This protocol describes methods to identify, purify and culture CD1d restricted invariant natural killer T (iNKT) cells from mouse tissue or human blood samples. The methods for identification and purification of iNKT cells are based on the interaction between iNKT cell receptor and its ligand. The iNKT cell receptor is composed of the invariant V alpha 14 J alpha 18/V beta 8.2 in mice or V alpha 24 J alpha 18/V beta 11 in humans and is expressed only on iNKT cells but not on conventional T cells. The iNKT cell antigen receptor in both species recognizes alpha-galactosylceramide (alpha-GalCer) presented by the MHC class I-like CD1d. Thus, alpha-GalCer-loaded CD1d dimer can be used for analysis and purification by fluorescence-activated cell sorting (FACS). Isolation of 1 x 10(6) purified iNKT cells from mouse thymus, spleen or liver requires 5-6 mice and takes 1-2 h for mononuclear cell preparation from mouse tissues, 1.5 h for enrichment by magnetic beads and 4 h for detection and purification of the iNKT cells by FACS. In the case of isolation of human peripheral blood mononuclear cells (PBMCs) from whole blood, it takes 2 h and requires 5 ml of blood to obtain 5 x 10(6) PBMCs, which contain 500-25,000 iNKT cells.

T helper type 2 (Th2) cytokines, IL-4 and IL-13, attenuate the expression of genes that regulate epidermal cellular structures and the barrier function at the terminal stage of keratinocyte differentiation. However, whether these Th2 cytokines act at earlier stages remains unknown. We investigated the roles of cytokines in expression levels of mRNAs and/or proteins in primary mouse keratinocytes and human keratinocyte HaCaT cells at earlier stages. We showed that IL-4 downregulated the expression levels of Krt1, Krt10, Dsg1, and Dsc1 via IL-4Rα- and signal transducer and activator of transcription factor 6 (STAT6)-dependent mechanisms in differentiating mouse keratinocytes at early stages. As the expression levels of keratin-1 and -10 in the keratinocytes transiently expressing an active form of STAT6 were not downregulated, STAT6 and other IL-4-induced molecules may synergistically regulate this expression. The restoration of the downregulated expression levels of Krt1 and Krt10 induced by IL-4 with the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase) inhibitor U0126 indicated the involvement of the p44/42 MAPK signaling pathway in the attenuated expression. IL-13 also downregulated the expression of the four genes. Furthermore, IL-4 or IL-13 caused the downregulation of these genes in HaCaT cells and promoted the fragmentation of cell sheets with mechanical stress. Our results showed that IL-4 or IL-13 acted on differentiating keratinocytes in vitro at early stages to attenuate the gene expression.

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.

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and tosaka@research.twmu.ac.jp a N.M., T.O., and I.H. contributed equally to this work. AbstractThe incidence of non-alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non-alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF-and HCD-fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.