RESUMEN
An immunoregulatory role of stem cells, often mediated by their secretome, has been claimed by several studies. Stem cell-derived extracellular vesicles (EVs) are crucial components of the secretome. EVs, a heterogeneous group of membranous vesicles released by many cell types into the extracellular space, are now considered as an additional mechanism for intercellular communication. In this study, we aimed at investigating whether human amniotic stem cell-derived extracellular vesicles (HASC-EVs) were able to interfere with inflammasome activation in the THP-1 cell line. Two subsets of HASC-EVs were collected by sequential centrifugation, namely HASC-P10 and HASC-P100. We demonstrated that HASC-EVs were neither internalized into nor undertake a direct interaction with THP-1 cells. We showed that HASC-P10 and P100 were able to intrinsically produce ATP, which was further converted to adenosine by 5'-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39). We found that THP-1 cells conditioned with both types of HASC-EVs failed to activate the NLRP3/caspase-1/inflammasome platform in response to LPS and ATP treatment by a mechanism involving A2a adenosine receptor activation. These results support a role for HASC-EVs as independent metabolic units capable of modifying the cellular functions, leading to anti-inflammatory effects in monocytic cells.
Asunto(s)
Líquido Amniótico/citología , Antiinflamatorios/farmacología , Vesículas Extracelulares/metabolismo , Inflamasomas/antagonistas & inhibidores , Inflamación/prevención & control , Monocitos/citología , Células Madre/citología , Adenosina/metabolismo , Líquido Amniótico/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Monocitos/metabolismo , Antagonistas de Receptores Purinérgicos P1/farmacología , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/metabolismo , Células Madre/metabolismo , Células THP-1RESUMEN
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.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Autofagia/efectos de los fármacos , Ácidos Cólicos/farmacología , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares , Receptores Acoplados a Proteínas G , Animales , Autofagia/genética , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Adipogenesis is a finely orchestrated program involving a transcriptional cascade coordinated by CEBP and PPAR family members and by hormonally induced signaling pathways. Alterations in any of these factors result into impaired formation of fully differentiated adipocytes. Tm7sf2 gene encodes for a Δ(14)-sterol reductase primarily involved in cholesterol biosynthesis. Furthermore, TM7SF2 modulates the expression of the master gene of adipogenesis PPARγ, suggesting a role in the regulation of adipose tissue homeostasis. We investigated the differentiation of Tm7sf2-/- MEFs into adipocytes, compared to Tm7sf2+/+ MEFs. Tm7sf2 expression was increased at late stage of differentiation in wild type cells, while Tm7sf2-/- MEFs exhibited a reduced capacity to differentiate into mature adipocytes. Indeed, Tm7sf2-/- MEFs had lower neutral lipid accumulation and reduced expression of adipogenic regulators. At early stage, the reduction in C/EBPß expression impaired mitotic clonal expansion, which is needed by preadipocytes for adipogenesis induction. At late stage, the expression and activity of C/EBPα and PPARγ were inhibited in Tm7sf2-/- cells, leading to the reduced expression of adipocyte genes like Srebp-1c, Fasn, Scd-1, Adipoq, Fabp4, and Glut4. Loss of the acquisition of adipocyte phenotype was accompanied by a reduction in the levels of Irs1, and phosphorylated Akt and ERK1/2, indicating a blunted insulin signaling in differentiating Tm7sf2-/- cells. Moreover, throughout the differentiation process, increased expression of the antiadipogenic Mmp3 was observed in MEFs lacking Tm7sf2. These findings indicate Tm7sf2 as a novel factor influencing adipocyte differentiation that could be relevant to adipose tissue development and maintenance of metabolic health.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular/genética , Oxidorreductasas/genética , PPAR gamma/genética , Células 3T3-L1 , Adipocitos/citología , Adipogénesis/genética , Tejido Adiposo/crecimiento & desarrollo , Tejido Adiposo/metabolismo , Animales , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica/genética , Resistencia a la Insulina/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Fosforilación/genética , Transducción de Señal/genéticaRESUMEN
Prostate-derived extracellular vesicles (pEVs) may represent a way to selectively transport cargo molecules from the producing cells to the target cells to allow biological events, both in physiological and pathological circumstances. pEVs cargo participates in the modulation of the inflammatory responses in physiological conditions and during cancer progression. In the present study, we examined the expression levels of miRNA Let-7b, in both precursor and mature forms, in noncancerous and cancerous prostate cell lines, PNT2 and PC3 respectively, and in their extracellular vesicles (EVs) using reverse-transcription quantitative PCR strategies. We showed that miRNA Let-7b was highly expressed in noncancerous cells and strongly decreased in cancerous PC3 cells, while the opposite was observed in the respective EVs, thus supporting the tumor suppressor role of miRNA Let7-b. We also demonstrated that miRNA Let-7b can be transferred to THP-1 cells via EVs, which are known to induce TAM-like polarization. Our results support the view that miRNA Let-7 b, contained in PC3-derived EVs, is associated with the increase in the miRNA Let7-b observed in TAM-like macrophages. Overall, our results indicate that circulating EV-loaded miRNA might be useful biomarkers for prostate cancer progression and might also support a possible use of pEVs as targets for prostate cancer therapy.
Asunto(s)
Comunicación Celular , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , MicroARNs/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Neoplásico/metabolismo , Vesículas Extracelulares/patología , Humanos , Macrófagos/patología , Masculino , Células PC-3 , Neoplasias de la Próstata/patología , Células THP-1RESUMEN
The reproductive fitness of the Anopheles gambiae mosquito represents a promising target to prevent malaria transmission. The ecdysteroid hormone 20-hydroxyecdysone (20E), transferred from male to female during copulation, is key to An. gambiae reproductive success as it licenses females to oviposit eggs developed after blood feeding. Here we show that 20E-triggered oviposition in these mosquitoes is regulated by the stress- and immune-responsive c-Jun N-terminal kinase (JNK). The heads of mated females exhibit a transcriptional signature reminiscent of a JNK-dependent wounding response, while mating-or injection of virgins with exogenous 20E-selectively activates JNK in the same tissue. RNAi-mediated depletion of JNK pathway components inhibits oviposition in mated females, whereas JNK activation by silencing the JNK phosphatase puckered induces egg laying in virgins. Together, these data identify JNK as a potential conduit linking stress responses and reproductive success in the most important vector of malaria.
Asunto(s)
Anopheles/fisiología , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Mosquitos Vectores/fisiología , Oviposición/genética , Animales , Copulación/efectos de los fármacos , Ecdisterona/farmacología , Femenino , Malaria/parasitología , Malaria/transmisión , Masculino , Proteína Quinasa 8 Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Plasmodium , Interferencia de ARNRESUMEN
Prostate cancer (PCa) progression is strictly associated with microenvironmental conditions, which can be modified by cancer-released extracellular vesicles (EVs), important mediators of cell-cell communication. However, the role of EVs in the inflammatory cross-talk between cancer cells and microenvironment-residing cells remains largely unknown. To evaluate the role of EVs in the tumour microenvironment, we treated the non-cancerous prostate cell line PNT2 with EVs isolated from advanced-stage prostate cancer PC3 (PC3-EVs). Caspase-1-mediated IL-1ß maturation was evaluated after 24 h incubation with EVs. Moreover, the effect of PC3-EVs on differentiated macrophagic THP-1 cells was assessed by analyzing cytokine expression and PC3 cells migration and proliferation profiles. We illustrated that PC3 cells contain active NLRP3-inflammasome cascade and secrete IL-1ß. PC3-EVs affect the PNT2 inflammatory response, inducing caspase-1-mediated IL-1ß maturation via ERK1/2-mediated lysosomal destabilization and cathepsin B activation. We also verified that PC3-EVs induce a functional TAM-like polarization in differentiated THP-1 cells. Our results demonstrated that cancer-derived EVs induce an inflammatory response in non-cancerous prostate cells, while inducing an immunomodulatory phenotype in immune cells. These apparently contradictory effects are both committed to strengthening the tumour-promoting microenvironment.
RESUMEN
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 12â¯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.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Disbiosis/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Ácido Ursodesoxicólico/uso terapéutico , Animales , Ácido Quenodesoxicólico/uso terapéutico , Dieta/efectos adversos , Disbiosis/etiología , Disbiosis/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Células T Asesinas Naturales/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colestanoles/efectos adversos , Concanavalina A/efectos adversos , Modelos Animales de Enfermedad , Galactosilceramidas/efectos adversos , Células Hep G2 , Hepatitis , Humanos , Interleucina-10/metabolismo , Masculino , Ratones , Células T Asesinas Naturales/citología , Células RAW 264.7 , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Neuroinflammation, i.e. self-propelling progressive cycle of microglial activation and neuron damage, as well as improper protein folding, are recognized as major culprits of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Mutations in several proteins have been linked to ALS pathogenesis, including the G93A mutation in the superoxide dismutase 1 (SOD1) enzyme. SOD1(G93A) mutant is prone to aggregate thus inducing both oxidative stress and neuroinflammation. In this study we used hSOD1(G93A) microglial cells to investigate the effects of the antioxidant and anti-inflammatory cyclic dipeptide (His-Pro) on LPS-induced inflammasome activation. We found that cyclo(His-Pro) inhibits NLRP3 inflammasome activation by reducing protein nitration via reduction in NO and ROS levels, indicative of lower peroxynitrite generation by LPS. Low levels in peroxynitrite are related to NF-κB inhibition responsible for iNOS down-regulation and NO dampening. On the other hand, cyclo(His-Pro)-mediated ROS attenuation, not linked to Nrf2 activation in this cellular model, is ascribed to increased soluble SOD1 activity due to the up-regulation of Hsp70 and Hsp27 expression. Conclusively, our results, besides corroborating the anti-inflammatory properties of cyclo(His-Pro), highlight a novel role of the cyclic dipeptide as a proteostasis regulator, and therefore a good candidate for the treatment of ALS and other misfolding diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Antiinflamatorios/farmacología , Inflamasomas/efectos de los fármacos , Microglía/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Inflamasomas/metabolismo , Ratones , Ratones Transgénicos , Microglía/metabolismo , Neuronas Motoras/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/farmacologíaRESUMEN
Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs. Rat-transformed EGCs were treated with 10â¯ng/ml TcdB for 6â¯h-48â¯h, or for 48â¯h, followed by incubation for additional 4 or 11â¯days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays. TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-ßgalactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, cMyc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin2 and Sirtuin3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways. In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.
Asunto(s)
Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Clostridioides difficile/patogenicidad , Neuroglía/citología , Animales , Puntos de Control del Ciclo Celular , Células Cultivadas , Senescencia Celular , Clostridioides difficile/metabolismo , Daño del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/microbiología , Ratas , Transducción de SeñalRESUMEN
Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.
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Citocromos c/metabolismo , Membranas Mitocondriales/metabolismo , Neuroglía/metabolismo , Palmitatos/metabolismo , Animales , Apoptosis , Cardiolipinas/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Intestinos/citología , Intestinos/inervación , Intestinos/patología , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In this study, we demonstrate, for the first time, that Saccharomyces cerevisiae-based probiotic shows an inhibitory effect on Gardnerella vaginalis infection. This effect is likely due to several actions: direct interference with adherence to vaginal tissues, inhibition of sialidase activity, reduction of vaginal epithelial exfoliation. Gardnerella vaginalis does not induce vaginal inflammation and no inflammatory cytokines were, indeed, produced, by the mouse vagina, neither by Gardnerella vaginalis and by the probiotic. Collectively, our data incite to further investigations on Saccharomyces cerevisiae probiotic as a potential prophylactic or therapeutic agent in the vaginosis caused by Gardnerella vaginalis.
Asunto(s)
Gardnerella vaginalis/fisiología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Probióticos/administración & dosificación , Saccharomyces cerevisiae/fisiología , Vaginosis Bacteriana/tratamiento farmacológico , Animales , Antibiosis , Femenino , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Ratones , Ratones Endogámicos C57BL , Vagina/microbiología , Vaginosis Bacteriana/microbiologíaRESUMEN
Liver fibrosis, a major health concern worldwide, results from abnormal collagen deposition by activated hepatic stellate cells (HSCs) in an injured liver. The farnesoid-x-receptor (FXR) is a bile acid sensor that counteracts HSCs transdifferentiation. While targeting FXR holds promise, 6-ethyl-CDCA known as obeticholic acid, the first in class of FXR ligands, causes side effects, partially because the lack of selectivity toward GPBAR1, a putative itching receptor. Here, we describe the 3-deoxy-6-ethyl derivative of CDCA, BAR704, as a highly selective steroidal FXR agonist. METHODS: Liver Fibrosis was induced in mice by carbon tetrachloride (CCl4). MAIN RESULTS: In transactivation assay BAR704 activated FXR with and EC50 of 967â¯nM while exerted no agonistic activity on other receptors including GPBAR1. In naïve mice, BAR704 modulated the expression of FXR target genes in the liver of wild type mice but not in FXR-/- mice. In cirrhotic mice, administration of BAR704, 15â¯mg/kg for 9 weeks, spared the liver biosynthetic activity (bilirubin and albumin plasma levels), reduced liver fibrosis score (Sirius red staining), expression of pro-fibrogenetic (Colα1α, TGFß and αSMA) and inflammatory genes (IL-1ß, TNFα) and portal pressure. From mechanistic stand point, we have found that exposure of LX2 cells, a human HSCs line, to BAR704 increased the transcription of the short heterodimer partner (SHP) and induced the binding of this nuclear receptor to SMAD3, thus abrogating the binding of phosho-SMAD3 to the TGFß promoter. CONCLUSIONS AND APPLICATIONS: BAR704 is a selective FXR agonist that reduces liver fibrosis by interfering with the TGFß-SMAD3 pathway in HSCs. Selective FXR agonists may represent an attractive strategy for the treatment of liver fibrosis.
Asunto(s)
Colanos/uso terapéutico , Cirrosis Hepática/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Transducción de Señal/efectos de los fármacos , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Neuroinflammation, characterized by the appearance of reactive microglial and astroglial cells, is one of the several pathogenic mechanisms of amyotrophic lateral sclerosis (ALS), a fast-progressing and fatal neurodegenerative disease. Cerebrospinal fluid and spinal cord of ALS patients and SOD1 mutant mice show high concentrations of IL-1ß. This interleukin, expressed as an inactive precursor, undergoes a proteolytic maturation by caspase1, whose activation, in turn, depends on inflammasomes. Whether and how inflammasome is activated in ALS models is still to be clarified. The mechanism of inflammasome activation was studied in murine microglial cells overexpressing hSOD1(G93A) and verified in the spinal cord of hSOD1(G93A) mice. Murine microglial hSOD1(G93A) cells express all the inflammasome components and LPS activates caspase1 leading to an increase in the secretion of IL-1ß. By activating NF-κB, LPS increases ROS and NO levels that spontaneously react to form peroxynitrite, thus leading to protein nitration. Reduction in peroxynitrite levels results in a decrease in caspase1 activity. Protein nitration and caspase1 activity are concomitantly increased in the spinal cord of pre-symptomatic SOD1(G93A) mice. Oxidative/nitrosative stress induces peroxynitrite formation that may be a key trigger of caspase1/inflammasome activation. Peroxynitrite formation may play a critical role in inflammasome activation and might be exploited as potential therapeutic target for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Modelos Animales de Enfermedad , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular Transformada , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Superóxido Dismutasa-1/genéticaRESUMEN
In prostate cancer, oxidative stress and the subsequent Nrf2 activation promote the survival of cancer cells and acquired chemoresistance. Nrf2 links prostate cancer to endoplasmic reticulum stress, an event that triggers the unfolded protein response, aiming to restore cellular homeostasis as well as an adaptive survival mechanism. Glucose-regulated protein of 78 kD /immunoglobulin heavy chain binding protein (GRP78/BiP) is a key molecular chaperone in the endoplasmic reticulum that, when expressed at the cell surface, acts as a receptor for several signaling pathways enhancing antiapoptotic and proliferative signals. We showed GRP78/BiP translocation to PC3 cell surface in the presence of tunicamycin, an ER stress inductor, and demonstrated the existence of a GRP78/BiP-dependent non-canonical Nrf2 activation, responsible for increased resistance to ER-stress induced apoptosis. We found that, even in the absence of ROS production, tunicamycin causes Nrf2 activation, and activates Akt signaling, events bulnted by anti-GRP78/BiP antibody treatment. The presence of GRP78/BiP at the cell surface might be exploited for the immunotherapeutic strategy of prostate cancer since its blockage by anti-GRP78/BiP antibodies might promote cancer death by suppressing some of the several molecular protective mechanisms found in aggressive cancer cells.
RESUMEN
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 6ß-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.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Células 3T3 , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/patología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/patología , Animales , Colestanoles/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hígado Graso/patología , Fructosa , Resistencia a la Insulina/fisiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , Sustancias Protectoras/farmacología , Distribución Aleatoria , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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-1ß, and IL-6 mRNAs, while increased the expression of TGFß 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.
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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.
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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 (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.
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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
Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners.