Pubblicazioni 2017

Biagioli M, Mencarelli A, Carino A, Cipriani S, Marchianò S, Fiorucci C, Donini A, Graziosi L, Baldelli F, Distrutti E, Costantino G, Fiorucci S. Genetic and Pharmacological Dissection of the Role of Spleen Tyrosine Kinase (Syk) in Intestinal Inflammation and Immune Dysfunction in Inflammatory Bowel Diseases. Inflamm Bowel Dis. 2017 Dec 19;24(1):123-135.

Abstract
Background: The DNAX adaptor protein 12 (DAP12) is a transmembrane adaptor molecule that signals through the activation of Syk (Spleen Tyrosine Kinase) in myeloid cells. The purpose of this study is to investigate the role of DAP12 and Syk pathways in inflammatory bowel diseases (IBDs).

Methods: DAP12 deficient and DAP12 transgenic, overexpressing an increased amount of DAP12, mice and Syk deficient mice in the C57/BL6 background were used for these studies. Colitis was induced by administering mice with dextran sulfate sodium (DSS), in drinking water, or 2,4,6-trinitrobenzene sulfonic acid (TNBS), by intrarectal enema.

Results: Abundant expression of DAP12 and Syk was detected in colon samples obtained from Crohn’s disease patients with expression restricted to immune cells infiltrating the colonic wall. In rodents development of DSS colitis as measured by assessing severity of wasting diseases, global colitis score,and macroscopic and histology scores was robustly attenuated in DAP12-/- and Syk-/- mice. In contrast, DAP12 overexpression resulted in a striking exacerbation of colon damage caused by DSS. Induction of colon expression of proinflammatory cytokines and chemokines in response to DSS administration was attenuated in DAP12-/- and Syk-/- mice, whereas opposite results were observed in DAP12 transgenic mice. Treating wild-type mice with a DAP-12 inhibitor or a Syk inhibitor caused a robust attenuation of colitis induced by DSS and TNBS.

Conclusions: DAP12 and Syk are essential mediators in inflammation-driven immune dysfunction in murine colitides. Because DAP12 and Syk expression is upregulated in patients with active disease, present findings suggest a beneficial role for DAP12 and Syk inhibitors in IBD.

Carino A, Cipriani S, Marchianò S, Biagioli M, Scarpelli P, Zampella A, Monti MC, Fiorucci S. Gpbar1 agonism promotes a Pgc-1α-dependent browning of white adipose tissue and energy expenditure and reverses diet-induced steatohepatitis in mice. Sci Rep. 2017 Oct 20;7(1):13689.

Abstract
Gpbar1 is a bile acid activated receptor for secondary bile acids. Here we have investigated the mechanistic role of Gpbar1 in the regulation of adipose tissues functionality in a murine model of steatohepatitis (NASH). Feeding wild type and Gpbar1-/- mice with a high fat diet-fructose (HFD-F) lead to development of NASH-like features. Treating HFD-F mice with 6β-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective Gpbar1-ligand, reversed insulin resistance and histologic features of NASH, increased the weight of epWAT and BAT functionality and promoted energy expenditure and the browning of epWAT as assessed by measuring expression of Ucp1 and Pgc-1α. The beneficial effects of BAR501 were lost in Gpbar1-/- mice. In vitro, BAR501 promoted the browning of 3T3-L1 cells a pre-adipocyte cell line and recruitment of CREB to the promoter of Pgc-1α. In conclusion, Gpbar1 agonism ameliorates liver histology in a rodent model of NASH and promotes the browning of white adipose tissue.

Biagioli M, Laghi L, Carino A, Cipriani S, Distrutti E, Marchianò S, Parolin C, Scarpelli P, Vitali B, Fiorucci S. Metabolic Variability of a Multispecies Probiotic Preparation Impacts on the Anti-inflammatory Activity. Front Pharmacol. 2017 Jul 28;8:505.

Abstract
Background: In addition to strain taxonomy, the ability of probiotics to confer beneficial effects on the host rely on a number of additional factors including epigenetic modulation of bacterial genes leading to metabolic variability and might impact on probiotic functionality. Aims: To investigate metabolism and functionality of two different batches of a probiotic blend commercialized under the same name in Europe in models of intestinal inflammation. Methods: Boxes of VSL#3, a probiotic mixture used in the treatment of pouchitis, were obtained from pharmacies in UK subjected to metabolomic analysis and their functionality tested in mice rendered colitis by treatment with DSS or TNBS. Results: VSL#3-A (lot DM538), but not VSL#3-B (lot 507132), attenuated “clinical” signs of colitis in the DSS and TNBS models. In both models, VSL#3-A, but not VSL#3-B, reduced macroscopic scores, intestinal permeability, and expression of TNFα, IL-1β, and IL-6 mRNAs, while increased the expression of TGFβ and IL-10, occludin, and zonula occludens-1 (ZO-1) mRNAs and shifted colonic macrophages from a M1 to M2 phenotype (P < 0.05 vs. TNBS). In contrast, VSL#3-B failed to reduce inflammation, and worsened intestinal permeability in the DSS model (P < 0.001 vs. VSL#3-A). A metabolomic analysis of the two formulations allowed the identification of two specific patterns, with at least three-folds enrichment in the concentrations of four metabolites, including 1-3 dihydroxyacetone (DHA), an intermediate in the fructose metabolism, in VSL#3-B supernatants. Feeding mice with DHA, increased intestinal permeability. Conclusions: Two batches of a commercially available probiotic show divergent metabolic activities. DHA, a product of probiotic metabolism, increases intestinal permeability, highlighting the complex interactions between food, microbiota, probiotics, and intestinal inflammation.

Guryanov I, Cipriani S, Fiorucci S, Zashikhina N, Marchianò S, Scarpelli P, Korzhikov-Vlakh V, Popova E, Korzhikova-Vlakh E, Biondi B, Formaggio F, Tennikova T. Nanotraps with biomimetic surface as decoys for chemokines. Nanomedicine. 2017 Nov;13(8):2575-2585.

Abstract
A creation of nanotraps that could selectively recognize the chemotactic mediators of leukocyte adhesion and eliminate them from the bloodstream and tissue intercellular matrix is a promising approach for the treatment of various inflammatory and autoimmune diseases. We designed nanotraps as artificial decoy receptors based on poly(lactic acid) (PLA) nanoparticles covered by heparin and bearing on the surface two fragments of CCR5 receptor (N-terminal domain, Nt, and second extracellular loop, ECL2), responsible for chemokine binding. In order to attach Nt and ECL2 to the heparin shell, the corresponding peptides were modified with N- and/or C-terminal oligolysines. The presence of the nanotraps in the cell medium completely eliminated the activating effect of a CCR5 ligand, chemokine Rantes, while strongly decreasing the adhesion of monocytes to the human endothelial cells. We found that the modified ECL2 alone was also able to prevent monocyte adhesion, thus acting as a decoy receptor itself.

Biagioli M, Carino A, Cipriani S, Francisci D, Marchianò S, Scarpelli P, Sorcini D, Zampella A, Fiorucci S. The Bile Acid Receptor GPBAR1 Regulates the M1/M2 Phenotype of Intestinal Macrophages and Activation of GPBAR1 Rescues Mice from Murine Colitis. J Immunol. 2017 Jul 15;199(2):718-733.

Abstract
GPBAR1 (TGR5 or M-BAR) is a G protein-coupled receptor for secondary bile acids that is highly expressed in monocytes/macrophages. In this study, we aimed to determine the role of GPBAR1 in mediating leukocyte trafficking in chemically induced models of colitis and investigate the therapeutic potential of BAR501, a small molecule agonist for GPBAR1. These studies demonstrated that GPBAR1 gene ablation enhanced the recruitment of classically activated macrophages in the colonic lamina propria and worsened the severity of inflammation. In contrast, GPBAR1 activation by BAR501 reversed intestinal inflammation in the trinitrobenzenesulfonic acid and oxazolone models by reducing the trafficking of Ly6C+ monocytes from blood to intestinal mucosa. Exposure to BAR501 shifted intestinal macrophages from a classically activated (CD11b+, CCR7+, F4/80-) to an alternatively activated (CD11b+, CCR7-, F4/80+) phenotype, reduced the expression of inflammatory genes (TNF-α, IFN-γ, IL-1β, IL-6, and CCL2 mRNAs), and attenuated the wasting syndrome and severity of colitis (≈70% reduction in the Colitis Disease Activity Index). The protective effect was lost in Gpbar1-/- mice. Exposure to BAR501 increased the colonic expression of IL-10 and TGF-β mRNAs and the percentage of CD4+/Foxp3+ cells. The beneficial effects of BAR501 were lost in Il-10-/- mice. In a macrophage cell line, regulation of IL-10 by BAR501 was GPBAR1 dependent and was mediated by the recruitment of CREB to its responsive element in the IL-10 promoter. In conclusion, GPBAR1 is expressed in circulating monocytes and colonic macrophages, and its activation promotes a IL-10-dependent shift toward an alternatively activated phenotype. The targeting of GPBAR1 may offer therapeutic options in inflammatory bowel diseases.

Festa C, De Marino S, Carino A, Sepe V, Marchianò S, Cipriani S, Di Leva FS, Limongelli V, Monti MC, Capolupo A, Distrutti E, Fiorucci S, Zampella A. Targeting Bile Acid Receptors: Discovery of a Potent and Selective Farnesoid X Receptor Agonist as a New Lead in the Pharmacological Approach to Liver Diseases. Front Pharmacol. 2017 Mar 30;8:162.

Abstract
Bile acid (BA) receptors represent well-defined targets for the development of novel therapeutic approaches to metabolic and inflammatory diseases. In the present study, we report the generation of novel C-3 modified 6-ethylcholane derivatives. The pharmacological characterization and molecular docking studies for the structure-activity rationalization, allowed the identification of 3β-azido-6α-ethyl-7α-hydroxy-5β-cholan-24-oic acid (compound 2), a potent and selective FXR agonist with a nanomolar potency in transactivation assay and high efficacy in the recruitment of SRC-1 co-activator peptide in Alfa Screen assay. In vitro, compound 2 was completely inactive towards common off-targets such as the nuclear receptors PPARα, PPARγ, LXRα, and LXRβ and the membrane G-coupled BA receptor, GPBAR1. This compound when administered in vivo exerts a robust FXR agonistic activity increasing the liver expression of FXR-target genes including SHP, BSEP, OSTα, and FGF21, while represses the expression of CYP7A1 gene that is negatively regulated by FXR. Collectively these effects result in a significant reshaping of BA pool in mouse. In summary, compound 2 represents a promising candidate for drug development in liver and metabolic disorders.

De Marino S, Carino A, Masullo D, Finamore C, Marchianò S, Cipriani S, Di Leva FS, Catalanotti B, Novellino E, Limongelli V, Fiorucci S, Zampella A. Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists. Sci Rep. 2017 Feb 24;7:43290.

Abstract
Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders.

Carino A, Cipriani S, Marchianò S, Biagioli M, Santorelli C, Donini A, Zampella A, Monti MC, Fiorucci S. BAR502, a dual FXR and GPBAR1 agonist, promotes browning of white adipose tissue and reverses liver steatosis and fibrosis. Sci Rep. 2017 Feb 16;7:42801.

Abstract
Non-alcoholic steatohepatitis (NASH) is a highly prevalent chronic liver disease. Here, we have investigated whether BAR502, a non-bile acid, steroidal dual ligand for FXR and GPBAR1, reverses steato-hepatitis in mice fed a high fat diet (HFD) and fructose. After 9 week, mice on HFD gained ≈30% of b.w (P < 0.01 versus naïve) and were insulin resistant. These overweighting and insulin resistant mice were randomized to receive HFD or HFD in combination with BAR502. After 18 weeks, HFD mice developed NASH like features with severe steato-hepatitis and fibrosis, increased hepatic content of triacylglycerol and cholesterol and expression of SREPB1c, FAS, ApoC2, PPARα and γ, α-SMA, α1 collagen and MCP1 mRNAs. Treatment with BAR502 caused a ≈10% reduction of b.w., increased insulin sensitivity and circulating levels of HDL, while reduced steatosis, inflammatory and fibrosis scores and liver expression of SREPB1c, FAS, PPARγ, CD36 and CYP7A1 mRNA. BAR502 increased the expression of SHP and ABCG5 in the liver and SHP, FGF15 and GLP1 in intestine. BAR502 promoted the browning of epWAT and reduced liver fibrosis induced by CCl4. In summary, BAR502, a dual FXR and GPBAR1 agonist, protects against liver damage caused by HFD by promoting the browning of adipose tissue.

Cipriani S, Carino A, Masullo D, Zampella A, Distrutti E, Fiorucci S. Decoding the role of the nuclear receptor SHP in regulating hepatic stellate cells and liver fibrogenesis. Sci Rep. 2017 Jan 24;7:41055.

Abstract
The small heterodimer partner (SHP) is an orphan nuclear receptor that lacks the DNA binding domain while conserves a putative ligand-binding site, thought that endogenous ligands for this receptor are unknown. Previous studies have determined that SHP activation protects against development of liver fibrosis a process driven by trans-differentiation and activation of hepatic stellate cells (HSCs), a miofibroblast like cell type, involved in extracellular matrix (ECM) deposition. To dissect signals involved in this activity we generated SHP-overexpressing human and rat HSCs. Forced expression of SHP in HSC-T6 altered the expression of 574 genes. By pathway and functional enrichment analyses we detected a cluster of 46 differentially expressed genes involved in HSCs trans-differentiation. Using a isoxazole scaffold we designed and synthesized a series of SHP agonists. The most potent member of this group, ISO-COOH (EC50: 9 μM), attenuated HSCs trans-differentiation and ECM deposition in vitro, while in mice rendered cirrhotic by carbon tetrachloride (CCl4) or α-naphthyl-isothiocyanate (ANIT), protected against development of liver fibrosis as measured by morphometric analysis and expression of α-SMA and α1-collagen mRNAs. In aggregate, present results identify SHP as a counter-regulatory signal for HSCs transactivation and describe a novel class of SHP agonists endowed with anti-fibrotic activity.

Fiorucci S, Zampella A, Cirino G, Bucci M, Distrutti E. Decoding the vasoregulatory activities of bile acid-activated receptors in systemic and portal circulation: role of gaseous mediators. Am J Physiol Heart Circ Physiol. 2017 Jan 1;312(1):H21-H32.

Abstract
Bile acids are end products of cholesterol metabolism generated in the liver and released in the intestine. Primary and secondary bile acids are the result of the symbiotic relation between the host and intestinal microbiota. In addition to their role in nutrient absorption, bile acids are increasingly recognized as regulatory signals that exert their function beyond the intestine by activating a network of membrane and nuclear receptors. The best characterized of these bile acid-activated receptors, GPBAR1 (also known as TGR5) and the farnesosid-X-receptor (FXR), have also been detected in the vascular system and their activation mediates the vasodilatory effects of bile acids in the systemic and splanchnic circulation. GPBAR1, is a G protein-coupled receptor, that is preferentially activated by lithocholic acid (LCA) a secondary bile acid. GPBAR1 is expressed in endothelial cells and liver sinusoidal cells (LSECs) and responds to LCA by regulating the expression of both endothelial nitric oxide synthase (eNOS) and cystathionine-γ-lyase (CSE), an enzyme involved in generation of hydrogen sulfide (H2S). Activation of CSE by GPBAR1 ligands in LSECs is due to genomic and nongenomic effects, involves protein phosphorylation, and leads to release of H2S. Despite that species-specific effects have been described, vasodilation caused by GPBAR1 ligands in the liver microcirculation and aortic rings is abrogated by inhibition of CSE but not by eNOS inhibitor. Vasodilation caused by GPBAR1 (and FXR) ligands also involves large conductance calcium-activated potassium channels likely acting downstream to H2S. The identification of GPBAR1 as a vasodilatory receptor is of relevance in the treatment of complex disorders including metabolic syndrome-associated diseases, liver steatohepatitis, and portal hypertension.