The MyoGravity project to study real microgravity effects on human muscle precursor cells and tissue.

Microgravity (µG) experienced during space flights promotes adaptation in several astronauts' organs and tissues, with skeletal muscles being the most affected. In response to reduced gravitational loading, muscles (especially, lower limb and antigravity muscles) undergo progressive mass loss and alteration in metabolism, myofiber size, and composition. Skeletal muscle precursor cells (MPCs), also known as satellite cells, are responsible for the growth and maintenance of muscle mass in adult life as well as for muscle regeneration following damage and may have a major role in µG-induced muscle wasting. Despite the great relevance for astronaut health, very few data are available about the effects of real µG on human muscles. Based on the MyoGravity project, this study aimed to analyze: (i) the cellular and transcriptional alterations induced by real µG in human MPCs (huMPCs) and (ii) the response of human skeletal muscle to normal gravitational loading after prolonged exposure to µG. We evaluated the transcriptomic changes induced by µG on board the International Space Station (ISS) in differentiating huMPCs isolated from Vastus lateralis muscle biopsies of a pre-flight astronaut and an age- and sex-matched volunteer, in comparison with the same cells cultured on the ground in standard gravity (1×g) conditions. We found that huMPCs differentiated under real µG conditions showed: (i) upregulation of genes related to cell adhesion, plasma membrane components, and ion transport; (ii) strong downregulation of genes related to the muscle contraction machinery and sarcomere organization; and (iii) downregulation of muscle-specific microRNAs (myomiRs). Moreover, we had the unique opportunity to analyze huMPCs and skeletal muscle tissue of the same astronaut before and 30 h after a long-duration space flight on board the ISS. Prolonged exposure to real µG strongly affected the biology and functionality of the astronaut's satellite cells, which showed a dramatic reduction of responsiveness to activating stimuli and proliferation rate, morphological changes, and almost inability to fuse into myotubes. RNA-Seq analysis of post- vs. pre-flight muscle tissue showed that genes involved in muscle structure and remodeling are promptly activated after landing following a long-duration space mission. Conversely, genes involved in the myelination process or synapse and neuromuscular junction organization appeared downregulated. Although we have investigated only one astronaut, these results point to a prompt readaptation of the skeletal muscle mechanical components to the normal gravitational loading, but the inability to rapidly recover the physiological muscle myelination/innervation pattern after landing from a long-duration space flight. Together with the persistent functional deficit observed in the astronaut's satellite cells after prolonged exposure to real µG, these results lead us to hypothesize that a condition of inefficient regeneration is likely to occur in the muscles of post-flight astronauts following damage.

The crosstalk between gut barrier impairment, mitochondrial dysfunction, and microbiota alterations in people living with HIV.

Functional and structural damage of the intestinal mucosal barrier significantly contribute to translocation of gut microbial products into the bloodstream and are largely involved in HIV-1 associated chronic immune activation. This microbial translocation is largely due to a progressive exhaustion of intestinal macrophage phagocytic function, which leads to extracellular accumulation of microbial derived components and results in HIV-1 disease progression. This study aims to better understand whether the modulation of gut microbiota promotes an intestinal immune restoration in people living with HIV (PLWH). Long-term virologically suppressed PLWH underwent blood, colonic, and fecal sampling before (T0) and after 6 months (T6) of oral bacteriotherapy. Age- and gender-matched uninfected controls (UC) were also included. 16S rRNA gene sequencing was applied to all participants' fecal microbiota. Apoptosis machinery, mitochondria, and apical junctional complex (AJC) morphology and physiological functions were analyzed in gut biopsies. At T0, PLWH showed a different pattern of gut microbial flora composition, lower levels of occludin (p = 0.002) and zonulin (p = 0.01), higher claudin-2 levels (p = 0.002), a reduction of mitochondria number (p = 0.002), and diameter (p = 0.002), as well as increased levels of lipopolysaccharide (LPS) (p = 0.018) and cCK18 (p = 0.011), compared to UC. At T6, an increase in size (p = 0.005) and number (p = 0.008) of mitochondria, as well as amelioration in AJC structures (p < 0.0001) were observed. Restoration of bacterial richness (Simpson index) and biodiversity (Shannon index) was observed in all PLWH receiving oral bacteriotherapy (p < 0.05). Increased mitochondria size (p = 0.005) and number (p = 0.008) and amelioration of AJC structure (p < 0.0001) were found at T6 compared to T0. Moreover, increased occludin and zonulin concentration were observed in PLWH intestinal tracts and decreased levels of claudin-2, LPS, and cCK18 were found after oral bacteriotherapy (T0 vs. T6, p < 0.05 for all these measures). Oral bacteriotherapy supplementation might restore the balance of intestinal flora and support the structural and functional recovery of the gut mucosa in antiretroviral therapy treated PLWH.

Discovery of Bile Acid Derivatives as Potent ACE2 Activators by Virtual Screening and Essential Dynamics.

The angiotensin-converting enzyme II (ACE2) is a key molecular player in the regulation of vessel contraction, inflammation, and reduction of oxidative stress. In addition, ACE2 has assumed a prominent role in the fight against the COVID-19 pandemic-causing virus SARS-CoV-2, as it is the very first receptor in the host of the viral spike protein. The binding of the spike protein to ACE2 triggers a cascade of events that eventually leads the virus to enter the host cell and initiate its life cycle. At the same time, SARS-CoV-2 infection downregulates ACE2 expression especially in the lung, altering the biochemical signals regulated by the enzyme and contributing to the poor clinical prognosis characterizing the late stage of the COVID-19 disease. Despite its important biological role, a very limited number of ACE2 activators are known. Here, using a combined in silico and experimental approach, we show that ursodeoxycholic acid (UDCA) derivatives work as ACE2 activators. In detail, we have identified two potent ACE2 ligands, BAR107 and BAR708, through a docking virtual screening campaign and elucidated their mechanism of action from essential dynamics of the enzyme observed during microsecond molecular dynamics calculations. The in silico results were confirmed by in vitro pharmacological assays with the newly identified compounds showing ACE2 activity comparable to that of DIZE, the most potent ACE2 activator known so far. Our work provides structural insight into ACE2/ligand-binding interaction useful for the design of compounds with therapeutic potential against SARS-CoV-2 infection, inflammation, and other ACE2-related diseases.

Bile Acids Activated Receptors in Inflammatory Bowel Disease.

Once known exclusively for their role in nutrients absorption, bile acids have emerged as signaling molecules, generated from cholesterol breakdown, acting on several immune cells by activating a variety of receptors including the G protein-coupled bile acid receptor 1 (GPABR1 or TGR5), the Farnesoid-X-receptor (FXR) and, as recently discovered, the retinoid-related orphan receptors (ROR)γt. GPBAR1, FXR, and RORγt are highly expressed in cells of the innate and adaptive immune system (i.e., dendritic cells (DCs), macrophages, innate lymphoid 3 cells (ILC3s), and T helper 17 (Th17) lymphocytes) and plays an important role in regulating intestinal and liver immunity, highlighting a role for various bile acid species in regulating immune responses to intestinal microbial antigens. While primary bile acids are generated from the cholesterol breakdown secondary bile acids, the GPBAR1 ligands, and oxo-bile acids derivatives, the RORγt ligands, are generated by the intestinal microbiota, highlighting the potential of these bile acids in mediating the chemical communication between the intestinal microbiota and the host. Changes in intestinal microbiota, dysbiosis, alter the composition of the bile acid pool, promoting the activation of the immune system and development of chronic inflammation. In this review, we focus on the molecular mechanisms by which an altered bile acid signaling promotes intestinal inflammation.

Analysis of Gastric Cancer Transcriptome Allows the Identification of Histotype Specific Molecular Signatures With Prognostic Potential.

Gastric cancer is the fifth most common malignancy but the third leading cause of cancer-associated mortality worldwide. Therapy for gastric cancer remain largely suboptimal making the identification of novel therapeutic targets an urgent medical need. In the present study we have carried out a high-throughput sequencing of transcriptome expression in patients with gastric cancers. Twenty-four patients, among a series of 53, who underwent an attempt of curative surgery for gastric cancers in a single center, were enrolled. Patients were sub-grouped according to their histopathology into diffuse and intestinal types, and the transcriptome of the two subgroups assessed by RNAseq analysis and compared to the normal gastric mucosa. The results of this investigation demonstrated that the two histopathology phenotypes express two different patterns of gene expression. A total of 2,064 transcripts were differentially expressed between neoplastic and non-neoplastic tissues: 772 were specific for the intestinal type and 407 for the diffuse type. Only 885 transcripts were simultaneously differentially expressed by both tumors. The per pathway analysis demonstrated an enrichment of extracellular matrix and immune dysfunction in the intestinal type including CXCR2, CXCR1, FPR2, CARD14, EFNA2, AQ9, TRIP13, KLK11 and GHRL. At the univariate analysis reduced levels AQP9 was found to be a negative predictor of 4 years survival. In the diffuse type low levels CXCR2 and high levels of CARD14 mRNA were negative predictors of 4 years survival. In summary, we have identified a group of genes differentially regulated in the intestinal and diffuse histotypes of gastric cancers with AQP9, CARD14 and CXCR2 impacting on patients' prognosis, although CXCR2 is the only factor independently impacting overall survival.

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.

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.

Bile acids and their receptors in metabolic disorders.

Bile acids are a large family of atypical steroids which exert their functions by binding to a family of ubiquitous cell membrane and nuclear receptors. There are two main bile acid activated receptors, FXR and GPBAR1, that are exclusively activated by bile acids, while other receptors CAR, LXRs, PXR, RORγT, S1PR2and VDR are activated by bile acids in addition to other more selective endogenous ligands. In the intestine, activation of FXR and GPBAR1 promotes the release of FGF15/19 and GLP1 which integrate their signaling with direct effects exerted by theother receptors in target tissues. This network is tuned in a time ordered manner by circadian rhythm and is critical for the regulation of metabolic process including autophagy, fast-to-feed transition, lipid and glucose metabolism, energy balance and immune responses. In the last decade FXR ligands have entered clinical trials but development of systemic FXR agonists has been proven challenging because their side effects including increased levels of cholesterol and Low Density Lipoproteins cholesterol (LDL-c) and reduced High-Density Lipoprotein cholesterol (HDL-c). In addition, pruritus has emerged as a common, dose related, side effect of FXR ligands. Intestinal-restricted FXR and GPBAR1 agonists and dual FXR/GPBAR1 agonists have been developed. Here we review the last decade in bile acids physiology and pharmacology.

The bile acid activated receptors GPBAR1 and FXR exert antagonistic effects on autophagy.

Autophagy is a highly conserved catabolic process activated by fasting and caloric restriction. FXR, a receptor for primary bile acids, reverses the activity of cAMP-response element binding protein (CREB) on autophagy-related genes (Atg)s and terminates autophagy in the fed state. GPBAR1, a receptor for secondary bile acids, exerts its genomic effects via cAMP-CREB pathway. By genetic and pharmacological approaches, we have obtained evidence that GPBAR1 functions as a positive modulator of autophagy in liver and white adipose tissue (WAT) in fasting. Mechanistically, we found that Gpbar1-/- mice lack the expression of Cyp2c70 a gene essential for generation of muricholic acids which are FXR antagonists, and have an FXR-biased bile acid pool. Because FXR represses autophagy, Gpbar1-/- mice show a defective regulation of autophagy in fasting. BAR501, a selective GPBAR1 agonist, induces autophagy in fed mice. Defective regulation of autophagy in Gpbar1-/- could be reversed by FXR antagonism, while repression of autophagy by feeding was partially abrogated by FXR gene ablation, and FXR activation repressed Atgs in the fast state. BAR501 reversed the negative regulatory effects of feeding and FXR agonism on autophagy and promoted the recruitment of CREB to a CRE on the LC3 promoter. In mice exposed to chronic high caloric intake, GPBAR1 agonism ameliorated insulin sensitivity and induced Atgs expression in the liver and WAT. In summary, GPBAR1 is required for positive regulation of autophagy in fasting and its ligands reverse the repressive effects exerted on liver and WAT autophagy flow by FXR in fed.

Bile Acid Signaling in Inflammatory Bowel Diseases.

Bile acids are a group of chemically different steroids generated at the host/microbial interface. Indeed, while primary bile acids are the end-product of cholesterol breakdown in the host liver, secondary bile acids are the products of microbial metabolism. Primary and secondary bile acids along with their oxo derivatives have been identified as signaling molecules acting on a family of cell membrane and nuclear receptors collectively known as "bile acid-activated receptors." Members of this group of receptors are highly expressed throughout the gastrointestinal tract and mediate the bilateral communications of the intestinal microbiota with the host immune system. The expression and function of bile acid-activated receptors FXR, GPBAR1, PXR, VDR, and RORγt are highly dependent on the structure of the intestinal microbiota and negatively regulated by intestinal inflammation. Studies from gene ablated mice have demonstrated that FXR and GPBAR1 are essential to maintain a tolerogenic phenotype in the intestine, and their ablation promotes the polarization of intestinal T cells and macrophages toward a pro-inflammatory phenotype. RORγt inhibition by oxo-bile acids is essential to constrain Th17 polarization of intestinal lymphocytes. Gene-wide association studies and functional characterizations suggest a potential role for impaired bile acid signaling in development inflammatory bowel diseases (IBD). In this review, we will focus on how bile acids and their receptors mediate communications of intestinal microbiota with the intestinal immune system, describing dynamic changes of bile acid metabolism in IBD and the potential therapeutic application of targeting bile acid signaling in these disorders.

Hijacking SARS-CoV-2/ACE2 Receptor Interaction by Natural and Semi-synthetic Steroidal Agents Acting on Functional Pockets on the Receptor Binding Domain.

The coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the severe acute respiratory syndrome coronavirus (SARS)-CoV-2. In light of the urgent need to identify novel approaches to be used in the emergency phase, we have embarked on an exploratory campaign aimed at repurposing natural substances and clinically available drugs as potential anti-SARS-CoV2-2 agents by targeting viral proteins. Here we report on a strategy based on the virtual screening of druggable pockets located in the central ß-sheet core of the SARS-CoV-2 Spike's protein receptor binding domain (RBD). By combining an in silico approach and molecular in vitro testing we have been able to identify several triterpenoid/steroidal agents that inhibit interaction of the Spike RBD with the carboxypeptidase domain of the Angiotensin Converting Enzyme (ACE2). In detail, we provide evidence that potential binding sites exist in the RBD of the SARS CoV-2 Spike protein and that occupancy of these pockets reduces the ability of the RBD to bind to the ACE2 consensus in vitro. Naturally occurring and clinically available triterpenoids such as glycyrrhetinic and oleanolic acids, as well as primary and secondary bile acids and their amidated derivatives such as glyco-ursodeoxycholic acid and semi-synthetic derivatives such as obeticholic acid reduces the RBD/ACE2 binding. In aggregate, these results might help to define novel approaches to COVID-19 based on SARS-CoV-2 entry inhibitors.

Discovery of a Novel Multi-Strains Probiotic Formulation with Improved Efficacy toward Intestinal Inflammation.

Dysbiosis is commonly detected in patients with inflammatory bowel disease (IBD), supporting the concept that a dysregulated immune reaction to bacterial antigens has a pathogenic role in the development of intestinal inflammation. In the present study, we have investigated the beneficial effects of a novel probiotic formulation assembled by combining four probiotics (Streptococcus thermophilus, Lactobacillus casei, Bifidobacterium breve) with Bacillus subtilis, a Gram-positive bacterium, with extensive bio-applications. Mice rendered colitic by administration of TNBS or DSS were administered with Bacillus subtilis alone, Vivomixx® or the novel Five strains formulation. Vivomixx® attenuated the severity of inflammation and reduced the development of signs and symptoms of colitis in both models. Adding Bacillus subtilis to Vivomixx® improved the beneficial effects of the bacterial therapy. The novel Five strains formulation was as effective as Vivomixx® in reducing the development of signs and symptoms of colitis and reduced the expression of pro-inflammatory mediators including Il-6 and Tnf-α while increased the expression of Il-10 mRNA and the number of Treg. In summary, we have shown that a novel Five strains probiotics formulation exerts beneficial effects on two chemical models of colitis, establishing Bacillus subtilis as a probiotic in rodent models of inflammation.

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.

GPBAR1 Activation by C6-Substituted Hyodeoxycholane Analogues Protect against Colitis.

GPBAR1 agonists have been identified as potential leads for the treatment of diseases related to colon inflammation such as Crohn's and ulcerative colitis. In this paper, we report the discovery of a small library of hyodeoxycholane analogues, decorated at C-6 with different substituents, as potent and selective GPBAR1 agonists. In vitro pharmacological assays showed that compound 6 selectively activates GPBAR1 (EC50 = 0.3 µM) and reduces the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) in THP1 cells. The binding mode of compound 6 in GPBAR1 was elucidated by docking calculations. Moreover, compound 6 protects against TNBS-induced colitis in Gpbar1+/+ rodent model, representing an intriguing lead for the treatment of these inflammatory disorders.

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.

Ursodeoxycholic acid is a GPBAR1 agonist and resets liver/intestinal FXR signaling in a model of diet-induced dysbiosis and NASH.

Obeticholic acid (OCA) is a farnesoid-X-receptor (FXR) ligand, shown effective in reducing steatosis and fibrosis in NASH patients. However, OCA causes major side effects including pruritus, while increases the risk for liver decompensation in cirrhotic patients. Ursodeoxycholic acid (UDCA), is a safe and unexpensive bile acid used in the treatment of liver disorders whose mechanism of action is poorly defined. Here we have compared the effects of OCA and UDCA in a mouse model of NASH. In mice exposed to a diet rich in fat/cholesterol and fructose (HFD-F), treatment with OCA or UDCA effectively prevented body weight gain, insulin resistance, as demonstrated by OGTT, and AST plasma levels. After 12 weeks HFD-F mice developed liver microvesicular steatosis, inflammation and mild fibrosis, increased expression of inflammatory (TNFα, IL6, F4/80) and fibrosis (αSma, Col1α1, Tgfß) markers, reduced liver expression of FXR, dysregulated liver FXR signaling and elevated levels of Tauro-α and ß-muricholic acid (T-α and ßMCA), two FXR antagonists in mice. Both compounds prevented these changes and improved liver histopathology. OCA reduced primary bile acid synthesis worsening the T-CA/T-ßMCA ratio. UDCA effectively transactivated GPBAR1 in vitro. By RNAseq analysis we found that among over 2400 genes modulated by the HFD-F, only 32 and 60 genes were modulated by OCA and UDCA, with only 3 genes (Dbp, Adh7, Osgin1) being modulated by both agents. Both agents partially prevented the intestinal dysbiosis. CONCLUSIONS: UDCA is a GPBAR1 ligand and exerts beneficial effects in a rodent model of NASH by activating non-overlapping pathway with OCA.

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.

Transcriptome Analysis of Dual FXR and GPBAR1 Agonism in Rodent Model of NASH Reveals Modulation of Lipid Droplets Formation.

Non-alcoholic steatohepatitis (NASH) is a progressive, chronic, liver disease whose prevalence is growing worldwide. Despite several agents being under development for treating NASH, there are no drugs currently approved. The Farnesoid-x-receptor (FXR) and the G-protein coupled bile acid receptor 1 (GPBAR1), two bile acid activated receptors, have been investigated for their potential in treating NASH. Here we report that BAR502, a steroidal dual ligand for FXR/GPBAR1, attenuates development of clinical and liver histopathology features of NASH in mice fed a high fat diet (HFD) and fructose (F). By RNAseq analysis of liver transcriptome we found that BAR502 restores FXR signaling in the liver of mice feed HFD-F, and negatively regulates a cluster of genes including Srebf1 (Srepb1c) and its target genes-fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec-involved in lipid droplets formation and triglycerides storage in hepatocytes. Additionally, BAR502 increased the intestinal expression of Fgf15 and Glp1 and energy expenditure by white adipose tissues. Finally, exposure to BAR502 reshaped the intestinal microbiota by increasing the amount of Bacteroidaceae. In conclusion, we have shown that dual FXR/GPBAR1 agonism might have utility in treatment of NASH.