Invariant V.ALPHA.14 Natural Killer T Cell Activation by Edible Mushroom Acidic Glycosphingolipids

Abstract

Natural killer T (NKT) cells recognize CD1d loaded with glycosphingolipid (GSL) antigens. After being stimulated by GSL antigens, NKT cells immediately secrete cytokines. 2-4)The variety of cytokines release from NKT cells can promote immunoresponses against tumors and microbial infections, and regulate autoimmune responses, 5,6) although a puzzling aspect of these responses is that both Th1 and Th2 cytokines can be secreted in response to the stimulation.The fungal glycolipids are exogenous antigens for NKT cells 7) ; therefore, we focused on acidic glycosphingolipids (AGLs) of edible mushrooms (Hypsizigus marmoreus and Pleurotus eryngii) 8,9) and reported that AGLs induced interferon-g (IFN-g) and interleukin-4 (IL-4) release from murine T cells in a CD11c-positive cell-dependent manner. 10)Invariant natural killer T (iNKT) cells, that are more than 80% of murine NKT cells, express an invariant T cell receptor Va14 chain in combination with certain Vb chains. In mice, CD1d-dependent NKT cells are either CD4CD8-double negative or CD4-positive. The significance of CD4 expression on NKT cells is ambiguous because there is no evidence of any interaction between CD4 and MHC class II molecules on antigen-presenting cells (APCs), although CD4 can directly interacts with CD1d and enhances NKT cell activation.11) Additionally, NKT cells mainly leave the thymus when NK1.1 is negative. 6) In this study, we reported that edible mushroom AGLs activated CD4-positive NK1.1-negative iNKT hybridoma cells that express Va14-Ja18/Vb8.2 12) in a CD1d-dependent manner and intravenous AGL injection elevated the cytokine level. MATERIALS AND METHODS MaterialsPurified AGLs of H. marmoreus and P. eryngii were structurally elucidated according to previous reports, as described briefly.10,13) Saponified and dialyzed extracts were fractionated on a DEAE-Sephadex A-25 column (acetate form) (Amersham Biosciences AB, Uppsala, Sweden), and further purification was performed using a silica gel-6RS-8060 Iatrobeads column (Iatron Laboratories Inc., Tokyo, Japan). The composition of fatty acids and sugars, identification of phosphate bonds, confirmation of inositolphosphate content, determination of linkages among sugars and between sugar and inositol in purified AGLs, and AGL purity was confirmed by treatment with the appropriate reagents and using assays including columns, thin layer chromatography, a microwave oven, gas chromatography, gas-liquid chromatography/mass spectrometry, matrix-assisted laser desorption ionization-time of flight/mass spectrometry and 1 H-NMR. a-Galactosylceramide (a-GalCer) (KRN7000: ALX-306-027) and isoglobotrihexosylceramide (iGb3) (ALX-306-028) were purchased from ALEXIS Biochemicals (Lausanne, Switzerland).Mice and Protocols for in Vivo Experiments AGLs were dissolved in 100% polysorbate-20, diluted with sterilized phosphate buffered saline (PBS) to 0.5% polysorbate-20, and then kept at 4°C until used. Stock solutions were further diluted to 200 and 100 mg/ml AGL or 5 mg/ml a-GalCer in PBS containing 0.025% polyso...

Inhibitory Effect of a Glycoprotein Isolated from Golden Oyster Mushroom (Pleurotus citrinopileatus) on the Lipopolysaccharide-Induced Inflammatory Reaction in RAW 264.7 Macrophage

“…are widespread throughout the hardwood forests of the world. Many bioactive components derived from Pleurotus are known as antitumor agents, cholesterol reducers, or antioxidative agents and have also been shown to modulate the immune system . Our recent investigations revealed the antitumor property of Pleurotus citrinopileatus .…”

A Potent Pharmacological Mushroom: Pleurotus eryngii

“…The antitumor activity increased in a concentrationdependent manner and reached to 61.4% at the concentration of 400 μg/ml. In addition, Ma et al [9] also found polysaccharides in Pleurotus eryngii could suppress the proliferation of HepG-2 cells and enhance lactate dehydrogenase (LDH) in a dose-and time-dependent manner [10][11][12][13][14]. A water-soluble polysaccharide isolated from Pleurotus eryngii significantly suppressed the tumor growth in Renca-bearing mice at the doses of 50, 100 and 200 mg/kg [2].…”

“…They found that the hotwater extract increased significantly in serum IFN-γ levels, the highlevel production of IFN-γ in the culture supernatant from can enhance immunity in the trial immunogen group mice [13]. Furthermore, purified acidic glycosphingolipids (AGLs) from Pleurotus eryngii were reported that AGL could induced interleukin-2 (IL-2) release from invariant natural killer T (iNKT) cells and induced prolonged retention of IL-4 in serum in vitro and in vivo [14]. These indicated that Pleurotus eryngii AGLs might be involved in the maintenance of immunohomeostasis through iNKT cell activation.…”

“…Immuno-modulating effects/ Hypolipidemic [13,14] Methanol extracts Antioxidant [22] Ethanol extracts Antimicrobial activity/Anti-Allergic Property, Estrogenlike activity [21,25,26,37,3 8,42]…”

Simple and rapid removal of the interference in gangliosides extracted from HPTLC spot on MALDI-TOF MS analysis

“…Apart from the AGLs, a-galactosylceramide (a-GalCer) and isoglobotriaosylceramide (iGb3, Gala1-3Galb1-4Glcb1-1 0 Cer) are also known to activate iNKT cells upon CD1d-mediated stimulation. [16][17][18][19][20] Although the AGLs in edible mushrooms were weaker antigens for iNKT cells than a-GalCer, they may contribute to enhance the immune system as immunomodulatory antigens.…”