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Structure-activity relationships provide insight into the binding interactions of beta-glycosphingolipids (GSLs) with both the TCR and the CD1d molecules, as well as the subsequent immunologic response of regulatory NKT cells., Aim: To determine the effects of synthetic GSL structures on their immune modulatory functions., Methods: GSLs of various structures were tested in vitro and in an animal model of Concanavalin A (ConA) immune-mediated hepatitis., Results: In vitro, using SV40 binding to live monkey CV1 cells, the l-threo stereoisomer of C8-β-LacCer inhibits caveolar internalization, reducing viral binding to the cell surface. In vivo, in the ConA model, LR172, which has a saturated C8 chain, and LR178, which has a trans double bond at C-2 in the C8 chain, suppressed the immune-mediated liver inflammation and reduced IFNγ levels in a dose dependent manner. The beneficial effects of LR172 and of LR178 are associated with suppression of liver apoptosis, increased phosphorylated STAT3 expression in the liver, and an increase in the NKT liver/spleen ratio., Summary: The assembly of GSLs determines their immunomodulatory effect and can serve as a method for structure-based design of immunotherapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Plasticity in biological systems is attributed to the combination of multiple parameters which determine function. These include genotypic, phenotypic, and environmental factors. While biological processes can be viewed as ordered and sequential, biological randomness was suggested to underline part of them. The present review looks into the concept of randomness in biological systems by exploring the glycosphingolipids-NKT cells example. NKT cells are a unique subset of regulatory lymphocytes which play a role in both inflammation and tolerance. Glycosphingolipids promote an immune balance by changing different arms of the immune system in opposing environments. Traditional immunology looks at skewing the immune system into different directions by different types of activation of the same cell stimulation of different cells subsets, use of different ligands, or different the effect of different immune environments. While these may explain some of the effects, the lack of consistency and opposing results under similar settings may involve randomness which may also be part of real life effects of immunomodulatory agents. It means that several of the biological processes, cannot be explained by simple linear models, and may involve more complex concepts. The application for these concepts for improving therapies to patients with Gaucher disease are discussed. SUMMARY   The use of different ligands that target a variety of cell subsets in different immune environments may underlie differences in the functionality of NKT cells and their variability in response to NKT-based therapies. The novel concept of randomness in biology means that several biological processes cannot be solely explained by simple linear models and may instead involve much more complicated schemes of random disorder. These may have implications on future design of therapeutic regimens for improving the response to current treatments.

Beta-glucosylceramide has been shown to affect natural killer T cell function in models of inflammation. We, therefore, investigated the effects of different beta-glycosphingolipids, including beta-glucosylceramide, on STAT (signal transducers and activators of transcription) signaling pathways and determined whether these effects were mediated by lipid raft microdomains and/or CD1d molecules. The effects of alpha- and beta-structured ligands on the lipid raft protein flotillin-2 were studied in both natural killer T hybridoma cells and leptin-deficient mice. To determine whether CD1d was involved in the effects of the beta-glycosphingolipids, an anti-CD1d blocking antibody was used in a cell proliferation assay system. The downstream effects on the protein phosphorylation levels of STAT1, STAT3, and STAT6 were examined in both immune-mediated hepatitis and hepatoma models. The effects of beta-glycosphingolipids on the STAT signaling pathways were found to be dependent on CD1d. Lipid rafts were affected by both the dose and ratio of the beta-glycosphingolipids and the acyl chain length, and these effects were followed by downstream effects on STAT proteins. Our results show that beta-glycosphingolipids have beneficial effects in natural killer T cell-dependent immune-mediated metabolic and malignant animal models in vivo.

The aim of this study was to determine the effect of beta-glycosphingolipids on intra-hepatic natural killer T (NKT) lymphocyte regulatory function and on lymphocyte trapping via alteration of cell membrane lipid rafts. Immune-mediated colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Mice were treated with beta-lactosylceramide (LC), beta-glucosylceramide (GC), beta-galactosylceramide, ceramide, or a combination of both GC and LC (IGL), or solvent alone. Lipid rafts were investigated by fluorescence-activated cell sorting analysis of ganglioside-GM1 and fluorescence microscopy of structure. Administration of beta-glycosphingolipids resulted in an increased intrahepatic/peripheral NKT ratio, increased intrahepatic CD8+ lymphocyte trapping, decreased serum interferon-gamma (IFN-gamma) levels and decreased serum IFN-gamma/interleukin-10 ratio. Administration of GC, LC, or IGL significantly altered the levels of GM1, a key marker of lipid rafts, on NKT regulatory lymphocytes. The immune modulatory effect of beta-glycosphingolipids was associated with increased survival and significant alleviation of colitis as determined by improvement in both the macroscopic and microscopic scores. In conclusion, administration of beta-glycosphingolipids increased NKT regulatory lymphocyte redistribution and intrahepatic CD8(+) T lymphocyte trapping, resulting in alleviation of immune-mediated colitis. The effects of these naturally occurring compounds were associated with modification of the T lymphocyte lipid raft structure, which is a site for immune modulation.

Natural killer T-cell (NKT) regulatory lymphocytes have been shown to behave differently in various immune settings. The aim of the present study was to determine the effect of microenvironmental signaling on NKT polarization and the process of active CD8 and NKT intrahepatic lymphocyte sequestration. In an in vitro assay, double negative (DN) NKT hybridoma cells were incubated with Hep3B hepatoma cells. This caused a significant increase in the secretion of alpha-fetoprotein (AFP) from Hep3B cells. When NKT cells were exposed to beta-glucoslyceramide (beta-GC) prior to incubation, Hep3B cells exhibited increased proliferation, increased IFN secretion, and reduced AFP secretion. In vivo, the adoptive transfer of naïve DN NKT cells into athymic nude-nu mice transplanted with human Hep3B hepatocellular carcinoma (HCC) caused accelerated tumor growth. This effect was inhibited by prior ex vivo exposure of DN NKT lymphocytes to beta-GC. To assess the effect of the immunological environment on NKT cells, immune mediated hepatitis and colitis were induced simultaneously in mice. Induction of TNBS colitis prior to administration of concanavalin A (Con A) hepatitis resulted in an aggravation of the liver damage caused by Con A hepatitis alone. This effect was associated with reduced intrahepatic CD8+ T cell trapping and an increase in intrahepatic NKT cells. The presence of different ligands altered host microenvironment signaling and influenced the fate and polarization of NKT cells and the sequestration of active intrahepatic lymphocytes. These data support the notion that NKT regulatory lymphocytes have an inherent plasticity that may be important for their regulatory function.

beta-Glycosphingolipids have emerged as a family of potential ligands for natural killer T (NKT)-regulatory lymphocytes. This subset of regulatory lymphocytes has been implicated in the regulation of autoimmune processes. The major histocompatibility complex (MHC) Class I-like CD1d glycoprotein is a member of the CD1 family of antigen-presenting molecules and is responsible for selection of NKT cells. beta-Glycolipids have been shown to alter immune responses in the opposing settings of autoimmune diseases or cancer. In this review, we discuss the potential use of beta-glycoshpingolipids for NKT-based immunotherapy.

• Microtubules (MTs) are tubular polymers of tubulin that are highly dynamic and found throughout the cytoplasm and play a role in the innate and adaptive immune systems. • Studies suggest an essential role for MTs in the gut. • Here, we propose a model that represents gut MTs as potential targets for immunotherapy. Microtubules (MTs) are tubular polymers of tubulin that are highly dynamic and found throughout the cytoplasm. MTs are involved in maintaining cell structure and, together with microfilaments and intermediate filaments, form the cytoskeleton. Recent findings on MT structure and function contributed to the understanding of their potential role as players in the innate and adaptive immune systems. Additionally, studies suggest an essential role for these cellular structures in the gut. Here, we review recent data on interactions between MT and various arms of the immune system and propose a model that represents gut MTs as potential targets for immunotherapy, and specifically for oral immunotherapy. [ABSTRACT FROM AUTHOR]

Plasticity in biological systems is attributed to the combination of multiple parameters which determine function. These include genotypic, phenotypic, and environmental factors. While biological processes can be viewed as ordered and sequential, biological randomness was suggested to underline part of them. The present review looks into the concept of randomness in biological systems by exploring the glycosphingolipids-NKT cells example. NKT cells are a unique subset of regulatory lymphocytes which play a role in both inflammation and tolerance. Glycosphingolipids promote an immune balance by changing different arms of the immune system in opposing environments. Traditional immunology looks at skewing the immune system into different directions by different types of activation of the same cell stimulation of different cells subsets, use of different ligands, or different the effect of different immune environments. While these may explain some of the effects, the lack of consistency and opposing results under similar settings may involve randomness which may also be part of real life effects of immunomodulatory agents. It means that several of the biological processes, cannot be explained by simple linear models, and may involve more complex concepts. The application for these concepts for improving therapies to patients with Gaucher disease are discussed. SUMMARY The use of different ligands that target a variety of cell subsets in different immune environments may underlie differences in the functionality of NKT cells and their variability in response to NKT-based therapies. The novel concept of randomness in biology means that several biological processes cannot be solely explained by simple linear models and may instead involve much more complicated schemes of random disorder. These may have implications on future design of therapeutic regimens for improving the response to current treatments. [ABSTRACT FROM AUTHOR]

The compounds of sphingomyelin-ceramide-glycosphingolipid pathways have been studied as potential secondary messenger molecules in various systems, along with liver function and insulin resistance. Secondary messenger molecules act directly or indirectly to affect cell organelles and intercellular interactions. Their potential role in the pathogenesis of steatohepatitis and diabetes has been suggested. Data samples collected from patients with Gaucher's disease, who had high levels of glucocerebroside, support a role for compounds from these pathways as a messenger molecules in the pathogenesis of fatty liver disease and diabetes. The present review summarizes some of the recent data on the role of glycosphingolipid molecules as messenger molecules in various physiological and pathological conditions, more specifically including insulin resistance and fatty liver disease. [ABSTRACT FROM AUTHOR]

Liver steatosis is a common characteristic of obesity and type 2 diabetes, and fatty liver disease is increasingly recognized as a major health burden. Accumulating evidence suggests that β-glycosphingolipids play an important role in insulin sensitivity and thus could affect hepatic steatosis. To determine the effect associated with β-glycosphingolipid-mediated amelioration of liver injury, seven groups of Psammomys obesus on a high-energy diet were studied. Animals were treated with daily injections of β-glucosylceramide, β-lactosylceramide, or a combination of both. β-glycosphingolipids ameliorated the hepatic injury manifested by decreased liver enzymes, liver weight, and hepatic fat, and improved liver histology. Administration of both β-glucosylceramide and β-lactosylceramide also decreased interferon (IFN)-γ serum levels. These effects were associated with improved serum cholesterol and triglyceride levels. These data suggest that β-glycosphingolipids ameliorate liver injury in an animal model of nonalcoholic steatohepatitis. [ABSTRACT FROM AUTHOR]

A link between altered levels of various gangliosides and the development of insulin resistance was described in transgenic mice. Naturally occurring glycosphingolipids were shown to exert immunomodulatory effects in a natural killer T (NKT) cell-dependent manner. This study examined whether glycosphingolipid-induced modulation of the immune system may reduce pancreatic and liver steatosis and stimulate insulin secre- tion in the Cohen diabetes-sensitive (CDS) rat, a lean model of non-insulin-resistant, nutritionally induced diabetes. Four groups of CDS rats fed a diabetogenic diet were treated with daily intraperito- neal injections of glycosphingolipids β-glucosylceramide, β-lactosylceramide, a combination of both (IGL), or vehicle (PBS) for up to 45 days. Immune modulation was assessed by fluorescence-activated cell sorting analysis of intrahepatic and intrasplenic lymphocytes. Steatosis was assessed by MRI imaging and histological examination of liver and pancreas, Blood glucose and plasma insulin concentrations were assessed during an oral glucose tolerance test. Administration of glycosphingolipids, particularly IGL, increased intrahepatic trapping of CD8 T and NKT lymphocytes. Pancreatic and liver histology were markedly improved and steatosis was reduced in all treated groups compared with vehicle-treated rats. Insulin secretion was restored after glycosphingolipid treatment, resulting in improved glucose tolerance. The immunomodulatory effect of 13-glycosphingolipids improved the p-cell function of the hyperglycemic CDS rat. Thus our results suggest a role for the immune system in the pathogenesis of diabetes in this model. [ABSTRACT FROM AUTHOR]

β-Glycosphingolipids have emerged as a family of potential ligands for natural killer T (NKT)- regulatory lymphocytes. This subset of regulatory lymphocytes has been implicated in the regulation of autoimmune processes. The major histocompatibility complex (MHC) Class I-like CD1d glycoprotein is a member of the CD1 family of antigen-presenting molecules and is responsible for selection of NKT cells. β-Glycolipids have been shown to alter immune responses in the opposing settings of autoimmune diseases or cancer. In this review, we discuss the potential use of β-glycoshpingolipids for NKT-based immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Natural killer T-cell (NKT) regulatory lymphocytes have been shown to behave differently in various immune settings. The aim of the present study was to determine the effect of microenvironmental signaling on NKT polarization and the process of active CD8 and NKT intrahepatic lymphocyte sequestration. In an in vitro assay, double negative (DN) NKT hybridoma cells were incubated with Hep3B hepatoma cells. This caused a significant increase in the secretion of alpha-fetoprotein (AFP) from Hep3B cells. When NKT cells were exposed to β-glucoslyceramide (β-GC) prior to incubation, Hep3B cells exhibited increased proliferation, increased IFN secretion, and reduced AFP secretion. In vivo, the adoptive transfer of naïve DN NKT cells into athymic nude-nu mice transplanted with human Hep3B hepatocellular carcinoma (HCC) caused accelerated tumor growth. This effect was inhibited by prior ex vivo exposure of DN NKT lymphocytes to β-GC. To assess the effect of the immunological environment on NKT cells, immune mediated hepatitis and colitis were induced simultaneously in mice. Induction of TNBS colitis prior to administration of concanavalin A (Con A) hepatitis resulted in an aggravation of the liver damage caused by Con A hepatitis alone. This effect was associated with reduced intrahepatic CD8+ T cell trapping and an increase in intrahepatic NKT cells. The presence of different ligands altered host microenvironment signaling and influenced the fate and polarization of NKT cells and the sequestration of active intrahepatic lymphocytes. These data support the notion that NKT regulatory lymphocytes have an inherent plasticity that may be important for their regulatory function. [Copyright &y& Elsevier]

Summary: Background: Non-responsiveness towards the currently used hepatitis B virus (HBV) vaccine is a major problem in attempts to protect against HBV infection. Several methods have been tested to overcome the lack of an effective immune response towards HBV antigens. Adjuvants that augment the immunologic reaction are essential components of the vaccines. β-Glycosphingolipids exert a natural killer T cell (NKT)-mediated immunomodulatory effect in various disorders. Aims: The aim of the present study was to test the ability of these compounds to augment the immune response towards HBV antigens, making them potential adjuvants for HBV vaccines. Six groups of mice were injected with different formulations of an HBV vaccine, along with various doses of β-glucosylceramide (β-GC), β-lactosylceramide (β-LC), or a combination of both (IGL) in different doses. The effect of β-glycosphingolipids on the immune response towards HBV was tested by fluorescence-activated cell sorting analysis of hepatic and splenic NKT and CD8 lymphocytes, and serum cytokine levels. Results: β-Sphingolipid treatment altered the hepatic NKT and CD8 lymphocyte distribution. β-LC, β-GC, and the combination of both augmented anti-HBV immunity, increasing both the anti-HBs titers and the percentage of mice exhibiting high titers. This effect was associated with altered hepatic NKT and CD8+ lymphocyte distribution. Conclusions: In summary, β-glycosphingolipids increased the anti-HBV immune response in association with an altered NKT and CD8 lymphocyte distribution, making β-glycosphingolipids potential potent adjuvants for overcoming non-responsiveness to HBV vaccination and augmenting the anti-viral immune response. [Copyright &y& Elsevier]