Analysis of the Targets and Glycosylation of Monoclonal IgAs From MGUS and Myeloma Patients.

Previous studies showed that monoclonal immunoglobulins G (IgGs) of "monoclonal gammopathy of undetermined significance" (MGUS) and myeloma were hyposialylated, thus presumably pro-inflammatory, and for about half of patients, the target of the monoclonal IgG was either a virus-Epstein-Barr virus (EBV), other herpes viruses, hepatitis C virus (HCV)-or a glucolipid, lysoglucosylceramide (LGL1), suggesting antigen-driven disease in these patients. In the present study, we show that monoclonal IgAs share these characteristics. We collected 35 sera of patients with a monoclonal IgA (6 MGUS, 29 myeloma), and we were able to purify 25 of the 35 monoclonal IgAs (6 MGUS, 19 myeloma). Monoclonal IgAs from MGUS and myeloma patients were significantly less sialylated than IgAs from healthy volunteers. When purified monoclonal IgAs were tested against infectious pathogens and LGL1, five myeloma patients had a monoclonal IgA that specifically recognized viral proteins: the core protein of HCV in one case, EBV nuclear antigen 1 (EBNA-1) in four cases (21.1% of IgA myeloma). Monoclonal IgAs from three myeloma patients reacted against LGL1. In summary, monoclonal IgAs are hyposialylated and as described for IgG myeloma, significant subsets (8/19, or 42%) of patients with IgA myeloma may have viral or self (LGL1) antigen-driven disease.

Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.

Intracellular metabolite ß-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity.

Sensing and reacting to tissue damage is a fundamental function of immune systems. Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. Notably, Mincle also recognizes glycolipid ligands on pathogens. To elucidate endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as ß-glucosylceramide (GlcCer), which is a ubiquitous intracellular metabolite. Synthetic ß-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated in Mincle-deficient cells. Sterile inflammation induced by excessive cell death in the thymus was exacerbated by hematopoietic-specific deletion of degrading enzyme of ß-GlcCer (ß-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1-deficient dendritic cells (DCs) in which ß-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas these responses were compromised in DCs from GBA1 × Mincle double-deficient mice. These results suggest that ß-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity.

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

Innate Invariant NKT Cell Recognition of HIV-1-Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell-mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

Clonal Immunoglobulin against Lysolipids in the Origin of Myeloma.

Antigen-driven selection has been implicated in the pathogenesis of monoclonal gammopathies. Patients with Gaucher's disease have an increased risk of monoclonal gammopathies. Here we show that the clonal immunoglobulin in patients with Gaucher's disease and in mouse models of Gaucher's disease-associated gammopathy is reactive against lyso-glucosylceramide (LGL1), which is markedly elevated in these patients and mice. Clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC). Substrate reduction ameliorates Gaucher's disease-associated gammopathy in mice. Thus, long-term immune activation by lysolipids may underlie both Gaucher's disease-associated gammopathies and some sporadic monoclonal gammopathies.

Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens.

Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d-α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1(+) type I NKT cell repertoire. These cells express a range of TCR α- and ß-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7-8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A'-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d-α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition.

Antigen Specificity of Type I NKT Cells Is Governed by TCR ß-Chain Diversity.

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR ß-chains, including Vß8, Vß7, and Vß2 in mice and Vß11 in humans, joined to a range of TCR Dß and Jß genes. In this study, we demonstrate that TCR ß-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vß7(+) NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vß8(+) NKT cells from mice. We show that the influence of the TCR ß-chain is due to a combination of Vß-, Jß-, and CDR3ß-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3ß-encoded residues determined Ag preference. These findings indicate that NKT TCR ß-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets.

The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α-linked glycosylceramides.

Glycosylceramides in mammalian species are thought to be present in the form of ß-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both ß-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

Characterization of Scedosporium apiospermum glucosylceramides and their involvement in fungal development and macrophage functions.

Scedosporium apiospermum is an emerging fungal pathogen that causes both localized and disseminated infections in immunocompromised patients. Glucosylceramides (CMH, GlcCer) are the main neutral glycosphingolipids expressed in fungal cells. In this study, glucosylceramides (GlcCer) were extracted and purified in several chromatographic steps. Using high-performance thin layer chromatography (HPTLC) and electrospray ionization mass spectrometry (ESI-MS), N-2'-hydroxyhexadecanoyl-1-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine was identified as the main GlcCer in S. apiospermum. A monoclonal antibody (Mab) against this molecule was used for indirect immunofluorescence experiments, which revealed that this CMH is present on the surface of the mycelial and conidial forms of S. apiospermum. Treatment of S. apiospermum conidia with the Mab significantly reduced fungal growth. In addition, the Mab also enhanced the phagocytosis and killing of S. apiospermum by murine cells. In vitro assays were performed to evaluate the CMHs for their cytotoxic activities against the mammalian cell lines L.929 and RAW, and an inhibitory effect on cell proliferation was observed. Synergistic in vitro interactions were observed between the Mab against GlcCer and both amphotericin B (AmB) and itraconazole. Because Scedosporium species develop drug resistance, the number of available antifungal drugs is limited; our data indicate that combining immunotherapy with the available drugs might be a viable treatment option. These results suggest that in S. apiospermum, GlcCer are most likely cell wall components that are targeted by antifungal antibodies, which directly inhibit fungal development and enhance macrophage function; furthermore, these results suggest the combined use of monoclonal antibodies against GlcCer and antifungal drugs for antifungal immunotherapy.

Identification of novel glycolipid ligands activating a sulfatide-reactive, CD1d-restricted, type II natural killer T lymphocyte.

Sulfatide-reactive CD1d-restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental auto-immune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self-lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self-lipids remains unknown. By isolating lipids from a CD1d-expressing, highly stimulatory antigen presenting cell, we identified isoforms of ß-glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide-reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated ß-galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingo-lipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d-dependent reactivity to antigen presenting cells lacking all GlcCer-based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady-state autoreactivity by type II NKT cells.

Detection of antibody against fungal glucosylceramide in immunocompromised patients: a potential new diagnostic approach for cryptococcosis.

We have developed an ELISA to determine the value of anti-glucosylceramide antibody for the prediction of disseminated cryptococcosis in immunocompromised subjects and performed a clinical prospective study at the Medical University of South Carolina. The study enrolled a total of 53 patients who were free of active fungal diseases at the time of enrollment but at risk of developing one because they were all immunocompromised, e.g., (1) patients positive for HIV and (2) patients post- or awaiting solid organ transplantation. Among 53 patients enrolled, two patients developed invasive cryptococcosis, and in both patients, IgM anti-GlcCer was detected in sera using the ELISA at least 6 weeks prior to the clinical presentation of the brain disease. These results were corroborated by a cryptococcal antigen lateral flow assay, which was also positive in serum prior to the development of meningoencephalitis. However, a high number of positive results were also detected in patients with no evidence of cryptococcosis. This study highlights the potential utility of this new assay in early diagnostic testing algorithms for patients at risk for cryptococcosis, but further investigations are needed to validate the sensitivity and specificity of the glucosylceramide ELISA as a predictor of cryptococcosis.

Impact of sugar stereochemistry on natural killer T cell stimulation by bacterial glycolipids.

Natural killer T (NKT) cells recognize glycolipids produced by Sphingomonas bacteria, and these glycolipids contain C6-oxidized sugars, either glucuronic acid or galacturonic acid, linked to ceramides. Glycolipids with gluco stereochemistry are the most prevalent. Multiple studies have demonstrated that galactosylceramides are more potent stimulators of NKT cells than their glucose isomers. To determine if this stereoselectivity is retained in the context of the C6-oxidized sugars found in bacterial glycolipids, we prepared two sets of gluco and galacto-glycolipids oxidized at their C6 positions and compared their NKT stimulatory properties. In the context of carboxylic acid groups at C6, gluco stereochemistry gave the more potent responses. We also prepared bacterial glycolipids containing more complex ceramide groups to determine if these chains impact NKT cell responses.

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

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 low-dose ERT and re-evaluating the use of ERT in asymptomatic patients.

Potential role of NKT regulatory cell ligands for the treatment of immune mediated colitis.

Natural killer T lymphocytes (NKT) have been implicated in the regulation of autoimmune processes in both mice and humans. In response to stimuli, this subset of cells rapidly produces large amounts of cytokines thereby provoking immune responses, including protection against autoimmune diseases. NKT cells are present in all lymphoid compartments, but are most abundant in the liver and bone marrow. They are activated by interaction of their T-cell receptor with glycolipids presented by CD1d, a nonpolymorphic, major histocompatibility complex class I-like molecule expressed by antigen presenting cells. Several possible ligands for NKT cells have recently been suggested. beta-glucosylceramide, a naturally occurring glycolipid, is a metabolic intermediate in the anabolic and catabolic pathways of complex glycosphingolipids. Like other beta-glycolipids, beta-glucosylceramide has an immunomodulatory effect in several immune mediated disorders, including immune mediated colitis. Due to the broad impact that NKT cells have on the immune system, there is intense interest in understanding how NKT cells are stimulated and the extent to which NKT cell responses can be controlled. These novel ligands are currently being evaluated in animal models of colitis. Here, we discuss strategies to alter NKT lymphocyte function in various settings and the potential clinical applications of natural glycolipids.

Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice.

In previous studies we showed that a Cryptococcus neoformans mutant lacking glucosylceramide (Deltagcs1) is avirulent and unable to reach the brain when it is administered intranasally into an immunocompetent mouse and is contained in a lung granuloma. To determine whether granuloma formation is key for containment of C. neoformans Deltagcs1, we studied the role of C. neoformans glucosylceramide in a T- and NK-cell-immunodeficient mouse model (Tgepsilon26) in which alveolar macrophages (AMs) are not activated and granuloma formation is not expected. The results show that Tgepsilon26 mice infected with Deltagcs1 do not produce a lung granuloma and that the Deltagcs1 mutant proliferates in the lungs and does disseminate to the brain, although its virulence phenotype is dramatically reduced. Since Deltagcs1 can grow only in acidic niches, such as the phagolysosome of AMs, and not in neutral or alkaline environments, such as the extracellular spaces, we hypothesize that in immunodeficient mice Deltagcs1 proliferates inside AMs. Indeed, we found that depletion of AMs significantly improved Tgepsilon26 mouse survival and decreased the dissemination of Deltagcs1 cells to the central nervous system. Thus, these results suggest that the growth of Deltagcs1 in immunodeficient mice is maintained within AMs. This study highlights the hypothesis that AMs may exacerbate C. neoformans infection in conditions in which there is severe host immunodeficiency.

Amelioration of hepatic fibrosis via beta-glucosylceramide-mediated immune modulation is associated with altered CD8 and NKT lymphocyte distribution.

BACKGROUND: While CD8 lymphocytes possess pro-fibrogenic properties and NK (non-T) cells are anti-fibrogenic, the role of NKT lymphocytes in liver fibrosis is still unclear. Beta-glucosylceramide (GC), a naturally occurring glycolipid, exerts modulatory effects on these cells. AIM: To explore the role of NKT cells in hepatic fibrosis via GC. METHODS: Hepatic fibrosis was induced by biweekly intra-peritoneal (IP) carbon tetrachloride (CCl(4)) administrations for 7 weeks in 5 groups (A-E) of male C57Bl/6 mice. Mice were treated with daily IP GC injections in groups A and C, or daily oral doses in groups B and D. GC was administered either for the duration of the study period (in groups A and B), or for the last 3 weeks of CCl(4) induction (groups C and D). GC-treated mice were compared with non-treated fibrotic controls (group E) and naive rodents (group F). Liver fibrosis, injury parameters and FACS analysis of lymphocytes were assessed. RESULTS: Marked amelioration (P < 0.0001) of hepatic fibrosis observed in all GC-treated mice without altering reactive oxygen species production. As determined by Sirius red-stained liver tissue sections and measured by Bioquant morphometry; all CCl(4)-administered groups significantly (P < 0.0001) increased the relative fibrosis area compared with naive animals. The increases were 14.4 +/- 1.03-fold in group A, 7.9 +/- 0.37-fold in group B, 5.2 +/- 0.2-fold in group C, 10.3 +/- 0.4-fold in group D and 23.8 +/- 1.9-fold in group E. Western blot analysis for alpha smooth muscle actin from liver extracts followed a similar pattern, increasing in groups A-E. A significant decrease in liver damage was observed in all GC-treated groups, as noted by a decrease in transaminase serum levels (P < 0.005). The beneficial effect of GC was associated with a significant decrease in the intra-hepatic NKT and CD8 lymphocytes as well as their attenuation of both T(h)1 and T(h)2 cytokines. CONCLUSIONS: Administration of GC had a significant anti-fibrotic effect following CCl(4) administration. This effect was associated with an altered NKT and CD8 lymphocyte distribution and a cytokine shift. Immune modulation using GC may have a role in the treatment of fibrosis and other immune-mediated disorders.

The biology of NKT cells.

Recognized more than a decade ago, NKT cells differentiate from mainstream thymic precursors through instructive signals emanating during TCR engagement by CD1d-expressing cortical thymocytes. Their semi-invariant alphabeta TCRs recognize isoglobotrihexosylceramide, a mammalian glycosphingolipid, as well as microbial alpha-glycuronylceramides found in the cell wall of Gram-negative, lipopolysaccharide-negative bacteria. This dual recognition of self and microbial ligands underlies innate-like antimicrobial functions mediated by CD40L induction and massive Th1 and Th2 cytokine and chemokine release. Through reciprocal activation of NKT cells and dendritic cells, synthetic NKT ligands constitute promising new vaccine adjuvants. NKT cells also regulate a range of immunopathological conditions, but the mechanisms and the ligands involved remain unknown. NKT cell biology has emerged as a new field of research at the frontier between innate and adaptive immunity, providing a powerful model to study fundamental aspects of the cell and structural biology of glycolipid trafficking, processing, and recognition.

Beta-glucosylceramide: a novel method for enhancement of natural killer T lymphoycte plasticity in murine models of immune-mediated disorders.

BACKGROUND: beta-Glucosylceramide, a naturally occurring glycolipid, exerts modulatory effects on natural killer T (NKT) lymphocytes. AIM: To determine whether beta-glucosylceramide can alter NKT function in opposite directions, colitis was induced by intracolonic installation of trinitrobenzenesulphonic acid, and hepatocellular carcinoma (HCC) was induced by transplantation of Hep3B cells. METHODS: The immunological effect of beta-glucosylceramide was assessed by analysis of intrahepatic and intrasplenic lymphocyte populations, serum cytokines and STAT protein expression. RESULTS: Administration of beta-glucosylceramide led to alleviation of colitis and to suppression of HCC, manifested by improved survival and decreased tumour volume. The beneficial effects were associated with an opposite immunological effect in the two models: the peripheral:intrahepatic CD4:CD8 lymphocyte ratio increased in the colitis model and decreased in the HCC group. The peripheral:intrahepatic NKT lymphocyte ratio decreased in beta-glucosylceramide-treated mice solely in the HCC model. The effect of beta-glucosylceramide was associated with decreased STAT1 and 4 expression, and with overexpression of STAT6, along with decreased interferon gamma levels in the colitis model, whereas an opposite effect was noted in the HCC model. CONCLUSIONS: beta-glucosylceramide alleviates immunologically incongruous disorders and may be associated with "fine tuning" of immune responses, by changes in plasticity of NKT lymphocytes.