31 Lack or loss of this regulatory subset of B cells has been demonstrated to

exacerbate symptoms in various experimental mice models with innate immunity disorders as well as autoimmunity.32–35 However, the precise role of this cell subset in the pathogenesis of CD has not been fully elucidated. SAMP1/Yit mice spontaneously develop transmural, patchy intestinal inflammation in the ileum and caecum, and are widely recognized as a murine model of CD.36–38 However, the disease is completely absent in mice reared under germ-free conditions.36 In the present study, we investigated the presence of a B-cell subset producing IL-10 KPT-330 research buy and TGF-β1 in the intestines of SAMP1/Yit mice, as well as its role in the pathogenesis of ileitis. Our results showed that intestinal regulatory B cells were mainly located in a population characterized by the cell surface markers CD1d+, while the production of IL-10 and TGF-β1 by TLR-activated intestinal B cells was significantly decreased in SAMP1/Yit mice compared with the control mice. These findings suggest that dysregulation of intestinal regulatory B cells in response to innate immune stimulation may be associated with the pathogenesis of CD. We used the

following antibodies for flow cytometry: fluorescein isothiocyanate-, phycoerythrin- (PE), and PE-Cy5-conjugated or purified anti-mouse MAPK inhibitor CD1d (1B1), CD5 (53-7.3), B220 (RA3-6B2), CD19 (1D3), immunoglobulin D (IgD; 11-26C.2a), IgM (R6-60.2),

IL-10 (JES5-16E3) (BD Biosciences-Pharmingen, San Jose, CA), TLR4/MD2 (UT41, recognizes both the antigens simultaneously), TLR9 (N/A), goat anti-rabbit IgG (Imgenex Biotech, Orissa, India), CD20 (AISB12), RP105 (RP/14), PDCA-1 (eBio927) (eBioscience, San Diego, CA) and TGF-β1 (9016) (R&D Systems, Minneapolis, AL), CD25/IL-2R (7D4) (Beckman Coulter, Brea, CA). We also used anti-mouse B220, CD90.1, and PDCA-1 microbeads (Miltenyi Biotec, Tau-protein kinase Auburn, CA). Ultra-pure Escherichia coli lipopolysaccharide (LPS; 0111:B4 strain) was obtained from Invivogen (San Diego, CA). Unmethylated CpG-DNA (5′-TGACTGTGAACGTTCGAGATGA-3′) was synthesized by Hokkaido System Science Co., Ltd (Sapporo, Japan). Enzyme-linked immunosorbent assay (ELISA) kits for Quantikine Mouse IL-10, IL-1β and interferon-γ (IFN-γ) Immunoassay, were from R&D Systems and a mouse TGF-β1 Immunoassay kit was from Invivogen. For measuring serum immunoglobulin, a rapid ELISA mouse antibody isotyping kit was obtained from Thermo Scientific (Yokohama, Japan). We obtained 7-week-old male specific pathogen-free BALB/c mice from Charles River (Yokohama, Japan).