RESUMO
Liver depleted of hepatic stellate cells (HSCs) is resistant to ischemia/reperfusion-, concanavalin A-, and acetaminophen-induced acute injury. Whether HSCs regulate carbon tetrachloride (CCl4 )-induced acute liver injury is not known. CCl4 treatment damages pericentral hepatocytes that express CCl4 -metabolizing Cyp2E1 and activates HSCs. We investigated whether HSC-depletion in mice transgenic for thymidine kinase under the glial fibrillary acidic protein promoter (GFAP-TK-Tg) confers resistance to injury and inflammation due to CCl4 rechallenge. GFAP-TK-Tg or wild type (WT) mice were administered 0.16 ml/kg CCl4 (3× at 3 days intervals), then 40 µg/g/day ganciclovir for 10 days. The treatment depletes ~70%-75% HSCs from GFAP-TK-Tg but not WT mice while the liver recovers from earlier CCl4 -induced injury. Mice were then administered CCl4 , and liver injury and inflammation were determined at 24 h. HSC-depleted and HSC-sufficient mice showed similar CCl4 -induced hepatocyte necrosis and oxidative stress. However, increase in F4/80+ macrophages, but not CD68+ cells, was greater in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. Expression of tumor necrosis factor-α (TNF-α), CCL2, and CXCL1 increased similarly, whereas increase in interleukin-6 (IL6), IL1ß, and IL10 expression was higher in CCl4 rechallenged HSC-depleted compared to HSC-sufficient mice. CCl4 rechallenge of HSC-sufficient mice rapidly activated HSCs causing significant fibrosis with increased expression of Col1a1, transforming growth factor ß1 (TGFß1), tissue inhibitors of metalloproteinases 1 (TIMP1); increase in TIPM1 was much lower and metalloproteinases 13 (MMP13) greater in CCl4 rechallenged HSC-depleted mice. Interestingly, hepatic recruitment of both profibrogenic (Ly6Chi ) and antifibrogenic restorative (Ly6Clo ) macrophages, and neutrophils was significantly greater in CCl4 rechallenged HSC-depleted mice. These data suggest that CCl4 directly damages hepatocytes but HSCs regulate inflammation. Rapid fibrogenesis in CCl4 rechallenged HSC-sufficient mice recovered from earlier injury indicates that even transiently activated HSCs that had reverted to the quiescent phenotype remain primed to become reactivated.
Assuntos
Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas , Camundongos , Animais , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Inflamação/patologia , Cirrose Hepática/genéticaRESUMO
BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ductos Biliares Intra-Hepáticos/patologia , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/patologia , Mutação , Proteínas de Peixe-Zebra/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose , Ductos Biliares Intra-Hepáticos/metabolismo , Estudos de Casos e Controles , Colestase Intra-Hepática/metabolismo , Doença Crônica , Feminino , Edição de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Sequenciamento do Exoma , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND AND AIMS: During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFßR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs. APPROACH AND RESULTS: We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl4 administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFßR, transforming growth factor beta receptor 1 (TGFßR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFßR, αSMA, TGFßR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ. CONCLUSIONS: In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.
Assuntos
Regulação da Expressão Gênica/imunologia , Células Estreladas do Fígado/imunologia , Lipídeo A/análogos & derivados , Cirrose Hepática Experimental/imunologia , Fígado/patologia , Animais , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Transdiferenciação Celular/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Regulação para Baixo , Inativação Gênica , Células Estreladas do Fígado/patologia , Humanos , Lipídeo A/imunologia , Lipídeo A/metabolismo , Fígado/citologia , Fígado/imunologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Knockout , Miofibroblastos/imunologia , Miofibroblastos/patologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myb/metabolismo , Ratos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína Smad7/genética , Proteína Smad7/metabolismo , Regulação para Cima/imunologiaRESUMO
In the multidrug resistance protein 2 (Mdr2)-/- mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2-/- mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14-30 of life. Antibody-mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro-fibrogenic cytokine. Depletion of intrahepatic Tregs with anti-CD25 antibody between days 7-30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti-interleukin 2 immune complexes (IL-2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL-2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro-inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2-/- mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg-associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL-2c, suggesting that Treg-directed low-dose Il-2 treatment may be considered as therapy for SC.
Assuntos
Ductos Biliares/patologia , Colangite Esclerosante/imunologia , Interleucina-2/imunologia , Fígado/imunologia , Linfócitos T Reguladores/imunologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Feminino , Fibrose/imunologia , Fibrose/patologia , Imunofluorescência , Humanos , Lactente , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise em MicrossériesRESUMO
UNLABELLED: Paracrine signalling mediated by cytokine secretion is essential for liver regeneration after hepatic resection, yet the mechanisms of cellular crosstalk between immune and parenchymal cells are still elusive. Interleukin-22 (IL-22) is released by immune cells and mediates strong hepatoprotective functions. However, it remains unclear whether IL-22 is critical for the crosstalk between liver lymphocytes and parenchymal cells during liver regeneration after partial hepatectomy (PH). Here, we found that plasma levels of IL-22 and its upstream cytokine, IL-23, are highly elevated in patients after major liver resection. In a mouse model of PH, deletion of IL-22 was associated with significantly delayed hepatocellular proliferation and an increase of hepatocellular injury and endoplasmic reticulum stress. Using Rag1(-/-) and Rag2(-/-) γc(-/) (-) mice, we show that the main producers of IL-22 post-PH are conventional natural killer cells and innate lymphoid cells type 1. Extracellular adenosine triphosphate (ATP), a potent danger molecule, is elevated in patients immediately after major liver resection. Antagonism of the P2-type nucleotide receptors, P2X1 and P2Y6, significantly decreased IL-22 secretion ex vivo. In vivo, specific inhibition of P2X1 was associated with decreased IL-22 secretion, elevated liver injury, and impaired liver regeneration. CONCLUSION: This study shows that innate immune cell-derived IL-22 is required for efficient liver regeneration and that secretion of IL-22 in the regenerating liver is modulated by the ATP receptor, P2X1. (Hepatology 2016;63:2004-2017).
Assuntos
Interleucinas/metabolismo , Células Matadoras Naturais/metabolismo , Regeneração Hepática , Receptores Purinérgicos P2X1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Estresse do Retículo Endoplasmático , Hepatectomia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
Fibrosing cholangiopathies, including biliary atresia and primary sclerosing cholangitis, involve immune-mediated bile duct epithelial injury and hepatic bile acid (BA) retention (cholestasis). Regulatory T-cells (Tregs) can prevent auto-reactive lymphocyte activation, yet the effects of BA on this CD4 lymphocyte subset are unknown. Gene regulatory networks for hepatic CD4 lymphocytes in a murine cholestasis model revealed Tregs are polarized to Th17 during cholestasis. Following bile duct ligation, Stat3 deletion in CD4 lymphocytes preserved hepatic Treg responses. While pharmacological reduction of hepatic BA in MDR2-/- mice prompted Treg expansion and diminished liver injury, this improvement subsided with Treg depletion. A cluster of patients diagnosed with biliary atresia showed both increased hepatic Treg responses and improved 2-year native liver survival, supporting that Tregs might protect against neonatal bile duct obstruction. Together, these findings suggest liver BA determine Treg function and should be considered as a therapeutic target to restore protective hepatic immune responses.
RESUMO
RATIONALE: The novel P2Y(12) antagonist ticagrelor inhibits ADP-induced platelet aggregation more rapidly and more potently than clopidogrel. Clinical trials have revealed dyspnea and asymptomatic ventricular pauses as side effects of ticagrelor. The mechanism behind these side effects is not known, but it is plausible that they are mediated by adenosine. OBJECTIVE: Ticagrelor is known to increase adenosine concentrations by inhibiting red blood cell reuptake, but the potency of this effect may be too low to fully explain the adenosine related effects. The purpose of the present study was to determine whether ticagrelor has other effects on red blood cells (RBCs) that could contribute to explain the pleiotropic effects seen with ticagrelor treatment. METHODS AND RESULTS: Using a luciferase-based bioluminescence assay, we studied ATP release in human blood. Human RBCs responded to ticagrelor in vitro by releasing substantial amounts of ATP in a dose-dependent manner (IC(50) 14µM). The rapid effect indicates release through membrane channels, which was supported by a depolarizing effect of ticagrelor and inhibition of ATP release by anion channel blockers. CONCLUSION: In conclusion, our data show that, in vitro, ticagrelor can induce ATP release from human RBCs, which is subsequently degraded to adenosine. Further studies are warranted to determine what role this mechanism may play in the clinical effects of ticagrelor.
Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/análogos & derivados , Eritrócitos/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Adenosina/farmacologia , Células Cultivadas , Eritrócitos/metabolismo , Humanos , Concentração Inibidora 50 , TicagrelorRESUMO
Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1ß and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1ß and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.
Assuntos
Colangite Esclerosante , Camundongos , Animais , Colangite Esclerosante/tratamento farmacológico , Linfócitos T , Ácidos e Sais Biliares , Fígado , MacrófagosRESUMO
Cholangiocytes are epithelial cells lining the intrahepatic and extrahepatic bile ducts. Cholangiocytes perform key physiological functions in the liver. Bile synthesized by hepatocytes is secreted into bile canaliculi, further stored in the gallbladder, and finally discharged into the duodenum. Due to liver injury, biliary epithelial proliferate in response to endogenous or exogenous signals leading to cholangiopathies, inflammation, fibrosis, and cholangiocarcinoma. Cholangiocytes exhibit anatomical and functional heterogeneity, and understanding such diversified functions will potentially help in finding effective therapies for various cholestatic liver diseases. To perform such functional studies, effective cholangiocyte isolation and culture procedures are needed. This protocol will aid in easy isolation and expansion of cholangiocytes from the liver.