The Bile Acid Receptor GPBAR1 Regulates the M1/M2 Phenotype of Intestinal Macrophages and Activation of GPBAR1 Rescues Mice from Murine Colitis This information is current as of September 28, 2021. Michele Biagioli, Adriana Carino, Sabrina Cipriani, Daniela Francisci, Silvia Marchianò, Paolo Scarpelli, Daniele Sorcini, Angela Zampella and Stefano Fiorucci J Immunol 2017; 199:718-733; Prepublished online 12 June 2017; Downloaded from doi: 10.4049/jimmunol.1700183 http://www.jimmunol.org/content/199/2/718 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/06/10/jimmunol.170018 Material 3.DCSupplemental References This article cites 40 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/199/2/718.full#ref-list-1

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

Michele Biagioli,* Adriana Carino,* Sabrina Cipriani,† Daniela Francisci,† Silvia Marchiano`,* Paolo Scarpelli,‡ Daniele Sorcini,x Angela Zampella,{ and Stefano Fiorucci‖

GPBAR1 (TGR5 or M-BAR) is a G protein–coupled receptor for secondary bile acids that is highly expressed in monocytes/ macrophages. In this study, we aimed to determine the role of GPBAR1 in mediating leukocyte trafficking in chemically induced models of colitis and investigate the therapeutic potential of BAR501, a small molecule agonist for GPBAR1. These studies demonstrated that GPBAR1 gene ablation enhanced the recruitment of classically activated macrophages in the colonic lamina Downloaded from propria and worsened the severity of inflammation. In contrast, GPBAR1 activation by BAR501 reversed intestinal inflammation in the trinitrobenzenesulfonic acid and oxazolone models by reducing the trafficking of Ly6C+ monocytes from blood to intestinal mucosa. Exposure to BAR501 shifted intestinal macrophages from a classically activated (CD11b+, CCR7+, F4/802)toan alternatively activated (CD11b+, CCR72, F4/80+) phenotype, reduced the expression of inflammatory genes (TNF-a, IFN-g, IL-1b, IL-6, and CCL2 mRNAs), and attenuated the wasting syndrome and severity of colitis (70% reduction in the Colitis Disease Activity Index). The protective effect was lost in Gpbar12/2 mice. Exposure to BAR501 increased the colonic expression of http://www.jimmunol.org/ IL-10 and TGF-b mRNAs and the percentage of CD4+/Foxp3+ cells. The beneficial effects of BAR501 were lost in Il-102/2 mice. In a macrophage cell line, regulation of IL-10 by BAR501 was GPBAR1 dependent and was mediated by the recruitment of CREB to its responsive element in the IL-10 promoter. In conclusion, GPBAR1 is expressed in circulating monocytes and colonic macro- phages, and its activation promotes a IL-10–dependent shift toward an alternatively activated phenotype. The targeting of GPBAR1 may offer therapeutic options in inflammatory bowel diseases. The Journal of Immunology, 2017, 199: 718–733.

nflammatory bowel diseases (IBDs) encompass two highly the lack of response or loss of response to drugs, because of in-

prevalent chronic disorders, Crohn’s disease and ulcerative tolerance or severe side effects that require cessation of therapy. by guest on September 28, 2021 I colitis, which result from a dysregulated intestinal immune Recently, novel approaches that target leukocyte trafficking to the response to Ags derived from the intestinal microbiota in geneti- gut by inhibiting integrins, chemokines, and chemokine receptors cally predisposed individuals (1, 2). IBDs have traditionally been (4) have been developed, but 30–40% of patients fail to respond to treated with nonspecific immunosuppressive drugs (e.g., cortico- these approaches in clinical trials. Therefore, there is a clinical steroids or azathioprine), anti-inflammatory agents (e.g., 5-amino- need for new treatments based on therapies that are able to se- salicylic acid), and, more recently, with anti–TNF-a agents (3). lectively regulate the intestinal immune response, overcoming the These treatments have shown efficacy in mild to severe disease; limitations of current therapies. however, a large percentage of patients with severe illness do not Bile acids, the end product of cholesterol metabolism, play an achieve complete remission or cannot maintain remission due to 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 *Department of Surgical and Biomedical Sciences, University of Perugia, Perugia lithocholic acid [LCA]), as well as their glycine and taurine 06132, Italy; †Department of Medicine, University of Perugia, Perugia 06132, Italy; ‡Department of Experimental Medicine, Laboratory of Biotechnology, University of conjugates, are signaling molecules that exert a variety of regu- Perugia, Perugia 06132, Italy; xSection of Pharmacology, Department of Medicine, latory functions by activating a family of cell surface and nuclear University of Perugia, Perugia 06132, Italy; {Department of Pharmacy, University of ‖ receptors collectively known as the bile acid–activated receptors Naples “Federico II,” Naples 80181, Italy; and Department of Surgical and Biomed- ical Sciences, University of Perugia, Perugia 06132, Italy (BARs) (7). The two best characterized members of the BAR ORCIDs: 0000-0003-2341-9528 (A.C.); 0000-0001-6583-3693 (P.S.). family are the G protein–coupled receptor GPBAR1 (also known Received for publication February 7, 2017. Accepted for publication May 16, 2017. as TGR5 or M-BAR) and the nuclear receptor FXR (8–11). Address correspondence and reprint requests to Prof. Stefano Fiorucci, Department GPBAR1 was the first transmembrane G protein–coupled receptor of Surgical and Biomedical Sciences, University of Perugia, Square L. Severi 1, shown to be activated by bile acids, with DCA and LCA, as well Palace B 3rd Floor, Perugia 06132, Italy. E-mail address: stefano.fi[email protected] as their taurine and glycine conjugates, functioning as bona fide The online version of this article contains supplemental material. physiological ligands (12, 13). In the human body, the expression Abbreviations used in this article: BAR, bile acid–activated receptor; CDAI, Colitis of GPBAR1 is essentially restricted to the small intestine, gall- Disease Activity Index; CDCA, chenodeoxycholic acid; ChIP, chromatin immuno- precipitation; CRE, cAMP-responsive element; DCA, deoxycholic acid; FMO, fluo- bladder, adipose tissues, and immune system. In response to li- rescence minus one; IBD, inflammatory bowel disease; LCA, lithocholic acid; M1, gands, GPBAR1 signals by increasing intracellular concentrations classically activated; M2, alternatively activated; siRNA, small interfering RNA; of cAMP, leading to rapid phosphorylation of downstream kinases TNBS, trinitrobenzenesulfonic acid; Treg, regulatory T cell. that are responsible for the nongenomic effects exerted by this Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 receptor (14). In addition, GPBAR1 exerts genomic effects that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700183 The Journal of Immunology 719 are mediated by binding of a CREB to a cAMP-responsive ele- brief, mice were fasted for 12 h overnight (day 21). The following day ment (CRE) in the promoter of target genes (15, 16). (day 0), under profound sedation induced by the administration of a In the immune system, GPBAR1 is primarily expressed by cells 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 of myeloid origin, whereas T and B cells express the receptor at from the anus, and 1 mg of trinitrobenzenesulfonic acid (TNBS) (Sigma- very low levels. In macrophages derived from peripheral blood and Aldrich, St. Louis, MO) in 50% ethanol was administered via the catheter liver macrophages, activation of GPBAR1 counteracts the activity into the colon lumen using a 1-ml syringe (injection volume of 100 ml). of CD14/TLR4, decreasing the phagocytic capacity and production Control mice received 50% ethanol alone. Animals were monitored daily for diarrhea, loss of body weight, presence of blood in the stool, and of proinflammatory cytokines TNF-a, IL-1a, IL-1b, and IL-6 (15, survival. At the end of the experiment, 4 d after the administration of 16). In the intestine, GPBAR1 oversees a variety of homeostatic TNBS, surviving mice were sacrificed, blood samples were collected by functions, and we have previously shown that mice harboring cardiac puncture, and the colon was excised, weighed, and evaluated for disrupted GPBAR1 have an altered intestinal morphology and macroscopic damage. When prompted by the experimental design, BAR501 (5 to 30 mg/kg/d) or dexamethasone (5 mg/kg/d) was administrated increased permeability and are more prone to develop intestinal orally or i.p. inflammation. Because the intestine hosts the highest concentra- For the oxazolone model, BALB/c mice were presensitized by appli- tion of bile acids in mammalian tissues, which fluctuates widely cation of 150 ml of 3% oxazolone (Sigma-Aldrich) in a 4:1 acetone/olive during the fast-to-feeding transition, GPBAR1 ligands might have oil solution to a 1.5 3 1.5-cm area of shaved skin on the back of the a role in regulating the recruitment and function of intestinal mouse. Seven days later, mice were anesthetized, and 150 mlof1% oxazolone solution in 50% ethanol was administered via the rectum. macrophages. Intestinal macrophages, the largest pool of tissue Mice were sacrificed 4 d after treatment, and clinical signs of colitis were macrophages, are made up of heterogeneous cells classified as scored. classically activated (M1) macrophages (high IL-12 and low IL-10 In both models, the severity of colitis was assessed each day for each Downloaded from producing), which are induced by IFN-g and microbial products, 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 and alternatively activated (M2) macrophages (low IL-12 and high Disease Activity Index (CDAI). The scoring system was as follows: per- IL-10 producing). The latter are induced by IL-4 or IL-13 and centage of body weight loss: none = 0, 1–5% = 1, 5–10% = 2, 10–20% = 3, display an anti-inflammatory phenotype, and it is thought that a and .20% = 4; stool consistency: normal = 0, soft but still formed = 1, wide transition between the two phenotypes exists (17, 18). The 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 composition of the intestinal macrophage pools in mice and hu- http://www.jimmunol.org/ bleeding with fresh blood around the anus, and very present in the mans changes considerably in conditions of perturbed homeosta- stool = 4. sis, leading to accumulation of the M1-bearing phenotype in the setting of inflammation. In this setting, M1 macrophages, in concert Isolation of lamina propria cells with other professional APCs, orchestrate the recruitment of cells The cells were isolated from the colon lamina propria using the Lamina of adaptive immunity and have a role in the initiation and main- Propria Dissociation Kit (Miltenyi Biotec; 130-097-401), according to the tenance of inflammation, making these cells a putative target to instructions. prevent immune activation in intestinal inflammatory states (19). Abs, flow cytometry, and cell sorting 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501) is a bile acid derivative that selectively activates GPBAR1 (20). In the Flow cytometry analyses were carried out using a two-laser standard by guest on September 28, 2021 current study, by using genetic and pharmacological approaches, configuration ATTUNE NxT (Life Technologies), and cell sorting was done using a two-laser standard configuration FACSAria. Data were analyzed we provide evidence that GPBAR1 ligation attenuates the using FlowJo software (TreeStar). The gates were set using a fluorescence inflammation-driven immune dysfunction that develops in two minus one (FMO) control strategy. FMO controls are samples that include rodent models of colitis by shifting macrophage polarization from all conjugated Abs present in the test samples except one. The channel in the M1 to the M2 phenotype. This effect is mediated by a genomic 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 circuit and involves GPBAR1-dependent recruitment of CREB to (RA3-6B2; eBioscience, San Diego, CA), CD4 allophycocyanin–eFluor the IL-10 promoter. To our knowledge, the present results are the 780 (GK1.5; eBioscience), CX3CR1 allophycocyanin (SA011F11; Bio- first evidence of fine-tuning of IL-10 expression/activity in intes- Legend), CCR7 Alexa Fluor 700 (4B12; eBioscience), CD11c Alexa Fluor tinal macrophages by bile acids and pave the way for the ex- 700 (N418; eBioscience), IL-6 PE (MP5-20F3; eBioscience), Foxp3 PE ploitation of GPBAR1 as a therapeutic target in the development (3G3; Tonbo Biosciences), IL-10 FITC (JES5-16E3; eBioscience), Ly-6C Alexa Fluor 488 (HK1.4; eBioscience), F4/80 PE-Cyanine7 (BM8; eBio- of gut-specific therapies for the treatment of IBDs. science), and CD8 PE-Cyanine7 (53-6.7; eBioscience).

Reverse transcription of mRNA and real-time PCR Materials and Methods Animals and colitis protocols Samples of the colon, 0.5 cm taken 1 cm from the anus, were immedi- ately frozen in liquid nitrogen and stored at 280˚C until used. The colon BALB/c mice were purchased from Charles River. GPBAR1-null mice on a samples were mechanically homogenized using a pestle, and the obtained C57BL/6NCrl background and C57BL/6NCrl congenic littermates were materials were resuspended in 1 ml of TRIzol Reagent (Thermo Fisher originally donated by Dr. Galya Vassileva (Schering-Plough Research In- Scientific). The RNA was extracted according to the manufacturer’s pro- stitute, Kenilworth, NJ). IL-10–null mice (B6.129P2-Il10tm1Cgn/J) were tocol. After purification from genomic DNA using DNase I (Thermo from The Jackson Laboratory. The colonies were maintained in the animal Fisher Scientific), 1 mg of RNA from each sample was reverse transcribed facility at the University of Perugia. Mice were housed under controlled using random hexamer primers with SuperScript II (Thermo Fisher Sci- temperatures (22˚C) and photoperiods (12:12-h light/dark cycle), were entific) in a 20-ml reaction volume; 10 ng of cDNA was amplified in a allowed unrestricted access to standard mouse chow and tap water, and 20-ml solution containing 200 nM each primer and 10 ml of SYBR Select were allowed to acclimate to these conditions for $5 d before inclusion in Master Mix (Thermo Fisher Scientific). All reactions were performed in an experiment. The study was conducted in agreement with Italian law, and triplicate using the following thermal cycling conditions: 3 min at 95˚C, the protocol was approved by a ethical committee at the University of followed by 40 cycles of 95˚C for 15 s, 56˚C for 20 s, and 72˚C for 30 s, Perugia and by a National Committee of Italian Ministry of Health (permit using a StepOnePlus system (Applied Biosystems). The relative mRNA number 42/2014/B). The health of the animals was monitored daily by the expression was calculated according to the D cycle threshold method. veterinarian in the animal facility. The study protocol caused minor suf- Primers were designed using Primer3 software (http://frodo.wi.mit.edu/ fering; however, animals that lost .25% of their initial body weight were primer3/) using published data obtained from the National Center for euthanized. Biotechnology Information database. Alternatively, for some genes, Taq- Only male mice were used in each experiment. Colitis was induced Man probes were used, with TaqMan GEX Master Mix (all from Thermo in BALB/c mice, Gpbar1+/+ and Gpbar12/2 mice, and IL-102/2 mice. In Fisher Scientific). The following primers were used (forward and reverse): 720 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. BAR501 counter-regulates macrophage immune activation in vitro. Spleen-derived macrophages were cell sorted from BALB/c mice and incubated with the appropriate agents, as described in Materials and Methods.(A) Quantitative real-time PCR analysis of GPBAR1 expression in B cells, CD4 T cells, CD8 T cells, and macrophages (MF). The data are normalized to Actb mRNA. Data are the mean 6 SEM of three (Figure legend continues) The Journal of Immunology 721

IFN-g (59-GCTTTGCAGCTCTTCCTCAT-39,59-ATCCTTTTGCCAGT- P-8833; Sigma-Aldrich) for 72 h. Silencing was then verified through real- 39), TNF-a (59-CCACCACGCTCTTCTGTCTA-39,59-AGGGTCTGG- time PCR using specific primers for the Il10 and Gpbar1 genes. GCCATAGAACT-39), IL-6 (59-CTTCACAAGTCGGAGGCTTA-39,59- Statistical analysis TTCTGCAAGTGCATCATCGT-39), IL-1b (59-GCTGAAAGCTCTC- CACCTCA-39,59-AGGCCACAGGTATTTTGTCG-39), TGF-b (59- ANOVA followed by the nonparametric Mann–Whitney U test or a two- TTGCTTCAGCTCCACAGAGA-39,59-TGGTTGTAGAGGGCAAGGA- tailed unpaired Student t test was used for statistical comparisons using C-39), IL-10 (59-CCCAGAAATCAAGGAGCATT-39,59-CTCTTCACCT- Prism 6.0 software (GraphPad). GCTCCACTGC-39), and Foxp3 (59-TCTTCGAGGAGCCAGAAGAG-39, 59-AGCTCCCAGCTTCTCCTTTT-39). Marker genes for M1 and M2 Results macrophage populations were Cd38 (Mm01220906_m1), Fpr2 (Mm00484464_ s1), Gpr18 (Mm02620895_s1), Egr2 (Mm00456650_m1), and c-myc GPBAR1 is expressed in myeloid cells, and its activation (Mm00487804_m1; all from Thermo Fisher Scientific). promotes the shift from M1 to M2 phenotype in vitro Histology We first investigated whether immune cells express GPBAR1. For this purpose, T and B cells and macrophages were sorted from the Samples of distal colon (2–3 cm from the anus) were fixed in buffered spleen of wild-type mice, and expression of GPBAR1 was assessed formalin, cut into 5-mm-thick sections (150 mm between each section, four to eight per fragment per colon), and stained with H&E. by real-time PCR As shown in Fig. 1A, GPBAR1 mRNA was detected at high levels only in macrophages, whereas expression Immunohistochemistry in other cells was minimal. Colon fragments were cryoprotected in 30% sucrose in PBS overnight at BAR501 is a recently discovered selective GPBAR1 agonist that 4˚C, embedded in OCT, and frozen. Colons were sectioned at 7 mm, activates the receptor with an EC50 of 1 mM (20). To investigate Downloaded from washed in PBS, and fixed in 4% paraformaldehyde in PBS for 15 min. the effects of GPBAR1 ligation on macrophage phenotype, After washing, sections were incubated with 10% goat serum, 2% BSA, spleen-derived macrophages were primed by exposure to LPS and 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 IFN-g, with or without increasing concentrations of BAR501. As 570 mAb (clone RM4-5; both from eBioscience) in PBS for 1 h, washed shown in Fig. 1B, although exposure to LPS/IFN-g increased the three times, and mounted with SlowFade Gold antifade reagent with DAPI expression of prototypical proinflammatory cytokines (IFN-g,IL-1b, (Invitrogen). Fluorescent images of tissue were acquired using an Axio- IL-6, TNF-a), this pattern was reversed by cotreating the cells http://www.jimmunol.org/ Vision.Z1 microscope equipped with an ApoTome filtering device and an AxioCam MRm digital camera (Carl Zeiss Microscopy) through a 203 with the GPBAR1 ligand, in a concentration-dependent manner Plan Apo objective. The ApoTome device, enabled for optical sectioning at (Fig. 1B). Additionally, exposure to the GPBAR1 ligand increased medium setting (digital noise reduction set to off), was used to remove the expression of anti-inflammatory genes IL-10 and TGF-b in the scattered light from underlying and overlying out-of-focus focal planes, same range of concentrations (Fig. 1C). Because exposure to LPS/ resulting in a high axial resolution and increased signal-to-noise ratio. IFN-g drives M1 polarization in macrophages (21), we have in- Images were visually optimized with a factor 140, 5-pixel sharpening mask and histogram equalization (Adobe Photoshop CC). vestigated whether BAR501 counteracts this effect and shifts macrophages toward an anti-inflammatory (M2) phenotype. To Cell culture, chromatin immunoprecipitation, and small this end, the expression of specific markers for the M1 and M2 interfering RNA phenotypes was investigated by real-time PCR (22). We found that by guest on September 28, 2021 RAW 264.7 cells were grown at 37˚C in DMEM containing 10% FBS, 1% expression of markers of the M1 phenotype (Cd38, Fpr2, and L-glutamine, and 1% penicillin/streptomycin. Cells were regularly pas- Gpr18) was upregulated in response to treatment with LPS/IFN-g saged to maintain exponential growth. Macrophages derived from the and was reversed by cotreating the cells with BAR501 (Fig. 1D). spleen of mice and RAW 264.7 cells were classically activated (M1 con- dition) with LPS (1 ng/ml, L2880; Sigma-Aldrich) + IFN-g (20 ng/ml; In addition, BAR501 rescued the downregulation of markers of eBioscience) for 16 h. the M2 phenotype (Egr2 and c-myc) caused by treatment with For the chromatin immunoprecipitation (ChIP) assay, by a promoter LPS/IFN-g (Fig. 1E). These in vitro results provide robust evi- analysis, we detected a CRE sequence 358 bp upstream of the exon 1 of the dence that GPBAR1 is expressed in macrophages, and its ligation gene encoding for murine IL-10. RAW 264.7 cells and RAW 264.7 cells promotes their polarization toward a M2 phenotype. silenced for GPBAR1 were exposed or not to BAR501 (50 mM) for 6 h and then cross-linked with formaldehyde. ChIP assays were performed BAR501 protects against colitis induced by TNBS according to the manufacturer’s protocols (EZ-ChIP; Upstate; cat. no. 17-371). Briefly, cell extracts were sonicated and divided. Abs (5 mg) Because macrophages are one of the most abundant leukocytes in against p-CREB and normal goat IgG were added for immunoprecipita- the intestinal mucosa and are implicated in the pathogenesis of tion. The immunoprecipitated chromatin was recovered and purified. The IL-10 promoter DNA was quantified by real-time PCR analysis using IBDs (23, 24), we have investigated whether treatment with primers around the CREB binding site in the proximal promoter of IL-10 BAR501 attenuates the inflammation and immune dysfunction in (forward 59-TGACTTCCGAGTCAGCAAGA-39, reverse 59-TATTTCCT- BALB/c mice treated with TNBS and modulates macrophage GAGGCAGACAGC-39). Primers spanning a region located ∼6800 kb recruitment/activity. The development and severity of TNBS- downstream of the IL-10 transcriptional start site (59-TCCCTACCTGT- induced colitis was attenuated by treatment with BAR501 in a TAAGGTTTATGG-39;rev59-ACACATGTCCACATGCAAGC-39) were used as control. At least three replicates of each group were performed. dose-dependent manner (Fig. 2A, 2B). At a dose of 30 mg/kg, For IL-10 RNA silencing, an IL-10 mouse short hairpin RNA plasmid BAR501 reversed the clinical signs and symptoms of colitis, as (pRFP-C-RS) and a GPBAR1 mouse short hairpin RNA plasmid (pGFP-V- measured by body weight loss and CDAI score, and attenuated the RS) were purchased from OriGene Technologies and were used to suppress severity of the macroscopic and microscopic features of colonic IL-10 and GPBAR1 expression. RAW 264.7 cells were seeded in a six-well plate and transfected with 1 mg of each plasmid using HD Transfection inflammation, as assessed by colon morphology, colon length, Reagent (Promega). The transfection was repeated for two consecutive colon weight, extent of ulcerations, and histological scores (p , days, and transfected cells were selected by exposure to puromycin (4 mg/ml, 0.01 versus TNBS, Fig. 2C–G). These changes were confirmed by

experiments. (B–E) Spleen-derived CD11b+ primary cells were incubated for 16 h with LPS and BAR501. BAR501 shifts the macrophage phenotype in vitro. Quantitative real-time PCR analysis of proinflammatory genes IFN-g, IL-1b, IL-6, and TNF-a (B), anti-inflammatory genes TGF-b and IL-10 (C), M1 macrophage markers Cd38, Fpr2, and Gpr18 (D), and M2 macrophage markers Egr2 and c-myc (E). The data are normalized to Actb mRNA. Results are the mean 6 SEM of three to five mice per group. *p , 0.05. 722 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. BAR501 reduces the severity of TNBS colitis in a dose-dependent manner. The experiment was carried out on BALB/c mice. Mice treated with TNBS were administered vehicle or BAR501 (5 to 30 mg/kg/d) by gavage from days 0 to 4. BAR501 attenuated the development of wasting disease (i.e., change in body weight) (*p , 0.05, TNBS versus TNBS + BAR501 (30 mg/kg), $p , 0.05, TNBS versus TNBS + BAR501 15 (mg/kg) (A) and CDAI score (B). (C) Photographs of colon from control mice and from mice treated with TNBS or TNBS plus various concentrations of BAR501. BAR501 reduced the intestinal inflammatory score: colon length (D), ratio of colon weight/ colonlength(E), and ulcer area (F). (G) H&E staining of colon sections from control mice, mice treated with TNBS, and mice treated with TNBS plus various concentrations of BAR501 (original magnification 310). Relative mRNA expression of IFN-g, IL-6,andIL-1b (H)andTGF-b, IL-10,andFoxp3 (I) genes in colon was assayed by real-time PCR. Data are normalized to Actb mRNA. Results are the mean 6 SEM of 8–14 mice per group from two independent experiments. *p , 0.05 (B and D–F). The Journal of Immunology 723 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. GPBAR1 ligand modulates the phenotype of lamina propria inflammatory cells. BALB/c mice were treated with TNBS and then were administered vehicle or BAR501 (30 mg/kg/d) by gavage from days 0 to 4. Quantitative real-time PCR analysis of the expression of M1 macrophage markers Cd38, Fpr2,and Gpr18 (A) and M2 macrophage markers Egr2 and c-myc (B) in total colon. The data were normalized to Actb mRNA. Data are mean 6 SEM of 6–10 mice per group. Number of cells in the lamina propria of colon (C) and WBCs (D) of mice at 4 d after TNBS administration. Frequency and number of immune cells in the colonic lamina propria: B220+ cells (E), CD8+ cells (F), CD4+ cells (G), CD4+Foxp3+ cells (H), CD11b+ cells (I), and CD11b+Gr1+ cells (J). (K) (Figure legend continues) 724 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 analysis of the expression of pro- and anti-inflammatory bio- disease (loss of body weight), although BAR501 was slightly markers in the colon. Thus, although we detected a robust increase more effective than dexamethasone. Furthermore, mice treated in the expression of proinflammatory cytokines (IFN-g, IL-6, with the GPBAR1 ligand exhibited a significantly lower CDAI IL-1b, and TNF-a) in response to TNBS by real-time PCR this compared with mice treated with dexamethasone (Supplemental effect was reversed by treatment with BAR501, which also in- Fig. 1B), and they had fewer ulcerations and a lower colon weight/ creased the expression of TGF-b, IL-10, and Foxp3 (Fig. 2H, 2I). length ratio in comparison with TNBS mice treated with dexa- To gain insights into the mechanisms that support the anti- methasone (Supplemental Fig. 1C–E). inflammatory activity of BAR501, as indicated by modulation of the expression of pro- and anti-inflammatory genes (data not GPBAR1 protects against colitis induced by oxazolone shown), we have checked whether exposure to the GPBAR1 ligand To investigate whether BAR501’s ability to attenuate intestinal shifts expression of the M1 and M2 macrophage recovered from the inflammation extends to other settings, we have administered the lamina propria of colon from mice administered TNBS in vivo. To GPBAR1 ligand to mice with oxazolone-induced colitis. Oxazolone this end, we measured mRNA expression levels of the M1 markers colitis is a Th2-mediated model of inflammation and exhibits CD38, Fpr2, and Gpr18 (Fig. 3A) and the M2 markers Egr2 and histologic features and a distribution similar to those observed in c-myc (Fig. 3B). The results of these experiments were consistent ulcerative colitis (29, 30). In these experiments, BALB/c mice with the above-mentioned in vitro findings and demonstrate that were administered oxazolone and were treated with vehicle or GPBAR1 activation effectively promotes the shift toward an M2 BAR501 (30 mg/kg/d). As shown in Fig. 4A and 4B, BAR501 phenotype in colitic mice. Analysis of the number of cells infil- effectively reduced the wasting disease and symptoms of colitis trating the colon lamina propria and the number of circulating (body weight loss and CDAI) caused by oxazolone. Furthermore, Downloaded from WBCs showed that BAR501 reversed the effect of TNBS by de- BAR501 reduced macroscopic (length of colon, weight/length creasing both populations (Fig. 3C, 3D). These findings were ratio of colon, and severity of ulcerations) and histologic fea- further confirmed by a detailed flow cytometry analysis of lamina tures of inflammation (Fig. 4C–G). Finally, BAR501 counteracted propria–infiltrating cells (Fig. 3E–J). To summarize these studies, the effects of oxazolone on markers of inflammation (IFN-g, IL-6, we have found that treatment with BAR501 reduced the per- and IL-1b) (Fig. 4H), whereas it increased the expression of TGF-b, + + centage and number of CD4 (Fig. 3G) and Mac1 macrophages IL-10, and Foxp3 (Fig. 4I). http://www.jimmunol.org/ in the colon (Fig. 3I), increased the percentage and number of CD4+Foxp3+ cells (Fig. 3H), and had no effect on the percentage Anti-inflammatory effects of BAR501 are lost in of B220+ cells (Fig. 3E), CD8+ cells (Fig. 3F), or Mac1+Gr1+ cells GPBAR1-deficient mice (Fig. 3J). Taken together, these data show that BAR501 reduces To tie the effect of BAR501 to GPBAR1, we next investigated inflammation and modulates the phenotype of lamina propria in- whether the anti-inflammatory and immunomodulatory activities flammatory cells in the TNBS model of colitis. of this agent are maintained in GPBAR1-knockout mice. For this Because GPBAR1 is highly expressed on macrophages, we have purpose, wild-type and Gpbar12/2 mice on a C57BL/6 back- further investigated the effect of in vivo treatment with BAR501 on ground were administered TNBS, with or without BAR501 these cells. Flow cytometry analysis of lamina propria cells revealed (30 mg/kg). As shown in Fig. 5A–E, the severity of wasting dis- by guest on September 28, 2021 that treating TNBS mice with BAR501 reduced the percentage of ease and intestinal inflammation induced by TNBS was exacer- inflammatory macrophages in the colon (CD11b+CX3CR1+ cells) bated by GPBAR1 gene ablation. In comparison with their (Fig. 3K, 3L). The further fractioning of these cells demonstrated congenic littermates, Gpbar12/2 mice developed more severe that, although TNBS treatment increases inflammatory macrophages disease and diffuse intestinal ulcerations. Treatment with BAR501 (CCR7+F4/802) (25, 26) and decreases the percentage of resident reversed these features in Gpbar1+/+ mice but failed to do the same macrophages (CCR72F4/80+) (21, 27), this pattern was reversed by in Gpbar12/2 mice (Fig. 5A–G). Finally, BAR501 attenuated the GPBAR1 activation. In contrast, treatment with BAR501 increased severity of disease in Gpbar1+/+ mice, but not in Gpbar12/2 mice, the percentage of resident macrophages and decreased the per- as measured by assessing the microscopic score (Supplemental centage of inflammatory macrophages. Fig. 2A). These findings were confirmed by analysis of circulating To gain insights into the role of GPBAR1 in regulating traf- monocytes (Fig. 3K, 3L). Thus, BAR501 reversed the effect that ficking toward the intestinal mucosa, we next carried out a detailed TNBS had on the number of total circulating monocytes (Fig. 3M, characterization of lamina propria–infiltrating cells in wild-type left panel), whereas it had no effect on the ratio of resident/ and Gpbar12/2 mice treated with TNBS. The results in Fig. 5H–L inflammatory monocytes (Fig. 3M, middle and right panels). demonstrated that GPBAR1 gene ablation worsens intestinal in- This ratio was not changed even by treatment with TNBS (28). flammation in this model and increases the expression of inflam- Because glucocorticoids are widely used in the treatment of matory biomarkers, such as IL-1b, in comparison with wild-type IBDs, we next investigated how BAR501 compares with dexa- mice (Fig. 5G). Furthermore, GPBAR1 gene ablation blunted the methasone, a synthetic glucocorticoid, in the treatment of TNBS ability of these mice to mount an anti-inflammatory response, colitis. We monitored the progress of the disease by recording body because expression of markers for the regulatory T cell (Treg) weight and CDAI daily. The data shown in Supplemental Fig. 1A phenotype (TGF-b, IL-10, and Foxp3) were dramatically reduced demonstrate that both compounds effectively attenuate wasting in these mice in comparison with their congenic littermates

+ + + 2 2 + Frequency of CD11b CX3CR1 cells in the colonic lamina propria (left panel) and frequency of CCR7 F4/80 cells (middle panel) or CCR7 F4/80 cells + + (right panel) in CD11b CX3CR1 cells from the colonic lamina propria. (L) Flow cytometry analysis of CD11b and CX3CR1 expression in cells derived + + from colonic lamina propria and flow cytometry analysis of F4/80 and CCR7 expression in CD11b CX3CR1 cells derived from colonic lamina propria. + + + 2 2 + (M) Frequency of CD11b CX3CR1 cells in blood (left panel) and frequency of CD11c Ly6C cells (middle panel) or CD11c Ly6C cells (right panel) in + + CD11b CX3CR1 cells in blood. (N) Flow cytometry analysis of CD11b and CX3CR1 expression in cells derived from blood of mice and flow cytometry + + analysis of CD11c and Ly6C expression in CD11b CX3CR1 cells derived from blood of mice. Data are the mean 6 SEM of 6–12 mice per group from two independent experiments. *p , 0.05. The Journal of Immunology 725 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. BAR501 reduces the severity of oxazolone colitis. After the presensitization step on skin, BALB/c mice were treated with oxazolone and then administered vehicle or BAR501 (30 mg/kg/d) by gavage from days 0 to 4. BAR501 attenuated the development of wasting disease (i.e., change in body weight) (A) (*p , 0.05, Oxa versus Oxa + BAR501 [30 mg/kg]) and CDAI score (B). (C) Photographs of the colon from control, oxazolone-treated, and oxazolone plus BAR501–treated mice. BAR501 reduced the intestinal inflammatory score: colon length (D), ratio of colon weight/colon length (E), and ulcer area (F). (G)H&E staining of colon sections from control, oxazolone-treated, and oxazolone plus BAR501–treated mice (original magnification 310). Quantitative real-time PCR analysis of expression of IFN-g, IL-6,andIL-1b (H)andTGF-b, IL-10,andFoxp3 (I) genes in colon. The data are normalized to Actb mRNA. Results are the mean 6 SEM of six to eight mice per group from two independent experiments. *p , 0.05 (B and D–F). 726 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. GPBAR1 deficiency exacerbates intestinal inflammation and prevents BAR501-mediated alleviation of TNBS colitis. Gpbar1+/+ and Gpbar12/2 mice were treated with TNBS and then administered vehicle or BAR501 (30 mg/kg/d) by gavage from days 0 to 4. BAR501 attenuated the development of wasting disease (i.e., change in body weight) only in Gpbar1+/+ mice (A)(*p , 0.05, Gpbar1+/+ TNBS versus Gpbar12/2 TNBS, #p , 0.05, Gpbar1+/+ TNBS + BAR501 [30 mg/kg] versus Gpbar12/2 TNBS + BAR501 [30 mg/kg]) and CDAI score (B). Colon length (C), ratio of colon weight/ colon length (D), and ulcer area (E) of Gpbar1+/+ and Gpbar12/2 mice. Number of cells in lamina propria of colon (F) and WBCs (G) in mice at 4 d after TNBS administration. Relative mRNA expression of IL-6, IL-1b, and TNF-a (H) and TGF-b, IL-10, and Foxp3 (I) in colon of Gpbar1+/+ and Gpbar12/2 mice was evaluated by real-time PCR. The data are normalized to Actb mRNA. Quantitative real-time PCR analysis of M1 macrophage markers Cd38, Fpr2,andGpr18 (J), M2 macrophage markers Egr2 and c-myc (K), and Ccl2 (L) in colon of Gpbar1+/+ and Gpbar12/2 mice presented as relative expression in comparison with Actb mRNA. Results are the mean 6 SEM of 6–10 mice per group from two independent experiments. *p , 0.05 (B–I).

(Fig. 5H). Treatment with BAR501 reduced the levels of proin- wild-type mice, the pattern of infiltration of the colonic lamina flammatory cytokines and increased the expression of anti- propria was very similar to that found in BALB/c mice; treating inflammatory genes in wild-type mice but failed to do the same these mice with BAR501 generated the same pattern of response in Gpbar12/2 mice (Fig. 5G, 5H). [i.e., reduced the expression of markers of the M1 phenotype in In addition, we found that GPBAR1 gene ablation exerts a the colon (Fig. 3A) and increased the expression of markers for profound influence on leukocyte trafficking toward the intestinal the M2 phenotype (Fig. 3B)]. In contrast, treating Gpbar12/2 mice mucosa and M1 and M2 polarization (Fig. 5I, 5J). In C57BL/6 with BAR501 failed to reproduce these effects. The Journal of Immunology 727

A similar pattern of regulation was observed for the chemokine ments (Supplemental Fig. 4A, 4C) demonstrated that GPBAR1 CCL2/MCP-1 (Fig. 5K), indicating that GPBAR1 mediates the gene expression was completely abrogated by this approach. In immunomodulatory activity of BAR501. wild-type cells cultured or not with 50 mM BAR501, the GPBAR1 These finding were further confirmed by flow cytometry analysis ligand increased the binding of CREB to CRE in the IL-10 pro- of lamina propria cells. The data shown in Fig. 6 demonstrate that, moter (Fig. 7A). The increase in the production of IL-10 in although treating wild-type mice with BAR501 effectively re- macrophages was also closely related to the activation of duced the percentage of infiltrating macrophages (CD11b+ GPBAR1, whereas no effect was observed in cells silenced for CX3CR1+) (Fig. 6A, left panel), this effect was lost in Gpbar12/2 GPBAR1. To gain further insights into the role of IL-10 in mice. The further characterization of macrophage subsets dem- GPBAR1 signaling in macrophages, we have the silenced the IL-10 onstrated that wild-type and Gpbar12/2 mice respond to TNBS gene in RAW 264.7 cells. Control experiments (Supplemental Fig. with an inflow of inflammatory macrophages (CCR7+F4/802) 4B–D) demonstrated that this approach resulted in an ∼70% re- (Fig. 6A, middle panel) and a decrease in resident macrophages duction in IL-10 mRNA. After priming with LPS and IFN-g,with (CCR72F4/80+) (Fig. 6A, right panel). However, although BAR501 or without BAR501 (50 mM), wild-type RAW 264.7 cells showed a administration blunted the recruitment of these cells in wild-type robust upregulation of Fpr2 (M1 phenotype) and TNF-a expression. mice, this protective effect was completely abrogated by GPBAR1 This pattern was reversed by cotreating the cells with the GPBAR1 gene ablation. ligand, which also increased the expression of Egr2 and TGF-b Because these data highlighted a very precise mechanism of (Fig. 7B). In contrast, the counterregulatory effects of BAR501 action for GPBAR1 in regulating macrophage differentiation and were lost in RAW 264.7 cells silenced for IL-10 or GPBAR1 (Fig. recruitment, we have further dissected the CD11b population by 7C, 7D), indicating that the upregulation of IL-10 is essential for the Downloaded from measuring the percentage of IL-6+ and IL-10+ CD11b+ cells by anti-inflammatory action of GPBAR1. flow cytometry (Fig. 6B, 6C). The data demonstrate that the inflow of CD11b+/IL-6+ cells was enhanced in Gpbar12/2 mice IL-10 is essential for the anti-inflammatory effects of BAR501 challenged with TNBS and that the modulatory effects of Because the above-mentioned data highlight a role for IL-10 in the BAR501 on the percentage of CD11b+/IL-6+ and CD11b+/IL-10+ beneficial effects exerted by GPBAR1 ligation, we have treated 2/2 cells were lost in mice harboring disrupted GPBAR1 (Fig. 6B, IL-10 mice rendered colitic by administration of TNBS with http://www.jimmunol.org/ middle and right panels). The analysis of circulating monocytes BAR501. For this purpose, wild-type and IL-102/2 mice on the (Fig. 6D, 6E) confirmed these findings, because BAR501 failed to C57BL/6 background were administered TNBS, with or without modulate the ratio of resident/inflammatory monocytes caused by BAR501 (30 mg/kg). As shown in Fig. 8A–E, the severity of TNBS administration in Gpbar12/2 mice, whereas it effectively wasting disease and the severity of intestinal inflammation in- + reduced the percentage of CD11b /CX3CR1 cells in wild-type mice duced by TNBS were exacerbated by IL-10 gene ablation. Further- (Fig. 5D, middle and right panels). Taken together, these data more in IL-102/2 mice, TNBS induced an increase in the number suggest that GPBAR1 ligation in this model attenuates inflamma- of lamina propria–infiltrating cells that was greater than that ob- tion by promoting a shift toward IL-10–producing macrophages served in wild-type animals (Fig. 8F). Again, BAR501 reversed (M2 phenotype). the effects of TNBS in IL-10+/+ mice but failed to do the same in by guest on September 28, 2021 These findings were confirmed by immunohistochemical anal- IL-102/2 mice. ysis of CD11b+ cells in the colonic lamina propria (Supplemental Analysis of the expression of mRNA for pro- and anti-inflammatory Fig. 2B). Thus, although TNBS administration increased the in- biomarkers by real-time PCR (Fig. 8G, 8H) showed that, in the flow of CD11b+ cells, this pattern was reversed by treating wild- absence of the IL-10 gene, after administration of TNBS the ex- type mice, but not Gpbar12/2 mice, with BAR501. pression levels of proinflammatory cytokines were higher than in A similar pattern of regulation was observed for CD4+ cells; IL-10+/+ mice. In addition, BAR501 failed to modulate markers of although the colonic expression of CD4 mRNA and protein (by inflammation in IL-102/2 mice, whereas it maintained its efficacy immunohistochemistry), a marker of Th cells (Supplemental Fig. in wild-type mice. In these mice, the GPBAR1 ligand reduced the 3A, 3B), increased in response to administration of TNBS in expression of proinflammatory cytokines (TNF-a, IL-1b, IL-6, Gpbar1+/+ and Gpbar12/2 mice, the magnitude of its regulation and IFN-g) and increased the expression of anti-inflammatory was significantly higher in Gpbar12/2 mice. This pattern was cytokines (TGF-b and IL-10). reversed by treating wild-type mice, but not Gpbar12/2 mice, with BAR501. Discussion The importance of bile acids and their receptors in regulating GPBAR1 regulates IL-10 gene expression in macrophages in a intestinal immunity has been confirmed in recent years by the promoter-dependent manner demonstration that FXR (8–11) and GPBAR1 (7) gene ablation In target tissues, activation of GPBAR1 increases the production of worsens the severity of intestinal inflammation. Furthermore, bile cAMP, leading to PKA-dependent phosphorylation of CREB and duct ligation, which blocks the inflow of bile acids into the in- allowing p-CREB to translocate into the nucleus and bind to the testine, worsens the severity of inflammation in mice with TNBS- CRE motif (31). Because in vivo data demonstrate that BAR501 induced colitis, highlighting a role for bile acids in maintaining increased the expression of IL-10 mRNA, we have investigated intestinal homeostasis. whether this effect involves direct regulation of IL-10 gene ex- Bile acid metabolism in mammals leads to the generation of pression by GPBAR1 (32). To reduce the number of mice used in several molecular species. In the liver, this evolutionarily conserved this study, the following experiments were carried out using RAW pathway generates primary bile acids from cholesterol. These bile 264.7 cells. Because analysis of the IL-10 promoter revealed the acids are preferential ligands for FXR, and CDCA regulates in- presence of CRE sequences (see Materials and Methods), we testinal and liver immunity in an FXR-dependent manner (10, 11). performed a ChIP assay on wild-type RAW 264.7 cells, a mac- Secondary bile acids, the natural ligands of GPBAR1, are gener- rophage cell line that expresses GPBAR1, and the same cells in ated in the intestine by the activity of intestinal microbiota (4, 6). which GPBAR1 expression was silenced by treatment with an During feeding, CDCA and cholic acid enter the small intestine anti-GPBAR1 small interfering RNA (siRNA). Control experi- and undergo a process of 7a/b dihydroxylation, leading to the 728 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. BAR501 reshapes the polarization of lamina propria macrophages. Gpbar1+/+ and Gpbar12/2 mice were treated with TNBS and then ad- + + ministered vehicle or BAR501 (30 mg/kg/d) by gavage from days 0 to 4. (A) Frequency of CD11b CX3CR1 cells in the colonic lamina propria (left panel) + 2 2 + + + and frequency of CCR7 F4/80 cells (middle panel) or CCR7 F4/80 cells (right panel) in CD11b CX3CR1 cells from the (Figure legend continues) The Journal of Immunology 729 generation of DCA and LCA, whose concentrations fluctuate GPBAR1 ablation contributes to shift macrophages toward an M1 widely in the intestine and blood according to the fast–fed cycle phenotype. (6). In mammals, GPBAR1 expression is restricted to a limited Because these data suggest that GPBAR1 might ground the number of tissues, including small intestine, colon, adipose tis- development of gut-specific anti-inflammatory therapies, we have sues, muscles, and lymphoid organs (12, 13). In immune cells, investigated whether treating mice with a small molecule agonist expression of GPBAR1 mRNA is essentially restricted to cells of for GPBAR1 (i.e., BAR501) attenuates colitis development in two myeloid origin (13). Because the intestines harbor the largest pool rodent models of inflammation: BALB/c mice administered TNBS of macrophages and contain the largest amount of secondary bile or oxazolone (33). Results from these experiments have shown acids, there was substantial background to support the notion that that GPBAR1 activation protects, in a dose-dependent manner, secondary bile acids might regulate intestinal innate immunity via from the development of signs and symptoms of colitis in both a GPBAR1-dependent mechanism (5, 6, 8). models. In both models, the beneficial effects of BAR501 were In this study, we have elaborated on this concept and demon- associated with a robust shift in the macrophage phenotype from strated that GPBAR1 is essential to counter-regulate immune re- M1 to M2. Importantly, although BAR501 effectively reduced the sponses in Th1 and Th2 models of colitis. The protective effect number of circulating monocytes, it failed to alter the ratio of exerted by GPBAR1 ligation is largely mediated by the reshaping Ly6C+/Ly6C2 cells, confirming that Ly6C expression per se does of leukocyte trafficking, which is associated with a robust shift of not affect the differentiation of monocytes toward a pro- or anti- intestinal macrophages from an M1 to an M2 phenotype. inflammatory phenotype and that the differentiation of Ly6C+ Thus, although Gpbar12/2 mice do not develop overt colitis monocytes, after they enter the tissues, depends on the organ mi- spontaneously, they were more likely to develop severe inflam- croenvironment (34, 35). These findings provide further support that Downloaded from mation when challenged with TNBS or dextran sulfate (7). Local GPBAR1 could be exploited for the development of gut-specific (diarrhea) and systemic (wasting disease) inflammation were therapies for IBDs without dampening the systemic immune system. worsened by GPBAR1 gene ablation. However, these different Treating mice with BAR501 reshaped the entire trafficking of clinical outcomes were not supported by detectable changes in the intestinal leukocytes. The results shown in Fig. 3 demonstrate that + + systemic immune response to inflammation; rather, they appear to GPBAR1 agonism reduced the percentages of CD11b /CX3CR1 + + + be caused by a defective regulation of the immune response that and CD4 cells, but not CD8 and B220 cells, whereas it in- http://www.jimmunol.org/ takes place in the intestine. This view is supported by the fact that creased the percentage of CD4/Foxp3 Tregs. Further fractioning the systemic response to TNBS was similar in Gpbar12/2 and of CD11b+ cells also revealed a robust reduction in the percentage wild-type mice. Both mice strains react to TNBS administration of these cells that emigrated in the lamina propria, as well as that with a robust increase in the number of total circulating white cells macrophages were shifted toward an M2 phenotype, as shown by + + + + and CD11b /CX3CR1 cells (monocytes); among these cells, the the robust increase (Fig. 3I) in the percentage of CD11b CX3CR1 relative percentage of “inflammatory” monocytes (i.e., CD11c+ CCR72F4780+ cells (M2 macrophages). Ly6C+) was similar. In contrast, the local immune response to Consistent with these findings, we have also demonstrated that TNBS (and oxazolone) was profoundly disturbed by Gpbar1 gene treating mice with BAR501 resets the level of intestinal inflam- ablation. Indeed, Gpbar12/2 mice displayed higher levels of mation, as demonstrated by the reversion of the induction of by guest on September 28, 2021 proinflammatory cytokines and chemokines (TNF-a,IFN-g, proinflammatory cytokines and chemokines (IL-1b, IL-6, IFN-g, IL-1b, IL-6, and CCL2), along with a strong reduction in the TNF-a and CCL2) caused by TNBS and oxazolone, as well as the expression of anti-inflammatory mediators, IL-10 and TGF-b induction of anti-inflammatory genes (IL-10 and TGF-b). Con- mRNAs, and the phenotype of leukocytes in the colonic lamina sistent with this view, exposure of colitic mice to BAR501 reduced propria was severely altered by the gene knockout. Specifically, in the intestinal expression of Cd38, Fpr2, and Gpr18, markers of the addition to slight changes in the expression of CD4 (mRNA and M1 phenotype, whereas it increased the expression of markers of histochemistry), the most relevant change that we detected was a the M2 phenotype (Egr2 and c-myc). marked upregulation of a subset of genes that was suggestive of an The essential role of GPBAR1 in shaping the intestinal mi- M1 phenotype (Cd38, Fpr2, and Gpr18), along with a profound croenvironment is well highlighted by the effect that BAR501 downregulation of Egr2 and c-myc (two markers of the M2 phe- exerts on CCL2/MCP1 gene expression in the colon (Fig. 5). CCL2 notype). Furthermore, fractioning of lamina propria–derived is a member of the CC chemokine family and is a potent che- CD11b+ cells by flow cytometry revealed that Gpbar1 gene ab- motactic factor for monocytes (MCP-1) (36). This chemokine and lation was associated with a higher percentage of CD11b+/IL-6+ its receptor, CCR5 (33, 34), are essential for monocyte recruitment cells (i.e., activated macrophages). Taken together, these data in the inflamed colon (37–41): thus, although treating mice with suggest that, although GPBAR1 does not regulate the trafficking TNBS dramatically increased the levels of CCL2 mRNA in the of monocytes from lymphoid organs or bone marrow to the blood colon, this pattern was reversed by BAR501, strongly supporting a (likely reflecting the lack of ligands in lymphoid organs), this counter-regulatory role for GPBAR1 in monocyte trafficking. The receptor is required for maturation of emigrated monocytes in the fact that these regulatory effects were lost in Gpbar12/2 mice intestine. In the presence of abundant concentrations of DCA and further ties the effects of BAR501 to this receptor. LCA in the intestine, emigrated macrophages are shifted toward Because the above-mentioned data suggest that GBAR1 drives the acquisition of an anergic phenotype (M2). In contrast, the development of a tolerogenic phenotype of intestinal macro-

colonic lamina propria. (B) Frequency of CD11b+ cells in the colonic lamina propria (left panel) and frequency of IL-6+ cells (middle panel) or IL-10+ cells (right panel) in CD11b+ cells from the colonic lamina propria. (C) Flow cytometry analysis of CD11b expression (top panel) and of IL-6 (middle panel) and + + + IL-10 (bottom panel) expression in CD11b cells derived from the lamina propria. (D) Frequency of CD11b CX3CR1 cells in blood (left panel) + 2 2 + + + and frequency of CD11c Ly6C cells (middle panel) or CD11c Ly6C cells (right panel) in CD11b CX3CR1 cells. (E) Flow cytometry analysis of CD11b + + and CX3CR1 expression in blood cells (upper panel) and flow cytometry analysis of CD11c and Ly6C expression (lower panel) in CD11b CX3CR1 blood cells. Results are the mean 6 SEM of four to eight mice per group from two independent experiments. *p , 0.05. 730 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. IL-10 supports the anti-inflammatory effects of BAR501. ChIP assay of the CREB binding IL10 promoter in RAW 264.7 cells and RAW 264.7 cells silenced for the GPBAR1 gene. (A) Cell lysates were immunoprecipitated with anti-CREB or control IgG, and the presence of specific regions in the immunoprecipitates was determined by real-time PCR. Quantitative real-time PCR analysis of Fpr2, TNF-a, Egr2,and TGF-b in RAW 264.7 cells (B), short hairpin GPBAR1 RAW 264.7 cells (C), and short hairpin IL-10 RAW 264.7 cells (D) under various culture conditions. The data are normalized to Actb mRNA. Results are the mean 6 SEM of three to five mice per group. *p , 0.05, **p , 0.005, ***p , 0.0005. The Journal of Immunology 731 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. IL-10 deficiency prevents BAR501-mediated alleviation of TNBS colitis. IL-10+/+ and IL-102/2 mice were treated with TNBS and then administered vehicle or BAR501 (30 mg/kg/d) by gavage from days 0 to 4. Treatment with BAR501 attenuated the development of wasting disease only in IL-10+/+ mice, as shown by changes in body weight (A)(*p , 0.05, WT+/+ TNBS + BAR501 [30 mg/kg] versus IL-102/2 TNBS + BAR501 [30 mg/kg]. #p , 0.005 WT+/+ TNBS versus WT+/+ TNBS + BAR501 [30 mg/kg]) and CDAI score (B). Colon length (C), ratio of colon weight/colon length (D), and ulcer area (E)inIL-10+/+ and IL-102/2 mice. (F) Number of cells of lamina propria of colon of mice at 4 d after TNBS administration. Relative mRNA expression of TNF-a, IL-1b, IL-6,andIFN-g (G)andTGF-b and IL-10 (H) in the colon of IL-10+/+ and IL-102/2 mice was evaluated by real-time PCR. The data are normalized to Actb mRNA. Results are the mean 6 SEM of five to eight mice per group. *p , 0.05. N.D., not detected. phages, and levels of TGF-b and IL-10 were markedly reduced in IL-10. By promoter analysis we detected a CRE motif in the Gpbar12/2 mice compared with their congenic littermates, we proximal promoter of the IL-10 gene. The functionality of these have further dissected signals that link GPBAR1 activation to CREs in terms of regulation of IL-10 transcription was examined 732 IL-10 MEDIATES REGULATION OF INTESTINAL MACROPHAGES BY TGR5 in detail in RAW 264.7 cells, a macrophage cell line. The results (TGR5) modulates integrity of intestinal barrier and immune response to ex- of these experiments demonstrate that the interaction between perimental colitis. [Published erratum appears in 2013 PLoS One 8(1).] PLoS One 6: e25637. BAR501 and GPBAR1 increases the binding of CREB to the 8. Renga, B., A. Mencarelli, S. Cipriani, C. D’Amore, A. Carino, A. Bruno, IL-10 promoter, driving the production of this cytokine. This D. Francisci, A. Zampella, E. Distrutti, and S. Fiorucci. 2013. The bile acid regulatory effect was abrogated by the silencing of GPBAR1 by an sensor FXR is required for immune-regulatory activities of TLR-9 in intestinal inflammation. PLoS One 8: e54472. anti-GPBAR1 siRNA. 9. Fiorucci, S., A. Mencarelli, E. Distrutti, and A. Zampella. 2012. Farnesoid X Because of the high levels of IL-10 in the colon, after treatment receptor: from medicinal chemistry to clinical applications. Future Med. Chem. with BAR501, monocytes and lymphocytes that reach the colon are 4: 877–891. 10. Mencarelli, A., B. Renga, M. Migliorati, S. Cipriani, E. Distrutti, L. Santucci, exposed to this anti-inflammatory cytokine, which is likely re- and S. Fiorucci. 2009. The bile acid sensor farnesoid X receptor is a modulator of sponsible for shifting macrophages from an M1 to an M2 phe- liver immunity in a rodent model of acute hepatitis. J. Immunol. 183: 6657–6666. notype. Because it is conceivable that, following treatment with the 11. Vavassori, P., A. 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