SUMMARY Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

The effect of 10-hydroxy-trans-2-decenoic acid (10H2DA), a major fatty acid component of royal jelly, was investigated on LPS-induced cytokine production in murine macrophage cell line, RAW264 cells. 10H2DA inhibited LPS-induced IL-6 production dose-dependently, but did not inhibit TNF-α production. 10H2DA inhibited LPS-induced NF-κB activation in a dose-dependent fashion. In addition, NF-κB activation induced by over-expression of either MyD88 or Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was also inhibited by 10H2DA. Degradation of IκB-α and phosphorylation of IκB kinase-α were not inhibited by 10H2DA. On the other hand, reduction of LPS-induced IκB-ζ expression was discovered. Production of lipocalin-2 and granulocyte colony-stimulating factor (G-CSF), which is dependent on IκB-ζ, was also inhibited by 10H2DA, whereas that of IκB-ζ-independent cytokines/chemokines, such as IFN-β, murine monocyte chemotactic protein-1 (JE), macrophage inflammatory protein (MIP)-1α and MIP-2, was not. Together, 10H2DA specifically inhibited LPS-induced IκB-ζ expression, followed by inhibition of IκB-ζ-dependent gene production. These results suggest that 10H2DA is one of the components of royal jelly to show anti-inflammatory effects and could be a therapeutic drug candidate for inflammatory and autoimmune diseases associated with IκB-ζ and IL-6 production.

Royal jelly acid, 10-hydroxy-trans-2-decenoic acid (10H2DA), is a major lipid component of royal jelly, which is the exclusive diet of queen honeybees. Previously, we showed partial inhibition of lipopolysaccharide (LPS)-induced NF-κB activation by 10H2DA. In this study, the ability of 10H2DA to inhibit LPS-induced nitric oxide (NO) production was investigated. LPS-induced NO production and inducible NO synthase (iNOS) gene transcription were inhibited by 10H2DA. LPS-stimulated interferon (IFN)-β production, IFN regulatory factor-1 induction and IFN-stimulated response element activation, which are required for iNOS induction, were unaffected by 10H2DA. IFN-β-induced NO production, however, was significantly inhibited by 10H2DA. Furthermore, IFN-β-induced nuclear factor (NF)-κB activation and tumour necrosis factor (TNF)-α production were significantly inhibited by 10H2DA, and TNF-α-induced NF-κB activation was also inhibited by 10H2DA. These results and our previous study suggest that 10H2DA inhibits LPS- and IFN-β-induced NO production via inhibition of NF-κB activation induced by LPS or IFN-β.

Shiga toxin (Stx)-1 and Stx-2 are involved in the pathogenesis of hemolytic uremic syndrome (HUS) and severe systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection in humans. [1][2][3][4] Stxs have an AB 5 structure, in which a single catalytic A subunit is associated with five identical B subunits. The B pentamer is responsible for the toxin binding to eukaryotic cell-surface glycolipid receptors such as galabiosyl (Gb 2 )-ceramide and globotriaosyl (Gb 3 )-ceramide. The susceptibility of target cells to Stxs is dependent on the presence of the functional glycolipid receptors. 5,6) After binding, Stxs are internalized into the cells, and then the A subunit exerts RNA N-glycosidase activity, resulting in inhibition of protein synthesis by catalytic inactivation of 28S rRNA. 7,8) Thus, the binding of Stxs to the receptor is the primary event causing Stx-mediated diseases.The binding of Stxs to the receptors is attributed to the interaction of the B pentamer with the saccharide moiety of the receptors. However, the Stxs do not strongly bind to the Gb 3 monomer.9,10) It has been shown that Gb 3 molecules are clustered and localized in lipid rafts on the cell membranes. 11)The clustering of Gb 3 molecules is important for strong binding to Stxs. 12) In light of these findings, many synthetic compounds mimicking the natural receptors have been investigated for eliminating Stxs from the intestine and/or neutralizing Stxs in the circulation, as a therapeutic strategy for protecting patients from serious Stx-mediated diseases. 9,10,[13][14][15][16][17][18][19][20][21][22] All of these previously reported compounds possess repeated Gb 3 mimic structures in a molecule with various backbones on expecting clustering effect of the saccharide moiety, although there was one exception of adamantyl-Gb 3 , 10) a Gb 3 monovalent derivative, that was speculated to assemble into small aggregates for activity.In the present study, we synthesized novel Gb 3 -and Gb 2 -conjugated derivatives with a phosphatidyl residue, a sugar unit monomer in the chemical structure, which would be expected to form liposomes and clusters of the sugar unit to neutralize Stx-1 and Stx-2. These compounds showed strong neutralizing activity against not only Stx-1 but also Stx-2. In the present paper, we propose monovalent Gb 3 -/Gb 2 -derivatives conjugated with phosphatidyl residue as a new type of Stx-neutralizing agent. MATERIALS AND METHODS CompoundsThe structures of the compounds used in this study are shown in Fig. 1. Gb 3 and Gb 2 conjugated with phosphatidylethanolamine dipalmitoyl (PEDP) residue are referred to as Gb 3 -PEDP and Gb 2 -PEDP, respectively. Gb 3 conjugated with dipalmitoyl phosphatidyl hexyl (DPPA) residue is referred to as Gb 3 -DPPA. These compounds were dissolved in dimethyl sulfoxide at a concentration of 2 mM, then diluted 10 times in phosphate-buffered saline (PBS, pH 7.4) and stored at 4°C. The stock solutions were diluted with culture medium just before use, and sonicated for 10 min in a water ...

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.