Synthesis and biological activity of ester and ether analogues of alpha-galactosylceramide (KRN7000).

Alpha-Galactosylceramide (alphaGalCer, KRN7000) has been identified as a modulator of immunological processes through its capacity to bind iNKT cells mediated by CD1d molecules. Some analogues in while the amide group in alphaGalCer is replaced with ester or ether groups were synthesized from d-arabinitol or l-ribose to evaluate their ability to activate iNKT cells. Ester analogues 30a, 31a, and 61 showed activity for IFNgamma and IL-4 production of iNKT cells, while ether (31b) and 4-methoxy ester (76) analogues of alpha-galactosylceramide were not active for iNKT cells.

Induction of Th1-biased cytokine production by alpha-carba-GalCer, a neoglycolipid ligand for NKT cells.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce Th1-biased cytokines.

Cyclitol [RCAI-37 (1), 59 (5), 92 (7), and 102 (2)] and carbasugar analogs [RCAI-56 (3), 60 (4), and 101 (6)] of KRN7000 were synthesized through coupling reactions of the corresponding cyclitol or carbasugar derivatives with a cyclic sulfamidate (9) as the key step. Bioassay showed RCAI-56 (3, carbagalactose analog of KRN7000), 59 (5, 1-deoxy-neo-inositol analog), and 92 (7, 1-O-methylated 5) to be remarkably potent stimulants of mouse lymphocytes to produce Th1-biased cytokines, such as interferon-gamma, in vivo. RCAI-60 (4, carbafucose analog) and RCAI-101 (6, 6-O-methylated 3) showed strong bioactivity, on the other hands, RCAI-37 (1, myo-inositol analog) and 102 (2, neo-inositol analog) induced little cytokine production.

A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity.

Airway hypersensitive reaction (AHR) is an animal model for asthma, which is caused or enhanced by environmental factors such as allergen exposure. However, the precise mechanisms that drive AHR remain unclear. We identified a novel subset of natural killer T (NKT) cells that expresses the interleukin 17 receptor B (IL-17RB) for IL-25 (also known as IL-17E) and is essential for the induction of AHR. IL-17RB is preferentially expressed on a fraction of CD4(+) NKT cells but not on other splenic leukocyte populations tested. IL-17RB(+) CD4(+) NKT cells produce predominantly IL-13 and Th2 chemokines upon stimulation with IL-25 in vitro. IL-17RB(+) NKT cells were detected in the lung, and depletion of IL-17RB(+) NKT cells by IL-17RB-specific monoclonal antibodies or NKT cell-deficient Jalpha18(-/-) mice failed to develop IL-25-dependent AHR. Cell transfer of IL-17RB(+) but not IL-17RB(-) NKT cells into Jalpha18(-/-) mice also successfully reconstituted AHR induction. These results strongly suggest that IL-17RB(+) CD4(+) NKT cells play a crucial role in the pathogenesis of asthma.

PDC-TREM, a plasmacytoid dendritic cell-specific receptor, is responsible for augmented production of type I interferon.

Type I interferons (IFNs) derived from plasmacytoid dendritic cells (PDCs) are critical for antiviral responses; however, the mechanisms underlying their production remain unclear. We have identified a receptor, PDC-TREM, which is associated with Plexin-A1 (PlxnA1) on the PDC cell surface and is preferentially expressed after TLR-stimulation. Limited TLR signals induced PDC-TREM expression but failed to induce IFN-alpha production. However, when coupled with Sema6D, a ligand for Plexin-A1, limited TLR-stimulation resulted in PDC-TREM-mediated DAP12-dependent phosphorylation of phosphoinositide 3-kinase (PI3K) and extracellular regulated kinase (Erk) 1/2 at 6-9 h, and IFN-alpha was produced. Inhibition of PDC-TREM expression by pdctrem-shRNA, blocking of PDC-TREM-binding with PlxnA1 by PDC-TREM mAb, and DAP12 deficiency all resulted in greatly reduced PDC-TREM-dependent activation of signaling molecules and IFN-alpha production. Thus, PDC-TREM is responsible for IFN-alpha production, whereas TLR signals are essential for PDC-TREM expression.

The anti-allergic effects of lactic acid bacteria are strain dependent and mediated by effects on both Th1/Th2 cytokine expression and balance.

BACKGROUND: There is growing interest in the immune-stimulating effect and in particular, the anti-allergic effect, of lactic acid bacteria (LAB). However, no comprehensive studies have been done that compare the immune-stimulating potential of LAB strains. METHODS: The in vitro immune-stimulating effects on Th1/Th2 balance of more than 100 LAB strains were compared in splenocytes from ovalbumin-sensitized Th2-polarized mice. The in vivo anti-allergic ability of strain KW3110 was studied in the Th2-polarized model by detecting serum IgE concentration, Th1/Th2 cytokine secretion from splenocytes, and the expression of co-stimulatory molecules on macrophages. RESULTS: In vitro studies from Th2-polarized splenocytes, using IL-12 as a Th1 parameter and IL-4 secretion as a Th2 parameter revealed a wide variety of IL-12-inducing and IL-4-repressing activities, depending on the strain of LAB, not depending on the species. However, evaluation of individual strains in vivo revealed that after exposure to Lactobacillus paracasei KW3110 strain, the serum IgE elevation elicited by repeated OVA injection of mice was strongly inhibited. Cytokine secretion from splenocytes 20 weeks after KW3110 administration showed increased IL-12 and decreased IL-4 expression. Both CD40 and B7-1 expression on macrophages was upregulated by administration of KW3110. CONCLUSIONS: Improving the consequences of the Th1/Th2 imbalance by administration of LAB was dependent upon the LAB strain rather than the LAB species. Oral KW3110 administration in the mouse allergy model directed the Th1/Th2 balance toward Th1 through the maturation of APCs and inhibition of serum IgE elevation.