RESUMEN
OBJECTIVES: The mechanisms underlying increased cardiovascular risk in primary Sjögren's syndrome (pSS) remain unclear. Since the recently discovered angiogenic T cells (Tang) may participate in endothelial repair by cooperating with endothelial progenitor cells (EPC), we aimed to quantify and characterise Tang in the peripheral blood and minor salivary glands (MSG) of pSS patients. METHODS: Tang (CD3+CD31+CXCR4+) and EPC (CD34+CD133+VEGFR-2+) were quantified by flow cytometry in peripheral blood samples from 36 pSS patients and 20 healthy donors. Tang subsets were assessed on the basis of CD4, CD8 and CD28 expression. Labial MSG sections from 10 pSS patients and 12 non-pSS sicca syndrome controls were subjected to immunofluorescence staining to investigate the presence of Tang and the expression of the CXCR4-ligand stromal cell-derived factor-1 (SDF-1)/CXCL12. RESULTS: Circulating Tang cells were expanded and directly correlated to EPC in pSS. Both Tang and EPC directly correlated with disease activity as calculated with the EULAR Sjögren's syndrome disease activity index (ESSDAI). In pSS, the majority of Tang cells were CD4-CD8- double negative (DN) and lacked CD28 revealing a senescent phenotype. A subset of CD4+, CD8+ and DN Tang cells produced interleukin-17. Immunohistology revealed the exclusive presence of periductal and perivascular infiltrating Tang cells along with increased SDF-1/CXCL12 expression in pSS MSG compared to non-pSS sicca syndrome controls. CONCLUSIONS: In pSS, Tang cells are expanded in peripheral blood and infiltrate MSG. Tang may be novel actors in pSS-related endothelial dysfunction and glandular neo-angiogenesis and inflammation.
Asunto(s)
Síndrome de Sjögren , Linfocitos T Reguladores/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inflamación , Glándulas Salivales/inmunología , Glándulas Salivales Menores , Transducción de Señal , Síndrome de Sjögren/sangre , Síndrome de Sjögren/etiología , Síndrome de Sjögren/inmunología , Linfocitos T Reguladores/citologíaRESUMEN
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-1ß, 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-ß 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.
Asunto(s)
Colitis/inmunología , Regulación de la Expresión Génica/inmunología , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antígenos Ly/genética , Antígenos Ly/inmunología , Línea Celular , Movimiento Celular , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Colestanoles/administración & dosificación , Colestanoles/farmacología , Colitis/inducido químicamente , Colitis/metabolismo , Inflamación/inmunología , Interleucina-10/deficiencia , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Ratones , Membrana Mucosa/inmunología , Oxazolona/administración & dosificación , Fenotipo , Regiones Promotoras Genéticas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Ácido Trinitrobencenosulfónico/administración & dosificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Platelets have the ability to influence the immune system and the inflammatory process and may be strongly involved in the whole pathogenic process of chronic inflammatory joint diseases, such as rheumatoid arthritis. They may play a significant role even before the clinical onset of the disease, contributing to the loss of tolerance of the immune system and the induction of autoimmunity. Subsequently, they can interact with the most important cellular players involved in autoimmunity and inflammation, namely innate immunity cells and T cells and eventually contribute to the building of inflammation in the synovium, thus inducing the activation, migration, and proliferation of fibroblasts that eventually lead to joint damage. Due to their peculiar features, studying the behavior of platelets is a challenging task; however, platelets may prove to be valuable therapeutic targets in the future.
Asunto(s)
Artritis Reumatoide/inmunología , Plaquetas/inmunología , Sinovitis/inmunología , Artritis Reumatoide/patología , Autoinmunidad/inmunología , Fibroblastos/inmunología , Humanos , Inmunidad Innata/inmunología , Inflamación/inmunología , Inflamación/patología , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Sinovitis/patología , Linfocitos T/inmunologíaRESUMEN
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.
Asunto(s)
Materiales Biomiméticos/química , Quimiocinas/aislamiento & purificación , Proteínas Inmovilizadas/química , Receptores CCR5/química , Biomimética , Adhesión Celular , Células Hep G2 , Heparina/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/terapia , Modelos Moleculares , Nanomedicina , Poliésteres/química , Propiedades de SuperficieRESUMEN
GPBAR1 is a bile acid-activated receptor (BAR) for secondary bile acids, lithocholic (LCA) and deoxycholic acid (DCA), expressed in the enterohepatic tissues and in the vasculature by endothelial and smooth muscle cells. Despite that bile acids cause vasodilation, it is unclear why these effects involve GPBAR1, and the vascular phenotype of GPBAR1 deficient mice remains poorly defined. Previous studies have suggested a role for nitric oxide (NO) in regulatory activity exerted by GPBAR1 in liver endothelial cells. Hydrogen sulfide (H2S) is a vasodilatory agent generated in endothelial cells by cystathionine-γ-lyase (CSE). Here we demonstrate that GPBAR1 null mice had increased levels of primary and secondary bile acids and impaired vasoconstriction to phenylephrine. In aortic ring preparations, vasodilation caused by chenodeoxycholic acid (CDCA), a weak GPBAR1 ligand and farnesoid-x-receptor agonist (FXR), was iberiotoxin-dependent and GPBAR1-independent. In contrast, vasodilation caused by LCA was GPBAR1 dependent and abrogated by propargyl-glycine, a CSE inhibitor, and by 5ß-cholanic acid, a GPBAR1 antagonist, but not by N(5)-(1-iminoethyl)-l-ornithine (l-NIO), an endothelial NO synthase inhibitor, or iberiotoxin, a large-conductance calcium-activated potassium (BKCa) channels antagonist. In venular and aortic endothelial (HUVEC and HAEC) cells GPBAR1 activation increases CSE expression/activity and H2S production. Two cAMP response element binding protein (CREB) sites (CREs) were identified in the CSE promoter. In addition, TLCA stimulates CSE phosphorylation on serine residues. In conclusion we demonstrate that GPBAR1 mediates the vasodilatory activity of LCA and regulates the expression/activity of CSE. Vasodilation caused by CDCA involves BKCa channels. The GPBAR1/CSE pathway might contribute to endothelial dysfunction and hyperdynamic circulation in liver cirrhosis.
Asunto(s)
Aorta/metabolismo , Ácidos y Sales Biliares/metabolismo , Cistationina gamma-Liasa/genética , Sulfuro de Hidrógeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Vasodilatación/genética , Animales , Aorta/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Ácido Quenodesoxicólico/farmacología , Ácidos Cólicos/farmacología , Cistationina gamma-Liasa/metabolismo , Células Endoteliales , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácido Litocólico/farmacología , Ratones Noqueados , Ornitina/análogos & derivados , Ornitina/farmacología , Péptidos/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: CCR5 plays an important role in atherosclerosis and ischemic cardiovascular diseases, as well as in HIV replication and diffusion. HIV infection is characterized by a high burden of cardiovascular diseases, particularly in subjects exposed to ritonavir-boosted protease inhibitors. Maraviroc, a CCR5 antagonist antiretroviral drug, might provide benefit for patients with M-tropic HIV infections at high risk for cardiovascular diseases. METHODS AND RESULTS: Exposure to maraviroc limits the evolution and associated systemic inflammation of ritonavir-induced atherosclerotic in ApoE(-/-) mice and inhibits plaques development in a late model of atherosclerosis in which dyslipidemia plays the main pathogenic role. In ritonavir-treated mice, maraviroc reduced plaque areas and macrophage infiltration; downregulated the local expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and interleukin-17A; and reduced tumor necrosis factor-α and RANTES (regulated on activation, normal T cell expressed, and secreted). Moreover, maraviroc counterregulated ritonavir-induced lipoatrophy and interlelukin-6 gene expression in epididymal fat, along with the splenic proinflammatory profile and expression of CD36 on blood monocytes. In the late model, maraviroc inhibited atherosclerotic progression by reducing macrophage infiltration and lowering the expression of adhesion molecules and RANTES inside the plaques. However, limited systemic inflammation was observed. CONCLUSIONS: In a mouse model of genetic dyslipidemia, maraviroc reduced the atherosclerotic progression by interfering with inflammatory cell recruitment into plaques. Moreover, in mice characterized by a general ritonavir-induced inflammation, maraviroc reversed the proinflammatory profile. Therefore, maraviroc could benefit HIV-positive patients with residual chronic inflammation who are at a high risk of acute coronary disease despite a suppressive antiretroviral therapy. To determine these benefits, large clinical studies are needed.
Asunto(s)
Antirretrovirales/efectos adversos , Aterosclerosis/inducido químicamente , Aterosclerosis/prevención & control , Antagonistas de los Receptores CCR5 , Ciclohexanos/uso terapéutico , Placa Aterosclerótica/inducido químicamente , Placa Aterosclerótica/prevención & control , Ritonavir/efectos adversos , Triazoles/uso terapéutico , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Movimiento Celular , Quimiocina CCL2/metabolismo , Quimiocina CCL5/metabolismo , Modelos Animales de Enfermedad , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-17/metabolismo , Macrófagos/patología , Masculino , Maraviroc , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/metabolismo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.
RESUMEN
The glucocorticoid receptor (GR) is a master gene orchestrating the activation of gluconeogenic genes in the liver in response to food withdrawal. Mechanisms of GR regulation by other nuclear receptors, however, are poorly defined. Here, we report that the farnesoid X receptor (FXR), a bile acid sensor, activates gluconeogenic pathways in the liver and regulates GR expression and activity. FXR-null mice are hypoglycemic in the unfed state and exhibit both a reduced hepatic production of glucose in response to the pyruvate challenge and a decreased expression of two rate-limiting enzymes involved in gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), along with blunted liver expression of GR. Treating wild-type mice with a semisynthetic FXR ligand (6E-CDCA) increases the liver expression of GR, PEPCK, and G6Pase. This effect was lost in fed animals, as well as in FXR(-/-) mice. The human and mouse GR promoters contain a conserved FXR-responsive element (an ER-8 sequence) whose activation by FXR ligation leads to GR transcription. GR silencing by siRNA in vitro or its pharmacological antagonism in vivo with mifepristone reverses the effect of FXR activation on expression of gluconeogenic genes. These findings demonstrate that an FXR-GR pathway regulates the activation of hepatic gluconeogenesis in the transition from the unfed to the fed state.
Asunto(s)
Ayuno/metabolismo , Gluconeogénesis/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/metabolismo , Ácido Quenodesoxicólico/farmacología , Gluconeogénesis/genética , Glucosa-6-Fosfatasa/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Ligandos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Transducción de SeñalRESUMEN
Glucocorticoids (GCs) are important endocrine regulators of a wide range of physiological processes ranging from immune function to glucose and lipid metabolism. For decades, synthetic glucocorticoids such as dexamethasone have been the cornerstone for the clinical treatment of inflammatory bowel diseases (IBD). A previous study has shown that farnesoid X receptor (FXR) enhances the transcription of NR3C1 gene, which encodes for human GR, by binding to a conserved FXR response element (FXRE) in the distal promoter of this gene. In the present study we demonstrate that FXR promotes the resolution of colitis in rodents by enhancing Gr gene transcription. We used the chromatin conformation capture (3C) assay to demonstrate that this FXRE is functional in mediating a head-to-tail chromatin looping, thus increasing Gr transcription efficiency. These findings underscore the importance of FXR/GR axis in the control of intestinal inflammation.
Asunto(s)
Cromatina/metabolismo , Colitis/genética , Regiones Promotoras Genéticas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Glucocorticoides/genética , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Células Cultivadas , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/antagonistas & inhibidores , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/uso terapéutico , Cromatina/química , Cromatina/genética , Dexametasona/farmacología , Dexametasona/uso terapéutico , Humanos , Ratones , Ratones Noqueados , Mifepristona/farmacología , Elementos de Respuesta/genética , Ácido TrinitrobencenosulfónicoRESUMEN
In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.
Asunto(s)
Antiinflamatorios , Artritis Reumatoide/tratamiento farmacológico , Colanos/farmacología , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Poríferos/química , Receptores de Esteroides/agonistas , Ésteres del Ácido Sulfúrico/farmacología , Animales , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/patología , Proteína C-Reactiva/metabolismo , Cartílago/patología , Quimiocina CCL3/metabolismo , Quimiocina CCL5/metabolismo , Colágeno Tipo II , Citocinas/sangre , Hepatocitos/efectos de los fármacos , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Conformación Molecular , Receptor X de Pregnano , Receptores de Esteroides/biosíntesis , Receptores de Esteroides/genética , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Multidrug resistance protein-4 (MRP4) is a member of the multidrug resistance associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive up-regulation in response to cholestatic injury or bile acid feeding. In this study we demonstrate that farnesoid X receptor (FXR) regulates MRP4 in vivo and in vitro. In vivo deletion of FXR induces MRP4 gene expression. In vitro treatment of HepG2 cells with FXR ligands, chenodeoxycholic acid (CDCA), cholic acid (CA) and the synthetic ligand GW-4064 suppresses basal mRNA level of the MRP4 gene as well as the co-treatment with CDCA and 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), an activator of constitutive androstane receptor (CAR). We found in the human MRP4 promoter a CAR responsive element (CARE) embedded within an FXR responsive element (FXRE). We cloned this region and found that FXR suppresses CAR activity in luciferase assay. Finally, we demonstrated that FXR competes with CAR for binding to this overlapping binding site. Our results support the view that FXR activation in obstructive cholestasis might worsen liver injury by hijacking a protective mechanism regulated by CAR and provides a new molecular explanation to the pathophysiology of cholestasis.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Secuencia de Bases , Ácidos y Sales Biliares/farmacología , Sitios de Unión , Células Cultivadas , Receptor de Androstano Constitutivo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Eliminación de Gen , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Cinética , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis/efectos de los fármacos , Co-Represor 1 de Receptor Nuclear/metabolismo , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Hepatic transport and metabolism of glutamate and glutamine are regulated by intervention of several proteins. Glutamine is taken up by periportal hepatocytes and is the major source of ammonia for urea synthesis and glutamate for N-acetylglutamate (NAG) synthesis, which is catalyzed by the N-acetylglutamate synthase (NAGS). Glutamate is taken up by perivenous hepatocytes and is the main source for the synthesis of glutamine, catalyzed by glutamine synthase (GS). Accumulation of glutamate and ammonia is a common feature of chronic liver failure, but mechanism that leads to failure of the urea cycle in this setting is unknown. The Farnesoid X Receptor (FXR) is a bile acid sensor in hepatocytes. Here, we have investigated its role in the regulation of the metabolism of both glutamine and glutamate. In vitro studies in primary cultures of hepatocytes from wild type and FXR(-/-) mice and HepG2 cells, and in vivo studies, in FXR(-/-) mice as well as in a rodent model of hepatic liver failure induced by carbon tetrachloride (CCl(4)), demonstrate a role for FXR in regulating this metabolism. Further on, promoter analysis studies demonstrate that both human and mouse NAGS promoters contain a putative FXRE, an ER8 sequence. EMSA, ChIP and luciferase experiments carried out to investigate the functionality of this sequence demonstrate that FXR is essential to induce the expression of NAGS. In conclusion, FXR activation regulates glutamine and glutamate metabolism and FXR ligands might have utility in the treatment of hyperammonemia states.
Asunto(s)
Glutamatos/metabolismo , Glutamina/metabolismo , Hiperamonemia/metabolismo , Cirrosis Hepática Experimental/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , N-Acetiltransferasa de Aminoácidos/genética , N-Acetiltransferasa de Aminoácidos/metabolismo , Animales , Western Blotting , Tetracloruro de Carbono/toxicidad , Núcleo Celular , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Hiperamonemia/inducido químicamente , Cirrosis Hepática Experimental/inducido químicamente , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Endothelial dysfunction contributes to increased cardiovascular (CV) disease in rheumatoid arthritis (RA). Angiogenic T cells (Tang) are a key regulator of vascular function via their interaction with endothelial progenitor cells (EPCs). Methotrexate (MTX) has been associated to reduced CV disease risk, but its effects on endothelial homeostasis have been poorly explored. We investigated MTX effects on endothelial homeostasis in early, treatment-naïve RA patients. METHODS: Fifteen untreated, early RA patients and matched healthy controls (HC) were enrolled. RA patients with long-standing disease in remission or low disease activity treated with MTX for at least 6 months were selected as controls. Circulating CD28+ and CD28null Tang cell, endothelial microparticle (EMP), EPC and soluble vascular cell adhesion molecule (sVCAM)-1 levels were measured. RESULTS: Tang percentage was higher in early RA than in HCs and significantly increased after 3-month MTX treatment. Tang cells in RA were characterized by higher percentage of CD28null and lower CD28-positive cells than HCs. MTX restored a Tang cell phenotype similar to HCs. Altered sVCAM-1, EMP and EPC were restored to levels similar to HCs after a 3-month MTX. Biomarker levels after 3 months of MTX were not different to those of patients with long-standing treatment. CONCLUSIONS: MTX has a positive effect on Tang, sVCAM-1, EPCs and EMPs in RA. Restoration of imbalance between CD28 + and CD28null Tang by MTX may be one of the mechanisms underlying its favourable effects on endothelial dysfunction. These effects seem to be long-lasting and independent from systemic inflammation reduction, suggesting a direct effect of MTX on the endothelium.
Asunto(s)
Antirreumáticos , Artritis Reumatoide , Humanos , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Biomarcadores , Antígenos CD28 , Endotelio , Metotrexato/uso terapéutico , Molécula 1 de Adhesión Celular VascularRESUMEN
OBJECTIVE: The regulation of hepatic stellate cells (HSCs) by bacterial lipopolysaccharide (LPS) represents a recently-discovered and novel mechanism for hepatic injury and fibrosis. Stimulation of HSCs with LPS results in a rapid and marked induction of interleukin (IL)1ß, IL6 and tumor necrosis factor α. These events lead to the development of the activated phenotype in the HSCs associated with fibrosis and inflammation in the injured liver. We have previously demonstrated that farnesoid X receptor (FXR) activation increases transcription of rat peroxisome proliferator-activated receptor-γ (PPARγ) gene in HSCs. We aimed at evaluating the molecular mechanism of the transcriptional regulation of the PPARγ gene by FXR. METHODS: Real-time PCR, ELISA, transactivations, EMSA and ChIP experiments were performed in HSC-T6 cells, in primary HSCs, in HEK293T cells and in CCl(4)-treated rats. RESULTS: In vivo and in vitro activation of FXR downregulates cytokines and collagen(α)1 while inducing PPARγ and small heterodimer partner (SHP). NUBIScan analysis of rat PPARγ promoter revealed the presence of a putative FXR response element. Cotransfection with FXR/retinoic acid receptor significantly enhanced chenodeoxycholic acid-induced luciferase activity. EMSA experiments demonstrated that FXR was able to bind to an inverted repeat-1 sequence and ChIP experiments confirmed that FXR is recruited on the PPARγ promoter. CONCLUSION: The present study provides a molecular basis for the physiological cross-talk between FXR and PPARγ pathways in HSCs.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Ácido Quenodesoxicólico/farmacología , PPAR gamma/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Tetracloruro de Carbono , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Interleucina-1beta/metabolismo , Masculino , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Fenotipo , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. In this study, we report that activation of the farnesoid X receptor (FXR), a member of the ligand-activated nuclear receptor superfamily and bile sensor highly expressed in the liver, attenuates liver injury in a model of autoimmune hepatitis induced by Con A. We found that FXR gene ablation results in a time-dependent increase of liver expression (up to 20-fold in a 9-mo-old mouse) of osteopontin, a NKT cell-derived extracellular matrix protein and immunoregulatory cytokine. In comparison to wild-type, FXR(-/-) mice are more susceptible to Con A-induced hepatitis and react to Con A administration by an unregulated production of osteopontin. Administering wild-type mice with a synthetic FXR agonist attenuated Con A-induced liver damage and liver expression of the osteopontin gene. By in vitro studies, we found that FXR is expressed by primarily isolated NKT cells and its ablation favors ostepontin production in response to Con A. Chromatin immunoprecipitation assay and coimmunoprecipitation experiments demonstrate that the short heterodimer partner (SHP), a nuclear receptor and FXR target, was expressed by NKT cell hybridomas and increased in response to FXR activation. FXR activates SHP that interacts with and inhibits c-Jun binding to the osteopontin promoter. These data indicate that in NKT cells, FXR activation causes a SHP-mediated inhibition of osteopontin production. These data support the notion that the bile acid sensor FXR regulates the activation of liver NKT cells.
Asunto(s)
Hepatitis/inmunología , Células T Asesinas Naturales/inmunología , Osteopontina/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Enfermedad Aguda , Animales , Ácidos y Sales Biliares/inmunología , Ácidos y Sales Biliares/metabolismo , Línea Celular , Concanavalina A/farmacología , Hepatitis/genética , Hepatitis/metabolismo , Interferón gamma/agonistas , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-4/agonistas , Interleucina-4/inmunología , Interleucina-4/metabolismo , Hígado/inmunología , Hígado/patología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitógenos/farmacología , Células T Asesinas Naturales/metabolismo , Osteopontina/agonistas , Osteopontina/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/inmunología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factor de Necrosis Tumoral alfa/agonistas , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The farnesoid X receptor (FXR) is a bile acid activated nuclear receptor. Zucker (fa/fa) rats, harboring a loss of function mutation of the leptin receptor, develop diabetes, insulin resistance, obesity, and liver steatosis. In this study, we investigated the effect of FXR activation by 6-ethyl-chenodeoxycholic acid, (6E-CDCA, 10 mg/kg) on insulin resistance and liver and muscle lipid metabolism in fa/fa rats and compared its activity with rosiglitazone (10 mg/kg) alone or in combination with 6E-CDCA (5 mg/kg each). In comparison to lean (fa/+), fa/fa rats on a normal diet developed insulin resistance and liver steatosis. FXR activation protected against body weight gain and liver and muscle fat deposition and reversed insulin resistance as assessed by insulin responsive substrate-1 phosphorylation on serine 312 in liver and muscles. Activation of FXR reduced liver expression of genes involved in fatty acid synthesis, lipogenesis, and gluconeogenesis. In the muscles, FXR treatment reduced free fatty acid synthesis. Rosiglitazone reduced blood insulin, glucose, triglyceride, free fatty acid, and cholesterol plasma levels but promoted body weight gain (20%) and liver fat deposition. FXR activation reduced high density lipoprotein plasma levels. In summary, FXR administration reversed insulin resistance and correct lipid metabolism abnormalities in an obesity animal model.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Hígado Graso/prevención & control , Hipolipemiantes/uso terapéutico , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Obesidad/complicaciones , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Ácido Quenodesoxicólico/administración & dosificación , Ácido Quenodesoxicólico/uso terapéutico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Hígado Graso/sangre , Hígado Graso/patología , Regulación de la Expresión Génica/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/uso terapéutico , Hipolipemiantes/administración & dosificación , Proteínas Sustrato del Receptor de Insulina/metabolismo , Metabolismo de los Lípidos/genética , Lípidos/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Obesidad/sangre , Fosforilación/efectos de los fármacos , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Zucker , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Rosiglitazona , Tiazolidinedionas/administración & dosificación , Tiazolidinedionas/uso terapéutico , Factores de TiempoRESUMEN
BACKGROUND: Hydrogen sulphide (H2S) is a gaseous neuro-mediator that exerts analgesic effects in rodent models of visceral pain by activating KATP channels. A body of evidence support the notion that KATP channels interact with endogenous opioids. Whether H2S-induced analgesia involves opioid receptors is unknown. METHODS: The perception of painful sensation induced by colorectal distension (CRD) in conscious rats was measured by assessing the abdominal withdrawal reflex. The contribution of opioid receptors to H2S-induced analgesia was investigated by administering rats with selective mu, kappa and delta opioid receptor antagonists and antisenses. To investigate whether H2S causes mu opioid receptor (MOR) transactivation, the neuronal like cells SKNMCs were challenged with H2S in the presence of MOR agonist (DAMGO) or antagonist (CTAP). MOR activation and phosphorylation, its association to beta arrestin and internalization were measured. RESULTS: H2S exerted a potent analgesic effects on CRD-induced pain. H2S-induced analgesia required the activation of the opioid system. By pharmacological and molecular analyses, a robust inhibition of H2S-induced analgesia was observed in response to central administration of CTAP and MOR antisense, while kappa and delta receptors were less involved. H2S caused MOR transactivation and internalization in SKNMCs by a mechanism that required AKT phosphorylation. MOR transactivation was inhibited by LY294002, a PI3K inhibitor, and glibenclamide, a KATP channels blocker. CONCLUSIONS: This study provides pharmacological and molecular evidence that antinociception exerted by H2S in a rodent model of visceral pain is modulated by the transactivation of MOR. This observation provides support for development of new pharmacological approaches to visceral pain.
Asunto(s)
Analgesia , Analgésicos/metabolismo , Sulfuro de Hidrógeno/metabolismo , Dolor/metabolismo , Receptores Opioides mu/metabolismo , Sulfuros/farmacología , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Canales KATP/agonistas , Canales KATP/antagonistas & inhibidores , Canales KATP/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Canales de Potasio/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Receptores Opioides mu/antagonistas & inhibidores , VíscerasRESUMEN
As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet ß-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr-/- mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25-/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.
Asunto(s)
Anticuerpos Monoclonales , Encapsulación Celular , Diabetes Mellitus Tipo 1/prevención & control , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Hibridomas , Linfocitos T Reguladores/fisiología , Animales , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , Ratones , Ratones Endogámicos NOD , Ratones NoqueadosRESUMEN
BACKGROUND: Experimental CCR5 antagonism with maraviroc in atherosclerosis-prone mice and preliminary data in humans suggest an anti-atherosclerotic effect of the drug. We assessed the impact of maraviroc treatment in persons living with HIV on subclinical indicators of atherosclerosis. METHODS: Persons living with HIV on effective antiretroviral therapy (ART) including only protease inhibitors were recruited if they had a Framingham risk score >20% and brachial flow-mediated dilation (bFMD) <4%, as indices of high cardiovascular risk. Maraviroc (300 mg per os for 24 weeks) was administered, in addition to ongoing ART, to all patients using a crossover design. Brachial FMD, carotid-femoral pulse wave velocity (cfPWV), and carotid intima-media thickness (cIMT) were measured as markers of atherosclerosis. Vascular competence-as expressed by the ratio of circulating endothelial microparticles (EMPs) to endothelial progenitor cells (EPCs)-and markers of systemic inflammation and monocyte and platelet activation were assessed. RESULTS: Maraviroc treatment significantly improved bFMD, cfPWV, and cIMT by 66%, 11%, and 13%, respectively (P = .002, P = .022, P = .038, respectively). We also found a beneficial effect of maraviroc on the EMP/EPC ratio (P < .001) and platelet/leucocyte aggregates (P = .013). No significant changes in markers of systemic inflammation, monocyte activation, and microbial translocation were observed. CONCLUSIONS: Maraviroc led to significant improvements in several markers for cardiovascular risk, endothelial dysfunction, arterial stiffness, and early carotid atherosclerosis, which was accompanied by an increase of vascular competence, without seeming to affect systemic inflammation. Our data support the need for larger studies to test for any effects of maraviroc on preventing atherosclerosis-driven pathologies.
RESUMEN
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.