were identified by staining with an antibody against CD3 and the PBS-57/CD1d tetramer. Results of representative examples (A) or mean ± SD (B, C, D). 4 weeks of age: n = 4 animals per group; 12 weeks of age: n = 10 wild type and 4 Fabry knockout mice (*, p b 0.05; **, p b 0.01; ***, p b 0.001, unpaired student's t test).

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Fig. 2. Fabry knockout mice show a reduction in splenic iNKT CD4+/CD4 − ratio Thymic, splenic and hepatic iNKT cells were analyzed in 12-week old animals for CD4 expression by flow cytometry. Results of representative examples (A) or mean ± SD (B, C). n = 10 wild type and 4 Fabry knockout mice; (*, p b 0.05; ***, p b 0.001; unpaired student's t test).

alterations in iNKT CD4+/CD4− ratio (Figs. 2 A, B). In sharp contrast, splenic CD4+/CD4− cell ratio of 12-week old animals is reduced by ≈50% (Figs. 2 A, B). This means that the reduction in splenic iNKT CD4+ and CD4− subsets is not equivalent. Indeed, analysis of splenic CD4+ and CD4− iNKT cell numbers showed a more pronounced reduction of the CD4+ subset (Fig. 2C). 3.2. Enzyme replacement therapy reduces Gb3 storage and prevents the progression of splenic iNKT cell deficiency in Fabry knockout mice To determine whether treatment of Fabry knockout mice with αGal A was effective in the correction of the abnormal Gb3 storage, reduced iNKT cell number and reduced CD4+/CD4 − splenic iNKT cell ratio, 4 week old Fabry knockout mice were treated weekly, for eight consecutive weeks, with 1.5 mg/kg of human α-Gal A. Mice injected with saline solution were used as controls. Animals were sacrificed one week after the last enzyme administration. 3.2.1. Two months treatment of Fabry knockout mice with α-Gal A drastically reduces Gb3 storage In order to examine if the ERT protocol used was effective in the delivery of the enzyme to the liver and spleen of treated Fabry knockout mice, immunofluorescent staining for human α-Gal A in hepatic and splenic sections of treated and untreated Fabry knockout mice was performed. Both liver and spleen showed intense fluorescence staining for α-Gal A in treated Fabry knockout mice (Fig. 3). To assess the functionality of the administered enzyme in treated Fabry knockout mice, enzymatic activity of α-Gal A and Gb3 accumulation was evaluated. α-Gal A activity in the liver of Fabry knockout mice treated with α-Gal A was approximately 4-fold increased relatively to wild type mice (Table 2). Splenic enzymatic activity in αGal A injected Fabry knockout mice was ≈30% of the activity observed in wild type mice (Table 2). Consistent with the results obtained for α-Gal A activity, UPLCMS/MS Gb3 quantification showed that liver Gb3 was highly reduced (≈99%) in α-Gal A treated Fabry knockout mice when compared to

untreated Fabry knockout mice (Table 1). The Gb3 levels dropped below those observed prior to treatment (4-week old Fabry knockout mice, ≈97% reduction) and were similar to the levels observed in 12week wild type mice (Table 1). Although the spleen showed reduced uptake of α-Gal A compared to the liver, the treatment protocol used was sufficient to reduce splenic Gb3 levels relatively to untreated 12week Fabry knockout mice by ≈86% (Table 1). Splenic Gb3 reduction relatively to Fabry knockout mice at the 4 weeks baseline was ≈75% (Table 1). However, when compared to 12-week wild type mice, αGal A treated mice still present a 11-fold elevation in spleen Gb3 content (Table 1). 3.2.2. Enzyme replacement therapy prevents the progression of splenic iNKT deficiency in Fabry knockout mice due to a specific effect on the iNKT CD4+ subset The treatment protocol used did not alter the total organ cell yields, as the number of splenocytes, thymocytes and purified liver leukocytes of treated and untreated Fabry knockout mice were similar (Table S2). In addition, the numbers of splenic and hepatic T cells were similar between untreated and treated Fabry knockout mice (Table S2). The above treatment protocol used revealed an organ dependent effect, which partially corrected splenic iNKT cell percentage when compared to untreated Fabry knockout mice and, did not alter the low thymic and hepatic iNKT cell levels (Figs. 4 A, B). Fabry knockout mice treated with α-Gal A had twice the percentage of splenic iNKT cells when compared to untreated mice (Figs. 4 A, B). The comparison between splenic iNKT percentage of wild type (1.30 ± 0.44) and treated Fabry knockout mice (0.64 ± 0.11) indicates that the ERT protocol used was not able to totally restore the normal percentage of splenic iNKT cells, as a ≈50% reduction was still present between wild type and treated mice. Interestingly, an equivalent difference was observed prior to the initiation of ERT (4 weeks of age) between wild type (1.08 ± 0.08) and Fabry knockout mice (0.55 ± 0.07). These results indicate that ERT prevents the progression of the splenic iNKT deficiency observed at the initiation of treatment.

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Fig. 3. Liver and spleen of α-Gal A injected Fabry knockout mice show immunoreactivity for α-Gal A one week after the last enzyme administration. Sections of spleen and liver from saline injected (A, B) and α-Gal A treated (C, D) Fabry knockout mice were subjected to immunofluorecent staining with anti-human α-galactosidase A antibody. Study of 3 animals gave similar results.

We next investigated the effect of ERT on iNKT subsets in 12-week old Fabry knockout mice as these mice showed reduced splenic CD4+/ CD4− iNKT cell ratio (Fig. 2B). The ERT protocol used was effective in increasing the splenic CD4+/CD4− iNKT cell ratio, having no effect on the thymic and hepatic iNKT CD4+/CD4− ratio (Figs. 4 C, D). Indeed, the comparison among the CD4+/CD4− iNKT cell ratio of wild type, treated and untreated Fabry knockout mice showed that ERT completely prevented the drastic reduction in the splenic CD4+/CD4− iNKT cell ratio that was observed in 12-week old untreated Fabry knockout Table 2 α-Galactosidase A activity in liver and spleen of wild type and Fabry knockout mice after perfusion with saline or α-Galactosidase A.

Tissue α-Gal A activity (nmol/h/mg protein) Liver Spleen

Strain

Wild type

Age (weeks)

12

Fabry 12

12

Treatment

Saline

Saline

α-Gal A

n = 10

n=4

(1.5 mg/kg) n=3

42.6 ± 9.6 47.7 ± 8.9

1.0 ± 0.0 1.2 ± 0.5

184.7 ± 46.7a 14.7 ± 2.5a

4-week old animals were perfused once a week with saline or α-Gal A (1.5 mg/Kg), for a total period of 8 weeks. Tissues were harvested one week after the last injection. Data are expressed as mean ± SD. Statistically significant differences between saline perfused Fabry and α-Gal A treated Fabry knockout mice were analyzed by unpaired student's t-test and are indicated by a, p b 0.001.

mice (Figs. 2 B and 4 D). This positive effect of ERT on splenic CD4+/CD4 − iNKT cell ratio was due to a specific effect on the iNKT CD4 + subset, as in treated mice there is a higher number of splenic CD4 + iNKT cells when compared to untreated Fabry knockout mice, with no alteration in the number of CD4 − iNKT cells (Fig. 4 E). 4. Discussion Fabry knockout mice have shown to be a valuable tool not only in the understanding of underlying pathophysiological mechanisms of Fabry disease but also in studies regarding the efficacy of ERT. In the present study, we have shown that 4-week old Fabry knockout mice already show accumulation of Gb3 and a decreased number of iNKT cells. Moreover, the progressive Gb3 accumulation occurring with age in major peripheral organs of iNKT cell homing of Fabry knockout mice is associated with an accentuated reduction of peripheral iNKT cells. Additionally, we show for the first time that there is an age dependent decrease in the splenic CD4+/CD4 − ratio of Fabry knockout mice. More importantly, ERT with α-Gal A reduces lipid deposition and has a positive organ and subset-specific effect on iNKT cells of Fabry knockout mice. In fact, ERT was able to prevent the reduction of splenic iNKT cell number that occurs with age in untreated mice, due to a specific effect on the CD4+ iNKT cell subset. Previous studies by Abe and co-workers analyzed Gb3 deposition in the heart, kidney and liver of 4 week-old Fabry knockout mice by HPTLC [46] and only detected substantial accumulation of Gb3 in

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Fig. 4. In vivo enzyme replacement therapy partially corrects the splenic iNKT deficiency in Fabry knockout mice. 4-week old Fabry knockout mice were treated weekly with αGalactosidase A, for 8 consecutive weeks, with an intravenous dose of 1.5 mg/kg. Control Fabry knockout mice received infusions of saline solution with the same periodicity. (A, B) Splenocytes, thymocytes, and purified liver leukocytes were analyzed by flow cytometry for the presence of iNKT cells. (C, D, E) iNKT cells were analyzed for the expression of CD4 by flow cytometry. Results of representative examples (A, C) or mean ± SD (B, D, E). n = 4 Fabry knockout mice and 3 Fabry knockout mice treated with α-Gal A. (*, p b 0.05; **, p b 0.01; unpaired student's t test).

the kidney and heart. Using a more sensitive UPLC-MS/MS method our findings show that 4-week old Fabry knockout mice already have elevated Gb3 tissue content, specifically in the liver and spleen. To our knowledge, this is the youngest age at which Gb3 accumulation was observed in Fabry knockout mice. This very early Gb3 accumulation is not surprising, since in utero lysosomal accumulation of Gb3 has previously been documented in humans [47]. We also observed an increase of Gb3 storage occurring from the fourth to the twelfth week of life of Fabry knockout mice, consistent with previous studies in adult (20- and 24-week old) and old (40–48 week old) age animals [14,15]. We found that the thymic selection of iNKT cells in Fabry knockout mice is compromised, consistent with previous results of Gadola and co-workers [34]. Moreover, our results showed that thymic selection of iNKT cells is similarly affected in 4- and 12-week old Fabry knockout mice. However, in the periphery, we observed an accentuated reduction in iNKT cell percentage occurring with age. Our results on the analyzes of the iNKT cell subsets in Fabry knockout mice showed that splenic iNKT cell reduction is mainly due to a decrease in the iNKT CD4+ subset. Interestingly, a reduction in CD4+/CD4− ratio among iNKT cells has also been previously

observed in the thymus of NPC2 knockout mice [39], that present low numbers of iNKT cells in several organs and a severe reduction in lipid antigen presentation [39,42]. CD4 is an important molecule for the activation of peptide specific T helper cells. The role of this molecule in iNKT cells is not clearly understood. Human CD4+ and CD4− iNKT cells have different cytokine expression profiles, with the CD4+ subset expressing both pro-inflammatory (Th1) and antiinflammatory (Th2) cytokines whereas CD4− produces mainly Th1 cytokines [48–50]. However, similar functional differences in these subsets have not been described in mice. Our observation strongly suggests that the splenic microenvironment of Fabry knockout mice has an unequal effect on iNKT subsets. These results may inspire additional studies on the development and function of the CD4+ and CD4− iNKT cells. As the best responses to ERT are observed in the early phases of the disease, we administered weekly injections of α-Gal A to juvenile 4-week old Fabry knockout mice for a period of 8 weeks and analyzed the effect of ERT on α-Gal A activity, Gb3 clearance, iNKT cell numbers and subsets one week after the last α-Gal A administration. Both liver and spleen showed a relatively high abundance and activity of α-Gal A one week after the last enzymatic administration. This suggests that

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α-Gal A levels remained elevated for at least one week in the liver and spleen of Fabry knockout mice and the α-Gal A treatment protocol that was used was effective in the correction of hepatic enzyme deficiency of Fabry knockout mice. Consistent with previous reports, splenic uptake of α-Gal A and Gb3 clearance was not as efficient [18,20]. Additionally, ERT had a stabilizing effect in the number of splenic iNKT cells in a subset dependent manner. More specifically, the treatment protocol used prevented the age associated decrease in the percentage of splenic iNKT cells due to a positive effect on the CD4+ subset. It is clear from this study that iNKT cells of Fabry knockout mice are unequally affected in different organs. More precisely, the effect of age is more pronounced in peripheral iNKT cells than in thymic iNKT cells; the CD4+/CD4 − iNKT cell ratio is decreased in the spleen, but not in the liver or thymus; ERT has a positive effect in splenic but not in hepatic or thymic iNKT cells. The organ dependent alterations in the iNKT cells and iNKT response to ERT may be related to organdependent abundance and type(s) of antigen presenting cells with which iNKT cells interact or organ-dependent alterations in the quantity and specificity of lipid antigens rather than purely related to the amount of Gb3 storage. Indeed, upon ERT the Gb3 storage in the liver is completed corrected and only partially corrected in the spleen, but it is in the spleen that the iNKT cell phenotype is partially corrected. Different antigen presenting cells are important for the survival and activation of iNKT cells in different organs. Differentiation of iNKT cells in the thymus depends on interaction with double positive thymocytes [28]. In the periphery, iNKT cells interact with many different types of antigen presenting cells. It is known from in vivo experiments that dendritic cells are crucial antigen presenting cells for splenic, but not for hepatic iNKT cells [51]. In the liver, Kupffer cells act as important antigen presenting cells to iNKT cells [51]. In this organ, CD1d is also expressed in endothelial cells lining liver sinusoids [52], and hepatocytes [53]. Moreover, in some LSDs, it was shown that lipid antigen presentation capacity is unequally affected in different antigen presenting cells of the same mouse models of LSDs [34,39]. Indeed, bone marrow derived dendritic cells from Sandhoff and GM1 gangliosidosis are less affected in their capacity to present lipid antigens than splenocytes or thymocytes [34,39]. Additionally, different quantities and specificities of lipid antigens are presented to iNKT cells in an organ-dependent manner. In summary, our results reveal that Fabry knockout mice present an age associated increase in Gb3 storage and a decrease in iNKT cell percentage. A positive effect of ERT on progressive Gb3 storage and iNKT cell deficiency was also demonstrated. In particular, age dependent alterations in splenic iNKT cells of Fabry knockout mice were partially prevented by ERT through a specific influence on CD4+ iNKT cells.

Acknowledgments This work was financially supported by Shire Human Genetics, USA, and the Piaget Institute, Portugal. The corporate sponsors had no influence on the conception, design and protocol of the study, as well as on the content of the article and on the decision to submit the article for publication. R Quinta was supported by a fellowship from Fundação para a Ciência e Tecnologia [SFRH/BD/33447/2008]. The authors would like to thank the National Institutes of Health (NIH), USA, for providing the CD1d-PBS57 tetramer, Professor R.O. Brady for kindly providing the Fabry knockout mice, and Shire Human Genetics for the α-Gal A antibody and agalsidase alfa (Replagal©). We would also like to thank Dr. Lorena G Rodrigues for critical discussions and management of mice colonies. We tank Dr. Carol Harley for reading the manuscript.

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