The Bile Acid Receptor GPBAR1 Modulates CCL2/CCR2 Signaling

The Bile Acid Receptor GPBAR1 Modulates CCL2/CCR2 Signaling at the Liver Sinusoidal/Macrophage Interface and Reverses Acetaminophen-Induced Liver This information is current as Toxicity of September 28, 2021. Michele Biagioli, Adriana Carino, Chiara Fiorucci, Silvia Marchianò, Cristina Di Giorgio, Martina Bordoni, Rosalinda Roselli, Monia Baldoni, Eleonora Distrutti, Angela Zampella

and Stefano Fiorucci Downloaded from J Immunol published online 25 March 2020 http://www.jimmunol.org/content/early/2020/03/24/jimmun ol.1901427 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 25, 2020, doi:10.4049/jimmunol.1901427 The Journal of Immunology

The Bile Acid Receptor GPBAR1 Modulates CCL2/CCR2 Signaling at the Liver Sinusoidal/Macrophage Interface and Reverses Acetaminophen-Induced Liver Toxicity

Michele Biagioli,* Adriana Carino,* Chiara Fiorucci,* Silvia Marchiano`,* Cristina Di Giorgio,* Martina Bordoni,* Rosalinda Roselli,† Monia Baldoni,‡ Eleonora Distrutti,x Angela Zampella,† and Stefano Fiorucci*

Drug-induced liver injury caused by acetaminophen (acetyl-para-aminophenol [APAP]) is the main cause of acute liver failure and liver transplantation in several Western countries. Whereas direct toxicity exerted by APAP metabolites is a key determinant for early hepatocytes injury, the recruitment of cells of innate immunity exerts a mechanistic role in disease progression, determining the clinical outcomes. GPBAR1 is a G protein–coupled receptor for secondary bile acids placed at the interface between liver Downloaded from sinusoidal cells and innate immunity. In this report, using genetic and pharmacological approaches, we demonstrate that whereas Gpbar1 gene deletion worsens the severity of liver injury, its pharmacological activation by 6b-ethyl-3a,7b-dihydroxy-5b-cholan- 24-ol rescues mice from liver injury caused by APAP. This protective effect was supported by a robust attenuation of liver recruitment of monocyte-derived macrophages and their repolarization toward an anti-inﬂammatory phenotype. Macrophage depletion by gadolinium chloride pretreatment abrogated disease development, whereas their reconstitution by spleen-derived macrophage transplantation restored the sensitivity to APAP in a GPBAR1-dependent manner. RNA sequencing analyses dem- http://www.jimmunol.org/ onstrated that GPBAR1 agonism modulated the expression of multiple pathways, including the chemokine CCL2 and its receptor, CCR2. Treating wild-type mice with an anti-CCL2 mAb attenuated the severity of liver injury. We demonstrated that negative regulation of CCL2 production by GPBAR1 agonism was promoter dependent and involved FOXO1. In conclusion, we have shown that GPBAR1 is an upstream modulator of CCL2/CCR2 axis at the sinusoidal cell/macrophage interface, providing a novel target in the treatment of liver damage caused by APAP. The Journal of Immunology, 2020, 204: 000–000.

cetaminophen (acetyl-para-aminophenol [APAP]) is the Western countries, accounting for up to 46% of all ALFs in the common ingredient of widely prescribed and over-the- United States and 40–70% in Europe (1–7). In addition, patients by guest on September 28, 2021 A counter analgesic and antipyretic drugs. Although safe with chronic liver diseases or children could develop a spectrum when administered at therapeutic dosages (4 g/d) to healthy adults, of clinically relevant liver diseases in response to therapeutic APAP might cause acute liver failure (ALF) when taken at dosage dosages of APAP, making APAP overdosing, intentional or unin- exceeding 10–12 g/d. APAP overdosing is a leading cause of ALF tentional, the most common cause of drug-induced liver injury and one of the major indications for liver transplantation in (DILI) in Western countries (1–5). Rodent models have proven useful in dissecting pathogenic mechanisms involved in the development of APAP-induced DILI. *Dipartimento di Scienze Biomediche e Chirurgiche, Universita` di Perugia, Perugia 06132, Italy; †Dipartimento di Farmacia, Universita` di Napoli Federico II, Naples At least in the case of ALF, the liver injury caused by APAP is 80131, Italy; ‡Dipartimento di Medicina, Universita` di Perugia, Perugia 06132, Italy; primarily due to the accumulation of toxic metabolites in hepa- x and Sezione di Gastroenterologia ed Epatologia, Azienda Ospedaliera di Perugia, tocytes. Indeed, although conventional dosages of APAP are me- Perugia 06132, Italy tabolized in the liver by phase II drug-metabolizing enzymes to ORCIDs: 0000-0002-2995-6896 (M. Biagioli); 0000-0003-2341-9528 (A.C.); 0000- 0002-6164-1923 (M. Baldoni); 0000-0003-3816-4222 (S.F.). generate APAP glucuronide and APAP sulfate, which are excreted through urine and feces via hepatobiliary drug transporters, in the Received for publication December 2, 2019. Accepted for publication March 2, 2020. setting of APAP overdose, a substantial portion of APAP is con- The transcriptomic data presented in this article have been submitted to Mendeley Data repository (http://dx.doi.org/10.17632/) under accession number dc2w3y2z47.1. verted to N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome Address correspondence and reprint requests to Prof. Stefano Fiorucci, Depart- P450 (Cyp450) enzymes. NAPQI, a toxic metabolite, reacts with ment of Surgical and Biomedical Science, University of Perugia, Piazza Lucio sulfhydryl groups on the glutathione (GSH) and other proteins, Severi 1, Sant’Andrea delle Fratte, 06132 Perugia, Italy. E-mail address: causing the formation of protein adducts and reactive oxygen stefano.fi[email protected] species, depleting GSH from hepatocytes, and impairing mito- The online version of this article contains supplemental material. chondrial bioenergetic, ultimately leading to centrilobular hepa- Abbreviations used in this article: ALF, acute liver failure; ALT, alanine aminotrans- tocyte death (8–10). This initial step is associated with an ferase; APAP, acetyl-para-aminophenol; AST, aspartate aminotransferase; BAR501, 6b-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol; ChIP, chromatin immunoprecipitation; inflammatory response mediated by cells of innate immunity, in- DILI, drug-induced liver injury; FMO, fluorescence minus one; GaCl3, gadolinium cluding liver-resident macrophages, the Kupffer cells, monocyte- chloride; GSH, glutathione; HUKCCS, cryopreserved human Kupffer cell; LSEC, liver sinusoidal cell; MoMF, monocyte-derived macrophage; NAPQI, N-acetyl-p- derived macrophages (MoMFs), dendritic cells, and NKT cells, benzoquinone imine; NT, not treated; o.s., oral administration, orally administered; along with different lymphoid derived immune cells (11, 12). In RNA-seq, RNA sequencing; SOD, superoxide dismutase; TAC, Transcriptome Anal- addition, NAPQI–protein complexes could be directly sensed by ysis Console. professional APC, leading to a cell-to-cell–mediated recruit- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 ment of Th and cytotoxic T cell subsets and to a Fas/FasL- and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901427 2 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY perforin-and-granzyme–mediated activation of death pathways in treatment. One hour after macrophage transfer, 500 mg/kg APAP was o.s., hepatocytes (13). This inflammatory/immune response that takes and the mice were sacrificed 24 h later. To investigate whether the same pathways were activated in different models of acute, hepatitis, Gpbar1+/+, place at the interface between liver parenchymal cells and endo- 2 2 and Gpbar1 / mice were treated by i.v. injection of 15 mg/kg Con A (28). thelial cells (known as liver sinusoidal cells [LSEC]), is orches- Mice were randomized to receive Con A alone or in combination with trated by a variety of chemical communications, including the BAR501 (30 mg/kg) daily, starting 3 d before induction of hepatitis. The various members of the chemokine superfamily and their recep- mice were sacrificed 8 or 24 h later. tors, and is thought to contribute to development of liver damage Histology and immunohistochemistry caused by APAP (11, 12). Accordingly, targeting the liver microenvironment at the sinusoidal cell/macrophage interface and Samples of liver were first fixed in buffered formalin, cut into 5-mm-thick sections (∼150 mm between each section, 4–8 mm/fragment/liver) and then the chemical communications that govern these cell-to-cell in- stained with H&E. Immunohistochemistry was performed on paraffin- teractions might have translational relevance in the treatment of embedded livers. In brief, Ag retrieval was achieved by incubation of APAP-induced DILI. the slides for 90 min in the hot (95˚C) sodium citrate buffer (pH 6.0) and GPBAR1, also known as TGR5, is a steroid-activated, G protein– 30 min of cooling at room temperature. Endogenous peroxidases were coupled membrane receptor (GPCRs). The physiological ligands quenched by incubating the slides with 3% H2O2 in methanol for 10 min. Nonspecific binding was blocked by treatment with BSA 8% plus Triton for GPBAR1 are secondary bile acids (i.e., lithocholic acid and 0.2% in PBS for 1 h. Anti-CCL2 (ab25124; Prodotti Gianni) was incubated deoxycholic acid and their taurine and glycine derivatives), al- overnight at 4˚C. After washing, anti-rabbit IgG (Vector Laboratories) was though the receptor could be activated by a variety of natural and incubated for 1 h. Sections were washed and incubated with VECTASTAIN dietetic compounds (14–16). Although GPBAR1 is not expressed ABC Reagent (Vector Laboratories) for 45 min. Color development was achieved by diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Sections Downloaded from by hepatocytes, several nonparenchymal cells, including liver- were then counterstained with hematoxylin, dehydrated through ethanol and resident macrophages, the Kupffer cells, monocytes and MoMFs, xylene, and coverslipped using a xylene-based mounting medium. NKT cells, and LSEC (17) express the receptor. Previous studies have shown that, once activated in these cells, GPBAR1 provides Cell culture essential regulatory signals to cells of innate immunity, contributing Raw264.7 cells, a murine macrophage cell line from American Type to the maintenance of a tolerogenic state toward microbial Ags Culture Collection (Manassas, VA), were cultured at 37˚C in DMEM supplemented with 10% FBS, 1% glutamine, and 1% penicillin/ derived from the intestinal microbiota (18–20). http://www.jimmunol.org/ streptomycin. Human LSEC were from Innoprot (catalog no. P10652; In the current study, by genetic and pharmacological approaches, Barcelona, Spain). LSEC were cultured in endothelial cell medium we demonstrated that GPBAR1 is an upstream modulator of (Innoprot) supplemented with 5% FBS, endothelial cell growth supplement chemokine production in the liver, and its activation resets the (Innoprot), and antibiotics. THP1 (TIB-202) and HepG2 (HB-8065) cell CCL2/CCR2 axis at the sinusoidal cell/macrophage interface, lines were from American Type Culture Collection. THP1 cells were thereby reducing macrophage recruitment and providing a novel cultured in RPMI 1640 supplemented with 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. HepG2 cells were cultured in Eagle’s target in the treatment of liver damage caused by APAP. MEM supplemented with 10% FBS, 1% glutamine, and 1% penicillin/ streptomycin. Cryopreserved human Kupffer cells (HUKCCS) were from Materials and Methods Invitrogen (Carlsbad, CA). HUKCCS cells were cultured in Kupffer Animals and hepatitis protocols monoculture medium (RPMI 1640 medium with GlutaMAX-I supplement by guest on September 28, 2021 and HEPES, supplemented with 10% FBS and 13 penicillin/streptomycin) C57BL6 mice were from Charles River Laboratories. GPBAR1 null mice at 37˚C according to the manufacturer’s instructions before use. on C57BL/6NCrl background and their C57BL/6NCrl congenic littermates For in vitro experiments, HepG2 (5 3 106) cells were plated and serum were originally donated by Dr. G. Vassileva (Schering-Plough Research starved, and after exposure to 10 mM APAP for 16 h, cell supernatants Institute, Kenilworth, NJ). The colonies were maintained in the animal were collected. Raw264.7 cells were plated in T75 flask (1.5 3 106 per facility of University of Perugia. Mice were housed under controlled flask) and starved overnight in 0.5% FBS-containing medium. Then, cells temperatures (22˚C) and photoperiods (12:12 h light/dark cycle), allowed were primed with conditioned medium from HepG2 treated with APAP unrestricted access to standard mouse chow and tap water and allowed to alone or in combination with BAR501 (20 mM) for 16 h. HUKCCS cells acclimate to these conditions for at least 5 d before inclusion in an ex- were thawed from liquid nitrogen and rapidly plated in 24-well plate periment. The study was conducted in agreement with the Italian law, and coated with collagen type I substrate (0.1 3 106 per well). After 24 h, cells the protocol was approved by the Ethical Committee of University of were primed with the conditioned medium harvested from HepG2 treated Perugia and by the National Committee of Italian Ministry of Health with APAP alone or in combination with BAR501 (20 mM) 16 h. Further permit no. 214/2017-PR. The health and body conditions of the animals on, THP1 and LSEC cells and conditioned medium from HepG2 were used were monitored daily by the veterinarian in the animal facility. Only male to perform a coculture experiment using 12-well Carrier Plate with Cell mice were used in each experiment. Hepatitis was induced in Gpbar1+/+ Culture Inserts, 0.4 mM (no. 141078; Nunc). Briefly, LSEC cells (3 3 105 and Gpbar12/2 mice by oral administration (o.s.) of APAP. In a prelimi- cells per well) were seeded on the bottom of culture plate with 2 ml of nary dosage-finding study, mice were administered orally with 150, 300, complete endothelial cell medium. After 24 h, the medium was replaced 500, and 750 mg/kg APAP, whereas in all the following studies, a dosage with 1 ml of complete RPMI 1640, and THP1 cells were plated on top of of 500 mg/kg APAP was used, and Gpbar1+/+ and Gpbar12/2 mice were the insert filter (7 3 105 cells) in 1 ml of RPMI 1640. Two milliliters of sacrificed at different time points (8, 24, 36, or 48 h). In some experimental conditioned medium from HepG2 treated with APAP were added to each groups, 6b-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501) was ad- well alone or in combination with BAR501 (20 mM). After 16 h, both cell ministrated orally at different dosages (5, 15, or 30 mg/kg), 45 min after types were separately collected to perform RNA extraction. APAP administration. Surviving mice were sacrificed 24 h after induction of hepatitis, blood samples collected by cardiac puncture, and the liver and Purification of primary mouse liver Kupffer and macrophages spleen collected and weighed (21, 22). +/+ +/+ Nonparenchymal mouse liver cells were isolated from the liver of Gpbar1 To investigate the involvement of CCL2 in the model, Gpbar1 and 2/2 2 2 and Gpbar1 mice using the Liver Dissociation Kit from Miltenyi Biotec Gpbar1 / male mice were pretreated with anti-CCL2 (clone 2H5, catalog (130-105-807) according to the manufacturer’s instructions. Kupffer/ no. BE0185; Bio X Cell) or anti-IgG Ab (catalog no. BE0091; Bio X Cell), macrophage cells were then purified by the Anti-F4/80 MicroBeads 200 mg/mouse i.p., 1 h before oral gavage 500 mg/kg APAP. The mice UltraPure Kit (Miltenyi Biotec) to obtain F4/80+ cells by a positive selection. were then sacrificed 24 h later (23). The purified cells were then plated in 10-mm plate (3 3 106 cells per plate) In another experimental setting, wild-type C57BL6 mice were pretreated for 2 h and then primed with conditioned medium obtained from HepG2 with 10 mg/kg gadolinium chloride (GaCl ) (catalog no. 439770-5G; 3 treated with APAP alone or in combination with BAR501 (20 mM) 16 h. Sigma-Aldrich) i.p. to deplete liver macrophages (24–26). As specifically indicated below, liver macrophage reconstitution in GaCl3-treated mice Cell adhesion assay was achieved by injection of 1 3 107 spleen-derived macrophages. For this purpose, C11b+/Gr12 cells were purified from the spleens of naive LSEC (5 3 104 cells per well) were plated on a 24-well plate (1 ml/well) +/+ 2/2 Gpbar1 or Gpbar1 mice (27) and injected i.v. 24 h after GaCl3 and primed with conditioned medium from HepG2 treated with APAP The Journal of Immunology 3 alone or in combination with BAR501 (20 mM) 24 h. THP1 monocytes (forward 59-ATATGGCTCGGACACCACTC-39;reverse59-TCCTTCGAGT- were primed with conditioned medium from HepG2 treated with APAP GACAAACACG-39), Cxcl7 (forward 59-CTGGGCCTGATCCTTGTTG-39; alone or in combination with BAR501 (20 mM) 24 h, then resuspended in reverse 59-TATTCGTACATCTGCAGCGC-39), Cxcl4 (forward 59-TGGAGGT- RPMI 1640 and starved for 2 h. Then, the medium was removed and GATCAAGGCAGG-39;reverse59-TATATAGGGGTGCTTGCCGG-39), Cxcl2 replaced with fresh medium containing 10 mM BCECF-AM. After 30 min, (forward 59-TCCAGAGCTTGAGTGTGACG-39;reverse59-GCTTCAGGGT- it was fluorescently labeled. THP1 cells were washed three times with CAAGGCAAAC-39), Ccr1 (forward 59-TGCAAGCTTCTCTCTGGGTT-39; PBS, supplemented with 1% FBS, and resuspended in endothelial medium. reverse 59-TGGACAATGGCCAGGTATCT-39), and Ccr2 (forward 59- The conditioned medium was removed from LSEC, and labeled THP1 ACCACATGTGCTAAGAATTGAAC-39;reverse59-GCCGTGGATGAACT- (1 ml, 5 3 105 cells) were added and incubated for 105 min at 37˚C. GAGGTA-39). The following TaqMan probes were also used: Cd38 Nonadherent monocytes were removed by gentle washing three times with (Mm01220906_m1; Thermo Fisher Scientific) and c-myc (Mm00487804_ PBS and supplemented with 10% of FBS. Then, 500 ml of lysis buffer m1; Thermo Fisher Scientific). (Tris buffer [pH 7.6] and 1% SDS) was added, and fluorescence intensity RT-PCR primers used in this study for human cell samples were as was measured (485-nm excitation and 520–560-nm emission) using a follows: GAPDH (forward 59-CAGCCTCAAGATCATCAGCA-39; reverse microplate reader. Fluorescence intensity was normalized using results 59-GGTCATGAGTCCTTCCACGA-39), CCL2 (forward 59-TAGCAGC- of MTS assay performed in parallel with the same culture and treatments CACCTTCATTCCC-39;reverse59-CTGCACTGAGATCTTCCTATTGG-39), conditions. CCR2 (forward 59-CCCATCATCTATGCCTTCGT-39;reverse59-ACTCCTG- GACCTCCACACAC-39), CXCL2 (forward 59-GCAGGGAATTCACCT- Chromatin immunoprecipitation CAAGA-39; reverse 59-GACAAGCTTTCTGCCCATTC-39), TNF-a (forward 59-AGCCCATGTTGTAGCAAACC-39; reverse 59-TGAGG- By a promoter analysis, we have preliminarily detected an FOXO1- TACAGGCCCTCTGAT-39), IL-1-b (forward 59-GTGGCAATGAG- responsive element sequence 384 bp upstream of the exon 1 of the gene GATGACTTG-39; reverse 59-GGAGATTCGTAGCTGGATGC-39), IL-6 encoding for murine Ccl2 and an FOXO1-responsive element sequence 459 (forward 59-AGGAGACTTGCCTGGTGAAA-39;reverse59-CAGAGGTGGT- bp upstream of the exon 1 of the gene encoding for murine Ccr2. Raw264.7 TATTCCATC-39), IL-10 (forward 59-TGCCTTCAGCAGAGTGAAGA- Downloaded from cells were exposed to conditioned medium from HepG2 treated with APAP 39; reverse 59-CTCAGACAAGGCTTGGCAAC-39), VCAM1 (forward alone or in combination with BAR501 (20 mM) for 16 h and then cross- 59-TGAGGATGGAAGATTCTGGA-39; reverse 59-GTCTCCAATCT- linked with formaldehyde. Chromatin immunoprecipitation (ChIP) assays GAGCAGCAA-39), ICAM1 (forward 59-TGATGGGCAGTCAACAGCTA- were performed according to the manufacturer’s protocols (EZ-ChIP, 39; reverse 59-ACCTGGCAGCGTAGGGTAAG-39), and E-SELECTIN catalog no. 17-371; Upstate). Briefly, cell extracts were sonicated and (forward 59-GCCACGGTGAATGTGTAGAG-39; reverse 59-GTGACTG- m divided. Abs (5 g) against FOXO1 and normal goat IgG were added for CAAACCAGGCTTC-39). immunoprecipitation. The immunoprecipitated chromatin was recovered and purified. The Ccl2 and Ccr2 promoters of DNA were quantified by AmpliSeq transcriptome (RNA sequencing) http://www.jimmunol.org/ real-time PCR analysis using primers around the FOXO1 binding site in the proximal promoter of Ccl2 (forward 59-GTCCTTTCCCAGCTGCAAG-39; The analysis of mouse liver samples was carried out using an Ion S5 reverse 59-AAAGGAAGTGGCCAAGGAAC-39) and in the proximal pro- sequencer with Torrent Suite Software v6. The analyses were per- moter of Ccr2 (forward 59-GGTCCCAGTGTAAATCTTTCCT-39;reverse formed with a range of fold ,22.0 and . +2.0, using Transcriptome 59-ATGCGCAGAGAGGTTTCACT-39). Primers spanning a region located Analysis Console Software (version 4.0.1) certified for AmpliSeq ∼5000 kb upstream of the Ccl2 transcriptional start site (forward 59- analysis (Thermo Fisher Scientific). High-quality RNA was extracted ATGTGTAGCTTGTGGTTGCG-39; reverse 59-AGGTGGGAAGCAGGTT- from the liver of mice using the PureLink RNA Mini Kit according to TACA-39)and∼4180 kb upstream of the Ccr2 transcriptional start site the manufacturer’s instructions. RNA quality and quantity were (forward 59-AGGAAATGGGGCAGATGGAA-39;reverse59-TGGCCATTGT- assessed with the Qubit RNA HS Assay Kit and a Qubit 3.0 Fluo- CACCAGTATT-39) were used as control. At least three replicates of each rometer (Invitrogen), followed by agarose gel electrophoresis. Li- group were performed. braries were generated using the Ion AmpliSeq Transcriptome Mouse by guest on September 28, 2021 Gene Expression Core Panel and Chef-Ready Kit (comprehensive Reverse transcription of mRNA and real-time PCR evaluation of AmpliSeq transcriptome and a whole-transcriptome RNA sequencing [RNA-seq] methodology). Briefly, 10 ng of RNA 2 Liver samples were immediately frozen in liquid nitrogen and stored at 80˚C was reverse transcribed with SuperScript VILO cDNA Synthesis Kit until use and mechanically homogenated with the aid of a pestle, and the before library preparation on the Ion Chef instrument. The resulting obtained materials were resuspended in 1 ml of TRIzol (Thermo Fisher cDNA was amplified to prepare barcoded libraries using the Ion Code Scientific). The RNA was extracted from liver samples and cells according PCR Plate and the Ion AmpliSeq Transcriptome Mouse Gene Ex- to the manufacturer’s protocol. After purification from genomic DNA by pression Core Panel and Chef-Ready Kit according to the manufac- DNase I treatment (Thermo Fisher Scientific), 1 mg of RNA from each turer’s instructions. Barcoded libraries were combined to a final sample was reverse transcribed using random hexamer primers with Su- concentration of 100 pM and used to prepare template-positive Ion perScript II (Thermo Fisher Scientific) in a 20-ml reaction volume; 10 ng Sphere Particles to load on Ion 540 Chips using the Ion 540 Kit-Chef. cDNA were amplified in a 20-ml solution containing 200 nM of each Sequencing was performed on an Ion S5 sequencer with Torrent Suite primer and 10 ml of SYBR Select Master Mix (Thermo Fisher Scientific). Software v6. The analyses were performed with a range of fold ,22.0 All reactions were performed in triplicate, and the thermal cycling con- and . +2.0, using Transcriptome Analysis Console Software (ver- ditions were as follows: 3 min at 95˚C, followed by 40 cycles of 95˚C for sion 4.0.1), certified for AmpliSeq analysis (Thermo Fisher Scien- 15 s, 56˚C for 20 s, and 72˚C for 30 s using a Step One Plus machine tific) (28). The transcriptomic data have been deposited as dataset on (Applied Biosystems). The relative mRNA expression was calculated ac- 2DCt Mendeley Data repository [Mendeley Data, v1 http://dx.doi.org/10. cordingly to the 2 method. Primers used in this study were designed 17632/dc2w3y2z47.1]. using the PRIMER3 (http://frodo.wi.mit.edu/primer3/) software using the National Center for Biotechnology Information database. Alternatively, Hepatic GSH and superoxide dismutase determinations for some genes, the TaqMan probes and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) were used. RT-PCR primers used in Frozen liver tissues were thaw rinsed with PBS. The tissues were then this study for mice samples were as follows: Ifn-g (forward 59-GCTT- homogenized in ice-cold MES buffer for GSH determination and in ice-cold TGCAGCTCTTCCTCAT-39; reverse 59-ATCCTTTTGCCAGT-39), Tnf-a PBS for superoxide dismutase (SOD) determination containing protease (forward 59-CCACCACGCTCTTCTGTCTA-39; reverse 59-AGGGTCTG- inhibitors for MDA determination. The homogenate was centrifuged at 3 GGCCATAGAACT-39), Il-6 (forward 59-CTTCACAAGTCGGAGGC- 1600 g at 4˚C for 10 min, and the supernatants were assayed for GSH TTA-39; reverse 59-TTCTGCAAGTGCATCATCGT-39), Il-1b (forward and SOD levels using the commercially available assay kits according the 59-GCTGAAAGCTCTCCACCTCA-39; reverse 59-AGGCCACAGGTA- manufacturer’s instructions (Glutathione Assay Kit no. 703002 and Su- TTTTGTCG-39), Tgf-b (forward 59-TTGCTTCAGCTCCACAGAGA-39; peroxide Dismutase Assay Kit no. 706002; Cayman Chemical). reverse 59-TGGTTGTAGAGGGCAAGGAC-39), Il-10 (forward 59-CC- Western blotting CAGAAATCAAGGAGCATT-39; reverse 59-CTCTTCACCTGCTCCAC- TGC-39), FoxP3 (forward 59-TCTTCGAGGAGCCAGAAGAG-39; reverse Total lysates were prepared by homogenization of Raw264.7 in E1A buffer 59-AGCTCCCAGCTTCTCCTTTT-39), Cd11b (forward 59-GTCAGA- containing phosphatase and protease inhibitors. Protein extracts were GTCTGCCTCCGTGT-39; reverse 59-CAGGGTCTAAAGCCAGGTCA- electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted 39), Ccl2 (forward 59-AAGAGGATCACCAGCAGCAG-39; reverse 59- to nitrocellulose membrane, and then incubated overnight with primary TCTGGACCCATTCCTTCTTG-39), Ccl3(forward 59-GATTCCACGC- Abs against Akt (C67E7 1:500; Cell Signaling), phospho-Akt (587F11 CAATTCATCG-39; reverse 59-CTTTGGAGTCAGCGCAGATC-39), Ccl5 1:500; Cell Signaling), FOXO1 (1:500; Invitrogen), phosho-FOXO1 4 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY

(1:500; Invitrogen), and GAPDH (D4C6R, 1:1000; Cell Signaling). treatment with BAR501 (Fig. 2A, 2B). These data were confirmed Primary Abs were detected with the HRP-labeled secondary Abs. Proteins by histological analysis (H&E) (Fig. 2C). Furthermore, as shown were visualized by Immobilon Western Chemiluminescent Reagent in Fig. 2D, 2E, whereas APAP reduced hepatic levels of GSH (MilliporeSigma) according to the manufacturer’s instructions. Quanti- tative densitometry analysis was performed using ImageJ software. and SOD, the liver contents of these two factors were preserved by treating wild-type APAP mice with BAR501 (Fig. 2D, 2E; Flow cytometry *p , 0.05). Flow cytometry analyses were carried out using a three-laser standard The key role of GPBAR1 in maintaining liver immunity was configuration Attune NxT (Life Technologies). Data were analyzed using confirmed by the analysis of the expression of pro- and anti- FlowJo software (Tree Star), and the gates were set using a fluorescence inflammatory cytokines/chemokines and macrophage markers in minus one (FMO) controls strategy. FMO controls are samples that include all conjugated Abs present in the test samples except one. The channel in the liver (Fig. 2F–I). Indeed, data shown in Fig. 2F–I demonstrated which the conjugated Ab is missing is the one for which the FMO provides a that, whereas exposure to APAP increased the expression of sev- gating control (29). The following mAbs were used: CD11b Alexa Fluor eral inflammatory markers, this pattern was amplified in mice 488 (M1/70; eBioscience), GR1 Brilliant Violet 510 (RB6-8C5; Bio- lacking Gpbar1 gene expression. Furthermore, although GPBAR1 Legend), CD49b allophycocyanin-eFluor 780 (DX5; eBioscience), CD3 agonism effectively reversed these changes in wild-type mice, it PerCP–Cyanine 5.5 (145-2C11; eBioscience), IFN-g eFluor 450 2/2 (XMG1.2; eBioscience), IL-10 Alexa Fluor 700 (JES5-16E3; eBio- was poorly effective/not effective in Gpbart1 mice (Fig. 2F–I). science), CD11c PE/Cy7 (N418; eBioscience), and CD206 allophy- Immunohistochemistry analysis with anti-CCL2 Ab on liver sec- cocyanin (MR6F3; eBioscience). tion (Fig. 3) highlighted a large increase in the concentration of CCL2 in the liver of Gpbar12/2 mice that was not reduced by Statistical analysis treatment with BAR501 (Fig. 3). Downloaded from The ANOVA followed by nonparametric Mann–Whitney U test or a two- To further dissect the mechanisms that support the anti- , tailed unpaired Student t test were used for statistical comparisons (*p inflammatory activity of BAR501 (Fig. 2), we then investigated 0.05) using the Prism 6.0 software (GraphPad). whether the GPBAR1 agonism impacts on the immune phenotype of the liver immune population by image construction–FACS Results analysis (Fig. 4, Supplemental Fig. 2). The count of the leuko- Gpbar1 gene deficiency exacerbates cytes infiltrating the liver demonstrated that exposure to APAP http://www.jimmunol.org/ acetaminophen-induced hepatitis effectively promotes a robust influx of immune cells in the liver In a preliminary experimental set shown in Fig. 1A–C, we first and that the deletion of the Gpbar1 gene exacerbates this effect investigated whether the liver damage caused by APAP was dos- (Fig. 4A). Treating APAP mice with BAR501 reduced the age dependent. Twenty-four-hour measurement of serum levels of number of infiltrating immune cells, but only in Gpbar1+/+ mice. aspartate aminotransferase (AST) and alanine aminotransferase The further characterization of immune cells subpopulations (ALT) in APAP-treated mice demonstrated a linear correlation indicated that the acute liver injury caused by APAP was asso- with the dosage of APAP administered. Exposure to APAP dos- ciated with recruitment of granulocytes (GR1+CD11b+), mac- ages of 500 and 750 mg/kg gave almost similar levels of AST/ rophages (GR12CD11b+), NKT cells (CD3+Cd49b+), and T cells ALT (Fig. 1A, 1B), but the dosage of 750 mg/kg induced a high (CD3+Cd49b2) (Fig. 4B). Only the NK cells showed no increase by guest on September 28, 2021 mortality rate (∼22%) (Fig. 1C). Thus, we have decided to carry in the number following the administration of APAP alone or in on the subsequent experiments using the intermediate dosage of combination with BAR501 (Fig. 4B). The number of all immune APAP (500 mg/kg). Similarly, we have carried out a dosage- cell subpopulations was decreased by the administration of finding study to determine the effective dosage of BAR501 that BAR501 in Gpbar1-dependent manner. protects against damage caused by 500 mg/kg APAP. The results Because T cells do not express GPBAR1 and their regulation is of this investigation demonstrated that 30 mg/kg BAR501 effec- bona fide indirect, we have characterized in detailed manner tively rescued from liver injury caused by APAP (Fig. 1D, 1E). macrophage and NKT cell subsets. Because several subsets of liver Thus, we have used this dosage in all the following investigations. macrophages have been identified with distinct phenotypes and To investigate whether liver toxicity was dependent on the functions, and M1 and M2 are considered only as the two ex- presence of GPBAR1, wild-type and Gpbar12/2 mice were ad- treme points of a varied population with high plasticity (13, 30–33), ministered 500 mg/kg APAP sacrificed at different time points (8, we have characterized the liver macrophages in more detail 24, 36, and 48 h). APAP administration induced a liver injury in using a flow cytometry approach. Results shown in Fig. 4C–G both Gpbar1+/+ and Gpbar12/2 mice with a different kinetic. In- and Supplemental Fig. 1A demonstrated that exposure to APAP deed, although damage caused by APAP peaked at 24 h in wild- increases the percentage of proinflammatory macrophages type mice, a very robust increase in AST/ALT plasma levels was (GR12CD11b+CD11c+CD2062) and that this finding was am- observed as early as 8 h in Gpbar12/2 mice (Fig. 1F). In general, plified in Gpbar12/2 mice (Fig. 4C, Supplemental Fig. 1A). Gpbar1 gene ablation resulted in higher levels of AST/ALT at each Of relevance, although exposure of wild-type mice to BAR501 time point (Fig. 1F). Furthermore, abnormal AST and ALT per- reduced the percentage of proinflammatory macrophages and sisted longer in Gpbar12/2, and significantly higher than normal increased the percentage of anti-inflammatory macrophages serum levels of AST and ALT were detected at 36 h after APAP (GR12CD11b+CD11c2CD206+) (Fig. 4C, Supplemental Fig. 1A), administration in these mice (Fig. 1F). These findings suggested these modulatory effects were lost in Gpbar12/2 mice. The ability that the maximum toxicity caused by APAP in both mice strains of BAR501 to promote the development of an anti-inflammatory occurred at 24 h. Accordingly, the rest of the experiments were phenotype of liver macrophages was also confirmed by the anal- performed using this time point. ysis of IL-10+ macrophages (Fig. 4D, 4E). The administration of To investigate the role of GPBAR1 in the pathogenesis of APAP- the APAP reduced the percentage of IL-10+ macrophages, but this induced hepatitis and in the beneficial effect of BAR501, wild-type effect was counteracted by the administration of the BAR501 in and Gpbar12/2 mice on C57Bl6 background were administered GPBAR1-dependent manner (Fig. 4D, 4E). Similar results were with 500 mg/kg APAP alone or in combination with 30 mg/kg derived from analysis of pro- and anti-inflammatory NKT cell BAR501. AST and ALT values demonstrated that Gpbar1 ablation subsets (Fig. 4F, 4G). Thus, although exposure to APAP increased exacerbated hepatic damage and prevented the beneficial effect of the percentage of INF-g+ NKT cells and reduced the frequency of The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. BAR501 reduces the severity of APAP-induced hepatitis in a dosage-dependent manner. Liver injury was induced in C57BL/6NCrl mice through o.s. of APAP at dosages of 150, 300, 500, and 750 mg/kg alone or in combination with BAR501 (5, 15, or 30 mg/kg). BAR501 was administered in a therapeutic manner 45 min after APAP. (A–C) Serum levels of (A) AST and (B) ALT and percentage of surviving mice (C). Serum levels of (D) AST and (E) ALT in mice administered with 500 mg/kg APAP alone or in combination with various dosages of BAR501 (5, 15, or 30 mg/kg). Liver damage was induced in Gpbar1+/+ and Gpbar12/2 mice by o.s. of 500 mg/kg APAP. (F) Serum levels of AST and ALT after 8, 24, 36, and 48 h from APAP administration. Results are the mean 6 SEM of 6–10 mice per group. *p , 0.05.

IL-10+ NKT cells (Fig. 4F, 4G), BAR501 reversed this pattern but and, 24 h after, we administered 500 mg/kg APAP by o.s. Mice only in Gpbar1+/+ mice (Fig. 4F, 4G). Furthermore, we found that were sacrificed 24 h after hepatitis induction (Fig. 5). AST and APAP administration reduced the percentage of IL-10+ T cells, and ALT values and histopathology analysis demonstrated that mac- this effect was reversed by BAR501 in wild-type mice (Supplemental rophage removal by GaCl3 protected mice from development of Fig.1B,1C).Gpbar12/2 mice had a significantly lower percentage APAP-induced hepatitis, thereby confirming the central pathoge- of IL-10+ T cells compared with wild-type mice, and treatment with netic role exerted by these cells in the model (Fig. 5A, 5B). BAR501 had no further effect (Supplemental Fig. 1B, 1C). The To tighten the role of GPBAR1 to the macrophage subsets, percentage of leukocyte subpopulations detected in the liver and the we then carried out a cell transfer experiment using spleen- gating strategy are shown in Supplemental Fig. 1D, 1E. purified macrophages obtained from Gpbar1+/+ and Gpbar12/2 Because many cytokines/chemokines modulated by APAP and mice (Fig. 5C–F, Supplemental Fig. 3). For these purposes, recip- BAR501 are largely placed at the interface between parenchymal ient wild-type C57BL/6N mice were treated with GaCl3 to deplete and nonparenchymal liver cells, and, more specifically, sinusoidal the macrophages, and 24 h later, were transferred with 1 3 107 cells and Kupffer/macrophage cells, we then investigated spleen-derived macrophages administered i.v. One hour later, whether macrophage removal by gadolinium (GaCl3) protected APAP was administered by oral gavage. As illustrated in Fig. 5C, mice from development of liver injury caused by APAP (24–26). AST and ALT and histopathology analysis of livers from recipient For this purpose, mice were pretreated with 10 mg/kg GaCl3 i.v. mice (Supplemental Fig. 2) confirmed that GaCl3 protected from 6 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Gpbar1 deficiency exacerbates APAP-induced DILI and abrogates the beneficial effects of BAR501. Liver damage was induced in Gpbar1+/+ and Gpbar12/2 mice by o.s. of 500 mg/kg APAP alone or in combination with BAR501 (30 mg/kg) 45 min after APAP administration. Serum levels of (A) AST and (B)ALT;(C) H&E staining on mice liver tissues (original magnification 34); hepatic levels of (D) GSH; (E) hepatic SOD activity. Each value represents the mean 6 SEM of five to eight animals per group. *p , 0.05. Relative mRNA expression levels of (F) Ifn-g, Il-6, Tnf-a, and Il-10; (G) macrophagic markers Cd11b, Cd38, and c-myc; (H) chemokine genes Ccl2, Ccl3, Ccl5, Cxcl7, Cxcl4, and Cxcl2; and (I) chemokine receptors Ccr1 and Ccr2 in total liver. The data are normalized to Gapdh mRNA. Results are the mean 6 SEM of 6–10 mice per group. *p , 0.05. The Journal of Immunology 7

Fig. 6G that encompassed a set of transcripts that were modulated by APAP in comparison with both naive and APAP-and-BAR501– treated mice (Mendeley Data, v1; http://dx.doi.org/10.17632/ dc2w3y2z47.1). Most of transcripts detected in this subset were inversely modulated by APAP and BAR501, confirming that the GPBAR1 agonism reversed the effect of APAP. The pathways analysis carried out by Transcriptome Analysis Console (TAC) software (Mendeley Data, v1; http://dx.doi.org/10.17632/dc2w3y2z47.1) demonstrated that exposure to APAP effectively regulated the expression of a number of genes in immunological and inflammatory pathways (Table I), including chemokines signaling pathways, integrin-mediated cell adhesion, and TNF-a–NF-kB signaling pathways (Fig. 6H). Conversely, BAR501 reversed the proinflammatory phenotype, downregulating all the genes involved in these pathways (Fig. 6H). Because previous studies have shown the involvement of Ccr2 and its ligand, Ccl2, in mediating liver damage caused by APAP (23), we have focused our analysis on the chemokine

pathways. As shown in Table II, exposure to APAP upregu- Downloaded from lated 10 genes of this pathway, including genes encoding for the chemokines Ccl2 (7.44-fold change versus mice not treated [NT]), Ccl5 (4.5-fold change versus NT), Cxcl7 (3.79-fold change versus NT), Ccl3 (3.28-fold change versus NT), Cxcl2 (2.97-fold change versus NT), and Cxcl4 (2.37-fold change

versus NT) and the receptors Ccr1 (2.32-fold change versus http://www.jimmunol.org/ NT) and Ccr2 (2.1-fold change versus NT). Treating mice with BAR501 completely reversed this pattern, reducing the ex- FIGURE 3. The absence of GPBAR1 exacerbates the expression of pression of all genes included in this pathway, in particular, CCL2 in the liver of mice after APAP administration. Hepatitis was in- Ccl2 (212.64-fold change versus APAP) and Ccr2 (23.64- +/+ 2/2 duced in Gpbar1 and Gpbar1 mice through o.s. of acetaminophen fold change versus APAP), confirming the hierarchic relevance (APAP) at 500 mg/kg alone or in combination with BAR501 (30 mg/kg) of these genes in the pathogenesis of liver injury caused by 45 min after APAP administration. The figure shows immunohistochem- APAP. Additionally, the analysis of metabolic and survival istry representative images of the liver of one mouse for each experimental pathways (Supplemental Fig. 3C, 3D) demonstrated that APAP group stained with anti-CCL2 Ab (original magnification 34, inset 310). and BAR501 inversely modulated the expression of several by guest on September 28, 2021 genes in these pathways. liver damage caused by APAP. However, macrophage transfer reinduced the disease, confirming the central role of these cells in CCL2 is involved in the pathogenesis of acute the pathogenesis of APAP-induced hepatitis. The biochemical data APAP-induced hepatitis combined with the analysis of cytokine expression by RT-PCR Because the data obtained from RT-PCR analyses (Fig. 2H, 2I) and (Fig. 5D–F) demonstrated that, in comparison with wild-type RNA-seq (Fig. 6G, 6H) highlighted a therapeutic potential for mice, the transfer of Gpbar12/2 macrophages resulted in very CCL2 (23), we have decided to further investigate the role of aggressive disease. CCL2 neutralization in this model. For this purpose, wild-type and Together, these data support a regulatory role for GPBAR1 in Gpbar12/2 mice were administered with an anti-CCL2–neutral- macrophage cells in the liver. To better understand the mechanisms izing Ab 1 h before the induction of hepatitis by APAP. The data involved in the pathogenesis of APAP-induced acute hepatitis and shown in Fig. 7 demonstrated that pretreatment with anti-CCL2 to investigate the genes modulated by GPBAR1 following the Ab prevents the development of APAP-induced hepatitis and ef- administration of BAR501, we performed a new experimental set fectively reduced AST and ALT values (Fig. 7A) and ameliorated on wild-type mice with RNA-seq analysis on the liver. AST and the liver histopathology (Fig. 7B, 7C). Moreover, the anti-CCL2 ALT levels (Fig. 6A), liver index and histopathology analysis Ab reduced the expression of proinflammatory cytokines Tnf-a, Il- (Fig. 6B, 6C), and the expression of several genes involved in liver 1b and Il-6, and Cd11b, a marker of macrophages (Fig. 6D–G). inflammation (Fig. 6D–F) confirmed the data obtained in wild- The effect of pretreatment with anti-CCL2 Ab in alleviating type mice in the first experiments (Fig. 2). APAP-induced hepatitis was lower than that exerted by BAR501, Therefore, to gain insights on the pathogenesis and on the indicating that the downregulation of CCL2 represents only one of therapeutic role of GPBAR1 agonism in this model, total RNA the mechanisms by which GPBAR1 agonism acts, as suggested by extracted from the livers of each group of mice were subjected to the many other genes whose expression was modulated in the RNA-seq analysis. As illustrated in Fig. 6G and 6H and Supplemental RNA-seq analysis (Fig. 6). We had already shown in 2017 that, for Fig. 3A and 3B, exposure to APAP modulated a wide set of genes: example, Gpbar1 positively modulates the expression of Il-10 in up to 1725 gene transcripts resulted in differential expression in macrophages in a mouse model of colitis (19). However, the the livers of APAP-treated mice compared with control (naive) protective effect of anti-CCL2 Ab was also maintained in mice (fold change # 22 and $ +2). As shown by Venn diagram Gpbar12/2 mice, although it was partially blunted compared with analysis, treating mice with BAR501 reshaped the landscape of that of Gpbar1+/+. gene transcripts, resulting in 1642 differentially expressed tran- Protection exerted by GPBAR1 agonism extended to other scripts in comparison with APAP-treated mice (Fig. 6G). By Venn models of acute liver injury. Thus, acute hepatitis caused by treating diagram analysis, we isolated a gene subset indicated as AB in wild-type and Gpbar12/2 mice with Con A was reversed by 8 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. BAR501 reshapes the polarization of the liver leukocytes. Liver damage was induced in Gpbar1+/+ and Gpbar12/2 mice by o.s. of APAP at 500 mg/kg alone or in combination with 30 mg/kg BAR501 45 min after APAP administration. Liver infiltrating cells were characterized by image construction–FACS analysis. Data shown are numbers of total leukocyte cells obtained from liver of mice of each experimental groups (A) and number of various immune cell subpopulations: granulocytes (GR1+CD11b+), macrophages (GR12CD11b+), NK cells (CD32CD49b+), NKT cells (CD3+Cd49b+), and T cells (CD3+Cd49b2)(B). Characterization of proinflammatory (CD11c+CD2062) and anti-inflammatory (CD11c2CD206+) macrophages (C). Characterization of IL-10+ macrophages (D) and flow cytometry analysis of IL-10 expression in macrophages recruited into the liver (E). Characterization of proinflammatory (INF-g+) and anti-inflammatory (IL-10+) NKT cells (F) and flow cytometry analysis of INF-g expression and IL-10 expression in NKT cells recruited into the liver (G). Results are the mean 6 SEM of 6–10 mice per group. *p , 0.05. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. The absence of GPBAR1 from macrophage cells is sufficient to aggravate APAP-induced hepatitis. Liver macrophages were depleted by treating C57BL6 mice with GaCl3 at a dosage of 10 mg/kg i.p. Mice were then treated with 500 mg/kg APAP o.s. 24 h later. Serum levels of (A) AST and ALT 24 h after APAP administration and H&E staining on mice liver tissues (original magnification 34) (B). In another experimental set, 24 h after administration of gadolinium, we injected i.v. 1 3 107 macrophages purified from spleen of naive Gpbar1+/+ or Gpbar12/2 mice and 1 h after we administered 500 mg/kg APAP by o.s. Serum levels of (C) AST and ALT 24 h after APAP administration. Relative mRNA expression levels of proinflammatory cytokines Il-1b, Il-6, and Tnf-a; anti-inflammatory cytokine Il-10 and chemokine Ccl2 and chemokine receptor Ccr2 on total liver (D). Relative mRNA expression levels of genes involved in inflammatory pathways on macrophage cells (E) and nonmacrophage immune cells (F) purified from liver of mice from each group. The data are normalized to Gapdh mRNA. Results are the mean 6 SEM of six to eight mice per group. *p , 0.05.

BAR501 in a Gpbar1-dependent manner, as demonstrated previ- microenvironment. For this purpose, we have developed an in vitro ously (28) (Supplemental Fig. 4). system to mimic the interface of the hepatic sinusoidal cell/ macrophage (Fig. 8A). Brieﬂy, using Transwell Plate (0.4 mm), BAR501 exerts a protective role at sinusoidal we have cocultured human LSEC, seeded on the bottom level, and cell/macrophage interface human monocytes, THP1, plated on top of the insert. Both cell Because hepatocytes do not express GPBAR1, we have investi- populations were stimulated with conditioned medium from gated the involvement of nonparenchymal cells found in the liver HepG2 cells treated with APAP (10 mM) alone or in combination 10 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. RNA-seq analysis of the effects of APAP and BAR501 in the liver. Liver damage was induced in C57BL6 mice by o.s. of 500 mg/kg APAP alone or in combination with BAR501 (30 mg/kg) 45 min after APAP administration. Serum levels of (A) AST and ALT; (B) liver index (C) H&E staining on mice liver tissues (original magnification 34); relative mRNA expression levels of (D) proinflammatory genes Tnf-a, Il-1b, Il-6, and Ifn-g;(E) anti- inflammatory genes Il-10 and Tgf-b;(F) phenotype macrophagic markers c-myc and Cd38; and (G) T regulatory (Figure legend continues) The Journal of Immunology 11

Table I. Immunological pathway analysis Downloaded from http://www.jimmunol.org/

Total RNA extracted from the livers of mice of each experimental group (NT, APAP, and APAP and BAR501 [APAP+BAR501]) were subjected to RNA-seq analysis. Pathway expression analysis was carried out by TAC software. with BAR501 (20 mM). We have therefore recreated the hepatic Conversely, treating cells with BAR501 completely reversed this microenvironment in vitro. The results shown in Fig. 8 demon- pattern (Fig. 9A). strated that exposure to HepG2-conditioned medium induced the These findings were reproduced by using liver-derived Kupffer/ by guest on September 28, 2021 expression of CCL2 both in LSEC and in THP1 cells (Fig. 8B, macrophage cells isolated from both Gpbar1+/+ and Gpbar12/2 8C). Furthermore, APAP increased the expression of adhesion mice. Thus, although exposure of these cells to supernatants molecules, such as VCAM1, ICAM1, and E-SELECTIN, in harvested from APAP-primed HepG2 resulted in a robust induc- LSEC (Fig. 8B); meanwhile, in THP1 monocytic cells, APAP tion in the expression of proinflammatory markers (Il-1b, Il-6, upregulated proinflammatory cytokine levels (IL-6, TNF-a,and Tnf-a, Ccl2, and Ccr2), treating HepG2 cells with BAR501 re- IL-1b) and the expression of CCL2, CXCL2 chemokine, and versed this pattern but only in Kupffer cells derived from Gpbar1 CCR2 chemokine receptors (Fig. 8C). APAP also downregulated wild-type mice (Fig. 9B). Additionally, in this setting, BAR501 the expression of anti-inflammatory cytokine IL-10. Treating reversed the negative regulation of IL-10 gene expression caused cells with BAR501 completely reversed these patterns both in by APAP (Fig. 9B). LSEC and THP1 cells (Fig. 8B, 8C), showing the protective role Previous studies have shown that the transcription factor FOXO1 of GPBAR1 agonism at sinusoidal cell/macrophage interface in modulates inflammation through regulation of a number of genes, the liver. These findings were confirmed in adhesion in vitro including Il-1b, Ccr2, and Ccl2 (34, 35). Furthermore, we have assay using THP1 and LSEC cells, both stimulated with condi- previously demonstrated that GPBAR1 activation results in a tioned medium harvested from HepG2 cells treated with APAP phosphorylation of Akt and FOXO1 proteins, leading to a down- (10 mM) alone or in combination with BAR501 (20 mM). As regulation of FOXO-1–downstream genes (22). To elaborate on shown in Fig. 8B, supernatants from APAP-primed HepG2 putative mechanisms by which GPBAR1 agonism counter- promoted THP1 adhesion to LSEC. This effect was robustly regulates cytokine/chemokine generation in macrophages, attenuated by cotreating LSEC/THP1 coculture with BAR501 Raw264.7, a murine macrophage cell line, was used. In these (Fig. 8D). experiments, we first verified that exposure of Raw264.7 to A similar counterregulatory effect of BAR501 was observed BAR501 reproduced the same effects observed using human using HUKCCS cells. As shown in Fig. 9A, HUKCCS cells ex- Kupffer cells and primary liver mouse macrophages. Indeed, as posed to conditioned medium harvested from APAP-primed shown in Fig. 10A, 10B, although exposure to APAP induced the HepG2 cells exhibited an increased expression of proin- expression of various cytokines and chemokines, including Ccl2, flammatory genes (IL-6, CCL2, and CCR2) as well as a strong and reduced the expression of Il-10, these effects were reversed by reduction of expression of anti-inflammatory genes, such as IL-10. BAR501.

(T-reg) marker Foxp3. The data are normalized to Gapdh mRNA. Results are the mean 6 SEM of ﬁve to eight mice per group. *p , 0.05. (G) Venn diagram of differentially expressed genes showing the overlapping regions (identiﬁed as A, AB, and B sets) between the three experimental groups of mice (fold change ,22or. 2). p , 0.05. (H) Pathway analysis on subset of genes included in region AB of the corresponding Venn diagram. 12 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY

Table II. Gene involved in chemokine signaling pathway

Total RNA extracted from the livers of mice of each experimental group (NT, APAP, and APAP and BAR501 [APAP+501]) were subjected to RNA-seq analysis. Pathway expression analysis was carried out by TAC software. Downloaded from

As shown in Fig. 10C, 10D, although exposure of Raw264.7 to In the current study, we have provided evidence that GPBAR1, a APAP impaired the Akt and FOXO1 phosphorylation, treating bile acid receptor for secondary bile acids, is an essential com- these cells with BAR501 reversed this pattern (Fig. 10C, 10D). ponent of the gate-keeper system that governs the maintenance of a Finally, to investigate whether the GPBAR1/Akt/FOXO1 axis was tolerogenic state in the liver and could be exploited for therapeutic

involved in the direct regulation of chemokine/chemokine recep- purposes, as shown by results obtained in a mouse model of APAP- http://www.jimmunol.org/ tor transcription, we have performed a ChIP assay on the pro- induced DILI. GPBAR1 is a cell membrane receptor belonging to moters of Ccl2 and its receptor, Ccr2, in Raw264.7 cells treated the functional family of bile acid-activated receptors, which in- with a conditioned medium harvested from APAP-primed HepG2. cludes nuclear receptors such as the farnesoid X receptor (FXR), Results shown in Fig. 10E, 10F demonstrated that although the main bile acid sensor, and other GPCRs such as the sphingosine treating Raw264.7 cells with this medium increased the recruit- 1-phosphate (36, 37). In contrast to FXR, which is activated by ment of FOXO1 on both Ccr2 and Ccl2 promoters, the effect was primary bile acids and whose expression is higher in hepatocytes, reversed by BAR501. GPBAR1 is expressed by LSEC, Kupffer cells, and cells of innate immunity, including NKT cells and MoMFs, but not by hepatic Discussion parenchymal cells (38). Previous studies have shown that by guest on September 28, 2021 APAP is the active ingredient of some of the most prescribed and GPBAR1 activation regulates macrophage/endothelial cell inter- over-the-counter anti-inflammatory and analgesic medicines actions in the systemic (39) and liver microcirculation (38) by a worldwide. In Western countries, its extensive use has emerged as mechanism that involves an AKT-dependent phosphorylation of one of the major causes of DILI, a spectrum of liver disorders endothelial NO synthase. ranging from self-resolving inflammation to ALF, a life-threatening In addition, to these nongenomic effects, GPBAR1 genomic condition that requires liver transplantation in ∼30% of patients. activities are mediated, at least in part, by the recruitment of cAMP- The spectrum of liver disorders caused by APAP is largely related response element (CRE)–binding proteins (CREBs) to CREs at the to the amount of drug to which the liver is exposed, but it is well promoter of target genes (19, 28). In LSEC, this leads to an en- established that dosages exceeding 10–12 g/d, taken usually in- hanced transcription of endothelial NO synthase and cys- tentionally for a suicidal scope, cause a massive centrilobular liver tathionine-g-lyase (CSE), two genes encoding for enzymes whose necrosis associated with a high rate of mortality. metabolic products, NO and hydrogen sulfide (H2S), exert robust Animal studies have revealed that, in addition to toxic metab- antiadhesive effects at the endothelium/macrophage interface (22). olites, an essential component of the APAP-damaging mecha- Because the genetic ablation of GPBAR1 results in a proin- nism(s) is provided by the involvement of the innate immune flammatory phenotype, this receptor is generally considered as one system, especially macrophages that are recruited into the liver of the components of the counterregulatory system that promotes microcirculation by chemoattractant gradients generated by the development of a tolerogenic phenotype in enterohepatic tis- damaged hepatocytes (11, 12). It is now relatively well established sues (18, 19, 28, 40). Indeed, as confirmed in this study, GPBAR1 that macrophages recruited in the liver following an injury are gene–ablated mice are more disease prone than congenic litter- derived essentially from two sources (i.e., liver-resident macro- mates and react to proinflammatory settings with an enhanced phages, Kupffer cells, and bone marrow–derived monocytes). recruitment of proinflammatory cells, including granulocytes, During acute liver injury, necrotic hepatocytes activate the Kupffer MoMFs, NKT cells, and T cells. In the current study, we have cells (and other nonparenchymal cells), which release chemokines confirmed that GPBAR1 agonism functions as a counterregulatory recruiting monocytes into the liver microcirculation. These cells signal for several inflammatory and immune cell subsets. How- will then differentiate into MoMFs. The MoMFs contribute to the ever, because GPBAR1 expression is restricted to cells of myeloid development of the liver injury but, at later stage, provide support origin and lymphoid cells lack the receptor, their modulation to liver repair, having both anti-inflammatory and prorestorative is indirect, as strongly demonstrated by results obtained in polarization in a time-resolved manner (30, 33). This established a macrophage-depleted animals. In aggregate, results obtained in continuum of cell MoMF phenotypes, with M1-like and M2-like this model of APAP-induced DILI highlight the concept that al- macrophages representing the two extreme points of this spectrum though MoMFs are essential to produce liver damage, GPBAR1 (13, 30–33). provides an essential component of the counterregulatory system The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Anti-CCL2 treatment attenuates acute liver injury caused by APAP. Gpbar1+/+ and Gpbar12/2 male mice were pretreated with anti-CCL2 or anti-IgG Ab (as a negative control) 200 mg/mice by i.p. 1 h before hepatitis induction. Then, the liver injury was induced by the administration of APAP at the concentration of 500 mg/kg o.s., and the mice were sacrificed 24 h later. Serum levels of AST and ALT (A); H&E staining on mice liver tissues (original magnification 34) (B) and evaluation of the area of necrosis (C); relative mRNA expression levels of proinflammatory genes Tnf-a (D), Il-1b (E), Il-6 (F), and macrophagic markers Cd11b (G). The data are normalized to Gadph mRNA. Results are the mean 6 SEM of five to eight mice per group. *p , 0.05. 14 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. BAR501 exerts a protective role at sinusoidal cell/macrophage interface. (A) THP1, LSEC cells, and conditioned medium from HepG2 treated with APAP were used to perform a coculture experiment using 12-well Carrier Plate with Cell Culture Inserts 0.4 mM (no. 141078; Nunc) as described in Materials and Methods section. (B) Relative mRNA expression levels of chemokine gene CCL2 and adhesion molecules VCAM, E-SELE, and ICAM1 in LSEC cells; (C) relative mRNA expression levels of cytokines (IL-6, IL-1b, TNF-a, and IL-10) and chemokines (CCL2, CCR2, and CXCL2) in THP1 cells. (D) THP1 adhesion to LSEC cells primed with conditioned medium of HepG2 cells treated with APAP with or without addition of BAR501 on THP1 and LSEC. The data are normalized to GAPDH mRNA (n = 3). *p , 0.05. that regulates immune cell activation at the endothelial cell/MoMF in rodent models of APAP (23, 41) and anti-CCL2/CCR2 strate- interface. By RT-PCR and RNA-seq analyses, we have found that gies, including the CCR2/CCR5 dual antagonist cenicriviroc, GPBAR1 agonism negatively regulates the expression of a number are currently actively investigated for their beneficial effects in of cytokines, chemokines, and their receptors, including Ccl2 and treating nonalcoholic steatohepatitis, we have focused our atten- Ccr2, both of which are highly represented by liver endothelial tion on this chemokine and found that GPBAR1 is a potent reg- cells and MoMFs. CCL2, also known as MCP-1, is a potent mac- ulator of the CCL2/CCR2 pathway. Thus, not only were the liver rophage activator, and its receptor, CCR2, which is highly expressed expressions of CCL2/CCR2, mRNAs, and protein (immunohis- by MoMFs and in the liver by Kupffer cells, is a potent driver of tochemistry) induced by APAP, but APAP was robustly enhanced macrophage recruitment/activation. In the liver, CCL2 is mainly by Gpbar1 gene ablation; treating mice with BAR501 reversed this generated by LSEC and Kupffer cells, and we have shown that its upregulation but only in mice with an intact receptor. However, it expression increases dramatically in response to APAP overdosing has to be noted that although treating mice with anti-CCL2 mAb but is robustly mitigated by BAR501, a selective GPBAR1 ligand. fully reversed the disease progression in wild-type mice, its efficacy Because genetic and pharmacological investigations have shown was partially blunted by Gpbar1 gene knockout. Together, these that CCL2/CCR2-based therapies effectively attenuate liver injury findings establish that regulation of the CCL2/CCR2 system is a The Journal of Immunology 15 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 9. BAR501 reversed the effect of APAP in both human and murine primary Kupffer/macrophage cells. (A) Human Kupffer cells, HUKCCS, were plated in 24-well plate coated with collagen type I substrate (0.1 3 106 per well) with Kupffer Monoculture Medium. After 24 h, cells were primed with conditioned medium from HepG2 treated with APAP alone or in combination with BAR501 (20 mM) for 16 h. (A) Relative mRNA expression levels of proinflammatory cytokine IL-6 and chemokine CCL2 and chemokine receptor CCR2 and anti-inflammatory cytokine IL-10 in HUKCCS cells. The data are normalized to GAPDH mRNA (n = 3). *p , 0.05. (B) Kupffer/macrophage cells were purified from Gpbar1+/+ and Gpbar12/2 mice liver, plated in 10-mm plate (3 3 106 cells per plate), and after 2 h, required for cell adhesion, were primed with conditioned medium from HepG2 treated with APAP alone or in combination with BAR501 (20 mM) for 16 h. (B) Relative mRNA expression levels of pro- and anti-inflammatory cytokines (Il-6, Tnf-a, Il-1b, and Il-10) and chemokines and chemokine receptors (Ccr1 and Ccr2). The data are normalized to Gapdh mRNA (n = 3). *p , 0.05. major mechanism involved in the anti-inflammatory and immuno- APAP. The results of these experiments demonstrated that super- modulatory effects of a GPBAR1 ligand, although it is not unique. natants obtained from HepG2 cells challenged with APAP in- We have further dissected interactions of GPBAR1 in liver creased the expression of CCL2 in the LSEC and CCL2 and nonparenchymal cells in vitro using a coculture system of human CCR2, along with IL-6 and TNF-a and adhesion molecules in LSEC and THP1, a human monocytes cell line. In these experi- THP1. GPBAR1 activation by BAR501 effectively reversed these ments, cocultures were challenged with supernatants from HepG2 patterns and reduced cell adhesion, firmly establishing that the cells, a human hepatocellular carcinoma cell line, and exposed to LSEC/macrophage interface is the likely target for the receptor. 16 GPBAR1 IN ACETAMINOPHEN-INDUCED LIVER TOXICITY Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 10. BAR501 counterregulates mouse–macrophage activation induced by APAP in vitro. Raw264.7 cells were plated in T75 flask (1.5 3 106 per flask), starved overnight in 0.5% FBS-containing medium, and then primed with conditioned medium from HepG2 treated with APAP alone or in combination with BAR501 (20 mM) for 16 h. Relative mRNA expression levels of (A) pro- and anti-inflammatory cytokines (Il-6, Tnf-a, Il-1b, and Il-10) and (B) chemokines and chemokine receptors (Ccr1, Ccr2, Ccl2, Ccl3, and Cxcl2). The data are normalized to Gapdh mRNA (n = 3). *p , 0.05. (C and D) Representative Western blots and densitometric analysis demonstrating phospho-Akt and phospho-FOXO1 in comparison with total Akt and FOXO1 in Raw264.7 cells. Data are presented as mean 6 SE of (C) phospho-Akt relative to Akt and (D) phospho-FOXO1 to FOXO1. GAPDH served as loading control. The blot shown is representative of another one showing the same pattern. *p , 0.05. ChIP assay of the Foxo1 binding on (E) Ccr2 and (F) Ccl2 promoters in Raw264.7 cells. Cell lysates were immunoprecipitated with anti-Foxo1 or control IgG, and the presence of specific regions in the immu- noprecipitates was determined by RT-PCR (n = 3). *p , 0.05. The Journal of Immunology 17

The molecular mechanisms that support the regulation of CCL2 16. Fiorucci, S., S. Cipriani, A. Mencarelli, B. Renga, E. Distrutti, and F. Baldelli. 2010. Counter-regulatory role of bile acid activated receptors in immunity and and CCR2 by GPBAR1 agonism were further investigated in inflammation. Curr. Mol. Med. 10: 579–595. Raw264.7 cells, a murine macrophage cell line. In this setting, 17. Keitel, V., R. Reinehr, P. Gatsios, C. Rupprecht, B. Go¨rg, O. Selbach, exposure to BAR501 prevented the recruitment of FOXO1 to the D. Haüssinger, and R. Kubitz. 2007. The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells. Hepatology 45: 695–704. binding sequence located in the promoter of both Ccl2 and Ccr2, 18. Fiorucci, S., M. Biagioli, A. Zampella, and E. Distrutti. 2018. Bile acids acti- thereby reducing the transcription of these two genes. These vated receptors regulate innate immunity. Front. Immunol. 9: 1853. findings are consistent with observation that BAR501, in liver 19. Biagioli, M., A. Carino, S. Cipriani, D. Francisci, S. Marchiano`, P. Scarpelli, D. Sorcini, A. Zampella, and S. Fiorucci. 2017. The bile acid receptor GPBAR1 endothelial cells, phosphorylates FOXO1 in an Akt-dependent regulates the M1/M2 phenotype of intestinal macrophages and activation of manner and causes its release from the endothelin-1 promoter, GPBAR1 rescues mice from murine colitis. J. Immunol. 199: 718–733. 20. Finamore, C., C. Festa, B. Renga, V. Sepe, A. Carino, D. Masullo, M. Biagioli, thus blocking the transcription of this protein (42). S. Marchiano`, A. Capolupo, M. C. Monti, et al. 2016. Navigation in bile acid In addition to modulation of CCL2/CCR2, BAR501 enhances chemical space: discovery of novel FXR and GPBAR1 ligands. Sci. Rep. 6: 29320. IL-10 gene expression in the whole liver and MoMFs, counter- 21. Festa, C., B. Renga, C. D’Amore, V. Sepe, C. Finamore, S. De Marino, A. Carino, S. Cipriani, M. C. Monti, A. Zampella, and S. Fiorucci. 2014. Ex- acting the negative effects exerted by APAP on this cytokine. ploitation of cholane scaffold for the discovery of potent and selective farnesoid Despite previous studies that have shown that IL-10 might exert X receptor (FXR) and G-protein coupled bile acid receptor 1 (GP-BAR1) beneficial effects in rodent models of APAP-induced toxicity, we ligands. J. Med. Chem. 57: 8477–8495. 22. Renga, B., S. Cipriani, A. Carino, M. Simonetti, A. Zampella, and S. Fiorucci. have been unable to observe an exacerbation of the severity of the 2015. Reversal of endothelial dysfunction by GPBAR1 agonism in portal hy- 2 2 disease in IL-10 / mice (data not shown), which suggests that pertension involves a AKT/FOXOA1 dependent regulation of H2S generation and endothelin-1. PLoS One 10: e0141082. regulation of this cytokine by BAR501 is an ancillary mechanism 23. Mossanen, J. C., O. Krenkel, C. Ergen, O. Govaere, A. Liepelt, T. Puengel, in comparison with the CCL2/CCR2 mechanism. F. Heymann, S. Kalthoff, E. Lefebvre, D. Eulberg, et al. 2016. Chemokine (C-C Downloaded from In summary, by using genetic and pharmacological approaches, motif) receptor 2-positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury. Hepatology 64: 1667–1682. we have validated the concept that GPBAR1 agonism exerts 24. Pendino, K. J., T. M. Meidhof, D. E. Heck, J. D. Laskin, and D. L. Laskin. 1995. beneficial effects when administered in a therapeutic manner in a Inhibition of macrophages with gadolinium chloride abrogates ozone-induced mouse model of APAP-induced DILI. Further on, we have shown pulmonary injury and inflammatory mediator production. Am. J. Respir. Cell Mol. Biol. 13: 125–132. that GPBAR1 exerts counterregulatory effects at the liver endo- 25. Laskin, D. L., C. R. Gardner, V. F. Price, and D. J. Jollow. 1995. Modulation of thelial cell/MoMF interface by exerting a promoter-dependent macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. http://www.jimmunol.org/ Hepatology 21: 1045–1050. regulation of CCL2/CCR2 transcription. Present results provide 26. Michael, S. L., N. R. Pumford, P. R. Mayeux, M. R. Niesman, and J. A. Hinson. a support for the development of GPBAR1-based strategies in the 1999. Pretreatment of mice with macrophage inactivators decreases acetamin- treatment of DILI. ophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology 30: 186–195. 27. Biagioli, M., A. Carino, C. Fiorucci, G. Annunziato, S. Marchiano`, M. Bordoni, Disclosures R. Roselli, C. D. Giorgio, F. Castiglione, P. Ricci, et al. 2019. The aryl hydro- carbon receptor (AhR) mediates the counter-regulatory effects of pelargonidins The authors have no financial conflicts of interest. in models of inflammation and metabolic dysfunctions. Nutrients 11: E1820. 28. Biagioli, M., A. Carino, C. Fiorucci, S. Marchiano`, C. Di Giorgio, R. Roselli, M. Magro, E. Distrutti, O. Bereshchenko, P. Scarpelli, et al. 2019. GPBAR1

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39.7 45.9 36.8

6.91 9.19 12.0

42.9 48.0 49.3

CD11c 5.43 6.94 7.09

CD206

C D

6 * 45 Granulocytes * T cells IL-10+ Macrophages * * 40 - NK cells 4 35 NKT cells 30

CD49b T cells * * + 20

2 % of T %cells of T

CD3 * % of live cells live of % 15 * * 10 * * * 5 0 0 APAP - + + - + + APAP - + + - + + - + + - + + - + + - + + - + + - + + - + + - + + BAR501 - - + - - + BAR501 - - + - - + - - + - - + - - + - - + - - + - - + - - + - - + Gpbar1+/+ Gpbar1-/- Gpbar1+/+ Gpbar1-/- Gpbar1+/+ Gpbar1-/- Gpbar1+/+ Gpbar1-/- Gpbar1+/+ Gpbar1-/- Gpbar1+/+ Gpbar1-/-

CD49b

Figure S1. IC-FACS analysis of liver infiltrating immune cells. Hepatitis was induced in Gpbar1+/+ and Gpbar1-/- mice through oral administration (o.s.) of acetaminophen (APAP) at 500 mg/kg alone or in combination with BAR501 (30 mg/kg), 45 minutes after APAP administration. Flow cytometry analysis of CD11c expression and CD206 expression in macrophages cells recruited into the liver (A).Data showed flow cytometry analysis and percentage of anti-inflammatory IL-10+ T cells (CD3+CD49b-IL-10+) (B-C) and percentage of various immune cells subpopulation: granulocytes (GR1+CD11b+), macrophages (GR1-CD11b+), NK cells (CD3-CD49b+), NKT cells (CD3+Cd49b+) and T cells (CD3+Cd49b-) (D). Results are the mean ± SEM of 6-10 mice per group. *p<0.05. (E) IC-FACS gating strategy. 4x

NT 10x

APAP 10x

GaCl + APAP 3 10x

GaCl3 + APAP + Mφ Gpbar1+/+ 10x

GaCl3 + APAP + Mφ Gpbar1-/- 10x

Figure S2. The absence of GPBAR1 from macrophage cells aggravates the histological damage induced by APAP. C57BL6 mice were pretreated with gadolinium (GaCl3) at a dose of 10 mg/kg i.p. 24 hours after administration of gadolinium we injected intravenously 107 macrophages purified from spleen of naive Gpbar1+/+ or Gpbar1-/- mice and one hour after we administered APAP 500 mg/kg o.s. Hematoxylin and eosin (H&E) staining on mice liver tissues of each experimental groups photographed at 4x and 10x.

A B

(log2)

Avg

APAP Avg (log2) Avg APAP APAP + BAR501 BAR501 + APAP

NT Avg (log2) APAP Avg (log2)

C D

Figure S3. RNAseq analysis of the effects of APAP and BAR501 in the liver. (A, B) Scatter plots of transcript expression data in each group of mice. Analysis of (C) metabolic pathways and (D) survival pathways. 20000 * 20000 * * * * * * * 15000 * * 15000 * * 10000 10000

6000 6000 ALT (U/L) ALT AST (U/L) AST 4000 4000

2000 2000

0 0 ConA - + + + + ConA - + + + + BAR501 - - + - + BAR501 - - + - +

8 h 24 h 8 h 24 h

Figure S4. BAR501 administration protects against acute hepatitis induced by Concanavallin A . Acute hepatitis was induced in C57BL6 male mice, GPBAR1+/+ and GPBAR1-/-, by intravenous injection of Con A (15 mg/kg) 8 or 24 hours. Mice were randomized to receive Con A alone or in combination with BAR501 (30 mg/kg) daily from three days before induction of hepatitis to the time of the sacrifice. Data shown are (A) plasmatic levels of AST and ALT in each group. Results are the mean ± SEM of 6-10 mice per group. *p<0.05.