RESUMO
BACKGROUND: Ruminococcus gnavus is a mucolytic commensal bacterium whose increased gut colonization has been associated with chronic inflammatory and metabolic diseases in humans. Whether R. gnavus metabolites can modulate host intestinal physiology remains largely understudied. METHODS: We performed untargeted metabolomic and bulk RNA sequencing analyses using R. gnavus mono-colonization in germ free mice. Based on transcriptome-metabolome correlations, we tested the impact of specific arginine metabolites on intestinal epithelial production of nitric oxide (NO) and examined the effect of NO on the growth of various strains of R. gnavus in vitro and in Nos2-deficient mice. RESULTS: R. gnavus produces specific arginine, tryptophan and tyrosine metabolites, some of which are regulated by the environmental richness of sialic acid and mucin. R. gnavus colonization promotes expression of amino acid transporters and enzymes involved in metabolic flux of arginine and associated metabolites into NO. R. gnavus induced elevated levels of Nitric Oxide Synthase 2 (NOS2) while Nos2 ablation resulted in R. gnavus expansion in vivo. The growth of various R. gnavus strains can be inhibited by NO. Specific R. gnavus metabolites modulate intestinal epithelial cell NOS2 abundance and reduce epithelial barrier function at higher concentrations. CONCLUSIONS: Intestinal colonization and interaction with R. gnavus are partially regulated by an arginine-NO metabolic pathway, whereby a balanced control by the gut epithelium may restrain R. gnavus growth in healthy individuals. Disruption in this arginine metabolic regulation will contribute to the expansion and blooming of R. gnavus.
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BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.
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Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Masculino , Camundongos , Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Proteína Forkhead Box O1 , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fenótipo , Inibidor p16 de Quinase Dependente de CiclinaRESUMO
OBJECTIVE: Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. GOAL: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (mdr2-/- ) mice and microbial profiles in PSC patient cohorts. DESIGN: We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. RESULTS: GF mdr2-/- mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients' clinical severity by Mayo risk scores. CONCLUSIONS: We identified novel functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.
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Escherichia coli , Vancomicina , Animais , Camundongos , Modelos Animais de Doenças , RNA Ribossômico 16S/genética , Inflamação , Cirrose Hepática , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , ClostridialesRESUMO
Chronic liver diseases encompass a wide spectrum of hepatic maladies that often result in cholestasis or altered bile acid secretion and regulation. Incidence and cost of care for many chronic liver diseases are rising in the United States with few Food and Drug Administration-approved drugs available for patient treatment. Farnesoid X receptor (FXR) is the master regulator of bile acid homeostasis with an important role in lipid and glucose metabolism and inflammation. FXR has served as an attractive target for management of cholestasis and fibrosis; however, global FXR agonism results in adverse effects in liver disease patients, severely affecting quality of life. In this review, we highlight seminal studies and recent updates on the FXR proteome and identify gaps in knowledge that are essential for tissue-specific FXR modulation. In conclusion, one of the greatest unmet needs in the field is understanding the underlying mechanism of intestinal versus hepatic FXR function.
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Colestase , Hepatopatias , Humanos , Amigos , Qualidade de Vida , Fígado/metabolismo , Colestase/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Hepatopatias/metabolismo , Ácidos e Sais Biliares/metabolismoRESUMO
BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.
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Cirrose Hepática Biliar , Secretina , Masculino , Feminino , Humanos , Camundongos , Animais , Recém-Nascido , Secretina/metabolismo , Cirrose Hepática Biliar/metabolismo , Bicarbonatos/metabolismo , Via Secretória , Regulador de Condutância Transmembrana em Fibrose Cística , Ductos Biliares/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Ácidos e Sais Biliares/metabolismo , RNA/metabolismo , Mucinas/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismoRESUMO
Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC.NEW & NOTEWORTHY We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.
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Colangite Esclerosante , Colestase , Doenças Inflamatórias Intestinais , Humanos , Animais , Camundongos , Colangite Esclerosante/tratamento farmacológico , Colangite Esclerosante/genética , Colangite Esclerosante/patologia , Ácidos e Sais Biliares , Histamina , Morfolinos/uso terapêutico , Fígado/metabolismo , Colestase/patologia , Cirrose Hepática/patologia , Inflamação/patologia , Proteínas de Membrana Transportadoras , Doenças Inflamatórias Intestinais/patologiaRESUMO
BACKGROUND AND AIMS: Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein-coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFß receptor type I (TGFßRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. APPROACH AND RESULTS: Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2-/- ) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2-/- mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin's interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFßR1 signaling, which was reduced by loss of MT1. CONCLUSIONS: Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFßR1 activation. Blocking GPR50/TGFßR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.
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Colestase , Melatonina , Animais , Colestase/complicações , Colestase/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/etiologia , Melatonina/metabolismo , Melatonina/farmacologia , Melatonina/uso terapêutico , Camundongos , Camundongos Knockout , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismoRESUMO
BACKGROUND AND AIMS: Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile-duct-ligated (BDL) MC-deficient mice (B6.Cg-KitW-sh /HNihrJaeBsmJ [KitW-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice. APPROACH AND RESULTS: In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in KitW-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2-/- ) mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. CONCLUSIONS: Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.
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Colangite Esclerosante/complicações , Colestase/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mastócitos/imunologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ductos Biliares/imunologia , Ductos Biliares/patologia , Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Colestase/patologia , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Mastócitos/metabolismo , CamundongosRESUMO
BACKGROUND AND AIMS: Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. APPROACH AND RESULTS: In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. CONCLUSION: The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.
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Hepatopatia Gordurosa não Alcoólica/genética , Receptores Acoplados a Proteínas G/genética , Receptores dos Hormônios Gastrointestinais/genética , Secretina/genética , Animais , Ductos Biliares/citologia , Ductos Biliares/metabolismo , Linhagem Celular , Senescência Celular/genética , Modelos Animais de Doenças , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados , Hepatócitos/metabolismo , Humanos , Lipogênese/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fenótipo , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Secretina/metabolismo , Regulação para CimaRESUMO
BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. CONCLUSIONS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.
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Sistema Biliar/patologia , Dieta Ocidental/efeitos adversos , Cirrose Hepática/imunologia , Mastócitos/imunologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aldeído Oxirredutases/genética , Animais , Sistema Biliar/imunologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/imunologia , Hepatócitos/patologia , Humanos , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Mastócitos/metabolismo , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Adulto JovemRESUMO
BACKGROUND AND AIMS: Following liver injury, mast cells (MCs) migrate into the liver and are activated in patients with cholestasis. Inhibition of MC mediators decreases ductular reaction (DR) and liver fibrosis. Transforming growth factor beta 1 (TGF-ß1) contributes to fibrosis and promotes liver disease. Our aim was to demonstrate that reintroduction of MCs induces cholestatic injury through TGF-ß1. APPROACH AND RESULTS: Wild-type, KitW-sh (MC-deficient), and multidrug resistance transporter 2/ABC transporter B family member 2 knockout mice lacking l-histidine decarboxylase were injected with vehicle or PKH26-tagged murine MCs pretreated with 0.01% dimethyl sulfoxide (DMSO) or the TGF-ß1 receptor inhibitor (TGF-ßRi), LY2109761 (10 µM) 3 days before sacrifice. Hepatic damage was assessed by hematoxylin and eosin (H&E) and serum chemistry. Injected MCs were detected in liver, spleen, and lung by immunofluorescence (IF). DR was measured by cytokeratin 19 (CK-19) immunohistochemistry and F4/80 staining coupled with real-time quantitative PCR (qPCR) for interleukin (IL)-1ß, IL-33, and F4/80; biliary senescence was evaluated by IF or qPCR for p16, p18, and p21. Fibrosis was evaluated by sirius red/fast green staining and IF for synaptophysin 9 (SYP-9), desmin, and alpha smooth muscle actin (α-SMA). TGF-ß1 secretion/expression was measured by enzyme immunoassay and qPCR. Angiogenesis was detected by IF for von Willebrand factor and vascular endothelial growth factor C qPCR. In vitro, MC-TGF-ß1 expression/secretion were measured after TGF-ßRi treatment; conditioned medium was collected. Cholangiocytes and hepatic stellate cells (HSCs) were treated with MC-conditioned medium, and biliary proliferation/senescence was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and qPCR; HSC activation evaluated for α-SMA, SYP-9, and collagen type-1a expression. MC injection recapitulates cholestatic liver injury characterized by increased DR, fibrosis/TGF-ß1 secretion, and angiogenesis. Injection of MC-TGF-ßRi reversed these parameters. In vitro, MCs induce biliary proliferation/senescence and HSC activation that was reversed with MCs lacking TGF-ß1. CONCLUSIONS: Our study demonstrates that reintroduction of MCs mimics cholestatic liver injury and that MC-derived TGF-ß1 may be a target in chronic cholestatic liver disease.
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Actinas/metabolismo , Colestase Intra-Hepática/metabolismo , Cirrose Hepática , Fígado/patologia , Mastócitos , Fator de Crescimento Transformador beta1 , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Ensaios de Migração Celular , Proliferação de Células , Senescência Celular , Descoberta de Drogas , Células Estreladas do Fígado , Histamina/sangue , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo , Regulação para CimaRESUMO
Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2-/- (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2-/- mice using vivo-morpholino. Wild-type and Mdr2-/- mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated ß-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-ß1, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-ß1 and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-/--mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC.
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Ductos Biliares/metabolismo , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Receptores Histamínicos H2/metabolismo , Animais , Ductos Biliares/patologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Morfolinos/farmacologia , Receptores Histamínicos H2/genéticaRESUMO
Activation of the substance P (SP)/neurokinin 1 receptor (NK1R) axis triggers biliary damage/senescence and liver fibrosis in bile duct ligated and Mdr2-/- (alias Abcb4-/-) mice through enhanced transforming growth factor-ß1 (TGF-ß1) biliary secretion. Recent evidence indicates a role for miR-31 (MIR31) in TGF-ß1-induced liver fibrosis. We aimed to define the role of the SP/NK1R/TGF-ß1/miR-31 axis in regulating biliary proliferation and liver fibrosis during cholestasis. Thus, we generated a novel model with double knockout of Mdr2-/- and NK1R-/ (alias Tacr1-/-) to further address the role of the SP/NK1R axis during chronic cholestasis. In vivo studies were performed in the following 12-week-old male mice: (i) NK1R-/-; (ii) Mdr2-/-; and (iii) NK1R-/-/Mdr2-/- (Tacr1-/-/Abcb4-/-) and their corresponding wild-type controls. Liver tissues and cholangiocytes were collected, and liver damage, changes in biliary mass/senescence, and inflammation as well as liver fibrosis were evaluated by both immunohistochemistry in liver sections and real-time PCR. miR-31 expression was measured by real-time PCR in isolated cholangiocytes. Decreased ductular reaction, liver fibrosis, biliary senescence, and biliary inflammation were observed in NK1R-/-/Mdr2-/- mice compared with Mdr2-/- mice. Elevated expression of miR-31 was observed in Mdr2-/- mice, which was reduced in NK1R-/-/Mdr2-/- mice. Targeting the SP/NK1R and/or miR-31 may be a potential approach in treating human cholangiopathies, including primary sclerosing cholangitis.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Ductos Biliares , Colangite Esclerosante , Cirrose Hepática , Receptores da Neurocinina-1/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Ductos Biliares/lesões , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Colangite Esclerosante/genética , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos Knockout , Receptores da Neurocinina-1/metabolismo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Our daily rhythmicity is controlled by a circadian clock with a specific set of genes located in the suprachiasmatic nucleus in the hypothalamus. Mast cells (MCs) are major effector cells that play a protective role against pathogens and inflammation. MC distribution and activation are associated with the circadian rhythm via two major pathways, IgE/FcεRI- and IL-33/ST2-mediated signaling. Furthermore, there is a robust oscillation between clock genes and MC-specific genes. Melatonin is a hormone derived from the amino acid tryptophan and is produced primarily in the pineal gland near the center of the brain, and histamine is a biologically active amine synthesized from the decarboxylation of the amino acid histidine by the L-histidine decarboxylase enzyme. Melatonin and histamine are previously reported to modulate circadian rhythms by pathways incorporating various modulators in which the nuclear factor-binding near the κ light-chain gene in B cells, NF-κB, is the common key factor. NF-κB interacts with the core clock genes and disrupts the production of pro-inflammatory cytokine mediators such as IL-6, IL-13, and TNF-α. Currently, there has been no study evaluating the interdependence between melatonin and histamine with respect to circadian oscillations in MCs. Accumulating evidence suggests that restoring circadian rhythms in MCs by targeting melatonin and histamine via NF-κB may be promising therapeutic strategy for MC-mediated inflammatory diseases. This review summarizes recent findings for circadian-mediated MC functional roles and activation paradigms, as well as the therapeutic potentials of targeting circadian-mediated melatonin and histamine signaling in MC-dependent inflammatory diseases.
Assuntos
Histamina/metabolismo , Mastócitos/metabolismo , Melatonina/metabolismo , Glândula Pineal/metabolismo , Animais , Ritmo Circadiano/fisiologia , Histidina Descarboxilase/metabolismo , Humanos , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Mastócitos/imunologia , Glândula Pineal/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Primary sclerosing cholangitis (PSC) is characterized by biliary damage and fibrosis. Multidrug resistance-2 gene knockout (Mdr2-/-) mice and PSC patients have increased histamine (HA) levels (synthesized by l-histidine decarboxylase, HDC) and HA receptor (HR) expression. Cholestatic HDC-/- mice display ameliorated biliary damage and hepatic fibrosis. The current study evaluated the effects of knockout of HDC-/- in Mdr2-/- mice (DKO) on biliary damage and hepatic fibrosis. WT, Mdr2-/- mice, and homozygous DKO mice were used. Selected DKO mice were treated with HA. We evaluated liver damage along with HDC expression and HA serum levels. Changes in ductular reaction were evaluated along with liver fibrosis, inflammation and bile acid signaling pathways. The expression of H1HR/PKC-α/TGF-ß1 and H2HR/pERK/VEGF-C was determined. In vitro, cholangiocyte lines were treated with HA with/without H1/H2 inhibitors before measuring: H1/H2HR, TGF-ß1, and VEGF-C expression. Knockout of HDC ameliorates hepatic damage, ductular reaction, fibrosis, inflammation, bile acid signaling and H1HR/PKC-α/TGF-ß1 and H2HR/pERK/VEGF-C signaling. Reactivation of the HDC/HA axis increased these parameters. In vitro, stimulation with HA increased HR expression and PKC-α, TGF-ß1, and VEGF-C expression, which was reduced with HR inhibitors. Our data demonstrate the key role for the HDC/HA axis in the management of PSC progression.
Assuntos
Colangite Esclerosante , Histamina/metabolismo , Histidina Descarboxilase , Cirrose Hepática , Transdução de Sinais/genética , Animais , Colangite Esclerosante/enzimologia , Colangite Esclerosante/metabolismo , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/enzimologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild-type and multidrug-resistant knockout (Mdr2-/- ) mice were treated by osmotic minipumps with saline, mepyramine, or ranitidine (10 mg/kg body weight/day) or a combination of mepyramine/ranitidine for 4 weeks. Liver damage was assessed by hematoxylin and eosin. We evaluated (1) H1/H2HR expression, (2) MC presence, (3) L-histidine decarboxylase/histamine axis, (4) cholangiocyte proliferation/bile duct mass, and (5) fibrosis/hepatic stellate cell activation. Nu/nu mice were implanted with Mz-ChA-1 cells into the hind flanks and treated with saline, mepyramine, or ranitidine. Tumor growth was measured, and (1) H1/H2HR expression, (2) proliferation, (3) MC activation, (4) angiogenesis, and (5) epithelial-mesenchymal transition (EMT) were evaluated. In vitro, human hepatic stellate cells were evaluated for H1HR and H2HR expression. Cultured cholangiocytes and CCA lines were treated with saline, mepyramine, or ranitidine (25 µM) before evaluating proliferation, angiogenesis, EMT, and potential signaling mechanisms. H1/H2HR and MC presence increased in human PSC and CCA. In H1/H2HR antagonist (alone or in combination)-treated Mdr2-/- mice, liver and biliary damage and fibrosis decreased compared to saline treatment. H1/H2HR antagonists decreased tumor growth, serum histamine, angiogenesis, and EMT. In vitro, H1/H2HR blockers reduced biliary proliferation, and CCA cells had decreased proliferation, angiogenesis, EMT, and migration. Conclusion: Inhibition of H1/H2HR reverses PSC-associated damage and decreases CCA growth, angiogenesis, and EMT; because PSC patients are at risk of developing CCA, using HR blockers may be therapeutic for these diseases. (Hepatology 2018).
Assuntos
Colangiocarcinoma/prevenção & controle , Colangite Esclerosante/tratamento farmacológico , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Antagonistas dos Receptores H2 da Histamina/uso terapêutico , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Colangiocarcinoma/etiologia , Colangite Esclerosante/complicações , Avaliação Pré-Clínica de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos H1/farmacologia , Antagonistas dos Receptores H2 da Histamina/farmacologia , Humanos , Fígado/efeitos dos fármacos , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neovascularização Patológica/prevenção & controle , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPAssuntos
Antagonistas dos Receptores Histamínicos/uso terapêutico , Hepatopatias/imunologia , Mastócitos/imunologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Quimases/antagonistas & inibidores , Quimases/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Liberação de Histamina/imunologia , Humanos , Imunidade Inata , Fígado/citologia , Fígado/imunologia , Fígado/patologia , Hepatopatias/tratamento farmacológico , Hepatopatias/patologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Receptores Histamínicos/metabolismo , Triptases/antagonistas & inibidores , Triptases/metabolismoAssuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Ductos Biliares Intra-Hepáticos , Biomarcadores , Proteínas do Domínio Duplacortina , Quinases Semelhantes a Duplacortina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos , Neuropeptídeos , Proteínas Serina-Treonina Quinases , Células-TroncoAssuntos
Ductos Biliares/metabolismo , Colestase/patologia , Fígado/patologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Secretina/metabolismo , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Doença Crônica , Modelos Animais de Doenças , Epitélio/patologia , Humanos , Transdução de SinaisRESUMO
Bile acids (BAs) are signaling molecules synthesized in the liver initially by CYP7A1 and CYP27A1 in the classical and alternative pathways, respectively. BAs are essential for cholesterol clearance, intestinal absorption of lipids, and endogenous modulators of farnesoid x receptor (FXR). FXR is critical in maintaining BA homeostasis and gut-liver crosstalk. Complex reactions in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs. In this study, we characterized the in vivo effects of three-day feeding of cholic acid (CA), deoxycholic acid (DCA), or ursodeoxycholic acid (UDCA) at physiological/non-hepatotoxic concentrations in a novel low-BA mouse model (Cyp7a1-/-/Cyp27a1-/-, DKO). Liver injury, BA levels and composition and BA signaling by the FXR-fibroblast growth factor 15 (FGF15) axis were determined. Overall, higher basal inflammation and altered lipid metabolism in DKO mice might be associated with low BAs. CA, DCA, and UDCA feeding activated FXR signals with tissue specificity. Dietary CA and DCA similarly altered tissue BA profiles to be less hydrophobic, while UDCA promoted a more hydrophobic tissue BA pool with the profiles shifted toward non-12α-OH BAs and secondary BAs. However, UDCA did not offer any overt protective effects as expected. These findings allow us to determine the precise effects of individual BAs in vivo on BA-FXR signaling and overall BA homeostasis in liver physiology and pathologies.