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. 2012 Jun;136(2):153–162. doi: 10.1111/j.1365-2567.2012.03554.x

Bile acids induce monocyte differentiation toward interleukin-12 hypo-producing dendritic cells via a TGR5-dependent pathway

Riko Ichikawa 1, Tetsuro Takayama 1, Kazuaki Yoneno 1, Nobuhiko Kamada 1, Mina T Kitazume 1, Hajime Higuchi 1, Katsuyoshi Matsuoka 1, Mitsuhiro Watanabe 2, Hiroshi Itoh 3, Takanori Kanai 1, Tadakazu Hisamatsu 1, Toshifumi Hibi 1
PMCID: PMC3403261  PMID: 22236403

Abstract

Dendritic cells (DCs) are known as antigen-presenting cells and play a central role in both innate and acquired immunity. Peripheral blood monocytes give rise to resident and recruited DCs in lymph nodes and non-lymphoid tissues. The ligands of nuclear hormone receptors can modulate DC differentiation and so influence various biological functions of DCs. The role of bile acids (BAs) as signalling molecules has recently become apparent, but the functional role of BAs in DC differentiation has not yet been elucidated. We show that DCs derived from human peripheral blood monocytes cultured with a BA produce lower levels of interleukin-12 (IL-12) and tumour necrosis factor-α in response to stimulation with commensal bacterial antigens. Stimulation through the nuclear receptor farnesoid X (FXR) did not affect the differentiation of DCs. However, DCs differentiated with the specific agonist for TGR5, a transmembrane BA receptor, showed an IL-12 hypo-producing phenotype. Expression of TGR5 could only be identified in monocytes and was rapidly down-regulated during monocyte differentiation to DCs. Stimulation with 8-bromoadenosine-cyclic AMP (8-Br-cAMP), which acts downstream of TGR5 signalling, also promoted differentiation into IL-12 hypo-producing DCs. These results indicate that BAs induce the differentiation of IL-12 hypo-producing DCs from monocytes via the TGR5-cAMP pathway.

Keywords: bile acid, cAMP, dendritic cell differentiation, interleukin-12, TGR5

Introduction

Dendritic cells (DCs) are classified as professional antigen-presenting cells and play a central role in both the innate and acquired immune responses. The DCs are a heterogeneous population of cells that can be divided into two major populations: (i) non-lymphoid tissue migratory and lymphoid tissue-resident DCs and (ii) plasmacytoid DCs. Migratory and resident DCs function in the maintenance of self-tolerance and the induction of specific immune responses against invading pathogens. The DCs act as antigen-presenting cells by phagocytosing pathogens and self antigens and then presenting the antigens on their cell surface to T and B cells. They also produce several cytokines in response to stimulation signals from pathogen-associated molecular patterns or whole bacteria. Hence, DCs contribute to immunological homeostasis by promoting inflammatory responses to pathogens, inducing tolerance to self antigen, and suppressing excessive immune responses.1,2 Dendritic cells play a critical role in the maintenance of immunological homeostasis and DC dysregulation can lead to autoimmune diseases and chronic inflammatory disorders. Abnormally excessive immune responses to commensal bacteria, food antigens and self antigens have been reported in the pathogenesis of these diseases. Therefore, conditioning DCs to display desirable properties, such as inducing an immunosuppressive DC phenotype, might represent a novel therapeutic strategy for these diseases. Recent studies have indicated that signalling through nuclear receptors, such as the retinoic acid receptor, the farnesoid X receptor (FXR) and the peroxisome proliferator-activated receptor-α, plays an important role in modulating the transcription of cytokine genes in innate immune cells.3 Interleukin-1 (IL-12) produced by DCs has been implicated in promoting a type 1 helper T cell immune response and contributing to the pathogenesis of several chronic inflammatory disorders.4–6 We previously demonstrated that Am80, a retinoic acid receptor agonist, promotes DC differentiation towards an IL-12 hypo-producing phenotype and that this molecule potentially represents a novel therapeutic molecule for inflammatory bowel disease.7 The identification of similar molecules that induce an IL-12 hypo-producing DC phenotype might allow the development of novel therapeutic molecules for chronic inflammatory disorders. We hypothesized that bile acids (BAs), which are ligands for FXR and TGR5, might regulate DC differentiation and so we examined whether a BA can induce an IL-12 hypo-producing DC phenotype.

Bile acids are a family of steroid molecules generated in the liver by cholesterol oxidation. They accumulate in the blood, intestine and liver via enterohepatic circulation. In addition to their role in nutrient absorption, BAs are signalling molecules that can regulate immune cell responses via FXR and TGR5.8 FXR is a member of the nuclear receptor superfamily of ligand-activated transcription factors8–12 and is primarily expressed in enterohepatic tissues. FXR is known to regulate genes involved in BA synthesis, detoxification and excretion, and an increase in intracellular BA concentrations promotes transcriptional activation of FXR.13–15 In addition, it has been reported that the FXR signalling pathway influences immunological responses such as cytokine production by immune cells.16 TGR5 is a member of the rhodopsin-like superfamily of transmembrane G-protein coupled receptors that transduces signals through G proteins, and is activated by bile acids.8,17

In the present study, we show that BA treatment alters DC differentiation in a way that induces an IL-12 hypo-producing DC phenotype. Importantly, we found that the BAs affected DC differentiation through the TGR5-cAMP pathway, but not through FXR signalling. We found TGR5 to be expressed on the surface of monocytes, but not on differentiated DCs. Hence, our study demonstrates for the first time that BAs have the potential for modulating immune cell differentiation through the newly discovered transmembrane BA receptor, TGR5.

Materials and methods

Reagents

Recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4 were purchased from R&D Systems (Minneapolis, MN). Gel filtration grade lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich (St Louis, MO). Taurochenodeoxycholic acid (TCDCA) was purchased from Calbiochem (San Diego, CA). 8-Bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cAMP; Sigma-Aldrich) was kept as a 50 mm stock solution at −20° and diluted into complete medium immediately before use. The FXR agonist Fexaramine was purchased from Tocris Bioscience (Ellisville, MO). The TGR5-specific agonist [benzyl 2-keto-6methyl-4-(2-thienyl)-1,2,3,4-tetra-hydropyrimidine-5-carboxylate] was kindly provided by Dr Mitsuhiro Watanabe.18

Bacterial heat-killed antigen

The Gram-positive strain Enterococcus faecalis (ATCC29212) was cultured in brain–heart infusion medium. Bacteria were harvested and washed twice with ice-cold PBS. Bacterial suspensions were then heated at 80° for 30 min, washed, resuspended in PBS and stored at −80°. Complete killing was confirmed by 24-hr incubation at 37° on solid growth medium.

Dendritic cell culture

Peripheral blood mononuclear cells were isolated from heparinized peripheral blood samples by density gradient centrifugation using Lymphoprep (Nycomed Pharma, Oslo, Norway). The cells were aspirated from the gradient interface, washed in PBS and resuspended at 1 × 106 cells/ml in RPMI-1640 medium (Sigma-Aldrich) containing 10% heat-inactivated fetal bovine serum (BioSource, Camarillo, CA), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, La Jolla, CA). Monocytes were purified using a magnetic cell separation system (MACS; Miltenyi Biotec, Auburn, CA) with anti-human CD14. Monocytes were seeded into six-well culture dishes at a density of 1 × 106 cells/well in 2 ml culture medium in the presence of GM-CSF (20 ng/ml) and IL-4 (20 ng/ml) to generate conventional immature DCs (cDCs). Identical cultures were prepared with the bile acid TCDCA at the indicated concentrations for 6 days. We refer to cells cultured in these conditions as BA-DCs. We also investigated the effect of adding the BA to cultures on day 0, 2 or 4 together with GM-CSF/IL-4 treatment. In some experiments, monocytes were differentiated into DCs in the presence of GM-CSF and IL-4 with FXR agonist, TGR5 agonist and/or 8-Br-cAMP for 6 days.

Dendritic cells were stimulated with heat-killed E. faecalis (multiplicity of infection = 100) or LPS for 24 hr. Supernatants from stimulated DCs were collected and stored at −80° until cytokine assays were performed.

Cell viability assay

PrestoBlue Cell Viability Reagent (Invitrogen), diluted 1 : 10 with medium, was added to generated DCs (2 × 105 cells/100 μl diluted solution) in a 96-well plate. Samples were then incubated for 30 min at 37°. PrestoBlue is reduced from blue resazurin to red resorufin in the presence of viable cells. We then read the fluorescence (excitation 570 nm, emission 600 nm) with a Benchmark plus (Bio-Rad Laboratories Inc., Hercules, CA).

Cytokine measurement

The supernatants of DC cultures were measured for cytokine content by cytometric bead array (CBA) assays. A human inflammation CBA kit (BD Pharmingen, San Jose, CA) was used to quantify IL-12p70 and tumour necrosis factor-α (TNF-α) levels. Samples were analysed using a FACS Caliber flow cytometer (BD Pharmingen).

Flow cytometry

Cell surface marker fluorescence intensity was assessed using a FACS Caliber analyser and analysed using CellQuest (BD Pharmingen) or FlowJo (TreeStar Inc., Ashland, OR) software. Dead cells were excluded with propidium iodide staining. Monoclonal antibodies against CD14, CD80, CD83, CD86, CD40, CD1a, CD209 and CD205 were purchased from BD Pharmingen. Anti-TGR5 monoclonal antibody was purchased from R&D Systems.

Quantitative real-time PCR analysis

Total RNA was extracted from cells using an RNeasy Micro kit (Qiagen, Hilden, Germany), and cDNA was synthesized using a Quantitect RT kit (Qiagen) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and on-demand gene-specific primers, designed using the DNA Engine Opticon 2 System (Bio-Rad Laboratories, Inc.) and analysed with Opticon monitor software (MJ Research, Waltham, MA). The primers were as follows: BSEP (Hs00184824_m1), NTCP (Hs00161820_m1), OATP (Hs00366488_m1), ASBT (Hs01001557_m1), TGR5 (Hs01937849_s1), TNFα (Hs00174128_m1), IL-12p35 (Hs00168405_m1) and IL-12p40 (Hs00233688_m1).

cAMP production assay

Monocytes (2 × 105 cells) were treated with lithocholic acid, TCDCA, glycoursodeoxycholic acid (GUDCA) and TGR5 agonist (5 μm) for 5 min in the presence of 1 mm 3-isobutyl-1-methylxanthine. The amount of cAMP was determined with a cAMP-Screen System (Applied Biosystems).

Phosphorylation of transcription factors

For intracellular phosphoprotein staining in monocytes we used a PhosFlow assay (BD Biosciences, Franklin Lakes, NJ). Cells in suspension were stimulated by TCDCA or with control medium for the indicated times, fixed with pre-warmed PhosFlow Cytofix solution for 10 min and permeabilized with ice-cold PhosFlow Perm buffer III for 30 min. Phycoerythrin-conjugated mouse anti-cAMP response element-binding protein (CREB) (pS133)/ATF-1 (pS63) or mouse anti-IgG isotype antibody was added to each tube and incubated at room temperature for 30 min in the dark. The cells were washed with 10 volumes of staining buffer and analysed by flow cytometry.

Statistical analysis

Statistical analysis was performed using GraphPadPrism software v. 4.0 (San Diego, CA). The statistical significance of differences between two groups was tested using a Student’s t-test. For comparison of more than two groups, Kruskal–Wallis one-way analysis of variance (anova) was used. If the anova was significant, the Tukey–Kramer test was used as a post hoc test. Differences of P < 0·05 were considered significant. All data are expressed as means ± SEM, *P < 0·05, **P < 0·01, ***P < 0·001.

Results

Morphology and expression of surface markers in BA-DCs, TGR5-DCs, cAMP-DCs and FXR-DCs

Conventional immature DCs were generated from monocytes by 6 days of culture with GM-CSF and IL-4. Other stimuli were added during the differentiation process; TCDCA (100 μm) for TCDCA-DCs, TGR5 agonist (20 μm) for TGR5-DCs, 8-Br-cAMP (10 μm) for cAMP-DCs, and fexaramine (100 μm) for FXR-DC. These DCs revealed different morphology and cell surface antigen expression (Fig. 1a,b). We observed BA-DCs, TGR5-DCs and FXR-DC expressing low levels of CD1a, but not cAMP-DCs. Expression of co-stimulatory molecules, CD80 and CD86, was increased in BA-DCs, TGR5-DCs, cAMP-DCs and FXR-DCs. These findings demonstrated that TCDCA, TGR5 agonist, cAMP and FXR agonist induce different types of DCs during the 6-day differentiation culture. The viability of cDC, TCDCA-DCs, and TGR5-DCs was also confirmed (see Supplementary material, Fig. S1).

Figure 1.

Figure 1

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Morphology and expression of surface markers in bile acid-treated dendritic cells (BA-DCs), TGR5-DCs, cAMP-DCs and farnesoid X receptor (FXR)-DCs. (a) Conventional immature (cDCs) were generated from monocytes by 6 days of culture with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Other stimuli were added during the differentiation process; taurochenodeoxycholic acid (TCDCA; 100 μm) for TCDCA-DCs, TGR5 agonist (20 μm) for TGR5-DCs, 8-Br-cAMP (10 μm) for cAMP-DCs and fexaramine (100 μm) for FXR-DC. (b) Differentiated DCs were harvested and the following surface markers and their isotype-matched monoclonal antibodies were analysed: CD83, CD86, CD80, CD40, CD14, CD1a, CD205 and CD209.

BA induces differentiation of IL-12 hypo-producing DCs in a dose-dependent manner

We have previously found that retinoic acid affects the differentiation of DCs from monocytes and induces anti-inflammatory DC differentiation.7 We hypothesized that BAs might also affect the differentiation of DCs. To assess this, we cultured DCs differentiated from monocytes in the presence (referred to as BA-DCs) or absence (referred to as cDCs) of a BA and measured the cytokine-producing ability of these cells following stimulation with heat-killed antigen from the commensal bacteria E. faecalis or LPS + interferon-γ. The BA-DCs produced significantly less of the pro-inflammatory cytokines IL-12p70 and TNF-α in response to bacterial antigen or LPS + interferon-γ stimulation than cDCs, in a manner that was dependent on the concentration of the BA (Fig. 2a,b).

Figure 2.

Figure 2

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Bile acids (BAs) promote differentiation of interleukin-12 (IL-12) hypo-producing dendritic cells (DCs). (a) Human peripheral blood monocytes (1 × 106 cells/well in 2 ml culture medium) were cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF; 20 ng/ml) and IL-4 (20 ng/ml) with or without taurochenodeoxycholic acid (TCDCA; 100 μm). After 6 days in culture, DCs were stimulated with Enterococcus faecalis (multiplicity of infection = 100) or lipopolysaccharide (LPS; 100 ng/ml) + interferon-γ (IFN-γ; 100 ng/ml). The cytokines in culture supernatants were measured by cytometric bead array. Results show means ± SEM, control-DC, n = 12, TCDCA-DC, n = 12. (b) Monocytes were cultured as described above, with or without 100 μm or 10 μm TCDCA. IL-12p70 production in culture supernatants was measured by cytometric bead array. Results show means ± SEM, n = 6. Statistical analysis was performed by Kruskal–Wallis one-way analysis of variance and Tukey–Kramer test for multiple comparisons. Experiments were repeated at least three times.

FXR agonist does not affect the differentiation of DCs from monocytes

We next investigated whether the FXR signalling pathway was involved in the DC differentiation process, using fexaramine, a powerful synthetic FXR agonist, in place of the BA during DC differentiation from monocytes. Unexpectedly, DCs differentiated in the presence of the FXR agonist did not show the same IL-12 hypo-producing DC phenotype as DCs differentiated in the presence of the BA (Fig. 3a,b). We also examined mRNA expression of BA transporters, bile salt export pump (BSEP), organic anion transporting polypeptide C (OATP), sodium taurocholate cotransporting polypeptide (NTCP) and apical sodium-dependent bile salt transporter (ASBT) on monocytes and DCs. As shown in Fig. 3(c), no transporters for BAs were expressed on peripheral blood monocytes. The transporter BSEP was expressed in DCs, but all other transporters were absent in both monocytes and DCs.

Figure 3.

Figure 3

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Effect of biles acids (Bas) on dendritic cell (DC) differentiation is independent of farnesoid X receptor (FXR). (a) DCs were differentiated from monocytes by treatment with granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and Fexaramine (100 μm or 1 mm) for 6 days (FXR-DC). Cytokine production after Enterococcus faecalis stimulation was measured. Results show means ± SEM, n = 6; differences were analysed by Kruskal–Wallis one-way analysis of variance. (b) FXR-DC (100 μm fexaramine) was stimulated with lipopolysaccharide (LPS; 100 ng/ml) and interferon-γ (IFN-γ; 100 ng/ml). Cytokine production was measured. Results show means ± SEM, n = 6; differences were analysed by Kruskal–Wallis one-way analysis of variance. Experiments were repeated at least three times. (c) No transporters for BAs are expressed on peripheral blood monocytes. We examined BA transporter expression in monocytes and DCs using real-time quantitative PCR. The transporter bile salt export pump (BSEP) was expressed in DCs, but all other transporters were absent in both monocytes and DCs. Statistical analysis was performed using paired t-tests n = 3.

IL-12 hypo-producing BA-DCs are induced through a TGR5 signalling pathway

As the FXR pathway did not appear to be involved in BA-DC differentiation, we next focused on TGR5. The DCs were differentiated from monocytes in the presence of a TGR5-specific agonist at several concentrations and IL-12 and TNF-α production in response to commensal bacterial antigen stimulation was measured. These TGR5-DCs produced less IL-12 and TNF-α than cDCs, in a similar manner to BA-DCs (Fig. 4a,b). We also measured the mRNA transcripts of TNF-α, IL-12p35 and IL-12p40 after stimulation with LPS and interferon-γ. We found that, at the mRNA level, expression of these pro-inflammatory cytokines was suppressed in TGR5-DCs (see Supplementary material, Fig. S2).

Figure 4.

Figure 4

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TGR5 agonist induces interleukin-12 (IL-12) hypo-producing dendritic cells (DCs). DCs were generated from monocytes by treatment with granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and TGR5 agonist (10 or 20 μm) for 6 days. Cytokine production in culture supernatants was analysed after 24-hr stimulation with Enterococcus faecalis (a) or lipopolysaccharide (LPS; 100 ng/ml) + interferon-γ (IFN-γ; 100 ng/ml) (b). Results show means ± SEM, TGR5 agonist 10 μm, n = 4, 20 μm, n = 6. Differences were analysed by Kruskal–Wallis one-way analysis of variance. Experiments were repeated at least three times.

cAMP, a downstream target of TGR5, induces IL-12 hypo-producing DCs

We next assessed the mechanism by which BAs modify the differentiation of DCs to give an anti-inflammatory phenotype. It is known that cAMP has an immunosuppressive effect in various cells, so we measured cAMP levels of monocytes cultured with BA or the TGR5-specific agonist at several points during their differentiation to DC. Consistent with previous reports, the concentration of cAMP in monocytes increased following the administration of either BA or TGR5 agonist (Fig. 5a).18 To test the hypothesis that this process induces anti-inflammatory DC differentiation, monocytes were treated with the cAMP analogue 8-Br-cAMP instead of the BA. The DCs obtained from this differentiation also produced lower levels of IL-12 and TNF-α than cDCs (Fig. 5b). Moreover, activation of CREB, a key molecule in cAMP downstream signalling,8 was observed in monocytes treated with BA (Fig. 5c).

Figure 5.

Figure 5

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TGR5 signalling increases cAMP levels in monocytes, which induces differentiation into interleukin-12 (IL-12) hypo-producing dendritic cells (DCs). (a) Monocytes were treated with the indicated compounds at 5 μm (LCA, lithocholic acid; GUDCA, glycoursodeoxycholic acid; TCDCA, taurochenodeoxycholic acid). Results show means ± SEM, n = 3. (b) cAMP induces IL-12 hypo-producing DCs in a concentration-dependent manner. DCs were generated from monocytes with granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and 8-Br-cAMP (10 and 50 μm). After 6 days in culture, cytokine levels in culture supernatants were analysed after 24-hr stimulation with Enterococcus faecalis. Results show means ± SEM, n = 6. Data were analysed by Kruskal–Wallis one-way analysis of variance and Tukey–Kramer test for multiple comparisons. (c) Bile acid activates cAMP response element binding protein (CREB) in monocytes. Cells in suspension were stimulated by TCDCA (100 μm) or with control medium for 30 or 60 min, treated for PhosFlow analysis, and analysed using anti-CREB (pS133)/ATF-1 (pS63) or mouse IgG isotype antibodies for flow cytometry. Experiments were repeated at least three times.

BA and TGR5 agonist can only influence the development of anti-inflammatory DCs from monocytes if present from the start of in vitro culture

Unexpectedly, the BA did not show any anti-inflammatory effect on terminally differentiated DCs (6 days after differentiation from monocyte) (Fig. 6a). To further investigate this discrepancy, we focused on the expression level of TGR5 on monocytes and DCs. We found TGR5 expression only in monocytes, and its expression was rapidly down-regulated over the course of differentiation to DCs, as assessed both by the surface expression of receptors and mRNA levels (Fig. 6b,c). Consistent with these results, the BA induced anti-inflammatory DCs when the BA was administrated on day 0, but not when the BA was added on day 2 or 4 after DC differentiation (Fig. 6d). Addition of the TGR5 agonist showed similar results (Fig. 6e). Next, we examined medium replacement experiments. As expected, DCs cultured in the presence of TGR5 agonist in the initial 3 days after DC differentiation (day 0–2) also showed an IL-12 hypo-producing phenotype (Fig. 6f).

Figure 6.

Figure 6

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Bile acids (BA) do not directly suppress cytokine production from human dendritic cells (DCs). (a) After monocytes were cultured with granulocyte–macrophage colony-stimulating factor (GM-CSF; 20 ng/ml) and interleukin-4 (IL-4; 20 ng/ml) for 6 days, DCs (5 × 105 cells/ml) were stimulated with Enterococcus faecalis (100 ng/ml) and lipopolysaccharide (LPS; 100 ng/ml) with or without bile acids (taurochenodeoxycholic acid; TCDCA; 100 μm). Cytokines in culture supernatants were measured by cytometric bead assay (CBA). Results show means ± SEM, n = 5. (b) Monocytes were differentiated into DCs by treatment with GM-CSF and IL-4 for 6 days and surface expression levels of TGR5 and its isotype-matched monoclonal antibodies on monocytes were analysed by flow cytometry on day 0, day 1, day 2 and day 4. (c) mRNA transcripts of TGR5 in CD14+ monocytes and DCs were measured by quantitative PCR. The transcript level of TGR5 was normalized to the level of β-actin transcript. Results show means ± SEM of fold induction of at least four individual experiments. (d) DCs were generated from monocytes with GM-CSF and IL-4. TCDCA (50 μm) was added on day 0, day 2 and day 4. Cytokine levels in culture supernatants on day 6 were measured by CBA after 24-hr stimulation with E. faecalis. Results show means ± SEM, n = 3. (e) DCs were generated from monocytes with GM-CSF, IL-4. TGR5 agonist (20 μm) was added on day 0 and day 3. Cytokine levels in culture supernatants on day 6 were measured by CBA after 24-hr stimulation with E. faecalis. Amount of IL-12p70 and tumour necrosis factor-α (TNF-α) in culture supernatant were indicated as relative percentage of control DCs with stimulation. Results show means ± SEM, n = 5. ***P < 0.001. (f) DCs were generated from monocytes with GM-CSF and IL-4 (day 0–6). TGR5 agonist (20 μm) was added only on day 0–2. Cytokine levels in culture supernatants on day 6 were measured by CBA after 24 hr of stimulation with LPS + interferon-γ (IFN-γ). Amounts of IL-12p70 and TNF-α in culture supernatant were indicated as relative percentage of control DCs with stimulation. Results show means ± SEM, n = 4. ***P < 0.001. Data were analysed by Kruskal–Wallis one-way analysis of variance and Tukey–Kramer test for multiple comparisons.

Discussion

Both primary and secondary BAs can activate TGR5 and FXR, and several BAs have been reported to be natural ligands of TGR5. Of these lithocholic acid and taurolithocholic acid activate the TGR5 with an EC50 of ∼ 600 and 300 nm, respectively, indicating that they can be considered physiological ligands for TGR5.8,17,19–23 Other BAs activate TGR5 at micromolar concentrations. Chenodeoxycholic acid, which activates FXR at an EC50 of ∼10 μm, is considered a physiological ligand for FXR. Other BAs can activate FXR at higher concentrations.8–12 We used 10–100 μm of TCDCA, a concentration range at which it can activate both FXR and TGR5.

TGR5 is expressed in several tissues, with the highest levels detected in the gall bladder, followed by the ileum and colon. TGR5 expression is not detectable in primary hepatocytes.8,19 In contrast, FXR is highly expressed in the liver, intestine, kidney and adrenal glands.8–10,13,24–27 FXR expression in immune cells, such as CD14+ monocytes, has also been reported, but its expression in these cells is relatively low compared with the expression of other nuclear receptors such as LXRα (Liver X Receptor alpha).3 In addition, we could not detect expression of BA transporter mRNA in monocytes. These findings are consistent with our demonstration that the FXR agonist did not influence DC differentiation in our experiments. In the present study, we found expression of TGR5 on CD14+ peripheral blood monocytes. Furthermore, the presence of the TGR5-specific agonist promoted the differentiation of IL-12 hypo-producing DC in a similar manner to that seen in the presence of BA. Taken together, these results suggest that BAs can regulate the DC differentiation process through TGR5 expressed on primary peripheral blood monocytes.

Expression of TGR5 was rapidly down-regulated during DC differentiation from monocytes, and differentiated DCs did not express detectable levels of cell surface TGR5. Although the mechanisms of TGR5 gene transcription regulation have not been identified, our study of mRNA transcription revealed that the amount of TGR5 mRNA transcript was dramatically reduced following GM-CSF and IL-4 stimulation. In addition, it has been reported that ligand stimulation causes cellular internalization of TGR5.8 These findings suggest that the binding of the BA to TGR5 on monocytes at the initial phase of differentiation is crucial if differentiation outcomes are to be influenced by the BA.

Activation of TGR5 leads to intracellular cAMP accumulation, which activates CREB.8,18 The CREB then transactivates target genes by binding to the cAMP response element in the promoter region of these genes.8,20,22,23 In our studies, stimulation of monocytes by BA or a TGR5-specific agonist led to up-regulated intracellular cAMP concentrations. It has been reported that intracellular cAMP concentration is an important modulator of pro-inflammatory cytokine transcription.28 Consistent with these observations, treatment of monocytes with cAMP also promoted cellular differentiation into IL-12 hypo-producing DC. The cAMP promotes the differentiation of CD14+ monocytes into CD1alow CD209+ DCs.29 We observed BA-DCs and TGR5-DCs, but not cAMP-DCs, expressing low levels of CD1a (Fig. 1), although all three DC types displayed a similarly low capacity to produce IL-12. Interestingly, FXR-DCs also showed a CD1a-positive DC phenotype, but FXR-DCs did not display an IL-12 hypo-producing phenotype.

The finding that BAs can induce the differentiation of IL-12 hypo-producing DCs through activation of the TGR5-cAMP pathway suggests that the TGR5 pathway may be a novel therapeutic target for T helper type 1 dominant chronic inflammatory disorders, such as Crohn’s disease and psoriasis.7,28,30 As BAs are part of the enterohepatic circulation, the ileum, mesenteric lymph node and liver may be candidates as sites where BAs act to modulate DC differentiation.

Acknowledgments

The authors thank T. Yajima, M. Uo, H. Naruse, S. Ando and Y. Wada for helpful discussions and critical comments. This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japan Society for the Promotion of Science, and the Keio University Medical Fund.

Glossary

ASBT

apical sodium-dependent bile salt transporter

BA

bile acid

BSEP

bile salt export pump

CBA

cytometric bead assay

CREB

cAMP response element binding protein

DC

dendritic cell

FXR

farnesoid X receptor

GM-CSF

granulocyte–macrophage colony-stimulating factor

GUDCA

glycoursodeoxycholic acid

IL

interleukin

LCA

lithocholic acid

LPS

lipopolysaccharide

NTCP

sodium taurocholate cotransporting polypeptide

OATP

organic anion transporting polypeptide C

qPCR

quantitative real-time polymerase chain reaction

TCDCA

taurochenodeoxycholic acid

TNF

tumour necrosis factor

Disclosure

The authors declare no conflict of interests.

Author contributions

RI, TT, KY performed the experiments. RI, TT, KY, NK, MK, HH, SO, MW, TK and HI designed the experiments, collected data and wrote the manuscript. T. Hisamatsu reviewed the manuscript and T. Hisamatsu and T. Hibi supervised and compiled the final version of the manuscript.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Cell viability of peripheral blood monocyte derived DCs.

Figure S2. mRNA transcript of proinflammatory cytokines in TGR5-DCs.

imm0136-0153-SD1.ppt (185KB, ppt)
imm0136-0153-SD2.ppt (234.5KB, ppt)

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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Supplementary Materials

imm0136-0153-SD1.ppt (185KB, ppt)
imm0136-0153-SD2.ppt (234.5KB, ppt)

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