Activation of GPBAR1 attenuates vascular inflammation and atherosclerosis in a mouse model of NAFLD-related cardiovascular disease.

While patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk to develop clinically meaningful cardiovascular diseases (CVD), there are no approved drug designed to target the liver and CVD component of NAFLD. GPBAR1, also known as TGR5, is a G protein coupled receptor for secondary bile acids. In this study we have investigated the effect of GPBAR1 activation by BAR501, a selective GPBAR1 agonist, in Apolipoprotein E deficient (ApoE-/-) mice fed a high fat diet and fructose (Western diet), a validated model of NAFLD-associated atherosclerosis. Using aortic samples from patients who underwent surgery for abdominal aneurism, and ex vivo experiments with endothelial cells and human macrophages, we were able to co-localize the expression of GPBAR1 in CD14+ and PECAM1+ cells. Similar findings were observed in the aortic plaques from ApoE-/- mice. Treating ApoE-/- mice with BAR501, 30 mg/kg for 14 weeks, attenuated the body weight gain while ameliorated the insulin sensitivity by increasing the plasma concentrations of GLP-1 and FGF15. Activation of GPBAR1 reduced the aorta thickness and severity of atherosclerotic lesions and decreased the amount of plaques macrophages. Treating ApoE-/- mice reshaped the aortic transcriptome promoting the expression of anti-inflammatory genes, including IL-10, as also confirmed by tSNE analysis of spleen-derived macrophages. Feeding ApoE-/- mice with BAR501 redirected the bile acid synthesis and the composition of the intestinal microbiota. In conclusion, GPBAR1 agonism attenuates systemic inflammation and improve metabolic profile in a genetic/dietetic model of atherosclerosis. BAR501 might be of utility in the treatment for NAFLD-related CVD.

Defective Bile Acid Signaling Promotes Vascular Dysfunction, Supporting a Role for G-Protein Bile Acid Receptor 1/Farnesoid X Receptor Agonism and Statins in the Treatment of Nonalcoholic Fatty Liver Disease.

BACKGROUND: Patients with nonalcoholic fatty liver disease are at increased risk to develop atherosclerotic cardiovascular diseases. FXR and GPBAR1 are 2 bile acid-activated receptors exploited in the treatment of nonalcoholic fatty liver disease: whether dual GPBAR1/FXR agonists synergize with statins in the treatment of the liver and cardiovascular components of nonalcoholic fatty liver disease is unknown. METHODS AND RESULTS: Investigations of human aortic samples obtained from patients who underwent surgery for aortic aneurysms and Gpbar1-/-, Fxr-/-, and dual Gpbar1-/-Fxr-/- mice demonstrated that GPBAR1 and FXR are expressed in the aortic wall and regulate endothelial cell/macrophage interactions. The expression of GPBAR1 in the human endothelium correlated with the expression of inflammatory biomarkers. Mice lacking Fxr and Gpbar1-/-/Fxr-/- display hypotension and aortic inflammation, along with altered intestinal permeability that deteriorates with age, and severe dysbiosis, along with dysregulated bile acid synthesis. Vasomotor activities of aortic rings were altered by Gpbar1 and Fxr gene ablation. In apolipoprotein E-/- and wild-type mice, BAR502, a dual GPBAR1/FXR agonist, alone or in combination with atorvastatin, reduced cholesterol and low-density lipoprotein plasma levels, mitigated the development of liver steatosis and aortic plaque formation, and shifted the polarization of circulating leukocytes toward an anti-inflammatory phenotype. BAR502/atorvastatin reversed intestinal dysbiosis and dysregulated bile acid synthesis, promoting a shift of bile acid pool composition toward FXR antagonists and GPBAR1 agonists. CONCLUSIONS: FXR and GPBAR1 maintain intestinal, liver, and cardiovascular homeostasis, and their therapeutic targeting with a dual GPBAR1/FXR ligand and atorvastatin holds potential in the treatment of liver and cardiovascular components of nonalcoholic fatty liver disease.

Modeling Inflammatory Bowel Disease by Intestinal Organoids.

Inflammatory bowel disease (IBD) is a chronic and relapsing disease caused by a dysregulated immune response to host intestinal microbiota that occurs in genetically predisposed individuals. IBD encompasses two major clinical entities: ulcerative colitis (UC), limited to the colonic mucosa, and Crohn's disease (CD), which might affect any segment of the gastrointestinal tract. Despite the prevalence of IBD increasing worldwide, therapy remains suboptimal, largely because of the variability of causative mechanisms, raising the need to develop individualized therapeutic approaches targeted to each individual patient. In this context, patients-derived intestinal organoids represent an effective tool for advancing our understanding of IBD's pathogenesis. Organoid 3D culture systems offer a unique model for dissecting epithelial mechanisms involved IBDs and testing individualized therapy, although the lack of a functional immune system and a microbiota, two driving components of the IBD pathogenesis, represent a major barrier to their exploitation in clinical medicine. In this review, we have examined how to improve the translational utility of intestinal organoids in IBD and how co-cultures of 3D or 2D organoids and immune cells and/or intestinal microbiota might help to overcome these limitations.

Combinatorial targeting of G-protein-coupled bile acid receptor 1 and cysteinyl leukotriene receptor 1 reveals a mechanistic role for bile acids and leukotrienes in drug-induced liver injury.

BACKGROUND AND AIM: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist. APPROACH AND RESULTS: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 . CONCLUSIONS: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI.

Repositioning Mifepristone as a Leukaemia Inhibitory Factor Receptor Antagonist for the Treatment of Pancreatic Adenocarcinoma.

Pancreatic cancer is a leading cause of cancer mortality and is projected to become the second-most common cause of cancer mortality in the next decade. While gene-wide association studies and next generation sequencing analyses have identified molecular patterns and transcriptome profiles with prognostic relevance, therapeutic opportunities remain limited. Among the genes that are upregulated in pancreatic ductal adenocarcinomas (PDAC), the leukaemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, has emerged as potential therapeutic candidate. LIF is aberrantly secreted by tumour cells and promotes tumour progression in pancreatic and other solid tumours through aberrant activation of the LIF receptor (LIFR) and downstream signalling that involves the JAK1/STAT3 pathway. Since there are no LIFR antagonists available for clinical use, we developed an in silico strategy to identify potential LIFR antagonists and drug repositioning with regard to LIFR antagonists. The results of these studies allowed the identification of mifepristone, a progesterone/glucocorticoid antagonist, clinically used in medical abortion, as a potent LIFR antagonist. Computational studies revealed that mifepristone binding partially overlapped the LIFR binding site. LIF and LIFR are expressed by human PDAC tissues and PDAC cell lines, including MIA-PaCa-2 and PANC-1 cells. Exposure of these cell lines to mifepristone reverses cell proliferation, migration and epithelial mesenchymal transition induced by LIF in a concentration-dependent manner. Mifepristone inhibits LIFR signalling and reverses STAT3 phosphorylation induced by LIF. Together, these data support the repositioning of mifepristone as a potential therapeutic agent in the treatment of PDAC.

Next-Generation Sequencing Analysis of Gastric Cancer Identifies the Leukemia Inhibitory Factor Receptor as a Driving Factor in Gastric Cancer Progression and as a Predictor of Poor Prognosis.

Gastric cancer (GC) is the third cause of cancer-related mortality worldwide. Nevertheless, because GC screening programs are not cost-effective, most patients receive diagnosis in the advanced stages, when surgical options are limited. Peritoneal dissemination occurs in approximately one-third of patients with GC at the diagnosis and is a strong predictor of poor outcome. Despite the clinical relevance, biological and molecular mechanisms underlying the development of peritoneal metastasis in GC remain poorly defined. Here, we report results of a high-throughput sequencing of transcriptome expression in paired samples of non-neoplastic and neoplastic gastric samples from 31 patients with GC with or without peritoneal carcinomatosis. The RNA-seq analysis led to the discovery of a group of highly upregulated or downregulated genes, including the leukemia inhibitory factor receptor (LIFR) and one cut domain family member 2 (ONECUT2) that were differentially modulated in patients with peritoneal disease in comparison with patients without peritoneal involvement. Both LIFR and ONECUT2 predicted survival at univariate statistical analysis. LIFR and its major ligand LIF belong to the interleukin-6 (IL-6) cytokine family and have a central role in immune system regulation, carcinogenesis, and dissemination in several human cancers. To confirm the mechanistic role of the LIF/LIFR pathway in promoting GC progression, GC cell lines were challenged in vitro with LIF and a LIFR inhibitor. Among several GC cell lines, MKN45 cells displayed the higher expression of the receptor, and their exposure to LIF promotes a concentration-dependent proliferation and epithelial-mesenchymal transition (EMT), as shown by modulation of relative expression of E-cadherin/vimentin along with JAK and STAT3 phosphorylation and acquisition of a migratory phenotype. Furthermore, exposure to LIF promoted the adhesion of MKN45 cells to the peritoneum in an ex vivo assay. These effects were reversed by the pharmacological blockade of LIFR signaling. Together, these data suggest that LIFR might have a major role in promoting disease progression and peritoneal dissemination in patients with GC and that development of LIF/LIFR inhibitors might have a role in the treatment of GC.

Discovery of a Potent and Orally Active Dual GPBAR1/CysLT1R Modulator for the Treatment of Metabolic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are two highly prevalent human diseases caused by excessive fat deposition in the liver. Although multiple approaches have been suggested, NAFLD/NASH remains an unmet clinical need. Here, we report the discovery of a novel class of hybrid molecules designed to function as cysteinyl leukotriene receptor 1 (CysLT1R) antagonists and G protein bile acid receptor 1 (GPBAR1/TGR5) agonists for the treatment of NAFLD/NASH. The most potent of these compounds generated by harnessing the scaffold of the previously described CystLT1R antagonists showed efficacy in reversing liver histopathology features in a preclinical model of NASH, reshaping the liver transcriptome and the lipid and energy metabolism in the liver and adipose tissues. In summary, the present study described a novel orally active dual CysLT1R antagonist/GPBAR1 agonist that effectively protects against the development of NAFLD/NASH, showing promise for further development.

GLP-1 Mediates Regulation of Colonic ACE2 Expression by the Bile Acid Receptor GPBAR1 in Inflammation.

BACKGROUND & AIMS: ACE2, a carboxypeptidase that generates Ang-(1-7) from Ang II, is highly expressed in the lung, small intestine and colon. GPBAR1, is a G protein bile acid receptor that promotes the release of the insulinotropic factor glucagon-like peptide (GLP)-1 and attenuates intestinal inflammation. METHODS: We investigated the expression of ACE2, GLP-1 and GPBAR1 in two cohorts of Crohn's disease (CD) patients and three mouse models of colitis and Gpbar1-/- mice. Activation of GPBAR1 in these models and in vitro was achieved by BAR501, a selective GPBAR1 agonist. RESULTS: In IBD patients, ACE2 mRNA expression was regulated in a site-specific manner in response to inflammation. While expression of ileal ACE2 mRNA was reduced, the colon expression was induced. Colon expression of ACE2 mRNA in IBD correlated with expression of TNF-α and GPBAR1. A positive correlation occurred between GCG and GPBAR1 in human samples and animal models of colitis. In these models, ACE2 mRNA expression was further upregulated by GPABR1 agonism and reversed by exendin-3, a GLP-1 receptor antagonist. In in vitro studies, liraglutide, a GLP-1 analogue, increased the expression of ACE2 in colon epithelial cells/macrophages co-cultures. CONCLUSIONS: ACE2 mRNA expression in the colon of IBD patients and rodent models of colitis is regulated in a TNF-α- and GLP-1-dependent manner. We have identified a GPBAR1/GLP-1 mechanism as a positive modulator of ACE2.

Organoids as ex vivo culture system to investigate infection-host interaction in gastric pre-carcinogenesis.

Advancements in stem cell research have enabled the establishment of three-dimensional (3D) primary cell cultures, known as organoids. These culture systems follow the organization of an in vivo organ, as they enclose the different epithelial cell lines of which it is normally composed. Generation of these 3D cultures has bridged the gap between in vitro models, made up by two-dimensional (2D) cancer cell lines cultures, and in vivo animal models, that have major differences with human diseases. Organoids are increasingly used as a model to study colonization of gastric mucosa by infectious agents and to better understand host-microbe interactions and the molecular events that lead to infection, pathogen-epithelial cells interactions and mechanisms of gastric mucosal injury. In this review we will focus on the role of organoids as a tool to investigate molecular interactions of Helicobacter (H.) pylori and Epstein Barr Virus (EBV) and gastric mucosa and how these infections, that affect ≈ 45% of the world population, might progress to gastric cancer, a highly prevalent cancer and the third leading cause of cancer death.

Structural Basis for Developing Multitarget Compounds Acting on Cysteinyl Leukotriene Receptor 1 and G-Protein-Coupled Bile Acid Receptor 1.

G-protein-coupled receptors (GPCRs) are the molecular target of 40% of marketed drugs and the most investigated structures to develop novel therapeutics. Different members of the GPCRs superfamily can modulate the same cellular process acting on diverse pathways, thus representing an attractive opportunity to achieve multitarget drugs with synergic pharmacological effects. Here, we present a series of compounds with dual activity toward cysteinyl leukotriene receptor 1 (CysLT1R) and G-protein-coupled bile acid receptor 1 (GPBAR1). They are derivatives of REV5901─the first reported dual compound─with therapeutic potential in the treatment of colitis and other inflammatory processes. We report the binding mode of the most active compounds in the two GPCRs, revealing unprecedented structural basis for future drug design studies, including the presence of a polar group opportunely spaced from an aromatic ring in the ligand to interact with Arg792.60 of CysLT1R and achieve dual activity.

Inverse Virtual Screening for the rapid re-evaluation of the presumed biological safe profile of natural products. The case of steviol from Stevia rebaudiana glycosides on farnesoid X receptor (FXR).

Nonnutritive sweeteners (NNSs) are widely employed as dietary substitutes for classical sugars thanks to their safety profile and low toxicity. In this study, a re-evaluation of the biological effects of steviol (1), the main metabolite from Stevia rebaudiana glycosides, was performed using the Inverse Virtual Screening (IVS) target fishing computational approach. Starting from well-known pharmacological properties of Stevia rebaudiana glycosides, this computational tool was employed for predicting the putative interacting targets of 1 and, afterwards, of its five synthetic ester derivatives 2-6, accounting a large panel of proteins involved in cancer and inflammation events. Applying this methodology, the farnesoid X receptor (FXR) was identified as the putative target partner of 1-6. The predicted ligand-protein interactions were corroborated by transactivation assays, specifically disclosing the agonistic activity of 1 and the antagonistic activities of 2-6 on FXR. The reported results highlight the feasibility of IVS as a fast and potent tool for predicting the interacting targets of query compounds, addressing the re-evaluation of their bioactivity. In light of the obtained results, the presumably safe profile of known compounds, such as the case of steviol (1), is critically discussed.

Discovery of a AHR pelargonidin agonist that counter-regulates Ace2 expression and attenuates ACE2-SARS-CoV-2 interaction.

The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.

Opposite effects of the FXR agonist obeticholic acid on Mafg and Nrf2 mediate the development of acute liver injury in rodent models of cholestasis.

The farnesoid-X-receptor (FXR) is validated target in the cholestatic disorders treatment. Obeticholic acid (OCA), the first in class of FXR agonist approved for clinical use, causes side effects including acute liver decompensation when administered to cirrhotic patients with primary biliary cholangitis at higher than recommended doses. The V-Maf avian-musculoaponeurotic-fibrosarcoma-oncogene-homolog-G (Mafg) and nuclear factor-erythroid-2-related-factor-2 (Nrf2) mediates some of the downstream effects of FXR. In the present study we have investigated the role of FXR/MafG/NRF2 pathway in the development of liver toxicity caused by OCA in rodent models of cholestasis. Cholestasis was induced by bile duct ligation (BDL) or administration of α-naphtyl-isothiocyanate (ANIT) to male Wistar rats and FXR-/- and FXR+/+ mice. Treating BDL and ANIT rats with OCA exacerbated the severity of cholestasis, hepatocytes injury and severely downregulated the expression of basolateral transporters. In mice, genetic ablation FXR or its pharmacological inhibition by 3-(naphthalen-2-yl)-5-(piperidin-4-yl)-1,2,4-oxadiazole rescued from negative regulation of MRP4 and protected against liver injury caused by ANIT. By RNAseq analysis we found that FXR antagonism effectively reversed the transcription of over 2100 genes modulated by OCA/ANIT treatment, including Mafg and Nrf2 and their target genes Cyp7a1, Cyp8b1, Mat1a, Mat2a, Gss. Genetic and pharmacological Mafg inhibition by liver delivery of siRNA antisense or S-adenosylmethionine effectively rescued from damage caused by ANIT/OCA. In contrast, Nrf2 induction by sulforaphane was protective. CONCLUSIONS: Liver injury caused by FXR agonism in cholestasis is FXR-dependent and is reversed by FXR and Mafg antagonism or Nrf2 induction.

Identification of cysteinyl-leukotriene-receptor 1 antagonists as ligands for the bile acid receptor GPBAR1.

The cysteinyl leukotrienes (CysLTs), i.e. LTC4, LTD4 and LTE4, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT1R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT1R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT1R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC50 of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT1R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL1R and GPBAR1.

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

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 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 6ß-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 demonstrated 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 Aryl Hydrocarbon Receptor (AhR) Mediates the Counter-Regulatory Effects of Pelargonidins in Models of Inflammation and Metabolic Dysfunctions.

Pelargonidins are anthocyanidins thought to be beneficial for the human health, although controversies exist over the doses needed and the unclear mechanism of action, along with poor systemic bioavailability. One putative target of pelargonidins is the aryl hydrocarbon receptor (AhR). A synthetic pelargonidin (Mt-P) was synthesized by the methylation of the pelargonidin (the natural compound indicated as P). Mt-P transactivated the AhR with an EC50 of 1.97 µM and was ~2-fold more potent than the natural compound. In vitro Mt-P attenuated pro-inflammatory activities of Raw264.7 macrophage cells in an AhR-dependent manner. In vivo, administration of the Mt-P in Balb/c mice resulted in a dose-dependent attenuation of signs and symptoms of colitis induced by TNBS. A dose of 5 mg/kg Mt-P, but not the natural compound P, reversed intestinal inflammation and increased expression of Tnf-α, Ifn-Æ´, and Il-6, while promoted the expansion of regulatory T cells and M2 macrophages. In C57BL/6J mice fed a high fat diet (HFD), Mt-P attenuated body weight gain, intestinal and liver inflammation, and ameliorated insulin sensitivity, while worsened liver steatosis by up-regulating the liver expression of Cd36 and Apo100b. These effects were abrogated by AhR gene ablation. Mt-P is a synthetic pelargonidin endowed with robust AhR agonist activity that exerts beneficial effects in murine models of inflammation and metabolic dysfunction.

GPBAR1 Functions as Gatekeeper for Liver NKT Cells and provides Counterregulatory Signals in Mouse Models of Immune-Mediated Hepatitis.

BACKGROUND & AIMS: GPBAR1, also known as TGR5, is a G protein-coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis. MATERIAL AND METHODS: Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s. RESULTS: In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1-/- mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model. CONCLUSION: Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity.