Glucocerebroside: an evolutionary advantage for patients with Gaucher disease and a new immunomodulatory agent

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1 (2009) 87, & 2009 Australasian Society for Immunology Inc. All rights reserved /09 $ REVIEW Glucocerebroside: an evolutionary advantage for patients with Gaucher disease and a new immunomodulatory agent Yaron Ilan 1, Deborah Elstein 2 and Ari Zimran 2 Gaucher disease (GD) is caused by the reduced activity of a lysosomal enzyme, glucocerebrosidase, leading to the accumulation of glucocerebroside (GC). The relatively high prevalence of this disease within an ethnic group is believed to reflect a selective advantage. Treatment with enzyme replacement therapy (ERT) is safe and effective in ameliorating the primary symptoms of the disease, yet there have been reports that some patients on ERT have developed type 2 diabetes or metabolic syndrome, malignancies and central nervous system disorders. A series of animal studies suggest that these complications may be related to the reduction of GC levels by the enzyme administered. GC has been shown to have an immunomodulatory effect through the promotion of dendritic cells, natural killer T cells, and regulatory T cells. The break down of GC to ceramide can underline part of these findings. Clinical trials suggested a beneficial effect of GC in type 2 diabetes or nonalcoholic steatohepatitis. This review of the data from animal models and humans proposes that the increased level of GC may provide an evolutionary advantage for patients with GD. Indirectly, these data support treating symptomatic patients with mild/moderate GD with lowdose ERT and re-evaluating the use of ERT in asymptomatic patients. (2009) 87, ; doi: /icb ; published online 16 June 2009 Keywords: Gaucher disease; regulatory T cells; natural killer T cells; glucocerbroside GAUCHER DISEASE: A STORAGE DISEASE WITH AN ETHNIC PREDILECTION Gaucher disease (GD) is the most common glycolipid storage disorder and is caused by the reduced activity of a lysosomal enzyme, glucocerebrosidase. This leads to the accumulation of the substrate, glucocerebroside (GC), in the cells of the reticuloendothelial system. 1 This autosomal recessive disease is pan ethnic, but it is especially prevalent among Ashkenazi Jews. Although hundreds of mutations have been reported in the glucocerebrosidase gene, five mutations account for 98% of the disease producing alleles. Of these, the N370S (or 1226G) mutation occurs in 1 of 17 Ashkenazi Jews, resulting in a disease frequency of 1 in 850 in this ethnic group. 2,3 IS THERE A SELECTIVE EVOLUTIONARY ADVANTAGE FOR PATIENTS WITH GD? The high prevalence of more than one glucocerebrosidase mutation among Ashkenazi Jews is believed to be caused by a selective advantage 4 beyond the predictable founder effect. 5 Yet, the nature of such an advantage has not been identified. Among the hypotheses proposed for this selective advantage are a greater resistance to tuberculosis, 6 superior intelligence 7 and increased fertility (associated with higher levels of prosaposin activator in Sertoli cells, Leydig cells and peritubular cells 8 ). However, these associations remain unproven. THE SELECTIVE ADVANTAGE OF PATIENTS WITH GD: LESSONS LEARNED FROM ENZYME THERAPY Enzyme replacement therapy (ERT) for patients with GD was first introduced in 1991 using a placenta-derived product that was replaced with a recombinant enzyme in Each has proven effective in the management of the key disease manifestations, including a reduction in organomegaly, improvements in the blood counts and biochemical parameters, a decrease in bone-related pain (but not reversal of existing pathology) and compensatory growth in children ERT has also been remarkably safe, and the few adverse effects that have been reported are typically mild and transient in nature There have been no reports of any obvious toxicity because of overdose. Hence, with a high efficacy and an excellent safety profile, the major disadvantages of ERT are the very-high cost of therapy and the apparent lifetime dependency on intravenous infusions. 21,24 36 In addition, the dosing issue has become a major source of controversy, with only few centers adopting the low-dose regimen and the majority of centers advocating high-dose ERT to 1 Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel and 2 Gaucher Clinic, Shaare Zedek Medical Center, Jerusalem, Israel Correspondence: Professor Y Ilan, Internal Medicine/Liver Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, Kiryat Hadassah, POB 12000, Jerusalem 91120, Israel. ilan@hadassah.org.il Received 25 February 2009; revised 13 May 2009; accepted 19 May 2009; published online 16 June 2009

2 515 ensure better clinical results. 42,43 Recently, however, a few studies have reported an increasing number of complications that are not directly associated with the known consequences of GD and that, paradoxically, may be related to the long-term use of ERT. In a recently published study, 44 the life expectancy of patients with GD type 1 (GD1) was evaluated by comparing the survival data from patients who were enrolled in the International Gaucher Registry (of the International Collaborative Gaucher Group (ICGG)) to those of the general US population using the standard life table methods. Among the 2876 patients with GD1, there were 102 reported deaths in the person-years of follow-up. The overall estimated life expectancy at birth for patients with GD1 was 68 years, B9 years less than the estimated 77 years for the reference population. The estimated life expectancy was 64 years for splenectomized patients with GD and 72 years for patients with an intact spleen. The causes of death for 63 of the 102 patients were malignancies (17/63), cardiovascular disease (11/ 63) and cerebrovascular disease (8/63). However, the malignancies did not contribute to shortened life expectancy. 44 These causes of death could not be attributed to the glycolipid storage deficiency or to the complications of GD that were typically seen in the pre-ert era, such as bleeding or infections. 45 These studies, along with the suggested evolutionary advantage and the recent data describing the potent immunomodulatory, anti-inflammatory and anti-malignant effect of GC, suggest that the long-term suppression of GC by ERT (possibly more so by the high-dose regimens) may impact the outcome of some patients with GD. ASSOCIATION OF GD AND SYSTEMIC DISORDERS The selective advantage of patients with GD can be extrapolated from recent studies (Table 1). Table 1 Data suggesting selective advantage by glucocerebroside in Gaucher disease Type of disease Supporting data from human trials Supporting data from animal models Diabetes Diabetes type 2 46,171 Ob/Ob mice 76 Cohen rats 77 Sand rats 78 Neurological disorder Parkinson Metabolic syndrome and weight gain Diabetes type 2 46,171 Ob/Ob mice 76 Cohen rats 77 Sand rats 78 Malignancy Solid malignant Hepatocellular carcinoma 72 disorders 49,172 Melanoma (unpublished data) Colitis Experimental colitis 72 Hepatitis Nonalcoholic liver disease Autoimmune disorders Hepatitis C virus ConA immune mediated infection 124 hepatitis 71 Nonalcoholic Ob/Ob mice 76 steatohepatitis 171 Cohen rats 77 Sand rats 78 Acute and chronic graft versus host disease 73 Asthma Fibrosis Acute and chronic liver fibrosis 74 Increased immunogenicity of vaccines Augmentation of the response to HBV vaccine 79 Diabetes mellitus Gaucher disease type 1 is associated with metabolic abnormalities, such as a high resting energy expenditure, a low circulating adiponectin level and peripheral insulin resistance. 46 ERT leads to a decrease in the resting energy expenditure. Furthermore, the prevalence of overweight individuals is lower in untreated GD patients than in the general population. 46 Long-term treatment with ERT results in a larger than average weight gain, so that, ultimately, the prevalence of overweight individuals in ERT-treated GD patients is similar to the general population. The prevalence of type 2 diabetes increases significantly with ERT, resulting in a comparable prevalence of type 2 diabetes among patients receiving ERT and the general population. 46 These results suggest that the decrease in the serum level of GC has a deleterious effect on the metabolic pathways associated with insulin resistance. Malignancies It has been suggested that the incidence of malignant disorders is increased in patients with GD1, but the mechanisms underlying the development of these diseases are not fully understood. 17 Some researchers have suggested that the increased risk of malignancies in patients with GD is a result of the chronic stimulation of the immune system and the lymphoproliferation associated with the storage of GC in tissue macrophages. Another proposed explanation for this increased risk is T-cell dysfunction as a result of high levels of ferritin and/or a factor released by monocytes. 47 The accumulated GC in the reticuloendothelial organs, which is histologically visible as Gaucher cells, is thought to be a chronic antigenic stimulus of the immune system. This chronic stimulation results in polyclonal hypergammaglobulinemia, and monoclonal populations of lymphocytes and plasma cells may arise from this proliferative state. 48 A recent study was undertaken to ascertain the prevalence of various cancers that are associated with GD1. In a retrospective study, the incidence of malignancies in a cohort of 505 patients with GD1 that were seen at a large referral clinic in Israel was compared with age-matched data from the Israeli National Cancer Registry. A total of 20 patients with GD (4.0%) had developed cancer. The most common malignancies were lymphoma and myelodysplastic syndrome (three cases, each) and two cases of multiple myeloma. There was no statistically significant difference in the incidence of cancer among patients with GD1 relative to the age-matched rates reported in the national Jewish Israeli and Ashkenazi Jewish Israeli registry records. These data suggested that there is no increased risk of hematological or other cancers among patients with GD1 relative to the overall age-matched Jewish population. 49 The results of this study were very similar to those of the larger International Registry Report (ICGG) that showed that patients with GD1 do not have an increased risk of cancer, with the exception of multiple myeloma; patients with GD1 had a significantly higher incidence of multiple myeloma than the general population. 49 In a larger registry study of 2742 patients with GD, the incidence of cancer was compared with the expected incidence in the US population of similar age and sex. In the ICGG cohort, there were 10 patients with multiple myeloma, resulting in an estimated relative risk of 5.9, and the overall relative risk of cancer was There was no statistically significant increase in the risk of breast, prostate, colon/rectal, or lung cancer or hematological malignancies, other than multiple myeloma. 47 However, neither the Israeli study nor the ICGG study stratified patients with respect to use of ERT (yes/no) or, in the case of treated patients, the dosage. A study examining the incidence of malignancies in a population of 1525 male veterans with GD identified an elevated risk of non- Hodgkin s lymphoma, malignant melanoma and pancreatic cancer in patients with GD. However, no significant association between GD and either cancer in general or other specific malignancies, including

3 516 multiple myeloma, was observed. 50 Unfortunately, because of systematic methodological errors, these findings are difficult to interpret. Interestingly, a case in which a patient with GD developed hepatocellular carcinoma (HCC) after the administration of high-dose ERT, even though no known HCC-predisposing factors were identified, has been described. 51 Carriers of GD are considered to be healthy subjects because there is no manifestation of the disease, but they may show signs of macrophage dysfunction. Studies have shown that carrier status does not seem to increase the risk of other diseases. 52 This data raise questions as to the role of the genetic mutation in the development of cancer in patients with GD. Additional studies have suggested an increased incidence of some cancers, especially hematopoietic malignancies, in patients with GD; however, how many of these patients were treated with ERT is unclear. 53 The only consistent finding is an association between GD and multiple myeloma. 56 However, compounding factors such as splenectomy, which is also associated with a more aggressive bone involvement; earlier exposure to ERT; and a predisposition to malignancy 57 make definitive conclusions difficult. Parkinson s disease Another very interesting observation is the association between GD and Parkinson s disease (PD) Mild neurological manifestations may also affect patients with GD1; however, treatment with ERT or SRT does not improve neurological function. 17 Studies have identified an increased risk of PD in carriers of glucocerebrosidase mutations as compared with the general population: carriers of the 84GG null allele have a 3.6-fold increased risk of PD, whereas carriers of the N370S allele, which is milder, have a 2.2-fold increased risk of PD. These findings suggest that even among carriers, the risk of PD is related to the higher GC level, which is probably associated with intracellular damage. In contrast, we rarely see PD in patients with severe GD1 genotypes (for example, N370S/84GG). Most of the cases of PD comorbidity are seen in patients with relatively mild GD1 or in patients who have been taking ERT for many years (AZ, personal observation). The data suggest that this difference may be related to the level of plasma GC, which may possibly be neuroprotective against the inflammatory processes that are associated with various neurodegenerative diseases. If this is the case, a profound decrease in the plasma GC level may result in an added risk of PD and PD-like disorders. Autoimmune and other inflammatory disorders Natural auto-antibodies have been observed in GD, but no overt increase in autoimmunity has been described. 68 Similarly, despite the low HDL cholesterol level in patients with GD, unlike in individuals who do not have GD, there is no increase in the risk of cardiovascular disease. 69 This suggests a protective effect for GC against the inflammatory cells that contribute to cardiovascular disease. Although the underlying pathophysiology of GD is not completely understood, it has become increasingly clear that an altered cytokine balance may be associated with a higher GC level. 70 The above described studies, suggesting a selective advantage of patients with GD, can be explained by a direct effect of GC. Alternatively, the break down of GC to ceramide can underline part of these findings. IMMUNE ADVANTAGES OF GC The effects of b-glycosphingolipids in immune-mediated disorders have been studied in several animal models that suggest that b-glycosphingolipids may have immunomodulatory, anti-inflammatory and anti-malignant functions (Table 1). Immune-mediated hepatitis Glucocerebroside has been found to alleviate ConA (concanavalin A)- induced hepatitis in mice, an effect associated with a decreased serum level of interferon (IFN)-g and a reduced expression of the transcription factor, STAT1. 71 ConA induces liver damage that is mediated by natural killer T (NKT) cells. Serum aspartate aminotransferase and alanine aminotransferase levels were markedly lower, and histological damage was attenuated, in GC-treated mice compared with untreated animals. The beneficial effect of GC was associated with a 20% decrease in the number of intrahepatic NKT cells, a significant decrease in the serum IFN-g level, and decreased STAT1 and STAT6 expressions. Intraperitoneally administered radioactive GC could be detected in the liver and bowel. 71 Furthermore, the in vitro treatment of NKT cells with GC led to a 42% decrease in cell proliferation in the presence, but not absence, of dendritic cells (s). Immune-mediated colitis In a murine Th1-mediated colitis model, b-glucosylceramide generated a Th2 response that was associated with the alleviation of colitis. 72 Hepatocellular carcinoma In mice with HCC, GC administration resulted in a Th1 immune shift that was associated with the suppression of tumor growth and improved survival. 72 The immunological effect of GC was assessed by the analysis of intrahepatic and intrasplenic lymphocyte populations, serum cytokine levels and STAT protein expression in colitis and hepatoma models. The administration of GC led to the alleviation of colitis and to the suppression of HCC, as measured by improved survival and decreased tumor volume. These beneficial effects were associated with an opposing immunological effect in the two models: the peripheral:intrahepatic CD4:CD8 lymphocyte ratio increased in the colitis model, whereas it decreased in the HCC model. The effect of GC was associated with decreased STAT1 and STAT4 expressions, the overexpression of STAT6, and a decreased IFN-g level in the colitis model, but an increased IFN-g level in the HCC model. Taken together, these data suggest that GC alleviates immunologically incongruous disorders and may be associated with the fine tuning of the immune response by changes in the plasticity of NKT lymphocytes. Graft-versus-host disease Glucocerebroside treatment was effective in alleviating semi-allogeneic acute and chronic graft-versus-host disease (GVHD) in mice. 73 Acute and chronic GVHD were generated by the transfer of splenocytes from C57BL/6 mice to (C57BL/6xBalb/c) F1 mice and from B10.D2 donor mice into Balb/c mice, respectively. The recipient mice were then treated daily with GC, and histological and immunological parameters of GVHD were assessed. The treatment with GC significantly alleviated GVHD in both models. The beneficial effect of GC was associated with an increase in the intrahepatic:peripheral NKT lymphocyte ratio in the semi-allogeneic acute model. However, this ratio was decreased in the chronic GVHD model. A significant increase in the number of intrahepatic CD8 lymphocytes was detected in the semi-allogeneic acute model, but the opposite was observed in the chronic GVHD model. The administration of GC led to a decreased serum IFN-g level and an increased serum IL-4 level in the Th1- mediated model, whereas this was reversed in the Th2-mediated models. These data suggest that GC can ameliorate GVHD in both Th1- and Th2-mediated murine models and that it is able to differentially affect the immune system. 73

4 517 Liver fibrosis To explore the role of NKT cells and GC in hepatic fibrosis, hepatic fibrosis was induced in male C57Bl/6 mice by biweekly intraperitoneal injections of carbon tetrachloride for 7 weeks. 74 Some mice were also treated with daily intraperitoneal GC injections. A significant amelioration of hepatic fibrosis was observed in all GC-treated mice without an alteration in the production of reactive oxygen species. As determined by Sirius red-stained liver tissue sections and measured by Bioquant morphometry, all carbon tetrachloride-treated groups presented significantly larger areas of fibrosis than the naive animals. A similar pattern was observed after the western blot analysis for smooth muscle a-actin of liver extracts. A significant decrease in liver damage was observed in all GC-treated groups, as detected by a decrease in the transaminase serum level. The beneficial effect of GC was associated with a significant decrease in the number of intrahepatic NKT cells and CD8 cells, as well as the attenuation of both Th1 and Th2 cytokines production. 74 Type 2 diabetes and metabolic syndrome The treatment of animal models of type 2 diabetes, nonalcoholic fatty liver disease 75 and hyperlipidemia with GC resulted in a significant amelioration of the various disease parameters. 76,77 To determine the effect of GC on the metabolic dysregulation and the immune profile in leptin-deficient mice, ob/ob mice were treated with daily injections of GC for 8 weeks. A significant amelioration of the metabolic changes characteristic of leptin-deficient mice was observed: the liver size and hepatic fat content were significantly decreased, glucose tolerance was nearly normalized and serum triglyceride levels decreased. Furthermore, a change in the peripheral and intrahepatic lymphocyte populations was detected by flow cytometry. A 33% decrease in the serum IFN-g level and a 2.6-fold increase in the serum IL-10 level were noted in the GC-treated mice. 76 A link between the altered level of various gangliosides and the development of insulin resistance was described in the transgenic mice. A recently published study examined whether glycosphingolipid-induced modulation of the immune system may reduce pancreatic and liver steatosis and stimulate insulin secretion in the Cohen diabetes-sensitive rat, a lean model of noninsulin-resistant, nutritionally induced diabetes. Four groups of Cohen diabetes-sensitive rats were fed a diabetogenic diet and were treated for 45 days with GC. The administration of GC increased the number of intrahepatic CD8 T cells and NKT cells. The pancreatic and liver histology were markedly improved and steatosis was reduced in all treated groups compared with the vehicle-treated groups. Insulin secretion was restored after glycosphingolipid treatment, resulting in improved glucose tolerance. 77 To determine the mechanism associated with b-glycosphingolipidmediated amelioration of liver injury, GC was administered to Psammomys obesus fed a high-energy diet. The administration of GC decreased STAT1 and STAT4 expressions and increased STAT5 expression in the spleen. These effects were associated with the alleviation of insulin resistance, a decreased serum glucose and insulin level, a decreased plasma free fatty acid level, an altered distribution of body fat and improved serum cholesterol and triglyceride levels. In addition, the GC ameliorated the hepatic injury, as indicated by decreased liver weight, hepatic fat, liver enzymes and improved liver histology. 78 Adjuvants for augmentation of immunogenicity of antigens Glucocerebroside increased the immune response against hepatitis B virus (HBV) in association with an altered distribution of NKT cells and CD8 cells, suggesting that GC could be used as potent adjuvant for overcoming non-responsiveness to the HBV vaccine and augmenting the antiviral immune response. 79 Non-responsiveness to the currently used HBV vaccine is a major problem in attempts to protect against infection. Adjuvants that augment the immune response are essential components of the vaccine. GC exerts a NKT cell-mediated immunomodulatory effect in various disorders. In a recent study, mice were injected with different formulations of an HBV vaccine, along with various doses of GC. The GC treatment altered the distribution of hepatic NKT cells and CD8 cells, and all treatments augmented the anti-hbv immunity, increasing both the anti-hbv antibody titers and the percentage of mice exhibiting high titers. This effect was associated with the altered distribution of hepatic NKT cells and CD8 cells. 79 MECHANISMS OF ACTION OF GC Glucocerebroside, a glycosphingolipid, can be presented to NKT cells and s through CD1 molecules. GC can exert an immunomodulatory effect directly on these target cells or indirectly by altering the cross talk between these cells and other subsets of cells of the immune system. Several mechanisms have been explored to explain the immunomodulatory effect of GC (Table 2). These include: altering the plasticity of NKT cells either by functioning as their natural ligand or replacing their yet-unidentified natural ligand(s); promoting regulatory T lymphocytes; altering lipid rafts, intracellular signaling machinery, or function; acting as an adjuvant for antigens to improve immunogenicity; functioning as a metabolic intermediate in insulin resistance; and promoting immune-dependent mucosal mechanisms. Natural killer T cells are a unique lineage of T cells that share properties with both NK cells and memory T cells, 80 and are unique in their invariant usage of the Va14 Ja18 T-cell receptor (TCR) a-chain, and their TCR b-chain is biased towards Vb8.2, Vb2 and Vb7. NKT cells are also unique in their glycolipid antigen reactivity and marked cytokine production. 81,82 The ability of NKT cells to generate either Table 2 Possible mechanisms of action of glucocerebroside Mechanism Altered plasticity of NKT cells Promotion of regulatory T lymphocytes Altered function of dendritic cells Altered lipid rafts Alteration of intracellular signaling machinery Adjuvant to disease target antigens: improved immunogenicity Metabolic intermediate in insulin resistance pathways Promotion of mechanisms of mucosal immunity Replacement of natural NKT cell ligands Supporting data from human trials Patients with type 2 diabetes and NASH 170 Patients with HCV 124 Healthy volunteers (unpublished data) Healthy volunteers (unpublished data) Patients with NASH 170,175 Improves insulin resistance in patients with type 2 diabetes 171 Supporting data from animal models Zigmond et al., 72 Ilan et al., 73 Kjer-Nielsen et al., 173 Rouhi et al. 174 GC promotes NKT and other subsets of Tregs 121,135 (unpublished data) Lalazar et al., 160 Lalazar et al. 161 Increased flotilin2 161 Adjuvant for augmentation of the anti HBV immunity 79 Improves insulin resistance 76 78,137 Oral GC as an immunomodulatory agent 135

5 518 Th1 or Th2 responses shows their importance as immunoregulatory cells and the complexity of their immunomodulatory machinery different responses can be generated by the same ligands. 72,83 Several glycosphingolipids and phospholipids derived from mammalian, bacterial, protozoan and plant species have been identified as possible natural ligands for NKT cells, 84,85 and phospholipids, such as phosphatidylcholine and phosphatidylinositol. 86,87 The semi-invariant TCRs can recognize igb3 (isoglobotrihexosylceramide), a mammalian glycosphingolipid, and a microbial a-glycuronylceramide found in the cell wall of Gram-negative, lipopolysaccharide-negative bacteria. 88 igb3 has been proposed as one of the molecules recognized by NKT cells under pathological conditions, such as cancer and autoimmune disease. 88,89 Studies in mice deficient in igb3 synthase showed that these mice developed, grew and reproduced normally, whereas exhibiting no overt behavioral abnormalities. 90 Several glycosphingolipids have been extracted from the Okinawan marine sponge, Agelas mauritanus. 91 These agelasphins, which consist of D-galactose and ceramide, showed antitumor activity in mice. 92 As only small amounts of agelasphins are present in each sponge, the modified derivative a- galactosylceramide (a-galcer) was produced. 93 Multimers of CD1d1 loaded with a-galcer or a-galcer analogs show cooperative engagement of the Va14Ja18 inkt-cell receptor. 92,94 a-galcer is a potent ligand for NKT cells after binding to CD1d expressed by antigenpresenting cells (APCs). The CD1d/a-GalCer complex is recognized by NKT cells through the Va14i TCR in mice and the Va24i TCR in humans. 95 The administration of a-galcer activates NKT cells, which rapidly produce cytokines that activate a variety of cells of the innate and adaptive immune systems. 91 Although a-galcer induces the secretion of both IL-4 and IFN-g, the repeated administration favors the production of Th2 cytokines (for example, IL-4). 96,97 A role for activated NKT cells in various types of pathology, including infections, autoimmunity and neoplastic conditions, has been described NKT cells have been shown to play a role in animal models of colitis, 101 ConA-induced hepatitis and in the induction of oral tolerance Furthermore, reduced NKT cell numbers and defective NKT cell function have been shown in nonobese diabetic mice. a-galcer-activated NKT cells can prevent pancreatic islet b-cell destruction. 104,105 The transplantation of NKT cells, the introduction of a Va14Ja281 transgene onto the non-obese diabetic background, or the activation of NKT cells by the administration of a-galcer ameliorates diabetes in this model. 98,106 The administration of a-galcer and OCH, a sphingosine-truncated analog of a-galcer, had protective effects in murine experimental autoimmune encephalomyelitis and collagen-induced arthritis, respectively. 96 inkt cells can skew adaptive immunity toward Th2 responses or can act directly on effector cells at mucosal surfaces in diseases, such as ulcerative colitis and bronchial asthma. 107 NKT cells promote airway hyperresponsiveness and inflammation, which results in asthma. 108 In mouse models of asthma, NKT cell-deficient strains fail to develop allergen-induced airway hyperreactivity, a cardinal feature of asthma. 109 In HBV-transgenic mice, NKT cell activation abolished HBV replication. 110 a-galcer-induced NKT cell activation has been shown to increase IFN-g production and augment local host resistance to Cryptococcus neoformans infection. 111,112 The administration of a- GalCer enhances malaria immunity in mice that had received a suboptimal dose of irradiated sporozoites or a recombinant virus expressing sporozoite antigens. 113 Interestingly, in an experimental autoimmune encephalomyelitis model, a-galcer-induced NKT cell activation either potentiated or prevented disease, depending on the NKT cell reaction: 114 IFN-g secretion was associated with disease exacerbation, whereas IL-4 production was protective against experimental autoimmune encephalomyelitis. NKT cells are capable of inducing tumor regression through the IL-13-mediated inhibition of tumor-specific cytotoxic T lymphocytes, 115 and the absence of NKT cells has been associated with an increased risk of tumorigenesis. 116 KRN7000, an a-galcer analog with a di-unsaturated twenty-carbon chain, has been shown to stimulate IL-4 production, yet inhibit IFN-g production. 117 In addition, a-c-galcer, a C-glycoside analog, has been shown to generate a longer-lasting reaction, with a cleaner Th1 response. 113,118 In addition to a-galcer and its analogs, b-galcer has also been reported to be a CD1d ligand capable of stimulating NKT cells. 119,120 GC as a ligand for NKT cells To date, a-anomeric D-glycosylceramides have not been detected in mammals. However, several recent studies have suggested that endogenous b-structured glycosphingolipids may be potent NKT ligands. 121 b-structured glycosphingolipids are normal constituents of the cell membrane There is circumstantial evidence from studies of patients with GD suggesting that GC is involved in NKT cell regulation. 124 Patients with GD have altered humoral and cellular immune profiles, including altered NKT cell number and function. The results from these patients suggest a direct effect of b-galcer on the cell membrane: some patients have increased red blood cell aggregation due, in part, to changes in the properties of the cell membrane. 125 In vitro, CD1d-bound GC inhibits NKT cell activation by a- GalCer. 119 Glucosylceramide synthase deficiency leads to defective ligand presentation by CD1d, thereby inhibiting NKT cell activation. b-galcer-deficient mice exhibit normal NKT cell development and function, and cells from these animals can stimulate NKT cell hybridomas. 126 In contrast, the same hybridomas fail to react to CD1d1 expressed by a b-d-glucosylceramide (b-d-glccer)-deficient cell line. The ectopic expression of human b-d-glccer synthase restored the recognition of the mutant cells expressing CD1d1 by Va14Ja18 NKT cell hybridomas. The suppression of b-d-glccer synthesis inhibits antigen presentation to inkt cells; however, b-d- GlcCer itself is unable to activate NKT cell hybridomas. 126 b-d-galcer efficiently decreases the number of detectable NKT cells in vivo without inducing cytokine expression. Binding studies have shown that both a-galcer and b-d-galcer are equally efficient in reducing the number of NKT cells. However, in contrast to a-galcer, b-d- GalCer is a poor inducer of IFN-g, TNF (tumor necrosis factor)-a, granulocyte macrophage colony-stimulating factor and IL-4 gene expression. 119 GC-dependent alteration of lipid rafts and intracellular signaling machinery In addition to having mechanisms that require the binding to CD1d, b-glycosphingolipids may affect NKT cell activation through alteration of the properties of the cell membrane; specifically, the properties of lipid rafts. 127 Lipid rafts are highly dynamic submicroscopic assemblies enriched with sphingolipids and cholesterol. Alteration of the raft properties may impair raft receptor localization without necessarily inhibiting the binding of the ligand to the receptor. 128 Raft disruption has been shown to inhibit IL-6/STAT3 and IFN-g/ STAT1 signaling. 128 As CD1d is also localized in lipid rafts, 129 the disruption of lipid rafts can inhibit NKT cell activation without impairing CD1d-ligand binding. 129 The administration of naturally occurring b-glycosphingolipids can alter the lipid raft composition and structure, thereby affecting the intracellular signaling machinery. 119,130 It has been reported that b-galcer can stimulate NKT

6 519 cells. 120 b-glycosphingolipids seem to affect NKT cells differently than does a-galcer; b-glycosphingolipids inhibit NKT cell proliferation without stimulating cytokine expression. 71 GC as a ligand for cells The CD1d molecules are constitutively expressed by several different cells, including APCs (for example, macrophages and s) and B and T cells in the thymus and liver. 131,132 This constitutive expression suggests that the activation of s by glycosphingolipids is a possible mechanism of the GC pathway. Indeed studies have shown that the immunomodulatory effect of GC is dependent on s. 71 Similarly, other glycospingolipids, such as a-galcer, have been shown to alter the -NKT cell cross talk or to affect other regulatory T cells (Tregs). 133,134 GC promotes regulatory T cells Recent studies have suggested that glycosphingolipids can promote Tregs. This effect can be mediated by s or by cross talk between NKT cells and Tregs. 135,136 Studies have shown that CD1d expression is required for NKT cell development, but is not absolutely necessary for the generation of polarized T-helper-cell responses. 100 Furthermore, after the administration of a-galcer, NKT cells expand and produce cytokines, trans-activate a variety of innate and adaptive immune cells, and promote Th2 responses that are capable of suppressing Th1-dominant autoimmunity. These findings indicate that NKT cells play a regulatory role in the immune response and that the specific activation of these cells can be exploited for therapeutic purposes. 100 Recent studies in animal models and in humans have shown that GC can promote Tregs through activation of s (unpublished data). GC as a metabolic intermediate in insulin resistance pathways Mammalian target of rapamycin (mtor) is a serine threonine kinase that is regulated by nutrients and growth factors. Activation of mtor and its downstream target, S6K1, results in the downregulation of insulin signaling. Inhibition of the mtor pathway may serve as a novel therapeutic target for patients with GD. Treatment with GC decreased mtor and S6Kp70 phosphorylation. This was associated with a significant increase in the expression of IRS1 (insulin receptor substrate 1) and decreased IRS1 serine 636/639 phosphorylation (corrected for total IRS1). Moreover, AKT/PkB phosphorylation was significantly increased, indicating that the action of the liver was enhanced. These effects were associated with a significant improvement in glucose tolerance, as manifested by a decrease in serum insulin. The alleviation of liver injury was detected by decreased liver enzymes and by a significantly reduced serum TNF-a level. Furthermore, the administration of GC led to reduced hepatic steatosis and improvement of the lipid profile. 137 Alteration of the plasticity of NKT regulatory cells by GC One of the most intriguing characteristics of NKT cells is their plasticity. This subset of lymphocytes generates both Th1 and Th2 responses on activation. 72 The plasticity of a regulatory cell may evolve from the use of different ligands or from signals in the immune microenvironment. 138 Alternatively, the plasticity may depend on cell cell interactions, cytokines, or co-stimulatory molecules. 139 Antigen presentation and APCs may play an important role in this setting. 140 In contrast, plasticity may simply be the result of the natural programs of different subsets of cells. 141 Different functions of lymphocytes depend on the membrane structure, specifically lipid rafts 7 and the potential functional effects of the integration of glycosphingolipids within the membrane structure. 8 Therefore, the generation of different immune responses triggered by the same ligand can be classified into two types of plasticity: type-i and type-ii. Type-I plasticity, or rue plasticity, occurs when identical cells and ligands produce different immune responses. Type-II plasticity, or apparent plasticity, occurs when the exposure to glycolipid ligands alters cellular properties such that the cell population in question is no longer homogenous; the net effect is a function of the balance between these cell sub-populations. 135 In light of the differential effects of a given ligand in vivo and in vitro, the net effect of NKT cell activation may result not from the binding of a single ligand, but rather from the sum of the effects of a variety of mediators. 135 In addition, organ-specific factors may play a role in NKT cell plasticity, with different responses generated in different organs by an identical stimulus. Alternatively, stimuli that were originally identical may reach NKT cells through different pathways through presentation by different APCs. 73,83 Furthermore, NKT cells are a heterogeneous population of cells that differ from one another in their CD1d reactivity and CD expression, which may contribute to their plasticity. Apart from the inherent heterogeneity between different NKT cell populations, changes in the properties of lipid rafts may affect raft-bound receptors, such as CD1d, and may add to the variety of responses. NKT cell plasticity may also be a result of different immunological reaction profiles and dictated by genetic heterogeneity. POTENTIAL ROLE FOR CERAMIDE The break down of GC to ceramide could be an important factor for interpretation of the data published in recent studies suggesting a clinical advantage in patients with GD. Ceramide has a role as signaling and regulatory molecule. It exerts an effect in the regulation of cell growth, death, senescence, adhesion, migration, inflammation, angiogenesis and intracellular trafficking. 142 Ceramide and the immune system The importance ceramide and of ceramide-1-phosphate on the development, activation and regulation of the immune system is well-recognized. 143 The processing and presentation of lipid antigens by APCs is important for defense against infection, tumor immunosurveillance and autoimmunity. 144 Ceramidases catalyze hydrolysis of ceramides to generate sphingosine, which is phosphorylated to form sphingosine-1-phosphate. Ceramide, sphingosine and sphingosine-1- phosphate are bioactive lipids that mediate cell proliferation, differentiation, apoptosis, adhesion and migration. 145 Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by the action of a specific nuclear kinase. 146 Both ceramide and ceramide kinase (CERK) have a role in various signal transduction pathways involved in inflammation. 147 Accumulation of ceramide mediate cellular responses to LPS, IL-1b and TNF-a. 148 Ceramide with respect to its interactions with lipids, inflammatory cytokines, homocysteine and matrix metalloproteinases is a potential powerful immune-modulatory target. 149 Ceramide can control the steroid hormone biosynthetic pathway at multiple levels, including regulating steroidogenic gene expression and activity, as well as acting as second messengers in signaling cascades, further supporting its potential role in the control of inflammatory processes. 150 Ceramide, the backbone of sphingolipids, is the key component that affects atherosclerotic changes through its important second-messenger role. 149 The generation of transport carriers moving from the Golgi complex to the plasma membrane relies on an interplay between proteins and lipids. 151 Several of these carrier proteins possess a glycolipid-transfer-protein homology

7 520 domain, and may play a pivotal role in the synthesis of complex glycosphingolipids. 152 Alteration in the ceramide-gc metabolic pathway may also be associated with changes of the carrier proteins, further affecting the immune system. Effect on lipid membranes and on the regulation of lipid metabolism Ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signaling events. 153 Sphingolipids, together with phospholipids and cholesterol are the key components of membrane lipid bilayers, contribute to specialized membrane domains called rafts and function as signaling molecules. 154 Alteration of lipid rafts components can exert a distinct role in the post-transcriptional regulation of the sterol-regulatory element binding proteins, key transcription factors of lipid synthesis. 154 The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. 153 These platforms serve to cluster receptor molecules and to organize intracellular signaling molecules to facilitate signal transduction through a receptor on stimulation. 153 The dynamicity of cell membrane plays some vital roles in cell survival and cell death, including protection, endocytosis, signaling and increases in mechanical stability during cell division, and cell separation from tissues for cancer metastasis. 155 Ceramide and cancer There is extensive evidence supporting the pivotal role of ceramide in cancer biology, 156 making it an attractive target for anticancer drug development within the sphingolipid metabolic pathway. Its function in apoptosis is best characterized. Ceramide has been shown to be capable to trigger apoptosis in almost any cell, including tumor cells. 157 Stimuli that trigger a release of ceramide to mediate apoptosis include CD95, TNF-receptor, DR5, g-irradiation, cytotoxic drugs, ultravoilet (UV)-light, bacteria, viruses, some forms of developmental death, anti-cd20 and disruption of the cell s contact with its matrix. 157 Ceramide may have an important role in several antitumor treatment modalities, such as g-irradiation and chemotherapy. 157 Recent data on the proteomics of lipid rafts/caveolae have highlighted the enigmatic role of various signaling proteins in cancer development. 155 These data suggest a possible mechanism by which ceramide may exerts its anti-cancerous effect by being involved in cell signaling, that when dysregulated promotes cell transformation and tumor progression alterion. 155 Ceramide and insulin resistance Ceramide as well as other sphingolipids may be relevant in several cellular events associated with diabetes and cardiovascular disease, including insulin resistance, pancreatic b-cell failure, cardiomyopathy and vascular dysfunction. 158 The potential contribution of ceramide to insulin resistance was recently reviewed. 159 It was suggested that both ceramide and glucosylceramides, although being a relatively minor component of the lipid milieu in most tissues, may be among the most pathogenic lipids in the onset of the sequelae associated with excess adiposity. 158 Circulating factors associated with obesity including saturated fatty acids and inflammatory cytokines, can selectively induce enzymes that promote sphingolipid synthesis thus affecting their role in. Pharmacological inhibition or genetic ablation of enzymes controlling sphingolipid synthesis in rodents was shown to ameliorates these conditions. 158 SITES FOR ACTION OF GC Taken together, these data suggest that GC has a profound immunomodulatory effect. The potential sites for its actions have been explored (Figure 1). GC can exert a systemic effect through alteration of lipid rafts and intracellular signaling, thereby altering the cross talk between NKT cells, s, Tregs and effector cells. 160,161 Alternatively, as NKT cells play a major role in liver-mediated tolerance, GC may exert it effects in the liver. 102, Finally, as CD1d molecules are highly expressed in the bowel, and as both s and NKT cells play a role in mucosal immunity in the bowel, GC may exert its effect on the gutassociated lymphoid tissue GC AS A THERAPEUTIC AGENT IN HUMANS An altered number or function of NKT cells has also been described in several human autoimmune diseases. 96 Patients with systemic lupus erythematosus, scleroderma, diabetes, multiple sclerosis, Sjögren syndrome and rheumatoid arthritis have been found to have lower numbers of peripheral blood NKT cells. Invariant Va24JaQ cells have also been found to have a regulatory role in patients with scleroderma. 98 In addition, CD1d-mediated recognition of a-galcer is highly conserved. Human NKT cells recognize a-galcer presented by mouse CD1d molecules and vice versa. 168 However, a-galcer has been shown to be hepatotoxic in mice, limiting its use in human testing. 169 Based on the preclinical data suggesting that GC improves insulin resistance, hepatic steatosis and metabolic syndrome in animal models of diabetes and NASH, the safety and efficacy of oral GC was tested in patients with these disorders. A safety study showed that oral administration of GC is safe and free of side effects. 170 In this open label phase I/II clinical study, patients with liver biopsy-proven NASH were treated with oral GC for 24 weeks and followed for 16 additional weeks. The safety was assessed in all patients by monitoring for adverse events during regularly scheduled visits. The efficacy was assessed by periodic evaluation of liver enzymes, comparison of preand posttreatment liver biopsies and MRIs, and a number of metabolic and immunological parameters. No treatment-related adverse events were observed during treatment or follow-up in any of the patients. Reduced aspartate aminotransferase, alanine aminotransferase and g-glutamyl transferase levels were observed, and the histological findings of the liver biopsies improved in some of the patients. Serum hemoglobin A1C, triglycerides and LDL cholesterol also improved. Glucose-stimulated insulin secretion, as measured using an intravenous glucose tolerance test, improved in some of the patients. The liver fat content, as assessed by MRI, was reduced. An increase in the percentage of peripheral blood NKT cells was noted is some of the treated patients. The results of this preliminary study indicate that b-gc may be a promising treatment modality for liver injury and metabolic syndrome in individuals with NASH. The efficacy and mode of administration of b-glucosylceramide for the treatment of NASH should be further determined in large-scale, randomized, placebo-controlled trials. LESSONS FOR THE FUTURE Is it risky to use ERT in patients with GD? Based on the above study, one can hypothesize that the increase in the GC serum level in patients with GD has a protective effect against several inflammatory and malignant disorders. Alternatively, the break down of GC to ceramide can underline part of these findings. ERT may stimulate a process to change the balance of GC to ceramide in treated patients. ERT, mainly at high doses, may be associated with an increased incidence of diabetes, PD, malignant disorders, and, to a lesser extent, atherosclerosis and autoimmunity. Although this hypothesis requires further research, it certainly provides a new approach to understanding the different phenotypes and outcomes

8 521 a GC Tregs Altered lipid rafts NKT Cross talk Altered expression of raft membrane proteins Flotilin2 NFkB T cell b Bowel mucosa Systemic effect GC Effector cells M cell Lamina Propria NKT GC Tregs Macrophage Interfollicular T cell area B cell follicule Perifollicular area NKT Th2 cell Tregs Th3 cell Tr1 Figure 1 Possible mechanisms for the immunomodulatory effect of glucocerebroside. (a) GC may exert an effect on lipid rafts, altering the intracellular inflammatory cascade, and in turn altering the cross talk among NKT cells, s and Tregs. (b) The effects of GC may be systemic or occur at the level of the bowel mucosa by altering the -NKT cell interaction or promoting Tregs. observed among populations of patients that are treated with highversus low-dose ERT. Significant suppression of the circulating level of GC, a natural protective molecule against inflammation, may thereby prevent activation of s, NKT cells and Tregs or alter lipid rafts on specific subsets of lymphocytes. These effects could negate the proposed selective advantage in patients with GD. It may also be associated with the alteration of the levels of ceramide, and of other sphingolipids, exerting an effect on several signaling and metabolic pathways. Therefore, it is reasonable to treat patients with mild GD with low-dose ERT in an attempt to decrease the potential risk for the development of malignancies and cardiovascular and cerebrovascular disorders associated with high-dose ERT. The data presented above suggest that the reduction of GC levels by high-dose ERT may be associated with an alteration of the immune balance. There is a possible association between ERT and increased rates of immunemediated disorders, and altered metabolic pathways that can lead to insulin resistance and diabetes. What may be a side effect of an otherwise critically important and absolutely necessary therapy for patients with moderate to severe GD, whose disease features may cause significant suffering and devastating multi-organ dysfunction, may not be justified in patients with an otherwise very mild disease. Should we use GC to treat patients with disorders in which the immune system in involved? The data from several animal models and the preliminary data available from recently conducted clinical trials suggest that GC should be considered as an immunomodulatory agent for the treatment of diabetes, metabolic syndrome, immune-mediated disorders and/or malignancy. Alteration of NKT cell-, Treg- and/or -dependent pathways by oral administration of GC may benefit patients with various disorders. However, further studies are required to determine its role as a potential therapy.