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Ehud Zigmond,1,* Oshrat Tayer-Shifman,1,* Gadi Lalazar,1 Ami Ben Ya'acov,1 Sarah Weksler-Zangen,2 David Shasha,1 Miriam Sklair-Levy,3 Lidya Zolotarov,1 Zvi Shalev,1 Rony Kalman,2 Ehud Ziv,2 Itamar Raz,2 Yaron Ilan1 1Liver Unit, 2Diabetes Unit, 3Department of Radiology, Hebrew University-Hadassah Medical Center, Jerusalem, Israel *These authors contributed equally to this workAbstract: 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.Keywords: NAFLD, glycolipids, STAT, NASH, insulin resistance, diabetes

Aim of the Study: Akt is involved in upregulating the insulin-signaling pathways essential for maintaining glucose metabolism. Glycosphingolipids are involved in the pathogenesis of glucose intolerance and associated target organ injury. On the other hand, oral administration of b-glucosylceramide (GC) has been shown to alleviate insulin resistance. The present study aimed to determine the effects of oral administration of insulin and GC, separately and in combination, on Akt expression and the subsequent effect on metabolic syndrome characteristics in leptin-deficient mice., Material and Methods: Four groups of leptin-deficient ob/ob mice were orally administered for four weeks: vehicle, GC, short-acting insulin, and GC combined with insulin. Mice were followed for hepatic Akt expression and changes in tumor necrosis factor a (TNF-a) level, hyperlipidemia, and liver damage., Results: In mice that received insulin or GC, particularly those that received both, the liver phosphorylation of Akt was significantly increased compared to those that received only vehicle. Serum TNF-a levels decreased in insulin-treated mice. These effects were associated with alleviating glucose intolerance and hyperlipidemia, as manifested by a significant glucose tolerance test improvement and reductions in serum triglyceride and cholesterol levels. Significant liver damage alleviation was noted by liver enzyme reductions in all treated groups, along with liver steatosis in the insulin-treated mice., Conclusions: These data established the potential use of oral insulin administration with glycosphingolipids to alleviate glucose intolerance and associated liver damage and hyperlipidemia via increased Akt expression in the liver. The data support targeting Akt as a potent therapeutic target for metabolic syndrome., Competing Interests: Y. Ilan is a consultant for Teva; ENZO; Protalix; Betalin Therapeutics; Immuron; SciM; Natural Shield; Oberon Sciences; Tiziana Pharma; Plantylight; Exalenz Bioscience. The authors declare no conflict of interest., (Copyright © 2023 Clinical and Experimental Hepatology.)

Introduction: Glucocorticoids contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Natural killer T cells play a role in the pathogenesis of NAFLD and response to steroids. The present study aimed to determine the role of CD1d in steroid-mediated metabolic derangement and the steroid-protective effect of glycosphingolipids., Methods: Ten groups of mice were studied. Steroids were orally administered to C57BL/6 mice to assess the therapeutic effect of β-glucosylceramide (GC) on the development of steroid-mediated liver damage and metabolic derangements. The role of CD1d in the pathogenesis of steroid-induced liver damage and in mediating the hepatoprotective effect of GC was studied in CD1d -/- mice., Results: A model of oral administration of steroids was established, resulting in insulin resistance, hyperinsulinemia, hypertriglyceridemia, liver steatosis, and hepatocellular injury. Steroid administration to CD1d -/- mice was associated with hyperglycemia and hypertriglyceridemia. However, CD1d -/- mice did not manifest marked steroid-induced steatosis. GC treatment alleviated steroid-associated metabolic derangements and liver injury independent of CD1d expression., Conclusion: A steroid-mediated model of NAFLD and metabolic derangements was established in which steroid-mediated steatosis was CD1d-dependent while steroid-induced liver necrosis, inflammation, and metabolic changes were CD1d-independent, which may support a dichotomy between steatosis and steatohepatitis in NAFLD., (© 2022. The Author(s).)

Background and Aims: Acetaminophen (APAP) and HMG-CoA reductase inhibitors are common causes of drug-induced liver injury (DILI). This study aimed to determine the ability to reduce APAP- and statins-mediated liver injury by using formulations that combine glycosphingolipids and vitamin E. Methods: Mice were injected with APAP or with statins and treated before and after with β-glucosylceramide (GC), with or without vitamin E. Mice were followed for changes in liver enzymes, liver histology, hepatic expression of JNK, STAT3 and caspase 3, as well as intrahepatic natural killer T cells (NKT) and the serum cytokine levels by flow cytometry. Results: Administration of GC before or after APAP alleviated the liver damage, as noted by a reduction of the liver enzymes, improvement in the liver histology and decreased hepatic caspase 3 expression. Beneficial effect was associated with a reduction of the intrahepatic NKT, JNK expression in the liver, and increased glutathione in the liver, and decreased TNF-α serum levels. Synergistic effect of co-administration of GC with vitamin E was observed. Similar protective effect of GC on statin-mediated liver damage was documented by a reduction in liver enzymes and improved liver histology, which was mediated by reduction of NKT, increased STAT3 expression in the liver, and reduced the TGF-β and IL17 levels. Conclusions: β-glycosphingolipids exert a hepatoprotective effect on APAP- and statins-mediated liver damage. Vitamin E exerted a synergistic effect to that of GC. The generation of "safer drug" formulations, which include an active molecule combined with a hepatoprotective adjuvant, may provide an answer to the real unmet need of DILI., Competing Interests: The authors have no conflict of interests related to this publication.

The orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) (n=6) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human antibody IgG1 domain., Aim: To evaluate the immune modulatory effect of the oral administration of plant cells expressing PRX-106., Methods: Mice treated with Concanavalin A (ConA) to induce immune hepatitis was orally treated with cells expressing PRX-106 containing 0.5 or 5μg PRX 106. In the colitis model, TNBS-colitis was induced in mice followed by the oral administration of plant cells expressing PRX-106. The immune modulatory effect was determined through follow-up to assess the clinical effect, histology, and serum cytokine levels and by FACS analysis for lymphocyte subsets., Results: The oral administration of BY-2 cells expressing PRX-106 alleviated immune-mediated liver injury. Serum AST and ALT levels decreased and were comparable to those of mice that had received high-dose steroids. The beneficial effect was also observed as a marked decrease in hepatic necrosis. In the colitis model, the oral administration of BY-2 plant cells expressing PRX-106 alleviated weight loss associated with immune-mediated colitis and improved bowel histology. A reduction in I-IkB-alpha phosphorylation in treated mice was also observed. These effects were associated with a significant alteration in the distribution of CD4+CD25+FOXP3+ cells., Conclusions: Plant cells expressing recombinant anti-TNF fusion protein show biological activity when orally administered, exerting an immune modulatory effect through the alleviation of immune-mediated hepatitis and immune-mediated colitis., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Several of the drugs in development for treatment of nonalcoholic steatohepatitis (NASH) target liver fibrosis or have side effects that prohibit their long-term use in patients with mild to moderate disease. Lunasin is a soy-derived peptide with anti-inflammatory properties. ADM's CardioAid™ is a plant sterol extract that exerts cholesterol- and triacylglycerol-lowering effects., Aim: To determine the immunomodulatory effects of CardioAid and lunasin in a high-fat diet (HFD) animal model of NASH., Methods: C57BL/6 mice on an HFD were orally administered CardioAid or lunasin for 25 weeks. The effects on the immune system, liver function, insulin resistance and lipid profile were studied., Results: Treatment with CardioAid and lunasin was associated with a significant decrease in the CD4/CD8 ratio and an increase in CD4+CD25+ lymphocytes. A decrease in interleukin 1-alpha serum levels and an increase in transforming growth factor beta serum levels were noted. These were associated with alleviation of liver damage as indicated by a significant decrease in liver enzymes and improvement in the histological nonalcoholic fatty liver disease activity score (NAS). Decreases in both serum triglyceride and serum glucose levels were observed in treated mice. A decrease in total body fat measured by EchoMRI was also observed in treated mice., Conclusions: CardioAid and lunasin exerted hepatoprotective and glucose-protective effects in an HFD NASH model. These data and the high-safety profiles of CardioAid and Lunasin support their use in patients in the early stages of NASH to prevent deterioration due to the disease., (© 2017 S. Karger AG, Basel.)

Vitamin D has been known for its anti-inflammatory properties. Extracts derived from Lentinula edodes (Shiitake) edible mushroom exert an anti-inflammatory effect. These extracts contain high levels of ergosterol, which converts into ergocalciferol (vitamin D2) following exposure to ultraviolet light, followed by absorption and hydroxylation into the active form 25-hydroxyvitamin D [25(OH)D]. To determine the anti-inflammatory effect of overexpression of vitamin D in edible mushrooms, L. edodes mushrooms were exposed to ultraviolet-B light, freeze-dried, followed by measurement of vitamin D2 contents, in their dry weight. C57B1/6 mice were orally treated with vitamin D2-enriched or nonenriched mushroom extract prior and during concanavalin A-immune-mediated liver injury. Exposure to ultraviolet light increased vitamin D2 content in Shiitake edible mushrooms. Following feeding of vitamin D-enriched mushroom extracts to mice with immune-mediated hepatitis, a significant decrease in liver damage was noted. This was shown by a decrease in alanine aminotransferase and aspartate aminotransferase serum levels, a decrease in proportion of mice with severe liver injury, and by improvement in liver histology. These effects were associated with a decrease in serum interferon gamma levels. A synergistic effect was noted between the anti-inflammatory effect of the mushroom extracts and that of vitamin D. Oral administration of vitamin D-enriched L. edodes edible mushroom exerts a synergistic anti-inflammatory effect in the immune-mediated hepatitis. The data support its potential use as safe immunomodulatory adjuvant for the treatment of HCV and nonalcoholic steatohepatitis.

Background: Gut-derived bacterial endotoxin is an important cofactor in the pathogenesis of IBD. Regulatory T cells (Tregs) are essential for maintenance of peripheral tolerance and can prevent and alleviate IBD. To determine the immune modulatory effect of anti-LPS enriched hyperimmune colostrum, its ability to induce Tregs and alleviate immune mediated colitis., Methods: Immune-mediated colitis was induced in mice by intra-colonic instillation of Trinitrobenzene Sulfonate (TNBS). Four groups of mice were orally administered with two dosages of IgG-enriched colostrum fractions. The fractions were harvested from cows immunized against LPS derived from intestinal Escherichia coli bacteria (Imm124E). Control mice received non-immunized colostrum or vehicle (PBS). Treatment was administered one day following sensitization and four additional days following the administration of TNBS. The following parameters in the mice were tracked: body weight, bowel histology, serum cytokine levels and regulatory T cells., Results: Oral administration of Imm124E hyperimmune colostrum ameliorated immune-mediated colitis. Significant amelioration of weight reduction was noted in treated mice. Oral administration of Imm124E improved bowel histology. Both the extent of the disease, inflammation score, and colitis damage and regeneration scores decreased in Imm-124E treated animals. These effects were associated with an increase in serum IL10 anti inflammatory cytokine levels, and an increase in CD4 + CD25+ and CD4 + Foxp3+ Tregs., Conclusions: Oral administration of Imm124E promoted Tregs and alleviated bowel inflammation in immune mediated colitis. The present data suggests that the microbiome may serve as a target for Tregs-based immunotherapy.

Hepatocellular carcinoma is the second leading cause of cancer death worldwide. DNA microarray analysis identified the ornithine aminotransferase (OAT) gene as a prominent gene overexpressed in hepatocellular carcinoma (HCC) from Psammomys obesus. In vitro studies demonstrated inactivation of OAT by gabaculine (1), a neurotoxic natural product, which suppressed in vitro proliferation of two HCC cell lines. Alpha-fetoprotein (AFP) secretion, a biomarker for HCC, was suppressed by gabaculine in both cell lines, but not significantly. Because of the active site similarity between GABA aminotransferase (GABA-AT) and OAT, a library of 24 GABA-AT inhibitors was screened to identify a more selective inhibitor of OAT. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidene)cyclopentane-1-carboxylic acid (2) was found to be an inactivator of OAT that only weakly inhibits GABA-AT, l-aspartate aminotransferase, and l-alanine aminotransferase. In vitro administration of 2 significantly suppressed AFP secretion in both Hep3B and HepG2 HCC cells; in vivo, 2 significantly suppressed AFP serum levels and tumor growth in HCC-harboring mice, even at 0.1 mg/kg. Overexpression of the OAT gene in HCC and the ability to block the growth of HCC by OAT inhibitors support the role of OAT as a potential therapeutic target to inhibit HCC growth. This is the first demonstration of suppression of HCC by an OAT inactivator.

It has been shown that inhibitors of the immune system reside in the spleen and inhibit the endogenous antitumor effects of the immune system. We hypothesized that splenectomy would inhibit the growth of relatively large non-small lung cancer (NSCLC) tumors by modulating the systemic inhibition of the immune system, and in particular Myeloid Derived Suppressor Cells (MDSC). The effect of splenectomy was evaluated in several murine lung cancer models. We found that splenectomy reduces tumor growth and the development of lung metastases, but only in advanced tumors. In immune-deficient NOD-SCID mice the effect of splenectomy on tumor growth and metastatic spread disappeared. Splenectomy significantly reduced the presence of MDSC, and especially monocytic-MDSC in the circulation and inside the tumor. Specific reduction of the CCR2+ subset of monocytic MDSC was demonstrated, and the importance of the CCL2-CCR2 axis was further shown by a marked reduction in CCL2 following splenectomy. These changes were followed by changes in the macrophages contents of the tumors to become more antitumorigenic, and by increased activation of CD8 + Cytotoxic T-cells (CTL). By MDSC depletion, and adoptive transfer of MDSCs, we demonstrated that the effect of splenectomy on tumor growth was substantially mediated by MDSC cells. We conclude that the spleen is an important contributor to tumor growth and metastases, and that splenectomy can blunt this effect by depletion of MDSC, changing the amount and characteristics of myeloid cells and enhancing activation of CTL.

The mechanisms by which tumor-associated neutrophils (TANs) affect tumor growth are to a large extent unknown. Regulatory T-cells (T-regs) are functionally immune-suppressive subsets of T-cells. Depletion or inhibition of T-regs can enhance antitumor immunity. We demonstrated both by RT-PCR and by ELISA that murine TANs secrete significant amounts of the T-regs chemoattractant, CCL17, much more than circulating or splenic neutrophils, and at a level progressively increasing during tumor development. Migration assays, both in vitro and in vivo, showed recruitment of T-regs by TANs, which was inhibited with anti-CCL17 monoclonal antibodies. Systemic neutrophil depletion in tumor-bearing mice using anti-Ly6G monoclonal antibodies reduced the migration of T-regs into the tumors. We further showed, using flow cytometry, that CCL17 secretion by TANs is not limited to mouse models of cancer but is also relevant to human TANs. Our results suggest a new indirect mechanism by which TANs may inhibit antitumor immune activity, thus promoting tumor growth. We further describe, for the first time, a clear link between TANs and T-regs acting together to impair antitumor immunity., (© 2014 UICC.)

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines-Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H2O2. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway.

Objective: To determine the metabolic and immunological effects of the oral administration of DT56a, an enzymatic isolate of soybeans., Methods: DT56a was orally administered to mice in three animal models: leptin deficiency, high-fat diet (HFD) supplementation and immune-mediated hepatitis. Liver damage and immunological status were assessed., Results: Oral administration of DT56a to leptin-deficient (ob/ob) and HFD mice led to a significant reduction in serum triglyceride (TG) and total cholesterol (TC) levels. DT56a-treated mice in both models exhibited a significant reduction in hepatic levels of TG and marked alleviation of glycemic control as indicated by significant decreases in fasting blood glucose levels and glucose tolerance tests. The levels of liver enzymes were reduced. These metabolic effects were associated with altered distributions of regulatory T (Tregs) and natural killer T (NKT) cells. DT56a suppressed the immune-mediated liver damage induced by concanavalin A indicated by decreased liver enzymes and serum interferon-γ levels and by improved histology and decreased hepatic apoptosis. Oral administration of DT56a also alleviated immune-mediated hepatitis and affected Tregs and NKT cells., Conclusions: Oral administration of DT56a promotes a hepatoprotective effect associated with an alteration in the distribution of Tregs and NKT cells., (© 2012 The Authors. Journal of Digestive Diseases © 2012 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

Introduction: Parenteral OKT3 is used to treat transplant rejection and a humanized anti-CD3 Mab has shown positive clinical effects in new onset diabetes. Oral administration of anti-CD3 has not been tested in humans, but suppresses autoimmunity in animal models. Beta-glucosylceramide enhances NKT cell and regulatory T cell activity and enhances the effects of oral anti-CD3 in animals., Materials and Methods: Fifteen healthy volunteers (three per group) received orally administered OKT3 over a dose range of 0.2 to 5.0 mg daily with or without beta-glucosylceramide 7.5 mg for 5 days. Safety and immune parameters were measured on days 5, 10, and 30., Results and Discussion: Oral OKT3 enhanced T cell proliferation, suppressed Th1 and Th17 responses by 43% and 41%, respectively, increased TGF-beta/IL-10 expression and decreased IL-23/IL-6 expression by dendritic cells, and affected the IgG repertoire as measured by antigen arrays. Co-administration of oral beta-glucosylceramide induced similar effects. No side effects were observed and no subjects developed human anti-mouse antibodies., Conclusion: These findings demonstrate that oral anti-CD3 monoclonal antibody is safe and biologically active in humans and presents a new avenue for the treatment of autoimmune diseases.

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.

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.

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.

Aim: To determine the immunomodulatory effect of Shiitake (a mushroom extract), we tested its effect on liver-mediated immune regulation in a model of immune-mediated colitis., Methods: Four groups of mice were studied. Colitis was induced by intracolonic instillation of TNBS in groups A and B. Groups A and C were treated daily with Shiitake extract, while groups B and D received bovine serum albumin. Mice were evaluated for development of macroscopic and microscopic. The immune effects of Shiitake were determined by FACS analysis of intra-hepatic and intrasplenic lymphocytes and IFN-gamma ELISPOT assay., Results: Administration of Shiitake resulted in marked alleviation of colitis, manifested by significant improvement in the macroscopic and microscopic scores, and by reduction in IFN-gamma-producing colonies in group A, compared to group B mice (1.5 pfu/mL vs 3.7 pfu/mL, respectively). This beneficial effect was associated with a significant increase in the intra-hepatic CD8(+) lymphocyte trapping, demonstrated by an increased intrasplenic/intrahepatic CD4/CD8 lymphocyte ratio. These effects were accompanied by a 17% increase in the number of intrahepatic natural killer T (NKT) cells. A similar effect was observed when Shiitake was administered to animals without disease induction., Conclusion: Shiitake extract affected liver-mediated immune regulation by altering the NKT lymphocyte distribution and increasing intrahepatic CD8(+) T lymphocyte trapping, thereby leading to alleviation of immune-mediated colitis.

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. Beta-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 beta-glucosylceramide (beta-GC), beta-lactosylceramide (beta-LC), or a combination of both (IGL) in different doses. The effect of beta-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: Beta-sphingolipid treatment altered the hepatic NKT and CD8 lymphocyte distribution. beta-LC, beta-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, beta-glycosphingolipids increased the anti-HBV immune response in association with an altered NKT and CD8 lymphocyte distribution, making beta-glycosphingolipids potential potent adjuvants for overcoming non-responsiveness to HBV vaccination and augmenting the anti-viral immune response.

Unlabelled: The liver was previously suggested as a site of lymphocyte clearance. Liver-associated lymphocytes that express NK1.1 marker (NKT LAL) play a role in immune modulation., Aim: To determine the role of the liver and of NKT LAL in determining the CD4+/CD8+ balance during tolerance induction., Methods: Colitis was induced in C57 mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Immune tolerance was induced via five oral feedings of colitis-extracted proteins (CEP) from TNBS-colitis colonic wall, starting on the day of colitis induction (group A). Control mice were fed with BSA (group B). To determine the role of NKT cells in immune modulation, NK1.1 depletion was performed in nonfed (group C) and fed (group D) mice. To further evaluate the role of NKT cells in this model, mice in group E were tolerized following NKT depletion. To determine the effect of NKT depletion in a tolerized environment, tolerized mice in group F were NKT depleted following tolerance induction. Peripheral and intrahepatic NK1.1+ and CD4+/CD8+ T cells were determined in all groups. Colitis was assessed by standard clinical and histologic scores. Serum cytokines levels were measured by ELISA., Results: Oral tolerance induction led to a marked alleviation of colitis as manifested by a significant improvement of the clinical, macroscopic, and microscopic scores of colitis (group A vs. group B). NK1.1+ depletion without tolerance induction had a favorable effect on colitis (C). Depletion of NKT LAL prevented the ability to induce tolerance (group D). However, induction of tolerance following NK1.1+ depletion, and NK1.1+ depletion following tolerance induction led to a marked improvement of colitis (groups E and F). Tolerance induction led to a significant increase in NKT LAL numbers. The peripheral CD4+/CD8+ ratio increased up to 3-fold in tolerized vs. non-tolerized mice. A similar increase was observed in NKT-depleted healthy mice in groups C, E, and F (P < 0.005). In contrast, NK1.1+ depletion in the presence of antigen in the bowel led to a reverse effect with a significant decrease in the peripheral CD4+/CD8+ ratio. An opposite effect was observed in the intrahepatic CD4+/CD8+. The peripheral/intrahepatic CD4+/CD8+ ratio increased significantly in tolerized and in healthy mice (A, D, E, F, P < 0.005). In contrast, NK1.1+ depleted fed mice in group C manifested a marked decrease in the peripheral/intrahepatic CD4+/CD8+ ratio. Induction of tolerance led to a marked increase in the IL-10/interferon gamma (IFNgamma) and IL-4/IFNgamma ratios., Conclusions: In the experimental colitis model, the liver is an important site for CD8+ accumulation during tolerance induction in a process that is independent of NK1.1+ cells. NK1.1+ cells play a dual role in the pro/anti-inflammatory balance. In the presence of antigen, these lymphocytes may be accountable for keeping an anti-inflammatory lymphocyte balance. However, in the absence of antigen, they may induce a pro-inflammatory shift.

T lymphocyte-expressing natural killer (NK) cell markers (NKT cells) play a role in immune regulation. Our aim was to evaluate the in vivo effect of adoptive transfer of immune-programmed NKT cells. Colitis was induced in C57/B6 mice by 2,4,6-trinitrobenzenesulfonic acid. NKT, CD4, CD8 lymphocytes, and dendritic cells (DC) were prepared from spleens of naive mice, animals with colitis, and animals with colitis that were orally tolerized. Subsets of splenocytes, NKT, CD4, and CD8 and NKT+CD4, NKT+CD8, and NKT+DC lymphocytes were prepared. Assessment of the T helper cell type 1 (Th1)/Th2 cytokine secretion paradigm in vitro was performed before and following exposure to the antigen. Adoptive transfer of ex vivo immune-programmed lymphocytes from each group was performed into recipient mice, followed by colitis induction. Ex vivo exposure of NKT cells harvested from mice with colitis-to-colitis proteins [colitis-extracted proteins (CEP)] led to a Th2 cytokine shift. The interleukin (IL)-4/interferon-gamma (IFN-gamma) ratio increased for NKT harvested from colitis-harboring mice following exposure to CEP. Adoptive transfer of NKT lymphocytes harvested from colitis-harboring mice, which were ex vivo-educated, significantly alleviated experimental colitis in vivo. Intrahepatic NKT lymphocytes increased significantly in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP, similar to mice transplanted with NKT lymphocytes harvested from tolerized donors. Exposure of NKT cells to the disease-target antigen induced a significant increase in the IL-4/IFN-gamma cytokine ratio. Adoptive transfer of a relatively small number of immune-programmed NKT cells induced a systemic Th1 to Th2-immune shift and alleviated immune-mediated colitis.