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. 2010 Dec;131(4):473–487. doi: 10.1111/j.1365-2567.2010.03318.x

Decreased production of interleukin-10 and transforming growth factor-β in Toll-like receptor-activated intestinal B cells in SAMP1/Yit mice

Yoshiyuki Mishima 1, Shunji Ishihara 1, Md Monowar Aziz 1, Akihiko Oka 1, Ryusaku Kusunoki 1, Aya Otani 1, Yasumasa Tada 1, Yong-Yu Li 1, Ichiro Moriyama 1, Naoki Oshima 1, Takafumi Yuki 2, Yuji Amano 2, Satoshi Matsumoto 3, Yoshikazu Kinoshita 1
PMCID: PMC2999799  PMID: 20561083

Abstract

A unique subset of B cells expressing interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) plays an essential role in preventing inflammation and autoimmunity. We investigated the presence of this cell subset in intestines and its role in the pathogenesis of ileitis using SAMP1/Yit and age-matched control AKR/J mice. Mononuclear cells were isolated from mesenteric lymph nodes (MLNs) and the expressions of B220, CD1d, CD5, Toll-like receptor 4 (TLR4) and TLR9 in isolated cells were analysed. Purified B cells were stimulated with lipopolysaccharide (LPS) or CpG-DNA, then IL-10 and TGF-β1 expressions were examined by enzyme immunoassay and flow cytometry. Production of IL-1β by TLR-mediated macrophages co-cultured with or without purified MLN B cells from SAMP1/Yit and AKR/J mice was evaluated. In addition, interferon-γ (IFN-γ) production in intestinal T cells co-cultured with MLN B cells were also assessed in SAMP1/Yit and AKR/J strains. The production levels of IL-10 and TGF-β1 stimulated by LPS and CpG-DNA were significantly lower in B cells separated from MLNs from the SAMP1/Yit strain. B cells expressing IL-10 and TGF-β1 were mainly located in a population characterized by the cell surface marker CD1d+. Interleukin-1β production by TLR-activated macrophages co-cultured with MLN B cells from SAMP1/Yit mice was significantly higher than that of those from AKR/J mice. Interestingly, IFN-γ production by T cells was noted only when they were co-cultured with SAMP1/Yit but not the AKR/J B cells. These results are the first to show that disorders of regulatory B-cell function under innate immune activation may cause disease pathogenesis in a murine model of Crohn's disease.

Keywords: Crohn's disease, interleukin-10, regulatory B cells, Toll-like receptor, transforming growth factor-β

Introduction

Crohn's disease (CD), an idiopathic inflammatory bowel disease, is characterized by a chronic intestinal immune-mediated disorder.1–4 Previous studies have demonstrated that interference with the normal interactions between intestinal mucosal cells and microbial flora is closely associated with the pathogenesis of CD.5–7 Various susceptible genes for CD have been recently identified in several genome-wide association studies,8–12 which further implicates their involvement in the development of CD by linking to disorders of the innate immune system. Studies focused on the innate immune system have been crucial for understanding the pathogenesis of CD.

Intestinal innate immunity is maintained by a variety of cells, including macrophages, dendritic cells, and epithelial cells, which express several pattern recognition receptors (PPRs) and can sense luminal pathogen-associated molecular patterns (PAMPs).13–17 Innate immune regulation and disorders of these cells have been widely investigated in numerous studies to elucidate the pathogenesis of CD.5–7 On the other hand, T and B lymphocytes are well recognized as antigen-specific effector immune cells that play a critical role in the adaptive immune response under physiological and pathological conditions.1,2,16–20 Although T- and B-cell-mediated adaptive immune regulation have been evaluated in great detail, the contribution of these lymphocytes in innate immune-related intestinal disorders such as CD has also been recognized.

Recent studies have shown that a unique subset of B cells expressing interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) plays an essential role in preventing immune responses.21–25 This subset is currently considered to consist of regulatory B cells that designate B cells with immunoregulatory properties. Regulatory B cells produce IL-10 and TGF-β after sensing PAMPs via PPRs, including toll-like receptors (TLRs),26,27 and modulate autoimmunity and inflammation by regulating T helper type 1 (Th1)/Th2 balance,28–30 down-regulating pro-inflammatory networks,23 and inducing apoptosis in effector T cells.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 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.

Materials and methods

Reagents

We used the following antibodies for flow cytometry: fluorescein isothiocyanate-, phycoerythrin- (PE), and PE-Cy5-conjugated or purified anti-mouse 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, 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).

Animals

We obtained 7-week-old male specific pathogen-free BALB/c mice from Charles River (Yokohama, Japan). SAMP1/Yit mice were kindly provided by Yakult Central Institute for Microbiological Research (Tokyo, Japan) and age-matched male control AKR/J mice were obtained from Kyudo (Kumamoto, Japan). All animals were housed in a specific pathogen-free facility under constant environmental conditions with circadian light–dark cycles. The animals were cared for and handled in accordance with guidelines from the National Institutes of Health and Institute for Animal Experimentation of Shimane University.

Cell isolation

Mononuclear cells were isolated from the lamina propria of the large intestine, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), spleen and peritoneal cavity (PerC), as described in the following. The MLNs and PPs were crushed through 70-μm filters into phosphate-buffered saline (PBS) with 2% fetal bovine serum (FBS; ICN Biomedicals, Aurora, OH). Spleens were mechanically dissociated and red blood cells were lysed in ammonium phosphate/chloride lysis buffer. The PerC cells were collected after intraperitoneal injection of Ca2+-free and Mg2+-free Hanks' balanced salt solution (HBSS; Gibco-Invitrogen, Carlsbad, CA) with 2% FBS. For isolation of colon lamina propria lymphocytes (LPLs), the large intestines were washed with cold PBS and all visible PPs were removed with scissors. The intestines were opened longitudinally, then cut into 5-mm pieces and incubated in 1 mm dithiothreitol (Sigma-Aldrich, St Louis, MO) in HBSS for 15 min at room temperature. Next, the tissues were incubated in 1 mm EDTA in HBSS for 20 min at 37° with shaking, which was repeated after a thorough washing. The cell suspensions were removed and remaining fragments were transferred to flasks containing HBSS with 1 mg/ml collagenase type 3 (Worthington Biochemical Corporation, Lakewood, NJ), 0·1 mg/ml DNAse I (Worthington Biochemical Corporation), and 1% penicillin–streptomycin (Gibco-Invitrogen), then stirred gently for 60 min at 37°. Cell suspensions containing LPLs were filtered through a nylon mesh and centrifuged, then the LPLs were purified using a 44–70% discontinuous Percoll gradient (GE Healthcare, Buckinghamshire, UK). After centrifugation at 800 g for 20 min at 22°, cells were collected from the interface, and washed and resuspended in PBS with 2% FBS. Isolated cells were analysed by flow cytometry.

B-cell and T-cell purification and cell cultures

To evaluate the TLR-mediated production of IL-10 and TGF-β in isolated B and T cells, mononuclear cells obtained from each part were purified magnetically by positive selection with anti-B220 (for B cells) and anti-CD90.1 (for T cells) microbeads. In addition, we also used anti-PDCA-1 microbeads to avoid contamination by B220+ plasmacytoid dendritic cells. The percentage of PDCA-1+ cells among B220+ cells in each sample was < 2·5% (data not shown). All selections were performed according to the manufacturer's instructions. Final B220+ cell fractions were confirmed to be > 95% pure by flow cytometry and cell viability was shown to be > 90% by eosin Y exclusion. Isolated B and T cells (5 × 105) were cultured at 200 μl/well (96-well plates) for 72 hr at 37° with 5% CO2, respectively The culture medium was RPMI-1640 (Gibco-Invitrogen) containing 10% FBS and 1% penicillin–streptomycin–amphotericin B (Gibco-Invitrogen), with LPS (100 ng/ml) and CpG-DNA (100 nm/ml), or without the ligands. Following the cell cultures, the supernatants were collected for measurements of IL-10 and TGF-β1 by enzyme immunoassay (EIA).

Flow cytometry

Three-colour flow cytometric analyses were performed at the optimal concentrations recommended by the manufacturer. Cells were stained with the appropriate antibodies for 15 min and washed three times with cold PBS, then analysed using an EPICS XL (Beckman Coulter, Tokyo, Japan), with 5000 events counted for each condition, and analysed using expo32™ software (Beckman Coulter). Isotype controls were used for all of the samples. For intracellular cytokine staining, brefeldin A (Sigma-Aldrich) was added to the medium during the last 4 hr of the culture period. The cells were first stained with appropriate fluorescence antibodies to detect cell surface markers, then fixed and permeabilized with Intraprep (Beckman Coulter, Fullerton, CA). Cells were stained intracellularly with PE-conjugated anti-IL-10 or -TGF-β1. After washing, the cells were immediately subjected to flow cytometric analysis.

Measurements of IL-10 and TGF-β1 levels

The contents of IL-10 and TGF-β1 in culture media were measured using EIA, according to the manufacturer's instructions. Briefly, appropriate sample amounts were transferred by pipette into the wells of anti-mouse IL-10- or TGF-β1-coated microtitre strips. Secondary biotinylated monoclonal antibodies were then added to the wells and incubated at room temperature for 90 min. After removing the excess secondary antibodies by washing, the samples were incubated with streptavidin-peroxidase. A substrate solution was added to produce colour directly proportional to the concentration of mouse IL-10 or TGF-β1 present in the sample. Quantitative results were obtained from a standard curve produced from the experimental findings.

RNA extraction and real time-polymerase chain reaction

Total RNA was extracted from each sample of purified B cells using Isogen (Nippon Gene, Tokyo, Japan), then equal amounts of RNA were reverse transcribed into complementary DNA (cDNA) using a QPCR cDNA kit (Stratagene, La Jolla, CA). All primers used were flanked by intron–exon junctions using the NCBI blast tool and primer3 software (Howard Hughes Medical Institute, MD). Primer sequences used for reverse transcription–polymerase chain reaction (RT-PCR) were as follows: IL-10; 5′-CAGCCGGGAAGACAATAACT-3′ and 5′-TCATTTCCGATAAGGCTTGG-3′, TGF-β1; 5′-TGCTTCAGCTCCACAGAGAA-3′ and 5′-TACTGTGTGTCCAGGCTCCA-3′, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH); 5′-ACCCAGAAGACTCTGGATGG-3′ and 5′-GGTCCTCAGTGTAGCCCAAG -3′. Quantitative real-time PCR was performed using an ABI PRISM 7700 sequence detection system with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. The levels of IL-10 and TGF-β1 were normalized to that of GAPDH using sequence detector software (Applied Biosystems).

Microarray assays

The CD220 sorted MLN B cells (2 × 107 cells/well) from SAMP1/Yit and AKR/J mice were treated with or without CpG-DNA for 18 hr then subjected to microarray analysis using a TLR-signalling pathway-specific PCR Array system (SA Biosciences, Frederick, MD) according to the manufacturer's protocol. Briefly, total RNA was isolated from the cells with an ArrayGrade total RNA isolation system, then purified using a spin column (SA Biosciences). The purity and quantity of the extracted RNA were checked with Nanodrop. A total of 1·5 μg RNA was reverse transcribed to cDNA, followed by real-time PCR (One step; Applied Bioscience, Foster City, CA) and data analyses was performed using the SA Bioscience Array expression analysis suite.

Histology for identification of ileitis of SAMP1/Yit mice

Terminal ileums were excised from SAMP1/Yit and AKR/J mice of various ages, then immersion-fixed in 10% formaldehyde for 48 hr. Next, the tissues were embedded in paraffin and cut into 6-μm sections, and stained with haematoxylin & eosin to visualize the general morphology under a phase contrast light microscope.

IL-1β production by TLR-mediated macrophages co-cultured with purified mouse MLN B cells

To verify the role of MLN B cells in IL-1β production by TLR-mediated macrophages, we conducted an in vitro experiment. Peritoneal macrophages (1 × 106 cells/well) isolated from AKR/J and SAMP1/Yit mice were co-cultured with purified MLN B cells (1 × 106 cells/well) from SAMP1/Yit or AKR/J mice in 24-well plates, then stimulated with LPS (100 ng/ml) or CpG-DNA (100 nm/ml) for 72 hr. IL-1β contents in the culture supernatants were examined by EIA.

IFN-γ production by TLR-mediated intestinal T cells co-cultured with purified mice MLN B cells

To understand the role of MLN B cells in IFN-γ production by TLR-mediated intestinal T cells, we conducted an in vitro experiment. MLN T cells (1 × 106 cells/well) isolated from AKR/J and SAMP1/Yit mice by using the pan-T-cell-specific marker CD90.1 microbeads were co-cultured with purified MLN B cells (1 × 106 cells/well) from both mice in 24-well plates, then stimulated with LPS (100 ng/ml) or CpG-DNA (100 nm/ml) for 72 hr. The IFN-γ content in the culture supernatants was examined by EIA.

Statistical analysis

All data are expressed as the mean ± standard error of the mean (SEM). Values were analysed using Student's t-test and Spearman's rank correlation with Stat-View 4.0 software (Abacus Concepts, Inc., Berkeley, CA). For comparisons of multiple values, analysis of variance was used. P values < 0·05 were considered significant.

Results

Cell surface markers of B cells isolated from BALB/c mice

Initially, we used BALB/c mice and examined cell surface markers of B cells isolated from several parts of the mice using flow cytometry, with representative results shown in Fig. 1. In the B cells isolated from the MLNs, PPs, colon lamina propria, and spleens, similar expression patterns of CD1dhigh, CD5low, CD11b−, TLR4/MD-2low and TLR9low were observed. In contrast, high expression levels of CD5, CD11b and IgM were found in B cells isolated from PerC. We also noticed a significant expression of RP105 in B cells isolated from various organs. RP105, which is associated with MD-1 protein, was the first leucine-rich repeat (LRR) protein found on the surface of B cells. The role of RP105/MD-1 in TLR2/TLR4 responses seems to vary among types of immune cells. Recent reports have shown that RP105-deficient B cells are defective in their response to TLR2 and TLR4 ligands, whereas it is likely that RP105/MD-1 positively regulate TLR2/TLR4 responses in B cells.39 In contrast, Divanovic et al.40 reported that RP105 negatively regulates LPS-induced responses in macrophages and dendritic cells. In the present study, we examined RP105 to ascertain the expression of innate immune-related molecules in B cells. The major population of peritoneal B cells has been well reported to be B-1a cells and the immune function of this subset is essentially different from that of the conventional B-cell subset (B-2 cells) that exists in other organs. The present results obtained by flow cytometry suggest that the major population of intestine-related B cells (MLNs, PPs, colon lamina propria) has a B-2 lineage.

Figure 1.

Figure 1

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Representative results showing the cell surface marker of B cells (B220+) on cells isolated from BALB/c mice. Mononuclear cells were isolated from lamina propria (LP) of the large intestine, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), spleen samples and the peritoneal cavity (PerC). The cells were stained with appropriate antibodies (antibodies) for 15 min and washed three times with cold phosphate-buffered saline, then analysed by flow cytometry, with 5000 events counted for each condition, and analysed using EXPO32™ software. Isotype controls were utilized for all of the samples.

Production of IL-10 and TGF-β1 in TLR-mediated B cells isolated from BALB/c mice

Next, we examined the production of IL-10 and TGF-β1 in TLR-mediated B cells. Mononuclear cells were isolated from several parts of BALB/c mice and magnetically purified using microbeads. Next, purified B cells (B220+ PDCA-1−) were cultured with or without TLR ligands, then cytokine concentrations in the culture supernatants were measured by EIA. The B-cell fractions used in the experiments were confirmed to be > 95% pure by flow cytometry (Fig. 2a). Although IL-10 production was induced in TLR ligand-mediated B cells, the level of production in CpG-DNA-stimulated cells was significantly higher than that in LPS-stimulated cells (Fig. 2b). In addition, IL-10 production by TLR-mediated PerC B cells was remarkably higher than that by B cells isolated from other parts of the mice. These results may have been dependent on the unique characteristics of PerC B cells derived from a B-1 lineage. However, when compared with the results of IL-10, lower production levels of TGF-β1 in response to TLR ligands were observed in all of the tested samples (Fig. 2b). In the body systems, TGF-β1 occurs in two physiological forms: latent and active. Although TGF-β1 is important in regulating crucial cellular activities, in most cases an activated TGF-β1 ligand will initiate the TGF-β1 signalling cascade. In our present system, the majority of TGF-β1 as assessed was solely inactive or latent. We also measured the active form of TGF-β1 but the amount was too low to demonstrate any effects of TLR ligands on their secretion (data not shown).

Figure 2.

Figure 2

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(a) Purification rates for B cells. Mononuclear cells were obtained from each part of BALB/c mice, then purified magnetically by selection with anti-B220 and anti-PDCA-1 microbeads. The final B220+ PDCA-1− cell fractions were > 95% pure, as shown by flow cytometry. (b) Production of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) by Toll-like receptor-activated purified B cells. Purified B cells (5 × 105) were cultured at 200 μl/well (96-well plates) for 72 hr with lipopolysaccharide (LPS; 100 ng/ml) and CpG-DNA (100 nm/ml), or without the ligands. After the cell cultures, supernatants were collected for measurements of IL-10 and TGF-β1 by enzyme immunoassay. Error bars indicate the standard error of mean values obtained from four independent experiments. **P < 0·01, *P < 0·05 versus non-stimulated cells.

Histology of ileitis and cell surface markers on MLN B cells from SAMP1/Yit mice

Following our experimental results, we investigated the presence of a regulatory B-cell subset producing IL-10 and TGF-β1 in the intestines of BALB/c mice. Furthermore, we conducted additional experiments to elucidate the role of this intestinal regulatory B-cell subset in the pathogenesis of CD using SAMP1/Yit mice. Development of ileitis in the SAMP1/Yit mice was confirmed by histological examinations. Ileal inflammation was not present in 5-week-old SAMP1/Yit mice, whereas typical ileitis with numerous inflammatory cells infiltrating the lamina propria was observed in 15- and 30-week-old SAMP1/Yit mice (Fig. 3a). Next, we examined several cell surface markers of MLN B cells isolated from 15-week-old SAMP1/Yit mice by flow cytometry. As shown in Fig. 3(b), there were no differences between cell surface markers from SAMP1/Yit and AKR/J mice. In addition, the expression patterns of MLN B cells in these mice were similar to those in BALB/c mice.

Figure 3.

Figure 3

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(a) Representative pictures showing histological evidence of ileitis in SAMP1/Yit mice. Terminal ileums were obtained from various aged SAMP1/Yit and AKR/J mice (5, 15, 30 weeks old). (Original magnification: × 200). (b) Representative results showing cell surface markers of B cells (B220+) isolated from SAMP1/Yit and AKR/J mice. Mononuclear cells were isolated from mesenteric lymph nodes. Cells were stained with suitable antibodies for 15 min and washed three times with cold phosphate-buffered saline, then analysed by flow cytometry, with 5000 events counted for each condition, and analysed using expo32™ software. Isotype controls were used for all of the samples.

Decreased production of IL-10 and TGF-β1 in TLR-mediated MLN B cells from SAMP1/Yit mice

To know whether innate immune responses by MLN B cells are associated with the pathogenesis of ileitis that develops in SAMP1/Yit mice, we examined the production of IL-10 and TGF-β1 by TLR-mediated MLN B cells isolated from SAMP1/Yit and AKR/J mice. To achieve this, at first the surface phenotypes of the sorted B cells were checked by their presence of the commonly encountered markers CD19, CD20, B220 and PDCA-1 (Fig. 4a). The CpG-DNA induced production of IL-10 by MLN B cells from all age groups of SAMP1/Yit mice, which were significantly lower than those from AKR/J mice (Fig. 4b). Interleukin-10 production in response to CpG-DNA was markedly higher than that in response to LPS. Although lower production of TGF-β1 after stimulation with TLR ligands was observed in all samples tested, CpG-DNA significantly induced TGF-β1 production by MLN B cells isolated from 15- and 30-week-old AKR/J mice (Fig. 4b). Interleukin-10 is expressed not only by regulatory B cells, but also by the monocytes and type 2 helper T cells (Th2), mast cells, regulatory T cells, and in a certain subset of activated T cells. Similarly, TGF-β1 has also been produced by a wide variety of cells to generate diverse immune-regulatory phenotypes. We therefore aimed to carry out experiments to estimate IL-10 and TGF-β1 contents in purified T cells after stimulation with LPS and CpG-DNA. To achieve this, MLN T cells from SAMP1/Yit and AKR/J mice were isolated using CD90.1 microbeads. According to our findings, in contrast to regulatory B cells (Fig. 4b), sorted T cells from both SAMP1/Yit and AKR/J mice produced very small quantities of IL-10 and TGF-β1 in both LPS-treated and CpG-DNA-treated conditions (Fig. 4c), which we think was a result of their weak innate immune responses when stimulated with those TLR ligands. In light of these findings, we conclude that the regulatory B cells produced copious amount of IL-10 and TGF-β1 which may generate immune modulating role during intestinal inflammation.

Figure 4.

Figure 4

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(a) The surface phenotype of the sorted B cells (CD19, CD20, B220, and PDCA-1) is shown. Protein levels of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) produced by Toll-like receptor-activated purified B cells (b) and T cells (c) from SAMP1/Yit and AKR/J mice at different age groups. Purified B cells (5 × 105) or T cells (5 × 105) were cultured at 200 μl/well (96-well plates) for 72 hr (enzyme immunoassay; EIA), or for 24 or 48 hr (real-time polymerase chain reaction), with lipopolysaccharide (LPS; 100 ng/ml) and/or CpG-DNA (100 nm/ml), or without the ligands. After the 72-hr cell cultures, supernatants were collected for measurements of IL-10 and TGF-β1 by EIA. Error bars indicate the standard error of mean values obtained from four independent experiments. **P < 0·01, *P < 0·05 versus SAMP1/Yit mice.

In terms of logistics, one important point is that stimulation with antigens or TLR ligands may sometimes induce apoptosis or immune tolerance in B cells. To address this, we duly checked B-cell apoptosis status in our system after stimulation with TLR ligands LPS and CpG-DNA and observed that an insignificant portion of B-cell population can undergo apoptosis upon LPS and CpG-DNA stimulation (data not shown). Beside these, we also assessed B-cell activation upon TLR stimulation by screening the B-cell activation marker CD25 in isolated B220+ cells from both AKR/J and SAMP1/Yit mice. According to our results, the cells treated with CpG-DNA directly increased the CD25+ B220 cell population in both AKR/J and SAMP1/Yit mice to the same extent regardless of strain variation (data not shown).

Identification of cell surface markers of MLN B cells producing IL-10 and TGF-β1

Based on the above findings, we next examined the intracellular expression of IL-10 and TGF-β1 in TLR-stimulated MLN B cells. Representative results of flow cytometry are shown in Fig. 5(a) for IL-10 and Fig. 6(a) for TGF-β1. Stimulation of TLR ligands increased the total number of B cells producing IL-10 and TGF-β1. In particular as seen from the bar diagram, CpG-DNA significantly increased the expressions of IL-10 and TGF-β1 in MLN B cells isolated from AKR/J mice (Figs 5b, 6b), compared with those from SAMP1/Yit mice. These findings confirmed our results obtained with EIA. Previous studies have shown that CD1d and CD5 are possible cell surface markers for identification of B cells producing IL-10 and TGF-β1,41 we therefore examined the expressions of these markers on MLN B cells stimulated by TLR ligands. Our flow cytometric results showed that B cells producing IL-10 and TGF-β1 were mainly contained in populations characterized by the cell surface markers CD1d+ from both SAMP1/Yit and AKR/J mice (Figs 5b, 6b). On the other hand, we observed the presence of the regulatory subset in both CD5+ and CD5− populations of MLN B cells. In addition, decreased expression of IL-10 and TGF-β1 in CpG-DNA-stimulated MLN B cells of SAMP1/Yit mice was confirmed by the results of real-time PCR (Figs 5c and 6c).

Figure 5.

Figure 5

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Intracellular staining for detection of interleukin-10 (IL-10) in Toll-like receptor-activated purified B cells from SAMP1/Yit and AKR/J mice (15 weeks old). Purified B cells (5 × 105) were cultured at 200 μl/well (96-well plates) for 72 hr with lipopolysaccharide (LPS; 100 ng/ml) and CpG-DNA (100 nm/ml), or without the ligands. After culturing, the cells were stained with appropriate fluorescence antibodies to detect cell surface markers, then fixed and permeabilized. Cells were also stained intracellularly with phycoerythrin-conjugated anti-IL-10. After washing, the cells were immediately subjected to flow cytometric analysis. (a) Representative results of flow cytometry showing intracellular staining of IL-10. (b) Numbers of intracellular IL-10-positive B cells. Error bars indicate the standard error of mean values obtained from three independent experiments. **P < 0·01, *P < 0·05 versus SAMP1/Yit mice. (c) The gene expressions of IL-10 in CpG-DNA-activated purified B cells were examined by real-time polymerase chain reaction. Error bars indicate the standard error of mean values obtained from four independent experiments. **P < 0·01, *P < 0·05 versus SAMP1/Yit mice.

Figure 6.

Figure 6

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Intracellular staining for detection of transforming growth factor-β1 (TGF-β1) in Toll-like receptor-activated purified B cells from SAMP1/Yit and AKR/J mice. Purified B cells (5 × 105) were cultured at 200 μl/well (96-well plates) for 72 hr with lipopolysaccharide (LPS; 100 ng/ml) and CpG-DNA (100 nm/ml), or without the ligands. After culturing, the cells were stained with appropriate fluorescence antibodies to detect cell surface markers, then fixed and permeabilized. Cells were stained intracellularly with phycoerythrin-conjugated anti-TGF-β1. After washing, the cells were immediately subjected to flow cytometric analysis. (a) Representative results of flow cytometry showing intracellular staining of TGF-β1. (b) Numbers of intracellular TGF-β1-positive B cells. Error bars indicate the standard error of mean values obtained from three independent experiments. *P < 0·05 versus SAMP1/Yit mice. (c) The gene expressions of TGF-β1 in CpG-DNA-activated purified B cells were examined by real-time polymerase chain reaction. Error bars indicate the standard error of mean values obtained from four independent experiments. *P < 0·05 versus SAMP1/Yit mice.

Significant variation in altering the TLR-signalling pathway was not evident in SAMP1/Yit B cells

Although the SAMP1/Yit B-cell functional problem has been demonstrated previously,42 the plausible mechanism underlying the alteration in cell signalling pathway had not been explored. However, it was anticipated that an enlarged MLN with increased numbers of pathogenic B cells in SAMP1/Yit mice might be involved in ileitis. In our present study, we noted an increase of CD5+/− CD1d+ IL-10+ or CD5+/− CD1d+ TGF-β1+ B-cell population in AKR/J as compared with the SAMP1/Yit mice (Figs 5a, 6a) and therefore, depending on this fact, we expect a possible ground for increased production of IL-10 and TGF-β1 produced by B cells from AKR/J mice treated with TLR ligands. However, to gain detailed insight into the cell signalling events, we stimulated isolated B cells from AKR/J and SAMP1/Yit strains with CpG-DNA, as this ligand exhibited a better response than LPS for both IL-10 and TGF-β1 secretions, after which a TLR pathway focused PCR array assay was performed using total extracted RNA. Although we observed that the B cells from both strains of mice were responsive to CpG-DNA, they did not exhibit any marked difference between the B-cell types from two different strains in terms of inducing the expression of some familiar TLR pathway-related genes, e.g., Myd88, TRAF6, IRAK-1/4 (Fig. 7a). Our results did not show that changes in IL-10 production in AKR/J versus SAMP1/Yit mice were dependent on altering the major TLR signalling components, but rather on their number, differentiation or maturation status.

Figure 7.

Figure 7

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(a) Polymerase chain reaction (PCR) array in CpG-DNA-treated SAMP1/Yit and AKR/J B cells. Purified B cells (2 × 107 cells/well) were cultured with or without CpG-DNA (100 nm/ml) for 18 hr, followed by the RNA extraction, complementary DNA preparation and PCR array was performed, as described in the Materials and methods. (b) Serum immunoglobulin subtypes assay in SAMP1/Yit and AKR/J mice. Blood was drawn from the axillary artery, and then immunoglobulin subtypes were assayed from the serum using the protocols as provided by the manufacturer. Error bars indicate the standard error of mean values obtained from three independent experiments. *P < 0·05 versus SAMP1/Yit mice.

Considering the B-cell-mediated pivotal role in the adaptive immune system, we next aimed to check the serum level of immunoglobulins in those two strains of mice. To achieve this, we assessed the serum level of immunoglobulin in age-matched and sex-matched AKR/J and SAMP1/Yit mice strains, and observed that the serum contents of immunoglobulin were almost similar, except that a minor decrease was noted in IgG3 of AKR/J mice compared with that of SAMP1/Yit mice (Fig. 7b). The SAMP1/Yit mice exhibit serious B-cell defects, so they may generate a differential pattern of adaptive immune functions by producing less serum immunoglobulin compared with AKR/J strain.

Increased production of IL-1β by TLR-mediated macrophages co-cultured with MLN B cells from SAMP1/Yit mice

A decreased production of regulatory cytokines was observed in TLR-stimulated MLN B cells from SAMP1/Yit mice; for this reason, we speculated that these B cells may fail to inhibit inflammation. To confirm our speculation of whether B cells from SAMP1/Yit mice can modulate inflammatory consequences, peritoneal macrophages were isolated from AKR/J mice, and co-cultured with purified MLN B cells from SAMP1/Yit or AKR/J mice, then stimulated with LPS and CpG-DNA. The IL-1β contents in culture supernatants were examined by EIA. As shown in Fig. 8(a), LPS and CpG-DNA did not stimulate IL-1β production by MLN B cells without peritoneal macrophages. Following the co-culture with peritoneal macrophages, significant amounts of IL-1β were observed in the supernatant of TLR ligand-stimulated cells. Moreover, we also noticed that the SAMP1/Yit B cells co-cultured with macrophages did not regulate/inhibit but rather enhanced IL-1β secretion by macrophages, which implies with Olson's findings that the SAMP1/Yit B cells might be pathogenic.43 On the other hand, in the case of AKR/J B cells when co-cultured with LPS or CpG-DNA-treated macrophages, they neither induced nor reduced but instead maintained a steady state of IL-1β content as produced by the macrophages treated with the respective ligands and without co-culture. We therefore conclude that the B cells from SAMP1/Yit mice were found to be solely pathogenic whereas those from AKR/J groups were non-pathogenic.

Figure 8.

Figure 8

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Production of interleukin-1-β (IL-1β) by Toll-like receptor ligand-mediated macrophages co-cultured with mesenteric lymph node (MLN) B cells. Peritoneal macrophages (1 × 106 cells/well) were isolated from AKR/J (a) or SAMP1/Yit (b) mice (15 weeks old) and co-cultured with or without purified MLN B cells (1 × 106 cells/well) from SAMP1/Yit (15 weeks old) or AKR/J (15 weeks old) mice in 24-well plates, then simulated with lipopolysaccharide (LPS; 100 ng/ml) or CpG-DNA (100 nm/ml) for 72 hr. The IL-1β contents in the culture supernatants were examined by enzyme immunoassay. Error bars indicate the standard error of mean values obtained from three independent experiments. *P < 0·05 versus AKR/J mice.

Apart from AKR/J macrophages, using the SAMP1/Yit or AKR/J B-cell co-culturing system we also tested our hypothesis in SAMP/Yit mouse macrophages co-cultured with B cells from both mice. With this co-culture system including the peritoneal macrophages from SAMP1/Yit and the B cells from both mice, the effects of LPS or CpG-DNA for IL-1β production by SAMP1/Yit macrophages was lower and the B cells from both the mouse strains were found to increase IL-1β production (Fig. 8b), which implies that the later system employing the diseased model of mouse peritoneal macrophages did not represent any conclusive data towards our proposed hypothesis.

SAMP1/Yit but not the AKR/J B cells augmented the production of IFN-γ by intestinal T cells

Interferon-γ is a Th1-type cytokine produced mainly by T cells upon inflammation. To evaluate the role of regulatory B cells on T-cell-mediated IFN-γ secretion, we co-cultured intestinal T cells from AKR/J or SAMP1/Yit mice with their B cells at various combinations and then after stimulation with TLR ligands, IFN-γ production was checked by EIA (Fig. 9). We observed that the intestinal T and B cells from both the mouse strains did not produce IFN-γ even when stimulated with TLR ligands, whereas a significant amount of IFN-γ was produced when the T and B cells were co-cultured and stimulated with TLR ligands, implying B-cell-dependent IFN-γ production by T cells. With this phenomenon, we revealed that the AKR/J T cells co-cultured with SAMP1/Yit B cells induced IFN-γ production, whereas this was not clearly observed in the co-culture system with AKR/J B cells (Fig. 9a). Interestingly, the pathogenic role of SAMP1/Yit B cells was clearly visible in the experiment using co-culture with the SAMP1/Yit T cells, but these effects were completely absent in the case of AKR/J B cells (Fig. 9b). Depending on these findings, we suggest that the SAMP1/Yit B cells were exclusively pathogenic in terms of exacerbating the production of IFN-γ by AKR/J and SAMP1/Yit intestinal T cells, whereas AKR/J B cells did not induce pathogenicity and maintained a homeostatic balance in both of these mouse strains.

Figure 9.

Figure 9

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Production of interferon-γ (IFN-γ) by Toll-like receptor ligand-mediated intestinal T cells co-cultured with mesenteric lymph node (MLN) B cells. Intestinal T cells (1 × 106 cells/well) were isolated from AKR/J (a) or SAMP1/Yit (b) mice (15 weeks old) and co-cultured with or without purified MLN B cells (1 × 106 cells/well) from SAMP1/Yit (15 weeks old) or AKR/J (15 weeks old) mice in 24-well plates, then simulated with lipopolysaccharide (LPS; 100 ng/ml) or CpG-DNA (100 nm/ml) for 72 hr. The IFN-γ contents in the culture supernatants were examined by enzyme immunoassay. Error bars indicate the standard error of mean values obtained from three independent experiments. **P < 0·01, *P < 0·05 versus AKR/J mice.

Discussion

In the present study, we investigated the presence of a regulatory subset of B cells expressing IL-10 and TGF-β1 in mouse intestines, and its role in the pathogenesis of ileitis in SAMP1/Yit mice. These B cells exist in mouse intestines, and produce IL-10 and TGF-β in response to LPS and CpG-DNA, which we found to be mainly located in a population characterized by the cell surface markers CD1d+ and CD5− in both SAMP1/Yit and AKR/J mice. We also observed decreased production of IL-10 by TLR-activated intestinal B cells in SAMP1/Yit mice, which may be associated with the development of chronic ileitis. We noticed that B cells from both mouse strains were responsive to TLR for the production of IL-10, and the bioactive or inactive form of TGF-β, whereas sorted T cells from those groups did not demonstrate those characteristics. Different populations of mononuclear cells play essential roles in innate immune function during disease pathogenesis. Interleukin-10 and TGF-β are also produced by other cell types upon stimulation with various TLR ligations. However, we investigated a distinct population of B cells and compared their immune modulating functions in terms of production of anti-inflammatory cytokines between those obtained from two different mouse strains. Similar studies of other subsets of immunoreactive cells for the production of anti-inflammatory cytokines may add additional important information to this field of innate immunity.

First, for a preliminary examination for the presence of B-cell surface markers in various mouse tissues, we considered using BALB/c mice as a normal disease-free model in our study (Fig. 1), because that strain is widely used in many studies for its easy maintenance and availability. However, we analysed commonly encountered surface markers on cells from pathogenic SAMP1/Yit mice as well as their control counterpart AKR/J strain MLN B mice (Fig. 3b). The phenotype and frequency of these populations of B cells from the BALB/c, SAMP1/Yit and AKR/J strains were found to be similar.

The TGF-β1 appears in two physiological forms: bioactive and inactive. In the present system, the majority of TGF-β1 assessed was either solely inactive or latent. We also measured the active form of TGF-β1; however, the amount was too low to determine any effects of TLR ligands on its secretion. Moreover, of the two immune-modulatory cytokines (IL-10 and TGF-β), TLR responses, especially by CpG-DNA ligation, for IL-10 production from the B cells was more striking than that for TGF-β. Therefore, the present findings mainly highlight the intriguing role of IL-10, rather than that of TGF-β.

B cells are widely considered to play pathogenic roles in adaptive immune responses through antibody production and effector T-cell activation, which leads to the development of various autoimmune diseases. In addition to the pathogenic role of conventional B cells, a subset of B cells that negatively regulates autoimmunity and inflammation has also been reported.32–35 The regulatory role of B cells was initially demonstrated in mice with experimental autoimmune encephalitis (EAE), which indicated that B-cell deficiency exacerbates disease outcome and severity, and EAE model mice did not fully recover from the disease compared with wild-type mice.43–45 Recent studies confirmed that the regulatory contribution of B cells during EAE was dependent on their IL-10 production ability.46,47 B cells function as negative regulators of immune responses and have also been studied in a variety of experimental autoimmune models with rheumatoid arthritis,30,48 lupus,49 non-obese diabetes50 and skin diseases.51 The regulatory B-cell subset is therefore currently considered to be a key cell population for modulation of the immune system.

Critical roles of regulatory B cells have been reported in recent studies that used a variety of experimental inflammatory bowel disease models. Chronic colitis in T-cell receptor α knockout (TCR-α KO) mice resembles human ulcerative colitis and its pathogenesis is associated with autoantibody production mediated by pathogenic B cells.52,53 Mizoguchi et al.54 also reported that B-cell-deficient TCR-α double KO mice develop more severe intestinal inflammation, indicating that the regulatory subset of B cells contributes to suppression of TCR-α KO-mediated colitis. In another experiment, evaluations of G protein α inhibitory subunit (Gαi2) KO mice showed that disorders of a Gαi2-dependent process in the maturation of IL-10-producing B cells were associated with a mechanism for inflammatory bowel disease susceptibility.55 Although these interesting findings show the regulatory functions of intestinal B cells, their role in innate immune-related pathogenesis of inflammatory bowel disease is not fully understood.

In the present study, we focused on the innate immune responses of regulatory B cells and evaluated their role in intestinal inflammation. Our experiments with BALB/c mice clearly revealed the presence of intestinal B cells expressing IL-10 in response to TLR ligands. Particularly, CpG-DNA was shown to be a potent stimulator of the production of IL-10. Based on these findings, we also examined the innate immune roles of regulatory B cells in the pathogenesis of ileitis in SAMP1/Yit mice. Although there were no differences in the cell surface markers between SAMP1/Yit and AKR/J mice, EIA, flow cytometry, and real-time PCR results clearly showed that the expression of IL-10 by TLR-mediated MLN B cells isolated from SAMP1/Yi mice was significantly lower than by those from AKR/J mice. Interestingly, a decreased production of IL-10 was also observed in CpG-DNA-stimulated MLN B cells isolated from 5-week-old SAMP1/Yit mice. Ileitis in SAMP1/Yit mice usually develops after 10 weeks of age. In the present study, we could not detect inflammatory lesions in histological sections of ileums from 5-week-old SAMP1/Yit mice (Fig. 3a). These findings suggest that disorders of maturation and differentiation of intestinal regulatory B cells may lead to the development of intestinal inflammation in those mice.

Regulatory B cells have a variety of functions. Particularly, IL-10 and TGF-β produced by this subset are major players in the modulation of inflammation and autoimmunity under various conditions.21–25 Interleukin-10 can suppress immune responses by regulating Th1/Th2 balance or Th17,28–30 as well as by inhibiting the production of pro-inflammatory cytokines including IL-1 and tumour necrosis factor-α.23 On the other hand, TGF-β was shown to suppress disease severity in non-obese diabetes model mice by inducing apoptosis in effector T cells.31 Among their numerous functions, we focused on the anti-inflammatory role of regulatory B cells and evaluated their relationship to ileitis pathogenesis in SAMP1/Yit mice. To clarify our findings, we co-cultured peritoneal macrophages isolated from AKR/J mice with purified MLN B cells from SAMP1/Yit or AKR/J mice, then examined the production of IL-1β by TLR ligand-stimulated macrophages. The level of IL-1β produced by macrophages co-cultured with MLN B cells from SAMP1/Yit mice was significantly higher than that of those from AKR/J mice. This result suggests that MLN B cells in SAMP1/Yit mice do not regulate excess and uncontrolled intestinal inflammatory responses induced by TLR signalling, which might be dependent on decreased production of IL-10 by the MLN B cells. Recently, Olson et al.43 demonstrated a distinct and serious B-cell defect in SAMP1/Yit mice that tends to exacerbate ileitis. In light of the mentioned study, we noticed an enlarged MLN in SAMP1/Yit mice, occupied by expanded amounts of B and T cells compared with their age-matched and sex-matched AKR/J counterparts. They also revealed that these elevated B cells in SAMP1/Yit mice exhibited pathogenic phenomena rather than a regulatory role by abrogating regulatory T-cell functions. Therefore, they speculate that the B cells may be the primary cell population responsible for over-riding anti-inflammatory or regulatory signals in vivo and promoting the development of SAMP1/Yit ileitis. With the essence of their speculation of impeding the regulatory signals, here we proceeded to focus on IL-10 production by B cells from SAMP1/Yit and compared it with that of control AKR/J mice and added a maiden finding of decreased production of IL-10 in TLR-activated intestinal B cells of SAMP1/Yit mice, which may alter the immune regulatory phenotypes leading to intestinal inflammation. Apart from this, other studies have found that a regulatory subset of MLN B cells is involved in intestinal immune regulation by recruiting regulatory T cells,56 so disorders of such functions of MLN B cells may also be associated with the pathogenesis of ileitis in SAMP1/Yit mice.

The notion of specific cell surface markers that characterize regulatory B cells is controversial. Potential cell surface markers, such as CD5+ (B-1a), CD11blow CD5− IgD+, CD1bhigh CD21high (marginal zone B cells), and CD21high CD23high (T2-marginal zone precursor B cells), have been reported to specifically identify the phenotype of IL-10-producing regulatory B cells.21,32,33 Recently, Tedder and colleagues evaluated spleen B cells and found a rare CD1dhigh CD5+ B subset (1–2% of spleen B cells) with IL-10-producing ability.33,42 Furthermore, that study also revealed that CD19-mediated signalling is required for the production of IL-10 by CD1dhigh CD5+ B cells in the spleen. In the present study, we observed that MLN B cells producing IL-10 and TGF-β were mainly located in a population characterized by the cell surface markers CD1d+ in both SAMP1/Yit and AKR/J mice. However, we could not specifically identify the regulatory subset of MLN B cells by evaluating cell surface expression of CD5. More recently, Yanaba et al.57 demonstrated that spleen B cells expressing IL-10 were also found in a CD1dhigh CD5− CD19+ subset, though the number of those cells was relatively low. Organ specificity, signalling pathways via CD19, CD40 and TLRs, and other unknown factors may influence the characterization of regulatory B cells producing IL-10. Additional investigations are necessary to clearly understand these issues.

In summary, we investigated the presence of a subset of regulatory B cells expressing IL-10 and TGF-β1 in mouse intestines, as well as its role in the pathogenesis of ileitis in SAMP1/Yit mice. A decreased level of production of IL-10 and TGF-β1 by TLR-activated intestinal B cells was observed in SAMP1/Yit mice, which failed to inhibit IL-1β production by macrophages. The present results are the first to show that disorders of regulatory B-cell function under innate immune activation may cause disease pathogenesis in a murine model of CD.

Acknowledgments

This work was supported in part by Health and Labour Sciences Research Grants for research on intractable diseases from Ministry of Health, Labour and Welfare of Japan.

Disclosures

None of the authors have any financial or other conflicts of interest.

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