The Bile Acid Receptor GPBAR1 Regulates the M1/M2 Phenotype of Intestinal Macrophages and Activation of GPBAR1 Rescues Mice from Murine Colitis

Inflammatory bowel diseases (IBDs) encompass two highly prevalent chronic disorders, Crohn’s disease and ulcerative colitis, which result from a dysregulated intestinal immune response to Ags derived from the intestinal microbiota in genetically predisposed individuals (1, 2). IBDs have traditionally been treated with nonspecific immunosuppressive drugs (e.g., corticosteroids or azathioprine), anti-inflammatory agents (e.g., 5-aminosalicylic acid), and, more recently, with anti–TNF-α agents (3). These treatments have shown efficacy in mild to severe disease; however, a large percentage of patients with severe illness do not achieve complete remission or cannot maintain remission due to the lack of response or loss of response to drugs, because of intolerance or severe side effects that require cessation of therapy. Recently, novel approaches that target leukocyte trafficking to the gut by inhibiting integrins, chemokines, and chemokine receptors (4) have been developed, but 30–40% of patients fail to respond to these approaches in clinical trials. Therefore, there is a clinical need for new treatments based on therapies that are able to selectively regulate the intestinal immune response, overcoming the limitations of current therapies.

Bile acids, the end product of cholesterol metabolism, play an essential role in maintaining liver and intestinal homeostasis (5, 6). Primary bile acids (chenodeoxycholic acid [CDCA] and cholic acid) and secondary bile acids (deoxycholic acid [DCA] and lithocholic acid [LCA]), as well as their glycine and taurine conjugates, are signaling molecules that exert a variety of regulatory functions by activating a family of cell surface and nuclear receptors collectively known as the bile acid–activated receptors (BARs) (7). The two best characterized members of the BAR family are the G protein–coupled receptor GPBAR1 (also known as TGR5 or M-BAR) and the nuclear receptor FXR (8–11). GPBAR1 was the first transmembrane G protein–coupled receptor shown to be activated by bile acids, with DCA and LCA, as well as their taurine and glycine conjugates, functioning as bona fide physiological ligands (12, 13). In the human body, the expression of GPBAR1 is essentially restricted to the small intestine, gallbladder, adipose tissues, and immune system. In response to ligands, GPBAR1 signals by increasing intracellular concentrations of cAMP, leading to rapid phosphorylation of downstream kinases that are responsible for the nongenomic effects exerted by this receptor (14). In addition, GPBAR1 exerts genomic effects that are mediated by binding of a CREB to a cAMP-responsive element (CRE) in the promoter of target genes (15, 16).

In the immune system, GPBAR1 is primarily expressed by cells of myeloid origin, whereas T and B cells express the receptor at very low levels. In macrophages derived from peripheral blood and liver macrophages, activation of GPBAR1 counteracts the activity of CD14/TLR4, decreasing the phagocytic capacity and production of proinflammatory cytokines TNF-α, IL-1α, IL-1β, and IL-6 (15, 16). In the intestine, GPBAR1 oversees a variety of homeostatic functions, and we have previously shown that mice harboring disrupted GPBAR1 have an altered intestinal morphology and increased permeability and are more prone to develop intestinal inflammation. Because the intestine hosts the highest concentration of bile acids in mammalian tissues, which fluctuates widely during the fast-to-feeding transition, GPBAR1 ligands might have a role in regulating the recruitment and function of intestinal macrophages. Intestinal macrophages, the largest pool of tissue macrophages, are made up of heterogeneous cells classified as classically activated (M1) macrophages (high IL-12 and low IL-10 producing), which are induced by IFN-γ and microbial products, and alternatively activated (M2) macrophages (low IL-12 and high IL-10 producing). The latter are induced by IL-4 or IL-13 and display an anti-inflammatory phenotype, and it is thought that a wide transition between the two phenotypes exists (17, 18). The composition of the intestinal macrophage pools in mice and humans changes considerably in conditions of perturbed homeostasis, leading to accumulation of the M1-bearing phenotype in the setting of inflammation. In this setting, M1 macrophages, in concert with other professional APCs, orchestrate the recruitment of cells of adaptive immunity and have a role in the initiation and maintenance of inflammation, making these cells a putative target to prevent immune activation in intestinal inflammatory states (19).

6β-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501) is a bile acid derivative that selectively activates GPBAR1 (20). In the current study, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation-driven immune dysfunction that develops in two rodent models of colitis by shifting macrophage polarization from the M1 to the M2 phenotype. This effect is mediated by a genomic circuit and involves GPBAR1-dependent recruitment of CREB to the IL-10 promoter. To our knowledge, the present results are the first evidence of fine-tuning of IL-10 expression/activity in intestinal macrophages by bile acids and pave the way for the exploitation of GPBAR1 as a therapeutic target in the development of gut-specific therapies for the treatment of IBDs.

BALB/c mice were purchased from Charles River. GPBAR1-null mice on a C57BL/6NCrl background and C57BL/6NCrl congenic littermates were originally donated by Dr. Galya Vassileva (Schering-Plough Research Institute, Kenilworth, NJ). IL-10–null mice (B6.129P2-Il10tm1Cgn/J) were from The Jackson Laboratory. The colonies were maintained in the animal facility at the University of Perugia. Mice were housed under controlled temperatures (22°C) and photoperiods (12:12-h light/dark cycle), were allowed unrestricted access to standard mouse chow and tap water, and were allowed to acclimate to these conditions for ≥5 d before inclusion in an experiment. The study was conducted in agreement with Italian law, and the protocol was approved by a ethical committee at the University of Perugia and by a National Committee of Italian Ministry of Health (permit number 42/2014/B). The health of the animals was monitored daily by the veterinarian in the animal facility. The study protocol caused minor suffering; however, animals that lost >25% of their initial body weight were euthanized.

Only male mice were used in each experiment. Colitis was induced in BALB/c mice, Gpbar1^+/+ and Gpbar1^−/− mice, and IL-10^−/− mice. In brief, mice were fasted for 12 h overnight (day −1). The following day (day 0), under profound sedation induced by the administration of a mixture of tiletamine hypochloride and zolazepam hypochloride/xylazine (50/5 mg/kg), a 3.5 F catheter was inserted into the colon for up to 4 cm from the anus, and 1 mg of trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich, St. Louis, MO) in 50% ethanol was administered via the catheter into the colon lumen using a 1-ml syringe (injection volume of 100 μl). Control mice received 50% ethanol alone. Animals were monitored daily for diarrhea, loss of body weight, presence of blood in the stool, and survival. At the end of the experiment, 4 d after the administration of TNBS, surviving mice were sacrificed, blood samples were collected by cardiac puncture, and the colon was excised, weighed, and evaluated for macroscopic damage. When prompted by the experimental design, BAR501 (5 to 30 mg/kg/d) or dexamethasone (5 mg/kg/d) was administrated orally or i.p.

For the oxazolone model, BALB/c mice were presensitized by application of 150 μl of 3% oxazolone (Sigma-Aldrich) in a 4:1 acetone/olive oil solution to a 1.5 × 1.5-cm area of shaved skin on the back of the mouse. Seven days later, mice were anesthetized, and 150 μl of 1% oxazolone solution in 50% ethanol was administered via the rectum. Mice were sacrificed 4 d after treatment, and clinical signs of colitis were scored.

In both models, the severity of colitis was assessed each day for each mouse by assessing body weight, fecal occult blood, and stool consistency. Each parameter was scored from 0 to 4, and the sum represents the Colitis Disease Activity Index (CDAI). The scoring system was as follows: percentage of body weight loss: none = 0, 1–5% = 1, 5–10% = 2, 10–20% = 3, and >20% = 4; stool consistency: normal = 0, soft but still formed = 1, very soft = 2, diarrhea = 3, and liquid stools that stick to the anus or anal occlusion = 4; and fecal blood: none = 0, visible in the stool = 2, severe bleeding with fresh blood around the anus, and very present in the stool = 4.

The cells were isolated from the colon lamina propria using the Lamina Propria Dissociation Kit (Miltenyi Biotec; 130-097-401), according to the instructions.

Flow cytometry analyses were carried out using a two-laser standard configuration ATTUNE NxT (Life Technologies), and cell sorting was done using a two-laser standard configuration FACSAria. Data were analyzed using FlowJo software (TreeStar). The gates were set using a fluorescence minus one (FMO) control strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in which the conjugated Ab is missing is the one for which the FMO provides a gating control. The following mAbs were used: B220 Alexa Fluor 700 (RA3-6B2; eBioscience, San Diego, CA), CD4 allophycocyanin–eFluor 780 (GK1.5; eBioscience), CX3CR1 allophycocyanin (SA011F11; BioLegend), CCR7 Alexa Fluor 700 (4B12; eBioscience), CD11c Alexa Fluor 700 (N418; eBioscience), IL-6 PE (MP5-20F3; eBioscience), Foxp3 PE (3G3; Tonbo Biosciences), IL-10 FITC (JES5-16E3; eBioscience), Ly-6C Alexa Fluor 488 (HK1.4; eBioscience), F4/80 PE-Cyanine7 (BM8; eBioscience), and CD8 PE-Cyanine7 (53-6.7; eBioscience).

Samples of the colon, ≈0.5 cm taken 1 cm from the anus, were immediately frozen in liquid nitrogen and stored at −80°C until used. The colon samples were mechanically homogenized using a pestle, and the obtained materials were resuspended in 1 ml of TRIzol Reagent (Thermo Fisher Scientific). The RNA was extracted according to the manufacturer’s protocol. After purification from genomic DNA using DNase I (Thermo Fisher Scientific), 1 μg of RNA from each sample was reverse transcribed using random hexamer primers with SuperScript II (Thermo Fisher Scientific) in a 20-μl reaction volume; 10 ng of cDNA was amplified in a 20-μl solution containing 200 nM each primer and 10 μl of SYBR Select Master Mix (Thermo Fisher Scientific). All reactions were performed in triplicate using the following thermal cycling conditions: 3 min at 95°C, followed by 40 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for 30 s, using a StepOnePlus system (Applied Biosystems). The relative mRNA expression was calculated according to the Δ cycle threshold method. Primers were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/) using published data obtained from the National Center for Biotechnology Information database. Alternatively, for some genes, TaqMan probes were used, with TaqMan GEX Master Mix (all from Thermo Fisher Scientific). The following primers were used (forward and reverse): IFN-γ (5′-GCTTTGCAGCTCTTCCTCAT-3′, 5′-ATCCTTTTGCCAGT-3′), TNF-α (5′-CCACCACGCTCTTCTGTCTA-3′, 5′-AGGGTCTGGGCCATAGAACT-3′), IL-6 (5′-CTTCACAAGTCGGAGGCTTA-3′, 5′-TTCTGCAAGTGCATCATCGT-3′), IL-1β (5′-GCTGAAAGCTCTCCACCTCA-3′, 5′-AGGCCACAGGTATTTTGTCG-3′), TGF-β (5′-TTGCTTCAGCTCCACAGAGA-3′, 5′-TGGTTGTAGAGGGCAAGGAC-3′), IL-10 (5′-CCCAGAAATCAAGGAGCATT-3′, 5′-CTCTTCACCTGCTCCACTGC-3′), and Foxp3 (5′-TCTTCGAGGAGCCAGAAGAG-3′, 5′-AGCTCCCAGCTTCTCCTTTT-3′). Marker genes for M1 and M2 macrophage populations were Cd38 (Mm01220906_m1), Fpr2 (Mm00484464_s1), Gpr18 (Mm02620895_s1), Egr2 (Mm00456650_m1), and c-myc (Mm00487804_m1; all from Thermo Fisher Scientific).

Samples of distal colon (2–3 cm from the anus) were fixed in buffered formalin, cut into 5-μm-thick sections (≈150 μm between each section, four to eight per fragment per colon), and stained with H&E.

Colon fragments were cryoprotected in 30% sucrose in PBS overnight at 4°C, embedded in OCT, and frozen. Colons were sectioned at 7 μm, washed in PBS, and fixed in 4% paraformaldehyde in PBS for 15 min. After washing, sections were incubated with 10% goat serum, 2% BSA, and 0.2% Triton X-100 for 40 min and then incubated with anti-mouse CD11b–Alexa Fluor 488 mAb (clone M1/70) or anti-mouse CD4–eFluor 570 mAb (clone RM4-5; both from eBioscience) in PBS for 1 h, washed three times, and mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen). Fluorescent images of tissue were acquired using an AxioVision.Z1 microscope equipped with an ApoTome filtering device and an AxioCam MRm digital camera (Carl Zeiss Microscopy) through a 20× Plan Apo objective. The ApoTome device, enabled for optical sectioning at medium setting (digital noise reduction set to off), was used to remove scattered light from underlying and overlying out-of-focus focal planes, resulting in a high axial resolution and increased signal-to-noise ratio. Images were visually optimized with a factor 140, 5-pixel sharpening mask and histogram equalization (Adobe Photoshop CC).

RAW 264.7 cells were grown at 37°C in DMEM containing 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin. Cells were regularly passaged to maintain exponential growth. Macrophages derived from the spleen of mice and RAW 264.7 cells were classically activated (M1 condition) with LPS (1 ng/ml, L2880; Sigma-Aldrich) + IFN-γ (20 ng/ml; eBioscience) for 16 h.

For the chromatin immunoprecipitation (ChIP) assay, by a promoter analysis, we detected a CRE sequence 358 bp upstream of the exon 1 of the gene encoding for murine IL-10. RAW 264.7 cells and RAW 264.7 cells silenced for GPBAR1 were exposed or not to BAR501 (50 μM) for 6 h and then cross-linked with formaldehyde. ChIP assays were performed according to the manufacturer’s protocols (EZ-ChIP; Upstate; cat. no. 17-371). Briefly, cell extracts were sonicated and divided. Abs (5 μg) against p-CREB and normal goat IgG were added for immunoprecipitation. The immunoprecipitated chromatin was recovered and purified. The IL-10 promoter DNA was quantified by real-time PCR analysis using primers around the CREB binding site in the proximal promoter of IL-10 (forward 5′-TGACTTCCGAGTCAGCAAGA-3′, reverse 5′-TATTTCCTGAGGCAGACAGC-3′). Primers spanning a region located ∼6800 kb downstream of the IL-10 transcriptional start site (5′-TCCCTACCTGTTAAGGTTTATGG-3′; rev 5′-ACACATGTCCACATGCAAGC-3′) were used as control. At least three replicates of each group were performed.

For IL-10 RNA silencing, an IL-10 mouse short hairpin RNA plasmid (pRFP-C-RS) and a GPBAR1 mouse short hairpin RNA plasmid (pGFP-V-RS) were purchased from OriGene Technologies and were used to suppress IL-10 and GPBAR1 expression. RAW 264.7 cells were seeded in a six-well plate and transfected with 1 μg of each plasmid using HD Transfection Reagent (Promega). The transfection was repeated for two consecutive days, and transfected cells were selected by exposure to puromycin (4 μg/ml, P-8833; Sigma-Aldrich) for 72 h. Silencing was then verified through real-time PCR using specific primers for the Il10 and Gpbar1 genes.

ANOVA followed by the nonparametric Mann–Whitney U test or a two-tailed unpaired Student t test was used for statistical comparisons using Prism 6.0 software (GraphPad).

We first investigated whether immune cells express GPBAR1. For this purpose, T and B cells and macrophages were sorted from the spleen of wild-type mice, and expression of GPBAR1 was assessed by real-time PCR As shown in Fig. 1A, GPBAR1 mRNA was detected at high levels only in macrophages, whereas expression in other cells was minimal.

BAR501 is a recently discovered selective GPBAR1 agonist that activates the receptor with an EC₅₀ of 1 μM (20). To investigate the effects of GPBAR1 ligation on macrophage phenotype, spleen-derived macrophages were primed by exposure to LPS and IFN-γ, with or without increasing concentrations of BAR501. As shown in Fig. 1B, although exposure to LPS/IFN-γ increased the expression of prototypical proinflammatory cytokines (IFN-γ, IL-1β, IL-6, TNF-α), this pattern was reversed by cotreating the cells with the GPBAR1 ligand, in a concentration-dependent manner (Fig. 1B). Additionally, exposure to the GPBAR1 ligand increased the expression of anti-inflammatory genes IL-10 and TGF-β in the same range of concentrations (Fig. 1C). Because exposure to LPS/IFN-γ drives M1 polarization in macrophages (21), we have investigated whether BAR501 counteracts this effect and shifts macrophages toward an anti-inflammatory (M2) phenotype. To this end, the expression of specific markers for the M1 and M2 phenotypes was investigated by real-time PCR (22). We found that expression of markers of the M1 phenotype (Cd38, Fpr2, and Gpr18) was upregulated in response to treatment with LPS/IFN-γ and was reversed by cotreating the cells with BAR501 (Fig. 1D). In addition, BAR501 rescued the downregulation of markers of the M2 phenotype (Egr2 and c-myc) caused by treatment with LPS/IFN-γ (Fig. 1E). These in vitro results provide robust evidence that GPBAR1 is expressed in macrophages, and its ligation promotes their polarization toward a M2 phenotype.

Because macrophages are one of the most abundant leukocytes in the intestinal mucosa and are implicated in the pathogenesis of IBDs (23, 24), we have investigated whether treatment with BAR501 attenuates the inflammation and immune dysfunction in BALB/c mice treated with TNBS and modulates macrophage recruitment/activity. The development and severity of TNBS-induced colitis was attenuated by treatment with BAR501 in a dose-dependent manner (Fig. 2A, 2B). At a dose of 30 mg/kg, BAR501 reversed the clinical signs and symptoms of colitis, as measured by body weight loss and CDAI score, and attenuated the severity of the macroscopic and microscopic features of colonic inflammation, as assessed by colon morphology, colon length, colon weight, extent of ulcerations, and histological scores (p < 0.01 versus TNBS, Fig. 2C–G). These changes were confirmed by analysis of the expression of pro- and anti-inflammatory biomarkers in the colon. Thus, although we detected a robust increase in the expression of proinflammatory cytokines (IFN-γ, IL-6, IL-1β, and TNF-α) in response to TNBS by real-time PCR this effect was reversed by treatment with BAR501, which also increased the expression of TGF-β, IL-10, and Foxp3 (Fig. 2H, 2I).

To gain insights into the mechanisms that support the anti-inflammatory activity of BAR501, as indicated by modulation of the expression of pro- and anti-inflammatory genes (data not shown), we have checked whether exposure to the GPBAR1 ligand shifts expression of the M1 and M2 macrophage recovered from the lamina propria of colon from mice administered TNBS in vivo. To this end, we measured mRNA expression levels of the M1 markers CD38, Fpr2, and Gpr18 (Fig. 3A) and the M2 markers Egr2 and c-myc (Fig. 3B). The results of these experiments were consistent with the above-mentioned in vitro findings and demonstrate that GPBAR1 activation effectively promotes the shift toward an M2 phenotype in colitic mice. Analysis of the number of cells infiltrating the colon lamina propria and the number of circulating WBCs showed that BAR501 reversed the effect of TNBS by decreasing both populations (Fig. 3C, 3D). These findings were further confirmed by a detailed flow cytometry analysis of lamina propria–infiltrating cells (Fig. 3E–J). To summarize these studies, we have found that treatment with BAR501 reduced the percentage and number of CD4⁺ (Fig. 3G) and Mac1⁺ macrophages in the colon (Fig. 3I), increased the percentage and number of CD4⁺Foxp3⁺ cells (Fig. 3H), and had no effect on the percentage of B220⁺ cells (Fig. 3E), CD8⁺ cells (Fig. 3F), or Mac1⁺Gr1⁺ cells (Fig. 3J). Taken together, these data show that BAR501 reduces inflammation and modulates the phenotype of lamina propria inflammatory cells in the TNBS model of colitis.

Because GPBAR1 is highly expressed on macrophages, we have further investigated the effect of in vivo treatment with BAR501 on these cells. Flow cytometry analysis of lamina propria cells revealed that treating TNBS mice with BAR501 reduced the percentage of inflammatory macrophages in the colon (CD11b⁺CX3CR1⁺ cells) (Fig. 3K, 3L). The further fractioning of these cells demonstrated that, although TNBS treatment increases inflammatory macrophages (CCR7⁺F4/80⁻) (25, 26) and decreases the percentage of resident macrophages (CCR7⁻F4/80⁺) (21, 27), this pattern was reversed by GPBAR1 activation. In contrast, treatment with BAR501 increased the percentage of resident macrophages and decreased the percentage of inflammatory macrophages.

These findings were confirmed by analysis of circulating monocytes (Fig. 3K, 3L). Thus, BAR501 reversed the effect that TNBS had on the number of total circulating monocytes (Fig. 3M, left panel), whereas it had no effect on the ratio of resident/inflammatory monocytes (Fig. 3M, middle and right panels). This ratio was not changed even by treatment with TNBS (28).

Because glucocorticoids are widely used in the treatment of IBDs, we next investigated how BAR501 compares with dexamethasone, a synthetic glucocorticoid, in the treatment of TNBS colitis. We monitored the progress of the disease by recording body weight and CDAI daily. The data shown in Supplemental Fig. 1A demonstrate that both compounds effectively attenuate wasting disease (loss of body weight), although BAR501 was slightly more effective than dexamethasone. Furthermore, mice treated with the GPBAR1 ligand exhibited a significantly lower CDAI compared with mice treated with dexamethasone (Supplemental Fig. 1B), and they had fewer ulcerations and a lower colon weight/length ratio in comparison with TNBS mice treated with dexamethasone (Supplemental Fig. 1C–E).

To investigate whether BAR501’s ability to attenuate intestinal inflammation extends to other settings, we have administered the GPBAR1 ligand to mice with oxazolone-induced colitis. Oxazolone colitis is a Th2-mediated model of inflammation and exhibits histologic features and a distribution similar to those observed in ulcerative colitis (29, 30). In these experiments, BALB/c mice were administered oxazolone and were treated with vehicle or BAR501 (30 mg/kg/d). As shown in Fig. 4A and 4B, BAR501 effectively reduced the wasting disease and symptoms of colitis (body weight loss and CDAI) caused by oxazolone. Furthermore, BAR501 reduced macroscopic (length of colon, weight/length ratio of colon, and severity of ulcerations) and histologic features of inflammation (Fig. 4C–G). Finally, BAR501 counteracted the effects of oxazolone on markers of inflammation (IFN-γ, IL-6, and IL-1β) (Fig. 4H), whereas it increased the expression of TGF-β, IL-10, and Foxp3 (Fig. 4I).

To tie the effect of BAR501 to GPBAR1, we next investigated whether the anti-inflammatory and immunomodulatory activities of this agent are maintained in GPBAR1-knockout mice. For this purpose, wild-type and Gpbar1^−/− mice on a C57BL/6 background were administered TNBS, with or without BAR501 (30 mg/kg). As shown in Fig. 5A–E, the severity of wasting disease and intestinal inflammation induced by TNBS was exacerbated by GPBAR1 gene ablation. In comparison with their congenic littermates, Gpbar1^−/− mice developed more severe disease and diffuse intestinal ulcerations. Treatment with BAR501 reversed these features in Gpbar1^+/+ mice but failed to do the same in Gpbar1^−/− mice (Fig. 5A–G). Finally, BAR501 attenuated the severity of disease in Gpbar1^+/+ mice, but not in Gpbar1^−/− mice, as measured by assessing the microscopic score (Supplemental Fig. 2A).

To gain insights into the role of GPBAR1 in regulating trafficking toward the intestinal mucosa, we next carried out a detailed characterization of lamina propria–infiltrating cells in wild-type and Gpbar1^−/− mice treated with TNBS. The results in Fig. 5H–L demonstrated that GPBAR1 gene ablation worsens intestinal inflammation in this model and increases the expression of inflammatory biomarkers, such as IL-1β, in comparison with wild-type mice (Fig. 5G). Furthermore, GPBAR1 gene ablation blunted the ability of these mice to mount an anti-inflammatory response, because expression of markers for the regulatory T cell (Treg) phenotype (TGF-β, IL-10, and Foxp3) were dramatically reduced in these mice in comparison with their congenic littermates (Fig. 5H). Treatment with BAR501 reduced the levels of proinflammatory cytokines and increased the expression of anti-inflammatory genes in wild-type mice but failed to do the same in Gpbar1^−/− mice (Fig. 5G, 5H).

In addition, we found that GPBAR1 gene ablation exerts a profound influence on leukocyte trafficking toward the intestinal mucosa and M1 and M2 polarization (Fig. 5I, 5J). In C57BL/6 wild-type mice, the pattern of infiltration of the colonic lamina propria was very similar to that found in BALB/c mice; treating these mice with BAR501 generated the same pattern of response [i.e., reduced the expression of markers of the M1 phenotype in the colon (Fig. 3A) and increased the expression of markers for the M2 phenotype (Fig. 3B)]. In contrast, treating Gpbar1^−/− mice with BAR501 failed to reproduce these effects.

A similar pattern of regulation was observed for the chemokine CCL2/MCP-1 (Fig. 5K), indicating that GPBAR1 mediates the immunomodulatory activity of BAR501.

These finding were further confirmed by flow cytometry analysis of lamina propria cells. The data shown in Fig. 6 demonstrate that, although treating wild-type mice with BAR501 effectively reduced the percentage of infiltrating macrophages (CD11b⁺CX3CR1⁺) (Fig. 6A, left panel), this effect was lost in Gpbar1^−/− mice. The further characterization of macrophage subsets demonstrated that wild-type and Gpbar1^−/− mice respond to TNBS with an inflow of inflammatory macrophages (CCR7⁺F4/80⁻) (Fig. 6A, middle panel) and a decrease in resident macrophages (CCR7⁻F4/80⁺) (Fig. 6A, right panel). However, although BAR501 administration blunted the recruitment of these cells in wild-type mice, this protective effect was completely abrogated by GPBAR1 gene ablation.

Because these data highlighted a very precise mechanism of action for GPBAR1 in regulating macrophage differentiation and recruitment, we have further dissected the CD11b population by measuring the percentage of IL-6⁺ and IL-10⁺ CD11b⁺ cells by flow cytometry (Fig. 6B, 6C). The data demonstrate that the inflow of CD11b⁺/IL-6⁺ cells was enhanced in Gpbar1^−/− mice challenged with TNBS and that the modulatory effects of BAR501 on the percentage of CD11b⁺/IL-6⁺ and CD11b⁺/IL-10⁺ cells were lost in mice harboring disrupted GPBAR1 (Fig. 6B, middle and right panels). The analysis of circulating monocytes (Fig. 6D, 6E) confirmed these findings, because BAR501 failed to modulate the ratio of resident/inflammatory monocytes caused by TNBS administration in Gpbar1^−/− mice, whereas it effectively reduced the percentage of CD11b⁺/CX₃CR₁ cells in wild-type mice (Fig. 5D, middle and right panels). Taken together, these data suggest that GPBAR1 ligation in this model attenuates inflammation by promoting a shift toward IL-10–producing macrophages (M2 phenotype).

These findings were confirmed by immunohistochemical analysis of CD11b⁺ cells in the colonic lamina propria (Supplemental Fig. 2B). Thus, although TNBS administration increased the inflow of CD11b⁺ cells, this pattern was reversed by treating wild-type mice, but not Gpbar1^−/− mice, with BAR501.

A similar pattern of regulation was observed for CD4⁺ cells; although the colonic expression of CD4 mRNA and protein (by immunohistochemistry), a marker of Th cells (Supplemental Fig. 3A, 3B), increased in response to administration of TNBS in Gpbar1^+/+ and Gpbar1^−/− mice, the magnitude of its regulation was significantly higher in Gpbar1^−/− mice. This pattern was reversed by treating wild-type mice, but not Gpbar1^−/− mice, with BAR501.

In target tissues, activation of GPBAR1 increases the production of cAMP, leading to PKA-dependent phosphorylation of CREB and allowing p-CREB to translocate into the nucleus and bind to the CRE motif (31). Because in vivo data demonstrate that BAR501 increased the expression of IL-10 mRNA, we have investigated whether this effect involves direct regulation of IL-10 gene expression by GPBAR1 (32). To reduce the number of mice used in this study, the following experiments were carried out using RAW 264.7 cells. Because analysis of the IL-10 promoter revealed the presence of CRE sequences (see 2Materials and Methods), we performed a ChIP assay on wild-type RAW 264.7 cells, a macrophage cell line that expresses GPBAR1, and the same cells in which GPBAR1 expression was silenced by treatment with an anti-GPBAR1 small interfering RNA (siRNA). Control experiments (Supplemental Fig. 4A, 4C) demonstrated that GPBAR1 gene expression was completely abrogated by this approach. In wild-type cells cultured or not with 50 μM BAR501, the GPBAR1 ligand increased the binding of CREB to CRE in the IL-10 promoter (Fig. 7A). The increase in the production of IL-10 in macrophages was also closely related to the activation of GPBAR1, whereas no effect was observed in cells silenced for GPBAR1. To gain further insights into the role of IL-10 in GPBAR1 signaling in macrophages, we have the silenced the IL-10 gene in RAW 264.7 cells. Control experiments (Supplemental Fig. 4B–D) demonstrated that this approach resulted in an ∼70% reduction in IL-10 mRNA. After priming with LPS and IFN-γ, with or without BAR501 (50 μM), wild-type RAW 264.7 cells showed a robust upregulation of Fpr2 (M1 phenotype) and TNF-α expression. This pattern was reversed by cotreating the cells with the GPBAR1 ligand, which also increased the expression of Egr2 and TGF-β (Fig. 7B). In contrast, the counterregulatory effects of BAR501 were lost in RAW 264.7 cells silenced for IL-10 or GPBAR1 (Fig. 7C, 7D), indicating that the upregulation of IL-10 is essential for the anti-inflammatory action of GPBAR1.

Because the above-mentioned data highlight a role for IL-10 in the beneficial effects exerted by GPBAR1 ligation, we have treated IL-10^−/− mice rendered colitic by administration of TNBS with BAR501. For this purpose, wild-type and IL-10^−/− mice on the C57BL/6 background were administered TNBS, with or without BAR501 (30 mg/kg). As shown in Fig. 8A–E, the severity of wasting disease and the severity of intestinal inflammation induced by TNBS were exacerbated by IL-10 gene ablation. Furthermore in IL-10^−/− mice, TNBS induced an increase in the number of lamina propria–infiltrating cells that was greater than that observed in wild-type animals (Fig. 8F). Again, BAR501 reversed the effects of TNBS in IL-10^+/+ mice but failed to do the same in IL-10^−/− mice.

Analysis of the expression of mRNA for pro- and anti-inflammatory biomarkers by real-time PCR (Fig. 8G, 8H) showed that, in the absence of the IL-10 gene, after administration of TNBS the expression levels of proinflammatory cytokines were higher than in IL-10^+/+ mice. In addition, BAR501 failed to modulate markers of inflammation in IL-10^−/− mice, whereas it maintained its efficacy in wild-type mice. In these mice, the GPBAR1 ligand reduced the expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) and increased the expression of anti-inflammatory cytokines (TGF-β and IL-10).

The importance of bile acids and their receptors in regulating intestinal immunity has been confirmed in recent years by the demonstration that FXR (8–11) and GPBAR1 (7) gene ablation worsens the severity of intestinal inflammation. Furthermore, bile duct ligation, which blocks the inflow of bile acids into the intestine, worsens the severity of inflammation in mice with TNBS-induced colitis, highlighting a role for bile acids in maintaining intestinal homeostasis.

Bile acid metabolism in mammals leads to the generation of several molecular species. In the liver, this evolutionarily conserved pathway generates primary bile acids from cholesterol. These bile acids are preferential ligands for FXR, and CDCA regulates intestinal and liver immunity in an FXR-dependent manner (10, 11). Secondary bile acids, the natural ligands of GPBAR1, are generated in the intestine by the activity of intestinal microbiota (4, 6). During feeding, CDCA and cholic acid enter the small intestine and undergo a process of 7α/β dihydroxylation, leading to the generation of DCA and LCA, whose concentrations fluctuate widely in the intestine and blood according to the fast–fed cycle (6). In mammals, GPBAR1 expression is restricted to a limited number of tissues, including small intestine, colon, adipose tissues, muscles, and lymphoid organs (12, 13). In immune cells, expression of GPBAR1 mRNA is essentially restricted to cells of myeloid origin (13). Because the intestines harbor the largest pool of macrophages and contain the largest amount of secondary bile acids, there was substantial background to support the notion that secondary bile acids might regulate intestinal innate immunity via a GPBAR1-dependent mechanism (5, 6, 8).

In this study, we have elaborated on this concept and demonstrated that GPBAR1 is essential to counter-regulate immune responses in Th1 and Th2 models of colitis. The protective effect exerted by GPBAR1 ligation is largely mediated by the reshaping of leukocyte trafficking, which is associated with a robust shift of intestinal macrophages from an M1 to an M2 phenotype.

Thus, although Gpbar1^−/− mice do not develop overt colitis spontaneously, they were more likely to develop severe inflammation when challenged with TNBS or dextran sulfate (7). Local (diarrhea) and systemic (wasting disease) inflammation were worsened by GPBAR1 gene ablation. However, these different clinical outcomes were not supported by detectable changes in the systemic immune response to inflammation; rather, they appear to be caused by a defective regulation of the immune response that takes place in the intestine. This view is supported by the fact that the systemic response to TNBS was similar in Gpbar1^−/− and wild-type mice. Both mice strains react to TNBS administration with a robust increase in the number of total circulating white cells and CD11b⁺/CX3CR1⁺ cells (monocytes); among these cells, the relative percentage of “inflammatory” monocytes (i.e., CD11c⁺Ly6C⁺) was similar. In contrast, the local immune response to TNBS (and oxazolone) was profoundly disturbed by Gpbar1 gene ablation. Indeed, Gpbar1^−/− mice displayed higher levels of proinflammatory cytokines and chemokines (TNF-α, IFN-γ, IL-1β, IL-6, and CCL2), along with a strong reduction in the expression of anti-inflammatory mediators, IL-10 and TGF-β mRNAs, and the phenotype of leukocytes in the colonic lamina propria was severely altered by the gene knockout. Specifically, in addition to slight changes in the expression of CD4 (mRNA and histochemistry), the most relevant change that we detected was a marked upregulation of a subset of genes that was suggestive of an M1 phenotype (Cd38, Fpr2, and Gpr18), along with a profound downregulation of Egr2 and c-myc (two markers of the M2 phenotype). Furthermore, fractioning of lamina propria–derived CD11b⁺ cells by flow cytometry revealed that Gpbar1 gene ablation was associated with a higher percentage of CD11b⁺/IL-6⁺ cells (i.e., activated macrophages). Taken together, these data suggest that, although GPBAR1 does not regulate the trafficking of monocytes from lymphoid organs or bone marrow to the blood (likely reflecting the lack of ligands in lymphoid organs), this receptor is required for maturation of emigrated monocytes in the intestine. In the presence of abundant concentrations of DCA and LCA in the intestine, emigrated macrophages are shifted toward the acquisition of an anergic phenotype (M2). In contrast, GPBAR1 ablation contributes to shift macrophages toward an M1 phenotype.

Because these data suggest that GPBAR1 might ground the development of gut-specific anti-inflammatory therapies, we have investigated whether treating mice with a small molecule agonist for GPBAR1 (i.e., BAR501) attenuates colitis development in two rodent models of inflammation: BALB/c mice administered TNBS or oxazolone (33). Results from these experiments have shown that GPBAR1 activation protects, in a dose-dependent manner, from the development of signs and symptoms of colitis in both models. In both models, the beneficial effects of BAR501 were associated with a robust shift in the macrophage phenotype from M1 to M2. Importantly, although BAR501 effectively reduced the number of circulating monocytes, it failed to alter the ratio of Ly6C⁺/Ly6C⁻ cells, confirming that Ly6C expression per se does not affect the differentiation of monocytes toward a pro- or anti-inflammatory phenotype and that the differentiation of Ly6C⁺ monocytes, after they enter the tissues, depends on the organ microenvironment (34, 35). These findings provide further support that GPBAR1 could be exploited for the development of gut-specific therapies for IBDs without dampening the systemic immune system.

Treating mice with BAR501 reshaped the entire trafficking of intestinal leukocytes. The results shown in Fig. 3 demonstrate that GPBAR1 agonism reduced the percentages of CD11b⁺/CX₃CR₁⁺ and CD4⁺ cells, but not CD8⁺ and B220⁺ cells, whereas it increased the percentage of CD4/Foxp3 Tregs. Further fractioning of CD11b⁺ cells also revealed a robust reduction in the percentage of these cells that emigrated in the lamina propria, as well as that macrophages were shifted toward an M2 phenotype, as shown by the robust increase (Fig. 3I) in the percentage of CD11b⁺CX₃CR₁⁺CCR7⁻F4780⁺ cells (M2 macrophages).

Consistent with these findings, we have also demonstrated that treating mice with BAR501 resets the level of intestinal inflammation, as demonstrated by the reversion of the induction of proinflammatory cytokines and chemokines (IL-1β, IL-6, IFN-γ, TNF-α and CCL2) caused by TNBS and oxazolone, as well as the induction of anti-inflammatory genes (IL-10 and TGF-β). Consistent with this view, exposure of colitic mice to BAR501 reduced the intestinal expression of Cd38, Fpr2, and Gpr18, markers of the M1 phenotype, whereas it increased the expression of markers of the M2 phenotype (Egr2 and c-myc).

The essential role of GPBAR1 in shaping the intestinal microenvironment is well highlighted by the effect that BAR501 exerts on CCL2/MCP1 gene expression in the colon (Fig. 5). CCL2 is a member of the CC chemokine family and is a potent chemotactic factor for monocytes (MCP-1) (36). This chemokine and its receptor, CCR5 (33, 34), are essential for monocyte recruitment in the inflamed colon (37–41): thus, although treating mice with TNBS dramatically increased the levels of CCL2 mRNA in the colon, this pattern was reversed by BAR501, strongly supporting a counter-regulatory role for GPBAR1 in monocyte trafficking. The fact that these regulatory effects were lost in Gpbar1^−/− mice further ties the effects of BAR501 to this receptor.

Because the above-mentioned data suggest that GBAR1 drives the development of a tolerogenic phenotype of intestinal macrophages, and levels of TGF-β and IL-10 were markedly reduced in Gpbar1^−/− mice compared with their congenic littermates, we have further dissected signals that link GPBAR1 activation to IL-10. By promoter analysis we detected a CRE motif in the proximal promoter of the IL-10 gene. The functionality of these CREs in terms of regulation of IL-10 transcription was examined in detail in RAW 264.7 cells, a macrophage cell line. The results of these experiments demonstrate that the interaction between BAR501 and GPBAR1 increases the binding of CREB to the IL-10 promoter, driving the production of this cytokine. This regulatory effect was abrogated by the silencing of GPBAR1 by an anti-GPBAR1 siRNA.

Because of the high levels of IL-10 in the colon, after treatment with BAR501, monocytes and lymphocytes that reach the colon are exposed to this anti-inflammatory cytokine, which is likely responsible for shifting macrophages from an M1 to an M2 phenotype. Because it is conceivable that, following treatment with the GPBAR1 ligand, M2 macrophages produce IL-10 that directs T cell differentiation toward the anti-inflammatory Treg phenotype, as demonstrated by the marked increase in Foxp3 gene expression in the colon, we used IL-10^−/− mice to examine whether the anti-inflammatory effects of BAR501 were IL-10 dependent. Effectively, the results of these studies demonstrated that IL-10 gene ablation abrogates the protection exerted by the GPBAR1 ligand in the TNBS model. IL-10 gene ablation completely abrogated the effect of BAR501 on macrophage polarization, as measured by assessing the colonic expression of markers for the M1 and M2 phenotypes. The mechanistic role of IL-10 in supporting the activity of BAR501 was confirmed by the results of experiments carried out in RAW 264.7 cells, in which the IL-10 gene was silenced by anti–IL-10 siRNAs. Again, IL-10 gene silencing completely reversed the anti-inflammatory activity exerted by GPBAR1. These data indicate that GPBAR1 activation in physiological conditions counteracts the development of inflammatory immune responses, driving macrophages to an anti-inflammatory phenotype, and that this effect is likely mediated by CREB-dependent binding to the IL-10 gene promoter.

In addition to acting on GPBAR1, BAR501 regulates the composition of the bile acid pool. Although we have not detailed this effect in the current study, there is evidence that changes in bile acid composition deeply affect intestinal immunity (4). It is likely that modification of intestinal bile acids explains some of the receptor-independent effects (see Fig. 5) observed in Gpbar1^−/− mice treated with BAR501.

In summary, we have shown that GPBAR1, a cell membrane receptor for secondary bile acids, is essential for maintaining intestinal immune homeostasis and that its activation in the setting of inflammation reverses the immune dysfunction that occurs in rodent models of colitis. The present observations pave the way for the development of GPBAR1-based anti-inflammatory agents for the treatment of IBDs.

The authors have no financial conflicts of interest.