RESUMO
UNLABELLED: Clinical evidence suggests that many cases of serious idiosyncratic drug-induced liver injury are mediated by the adaptive immune system in response to hepatic drug-protein adducts, also referred to as "drug-induced allergic hepatitis"; but detailed mechanistic proof has remained elusive due to the lack of animal models. We have hypothesized that drug-induced allergic hepatitis is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver. We provide evidence that immune tolerance can be overcome in a murine model of halothane-induced liver injury initiated by trifluoroacetylated protein adducts of halothane formed in the liver. Twenty-four hours after female Balb/cJ mice were initially treated with halothane, perivenous necrosis and an infiltration of CD11b(+) Gr-1(high) cells were observed in the liver. Further study revealed a subpopulation of myeloid-derived suppressor cells within the CD11b(+) Gr-1(high) cell fraction that inhibited the proliferation of both CD4(+) and CD8(+) T cells. When CD11b(+) Gr-1(high) cells were depleted from the liver with Gr-1 antibody treatment, enhanced liver injury was observed at 9 days after halothane rechallenge. Toxicity was associated with increased serum levels of interleukin-4 and immunoglobulins G1 and E directed against hepatic trifluoroacetylated protein adducts, as well as increased hepatic infiltration of eosinophils and CD4(+) T cells, all features of an allergic reaction. When hepatic CD4(+) T cells were depleted 5 days after halothane rechallenge, trifluoroacetylated protein adduct-specific serum immunoglobulin and hepatotoxicity were reduced. CONCLUSION: Our data provide a rational approach for developing animal models of drug-induced allergic hepatitis mediated by the adaptive immune system and suggest that impaired liver tolerance may predispose patients to this disease.
Assuntos
Antígeno CD11b/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Halotano/toxicidade , Hepatite/imunologia , Células Mieloides/metabolismo , Alanina Transaminase/metabolismo , Análise de Variância , Animais , Antígeno CD11b/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Hepatite/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/efeitos dos fármacos , Óxido Nítrico/metabolismo , Distribuição AleatóriaRESUMO
UNLABELLED: Liver eosinophilia has been associated with incidences of drug-induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane-induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil-associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin-4 (IL-4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL-4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A-induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR-deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL-4 and proinflammatory cytokines IL-1ß and tumor necrosis factor alpha. CONCLUSION: This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug.
Assuntos
Anestésicos Inalatórios/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Citocinas/metabolismo , Halotano/efeitos adversos , Interleucina-4/metabolismo , Animais , Concanavalina A , Feminino , Hepatite Animal/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Linfopoietina do Estroma do TimoRESUMO
Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.
Assuntos
Organoides , Humanos , Animais , Organoides/efeitos dos fármacos , Organoides/metabolismo , Avaliação Pré-Clínica de Medicamentos , Indústria FarmacêuticaRESUMO
Inflammation plays a major role in immune-mediated liver injury, and exposure to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been reported to alter the inflammatory response as well as affect immune cell activity. In this study, we tested the hypothesis that TCDD pretreatment exacerbates hepatotoxicity in a murine model of immune-mediated liver injury induced by concanavalin A (Con A) administration. Mice were pretreated with 30 µg/kg TCDD or vehicle control on day zero and then given either Con A or saline intravenously on day four. Mice treated with TCDD did not develop liver injury; however, TCDD pretreatment increased liver injury resulting from moderate doses of Con A (4-10 mg/kg). TCDD-pretreated mice had altered plasma concentrations of inflammatory cytokines, including interferon gamma (IFNγ), and TCDD/Con A-induced hepatotoxicity was attenuated in IFNγ knockout mice. At various times after treatment, intrahepatic immune cells were isolated, and expression of cell activation markers as well as cytolytic proteins was determined. TCDD pretreatment increased the proportion of activated natural killer T (NKT) cells and the percent of cells expressing Fas ligand (FasL) after Con A administration. In addition FasL knockout mice and mice treated with CD18 antiserum were both protected from TCDD/Con A-induced hepatotoxicity, suggesting a requirement for direct cell-cell interaction between effector immune cells and parenchymal cell targets in the development of liver injury from TCDD/Con A treatment. In summary, exposure to TCDD increased NKT cell activation and exacerbated immune-mediated liver injury induced by Con A through a mechanism involving IFNγ and FasL expression.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Concanavalina A/toxicidade , Poluentes Ambientais/toxicidade , Inflamação/induzido quimicamente , Dibenzodioxinas Policloradas/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Concanavalina A/administração & dosagem , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Proteína Ligante Fas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/imunologia , Inflamação/patologia , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismo , Dibenzodioxinas Policloradas/administração & dosagem , Fatores de TempoRESUMO
The use of the fluoroquinolone antibiotic trovafloxacin (TVX) was severely restricted in 1999 due to its association with idiosyncratic hepatotoxicity. Previously, we reported that a nontoxic dose of TVX interacts with a nontoxic dose of lipopolysaccharide (LPS) to cause robust hepatocellular injury in mice. This interaction with LPS was not seen in mice treated with levofloxacin (LVX), a fluoroquinolone not associated with hepatotoxicity in people. TVX/LPS-coexposure caused an increase in plasma alanine aminotransferase (ALT) activity as early as 4.5 h after LPS administration which progressed through 15 h.We examined the role of the hemostatic system in TVX/LPS-induced liver injury. At the onset of liver injury, coexposure to TVX/LPS, but not exposure to TVX, LVX, LPS or LVX/LPS, caused increased plasma concentration of thrombin-antithrombin dimers and decreased plasma circulating fibrinogen. LPS treatment induced a small increase in plasma plasminogen activator inhibitor-1 (PAI-1) concentration, and TVX pretreatment enhanced this effect. TVX/LPS coexposure also resulted in hepatic fibrin deposition. Anticoagulant heparin administration reduced TVX/LPS-induced hepatic fibrin deposition and liver injury. PAI-1-/- mice treated with TVX/LPS exhibited similar fibrin deposition to wild-type mice but had significantly reduced hepatocellular injury. PAI-1-/- mice, but not heparin-treated mice, had reduced plasma concentrations of several cytokines compared to TVX/LPS-treated controls. In summary, TVX/LPS-coexposure caused an imbalance in the hemostatic system, resulting in thrombin activation increased, plasma concentration of PAI-1 and hepatic fibrin deposition. Both thrombin activation and PAI-1 play critical roles in the progression of TVX/LPS-induced liver injury, but through different modes of action.
Assuntos
Fluoroquinolonas/toxicidade , Hemostasia/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Naftiridinas/toxicidade , Animais , Anticoagulantes/farmacologia , Citocinas/biossíntese , Heparina/farmacologia , Camundongos , Camundongos Knockout , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/fisiologiaRESUMO
Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress.
Assuntos
Antibacterianos/toxicidade , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Replicação do DNA/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fluoroquinolonas/toxicidade , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Naftiridinas/toxicidade , Fator de Necrose Tumoral alfa/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Ativação Enzimática , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Fígado/enzimologia , Fígado/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Idiosyncratic drug-induced liver injury (IDILI) continues to be a significant human health problem. IDILI is characterized as occurring in a minority of individuals exposed to a drug, yet it accounts for as much as 17% of all cases of acute liver failure. Despite these concerns, the mechanisms underlying IDILI remain unknown. Trovafloxacin (TVX), which causes IDILI in humans, also causes hepatocellular death in vitro when combined with tumor necrosis factor-alpha (TNF) treatment. However, the molecular mechanisms involved in this toxicity are not fully characterized. The purpose of this study was to identify mechanisms by which TVX and TNF interact to cause hepatocellular death, with a focus on a human hepatocyte cell line. TVX and TNF interacted to cause cytotoxicity in HepG2 cells at drug concentrations similar to those in people undergoing TVX therapy. TVX/TNF treatment caused apoptosis and DNA damage in HepG2 cells that depended on caspase activation. Prolonged activation of JNK occurred in TVX/TNF-induced cytotoxicity, and treatment with the JNK selective inhibitor SP600125 attenuated cytotoxicity. TVX/TNF cotreatment also caused cytotoxicity in isolated primary murine hepatocytes that was dependent on caspase activation. These results increase understanding of molecular signaling pathways involved in hepatocellular death caused by a drug with idiosyncratic liability in the presence of TNF.
Assuntos
Antibacterianos/toxicidade , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fluoroquinolonas/toxicidade , Hepatócitos/efeitos dos fármacos , Naftiridinas/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Inibidores de Proteínas Quinases , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
For many liver diseases, including viral and autoimmune hepatitis, immune cells play an important role in the development and progression of liver injury. Concanavalin A (Con A) administration to rodents has been used as a model of immune-mediated liver injury resembling human autoimmune hepatitis. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to alter the development of immune-mediated diseases. Mice pretreated with TCDD developed exacerbated liver injury in response to administration of a mild dose (6 mg/kg) of Con A. In the present study, we tested the hypothesis that TCDD pretreatment exacerbates Con A-induced liver injury by enhancing the activation and recruitment of accessory cell types including neutrophils, macrophages, and natural killer (NK) cells. Mice were treated with 0, 0.3, 3, or 30 µg/kg TCDD and 4 days later with Con A or saline. TCDD pretreatment with doses of 3 and 30 µg/kg significantly increased liver injury from Con A administration. The plasma concentrations of neutrophil chemokines were significantly increased in TCDD-pretreated mice after Con A administration. NKT cell-deficient (CD1d KO) mice were used to examine whether NKT cells were required for TCDD/Con A-induced liver injury. CD1d KO mice were completely protected from liver injury induced by treatment with Con A alone, whereas the injury from TCDD/Con A treatment was reduced but not eliminated. However, T-cell deficient (RAG1 KO) mice were protected from liver injury induced by Con A irrespective of pretreatment with TCDD. TCDD/Con A treatment increased the percentage of NK cells expressing the activation marker CD69. Depletion of NK cells prior to treatment resulted in significant reductions in plasma interferon-γ and liver injury from TCDD/Con A treatment. In summary, exposure to TCDD exacerbated the immune-mediated liver injury induced by Con A, and our findings suggest that NK cells play a critical role in this response.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Concanavalina A/toxicidade , Fígado/efeitos dos fármacos , Células T Matadoras Naturais/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Animais , Antígenos CD/metabolismo , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Quimiocinas/sangue , Relação Dose-Resposta a Droga , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mediadores da Inflamação/sangue , Lectinas Tipo C/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Fatores de TempoRESUMO
Severe halothane (HAL)-induced hepatotoxicity occurs in one in 6000-30,000 patients by an unknown mechanism. Female sex is a risk factor in humans and rodents. We tested the hypothesis that a sex difference in natural killer (NK) cell activity contributes to HAL-induced liver injury. HAL (15 mmol/kg, ip) treatment resulted in severe liver injury by 12 h in female, wild-type BALB/cJ mice, and the magnitude of liver injury varied with stage of the estrous cycle. Ovariectomized (OVX) mice developed only mild liver injury. Plasma interferon-gamma (IFN-γ) was elevated 10-fold in HAL-treated females compared with similarly treated male mice or with OVX female mice. IFN-γ knockout mice were resistant to severe HAL-induced liver injury. The deactivation of NK cells with anti-asialo GM1 treatment attenuated liver injury and the increase in plasma IFN-γ compared with immunoglobulin G-treated control mice. Mice with a mutated form of perforin, a protein involved in granule-mediated cytotoxicity, were protected from severe liver injury. Furthermore, HAL increased the activity of NK cells in vivo, as indicated by increased surface expression of CD69, an early activation marker. In response to HAL, NK cell receptor ligands on the surface of hepatocytes were expressed in a manner that can activate NK cells. These results confirm the sexual dimorphic hepatotoxic response to HAL in mice and suggest that IFN-γ and NK cells have essential roles in the development of severe HAL-induced hepatotoxicity.