RESUMEN
Nonalcoholic steatohepatitis (NASH) and alcoholic hepatitis (AH) affect a large part of the general population worldwide. Dysregulation of lipid metabolism and alcohol toxicity drive disease progression by the activation of hepatic stellate cells and the capillarization of liver sinusoidal endothelial cells. Collagen deposition, along with sinusoidal remodeling, alters sinusoid structure, resulting in hepatic inflammation, portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for NASH and AH. However, the success of such treatments is limited and unpredictable. We report a strategy for NASH and AH treatment involving the induction of integrin αvß3-mediated cell apoptosis using a rationally designed protein (ProAgio). Integrin αvß3 is highly expressed in activated hepatic stellate cells (αHSCs), the angiogenic endothelium, and capillarized liver sinusoidal endothelial cells (caLSECs). ProAgio induces the apoptosis of these disease-driving cells, therefore decreasing collagen fibril, reversing sinusoid remodeling, and reducing immune cell infiltration. The reversal of sinusoid remodeling reduces the expression of leukocyte adhesion molecules on LSECs, thus decreasing leukocyte infiltration/activation in the diseased liver. Our studies present a novel and effective approach for NASH and AH treatment.
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Células Endoteliales , Células Estrelladas Hepáticas , Hepatitis Alcohólica , Hígado , Enfermedad del Hígado Graso no Alcohólico , Células Estrelladas Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Células Endoteliales/metabolismo , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Hígado/metabolismo , Hígado/patología , Apoptosis , Humanos , Integrina alfaVbeta3/metabolismo , Masculino , RatonesRESUMEN
Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.
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Señalización del Calcio , Hepatitis Alcohólica , Hepatocitos , Neutrófilos , Elastasa Pancreática , Humanos , Neutrófilos/metabolismo , Neutrófilos/patología , Animales , Ratones , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Hepatitis Alcohólica/genética , Elastasa Pancreática/metabolismo , Masculino , Proliferación Celular , Canales de Calcio/metabolismo , Canales de Calcio/genética , FemeninoRESUMEN
INTRODUCTION: The aim of this study was to investigate the effect and mechanism of kaempferol on alcoholic steatohepatitis. METHODS: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for 6 weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1ß, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1ß and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling pathway activation. RESULTS: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1ß and tumor necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. CONCLUSION: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine.
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Mucosa Intestinal , Quempferoles , Ratones Endogámicos C57BL , MicroARNs , Transducción de Señal , Animales , MicroARNs/metabolismo , Quempferoles/farmacología , Quempferoles/uso terapéutico , Humanos , Transducción de Señal/efectos de los fármacos , Ratones , Masculino , Células CACO-2 , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Hepatitis Alcohólica/tratamiento farmacológico , Hepatitis Alcohólica/metabolismo , Ocludina/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Modelos Animales de Enfermedad , FN-kappa B/metabolismo , Lipopolisacáridos , Factor de Necrosis Tumoral alfa/metabolismo , Etanol , Interleucina-1beta/metabolismo , Funcion de la Barrera IntestinalAsunto(s)
Hepatitis Alcohólica , Interleucina-8 , Neutrófilos , Neutrófilos/inmunología , Neutrófilos/patología , Neutrófilos/metabolismo , Humanos , Hepatitis Alcohólica/inmunología , Hepatitis Alcohólica/patología , Hepatitis Alcohólica/metabolismo , Interleucina-8/metabolismo , Interleucina-8/inmunología , Inflamación/inmunología , Inflamación/patología , Masculino , Animales , RatonesRESUMEN
Background Intrahepatic infiltration of neutrophils is a character of alcoholic hepatitis (AH) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. The gut-liver axis has been thought to play a critical role in many liver diseases also including AH. However, whether NETs appear in AH and play role in AH is still unsure. Methods Serum samples from AH patients were collected and LPS and MPO-DNA were detected. WT, NE KO, and TLR4 KO mice were used to build the AH model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AH-related markers were detected. Results The serum MPO-DNA and LPS concentration was increased in AH patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AH mice. NETs formation decreased with antibiotic intervention and restored with antibiotic intervention plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AH-related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with a combined treatment with antibiotics plus LPS. But the AH-related markers did show a difference in TLR4 KO mice when they received the same treatment. Conclusion Intestinal-derived LPS promotes NETs formation in AH through the TLR4 pathway and further accelerates the AH process by NETs (AU)
Antecedentes La infiltración intrahepática de neutrófilos es una característica de la hepatitis alcohólica (AH, por sus siglas en inglés) y las trampas extracelulares de neutrófilos (NET, por sus siglas en inglés) son una estrategia importante para que los neutrófilos fijen y maten microorganismos invasores. Se ha pensado que el eje intestino/hígado desempeña un papel crítico en muchas enfermedades hepáticas, incluida la AH. Sin embargo, aún no está claro si las NET aparecen en la AH y desempeñan un papel en la misma. Métodos Se recogieron muestras de suero de pacientes con AH, y se detectaron LPS y MPO-ADN. Se utilizaron ratones WT, NE KO y TLR4 KO para construir el modelo de la AH, y las bacterias intestinales se eliminaron al mismo tiempo y se administró LPS. Luego se detectó la formación de NET y los marcadores relacionados con la AH. Resultados La concentración sérica de MPO-ADN y LPS aumentó en los pacientes con HA, y se reveló una correlación entre estos 2 índices. Se formaron más NET intrahepáticos en ratones con AH. La formación de las NET disminuyó con la intervención antibiótica, y se restauró con la intervención antibiótica más suplemento de LPS. Mientras que la formación de NET no pudo cambiar con el microbioma intestinal o combinar el suplemento de LPS en ratones TLR4 KO. A medida que probamos los caracteres relacionados con la AH, la lesión hepática, la deposición de grasa intrahepática, la inflamación y la fibrosis se aliviaron con el agotamiento de las NET. Estas marcas relacionadas también se atenuaron con la esterilización intestinal con antibióticos, y se recuperaron con un tratamiento combinado con antibióticos más LPS. Pero los marcadores relacionados con la AH mostraron una diferencia en los ratones TLR4 KO cuando recibieron el mismo tratamiento. Conclusión El LPS de origen intestinal promueve la formación de NET en la AH a través de la vía TLR4, y acelera aún más el proceso de AH por NET (AU)
Asunto(s)
Humanos , Animales , Ratones , Trampas Extracelulares/metabolismo , Hepatitis Alcohólica/metabolismo , ADN/metabolismo , Lipopolisacáridos/metabolismo , Neutrófilos/metabolismo , Modelos Animales de EnfermedadRESUMEN
Acute liver failure caused by alcoholic hepatitis (AH) is only effectively treated with liver transplantation. Livers of patients with AH show a unique molecular signature characterized by defective hepatocellular redox metabolism, concurrent to hepatic infiltration of neutrophils that express myeloperoxidase (MPO) and form neutrophil extracellular traps (NETs). Exacerbated NET formation and MPO activity contribute to liver damage in mice with AH and predicts poor prognosis in AH patients. The identification of pathways that maladaptively exacerbate neutrophilic activity in liver could inform of novel therapeutic approaches to treat AH. Whether the redox defects of hepatocytes in AH directly exacerbate neutrophilic inflammation and NET formation is unclear. Here we identify that the protein content of the mitochondrial biliverdin exporter ABCB10, which increases hepatocyte-autonomous synthesis of the ROS-scavenger bilirubin, is decreased in livers from humans and mice with AH. Increasing ABCB10 expression selectively in hepatocytes of mice with AH is sufficient to decrease MPO gene expression and histone H3 citrullination, a specific marker of NET formation. These anti-inflammatory effects can be explained by ABCB10 function reducing ROS-mediated actions in liver. Accordingly, ABCB10 gain-of-function selectively increased the mitochondrial GSH/GSSG ratio and decreased hepatic 4-HNE protein adducts, without elevating mitochondrial fat expenditure capacity, nor mitigating steatosis and hepatocyte death. Thus, our study supports that ABCB10 function regulating ROS-mediated actions within surviving hepatocytes mitigates the maladaptive activation of infiltrated neutrophils in AH. Consequently, ABCB10 gain-of-function in human hepatocytes could potentially decrease acute liver failure by decreasing the inflammatory flare caused by excessive neutrophil activity.
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Hepatitis Alcohólica , Fallo Hepático Agudo , Humanos , Animales , Ratones , Hepatitis Alcohólica/genética , Hepatitis Alcohólica/metabolismo , Biliverdina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Inflamación/genética , Inflamación/metabolismo , Histonas/metabolismo , Fallo Hepático Agudo/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
Alcohol-associated hepatitis (AH) is often diagnosed at advanced stages, and severe AH usually carries poor prognosis and high short-term mortality. In addition, it is challenging to understand the molecular mechanisms of immune dysregulation and inflammation in AH due to the cellular complexity and heterogeneity. Using single-cell RNA sequencing, previous studies found that AH causes dysfunctional innate immune response in monocytes, involving activation of pattern recognition receptors (PRRs) and cytokine signaling pathways. To better understand the coordinated systemic immune response in AH patients, we performed combined single-cell transcriptome, cell-surface protein, and lymphocyte antigen receptor analysis of peripheral blood mononuclear cell (PBMC) samples. Our results showed inflammatory cytokines and chemokines were highly expressed in AH, including IL-2, IL-32, CXC3R1 and CXCL16 in monocytes and NK cells, whereas HLA-DR genes were reduced in monocytes. In addition, we also found altered differentiation of T-helper cells (TH1 and TH17), which could further lead to neutrophil recruitment and macrophage activation. Lastly, our results also suggest impaired NK-cell activation and differentiation in AH with reduced gene expression of KLRC2 and increased gene expression of KLRG1. Our findings indicate different mechanisms may be involved in impaired immune and inflammatory responses for the cellular subtypes of the PBMCs in AH.
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Hepatitis Alcohólica , Leucocitos Mononucleares , Humanos , Leucocitos Mononucleares/metabolismo , Citocinas/metabolismo , Quimiocinas/metabolismo , Hepatitis Alcohólica/genética , Hepatitis Alcohólica/metabolismo , Perfilación de la Expresión Génica , Subfamília C de Receptores Similares a Lectina de Células NKRESUMEN
BACKGROUND: Chronic alcohol consumption impairs gut barrier function and perturbs the gut microbiome. Although shifts in bacterial communities in patients with alcohol-associated liver disease (ALD) have been characterized, less is known about the interactions between host metabolism and circulating microbe-derived metabolites during the progression of ALD. METHODS: A large panel of gut microbiome-derived metabolites of aromatic amino acids was quantified by stable isotope dilution liquid chromatography with online tandem mass spectrometry in plasma from healthy controls (n = 29), heavy drinkers (n = 10), patients with moderate (n = 16) or severe alcohol-associated hepatitis (n = 40), and alcohol-associated cirrhosis (n = 10). RESULTS: The tryptophan metabolites, serotonin and indole-3-propionic acid, and tyrosine metabolites, p-cresol sulfate, and p-cresol glucuronide, were decreased in patients with ALD. Patients with severe alcohol-associated hepatitis and alcohol-associated cirrhosis had the largest decrease in concentrations of tryptophan and tyrosine-derived metabolites compared to healthy control. Western blot analysis and interrogation of bulk RNA sequencing data from patients with various liver pathologies revealed perturbations in hepatic expression of phase II metabolism enzymes involved in sulfonation and glucuronidation in patients with severe forms of ALD. CONCLUSIONS: We identified several metabolites decreased in ALD and disruptions of hepatic phase II metabolism. These results indicate that patients with more advanced stages of ALD, including severe alcohol-associated hepatitis and alcohol-associated cirrhosis, had complex perturbations in metabolite concentrations that likely reflect both changes in the composition of the gut microbiome community and the ability of the host to enzymatically modify the gut-derived metabolites.
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Aminoácidos Aromáticos , Microbioma Gastrointestinal , Hepatopatías Alcohólicas , Hígado , Humanos , Aminoácidos Aromáticos/metabolismo , Hepatitis/metabolismo , Hepatitis/fisiopatología , Cirrosis Hepática Alcohólica/metabolismo , Cirrosis Hepática Alcohólica/fisiopatología , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/fisiopatología , Triptófano/metabolismo , Tirosina , Microbioma Gastrointestinal/fisiología , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/fisiopatología , Hígado/metabolismo , Hígado/fisiopatologíaRESUMEN
Metabolic-associated fatty liver disease (MAFLD) and alcoholic hepatitis (AH) are among the most prevalent liver diseases worldwide, and their coexistence is common in clinical practice. However, currently established models of MAFLD-AH coexistence do not fully replicate their pathological characteristics and require sophisticated experimental techniques. Therefore, we aimed to develop an easily replicable model that mimics obesity-induced MAFLD-AH in patients. Our goal was to establish a murine model that replicates MAFLD and AH coexistence, resulting in significant liver injury and inflammation. To this end, we administered a single ethanol gavage dose to ob/ob mice on a chow diet. The administration of a single dose of ethanol led to elevated serum transaminase levels, increased liver steatosis, and apoptosis in ob/ob mice. Furthermore, ethanol binge caused a significant increase in oxidative stress in ob/ob mice, as measured via 4-hydroxynonenal. Importantly, the single dose of ethanol also markedly exacerbated liver neutrophil infiltration and upregulated the hepatic mRNA expression of several chemokines and neutrophil-related proteins, including Cxcl1, Cxcl2, and Lcn2. Whole-liver transcriptomic analysis revealed that ethanol-induced changes in gene expression profile shared similar features with AH and MAFLD. In ob/ob mice, a single dose of ethanol binge caused significant liver injury and neutrophil infiltration. This easy-to-replicate murine model successfully mimics the pathological and clinical features of patients with coexisting MAFLD and AH and closely resembles the transcriptional regulation seen in human disease.
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Hepatitis Alcohólica , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ratones , Animales , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Modelos Animales de Enfermedad , Hígado/patología , Etanol/efectos adversos , Etanol/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND & AIMS: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood. METHODS: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity. RESULTS: Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression. CONCLUSIONS: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH. IMPACT AND IMPLICATIONS: Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair.
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Reprogramación Celular , Hepatitis Alcohólica , Animales , Ratones , Hepatitis Alcohólica/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Hígado/patologíaRESUMEN
BACKGROUND AND AIMS: Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. APPROACH AND RESULTS: Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- âwild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WTâ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice. CONCLUSION: Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.
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Hepatitis Alcohólica , Hepatopatías Alcohólicas , Ratones , Animales , Lipopolisacáridos/metabolismo , Hepatopatías Alcohólicas/metabolismo , Hígado/metabolismo , Etanol/toxicidad , Hepatitis Alcohólica/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Fagocitosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Ratones Endogámicos C57BL , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismoRESUMEN
OBJECTIVE: Alcoholic hepatitis (AH) reflects acute exacerbation of alcoholic liver disease (ALD) and is a growing healthcare burden worldwide. Interleukin-11 (IL-11) is a profibrotic, proinflammatory cytokine with increasingly recognised toxicities in parenchymal and epithelial cells. We explored IL-11 serum levels and their prognostic value in patients suffering from AH and cirrhosis of various aetiology and experimental ALD. DESIGN: IL-11 serum concentration and tissue expression was determined in a cohort comprising 50 patients with AH, 110 patients with cirrhosis and 19 healthy volunteers. Findings were replicated in an independent patient cohort (n=186). Primary human hepatocytes exposed to ethanol were studied in vitro. Ethanol-fed wildtype mice were treated with a neutralising murine IL-11 receptor-antibody (anti-IL11RA) and examined for severity signs and markers of ALD. RESULTS: IL-11 serum concentration and hepatic expression increased with severity of liver disease, mostly pronounced in AH. In a multivariate Cox-regression, a serum level above 6.4 pg/mL was a model of end-stage liver disease independent risk factor for transplant-free survival in patients with compensated and decompensated cirrhosis. In mice, severity of alcohol-induced liver inflammation correlated with enhanced hepatic IL-11 and IL11RA expression. In vitro and in vivo, anti-IL11RA reduced pathogenic signalling pathways (extracellular signal-regulated kinases, c-Jun N-terminal kinase, NADPH oxidase 4) and protected hepatocytes and murine livers from ethanol-induced inflammation and injury. CONCLUSION: Pathogenic IL-11 signalling in hepatocytes plays a crucial role in the pathogenesis of ALD and could serve as an independent prognostic factor for transplant-free survival. Blocking IL-11 signalling might be a therapeutic option in human ALD, particularly AH.
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Hepatitis Alcohólica , Hepatopatías Alcohólicas , Humanos , Ratones , Animales , Interleucina-11/metabolismo , Hepatopatías Alcohólicas/metabolismo , Hígado/metabolismo , Hepatitis Alcohólica/metabolismo , Etanol/toxicidad , Etanol/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Cirrosis Hepática/patología , Ratones Endogámicos C57BLRESUMEN
Introduction: Changes in the expression of cyto- and chemokines due to alcohol-associated liver disease (ALD) have been reported to be both protective and pathogenic. This study examined plasma levels of two key cytokines, Il-17 and Il-22, which construct the proinflammatory vs. anti-inflammatory axes across the spectrum of alcohol use disorder (AUD) and ALD including alcohol-associated hepatitis (AH) to determine the underlying status of the inflammation. Methods: Forty-two males and females aged 25-63 yrs. were grouped as healthy controls (HV[n=8]), AUD with no liver injury (AUDNLI [n=8]), AUD with liver injury (AUDLI [n=8]), non-severe alcohol-associated hepatitis (NSAH [n=9]), and severe alcohol-associated hepatitis (SAH [n=9]). Demographic, drinking, and clinical data were collected. Blood samples were collected at baseline (BL, all subjects) and during week 4 (W4, only patients) for IL-17 and IL-22; and statistically analyzed. Results: IL-17 was highly elevated in the SAH group both at BL and post-SOC. LTDH and BL IL-22 in non-severe AH patients were associated significantly. LTDH significantly predicted W4 IL-22 levels, positively (increasing) in NSAH and inversely (lowering) in SAH patients. BL and W4 IL-22 levels were significantly higher (4-fold, p≤0.001) in all AH patients compared to all AUD patients (AUROC=0.988, p≤0.001). IL-22 showed significant affinity with AST, AST: ALT ratio, total bilirubin, INR, and PT both at BL and W4. IL-22 was inversely associated with IL-1ß; and positively with TNF-α and IL-8 both at BL, and W4. BL IL-17 showed a positive correlation with MELD (p=0.017) in all AH patients. In SAH, > 2-fold W4 IL-17 level compared to BL showed significant within subjects' effects, p=0.006. In AUD patients without AH, the drop in IL-17 at W4 vs. BL showed a significant within subjects' effect, p=0.031. Discussion: Drinking chronicity predicted opposite effects in IL-22 levels in NSAH (antiinflammatory) and SAH (pro-inflammatory) patients at post-SOC. BL IL-22 levels differentiated AH patients robustly from the AUD patients (with or without liver injury); and showed corresponding increases stepwise with the stages of ALD. IL-22 was closely associated with progression and injury markers of the liver; and response to the cytokines of pro-inflammatory nature. Pro-inflammatory indicator of IL-17 cell axis, IL-17 showed a strong positive association with MELD, a severity indicator of AH.
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Alcoholismo , Hepatitis Alcohólica , Hepatopatías Alcohólicas , Femenino , Humanos , Masculino , Alcoholismo/complicaciones , Citocinas , Hepatitis Alcohólica/metabolismo , Interleucina-17 , Interleucina-22 , Adulto , Persona de Mediana EdadRESUMEN
Intrahepatic neutrophil infiltration has been implicated in severe alcoholic hepatitis (SAH) pathogenesis; however, the mechanism underlying neutrophil-induced injury in SAH remains obscure. This translational study aims to describe the patterns of intrahepatic neutrophil infiltration and its involvement in SAH pathogenesis. Immunohistochemistry analyses of explanted livers identified two SAH phenotypes despite a similar clinical presentation, one with high intrahepatic neutrophils (Neuhi), but low levels of CD8+ T cells, and vice versa. RNA-Seq analyses demonstrated that neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with hepatic inflammation and disease progression. To study specifically the mechanisms related to Neuhi in AH patients and liver injury, we used the mouse model of chronic-plus-binge ethanol feeding and found that myeloid-specific deletion of the Ncf1 gene abolished ethanol-induced hepatic inflammation and steatosis. RNA-Seq analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic hepatitis (AH) by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key antiinflammatory and antifibrotic microRNA). In conclusion, two distinct histopathological phenotypes based on liver immune phenotyping are observed in SAH patients, suggesting a separate mechanism driving liver injury and/or failure in these patients.
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Hepatitis Alcohólica , Hepatopatías Alcohólicas , Animales , Etanol/efectos adversos , Hepatitis Alcohólica/genética , Hepatitis Alcohólica/metabolismo , Inflamación/patología , Hígado/metabolismo , Hepatopatías Alcohólicas/genética , Hepatopatías Alcohólicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenotipo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl- channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.
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Hepatitis Alcohólica , Hepatitis , Pancreatitis Alcohólica , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Etanol/toxicidad , Hepatitis/metabolismo , Hepatitis Alcohólica/genética , Hepatitis Alcohólica/metabolismo , Humanos , Ratones , Pancreatitis Alcohólica/metabolismoRESUMEN
The present study was to investigate the therapeutical effects and mechanisms of Asiatic acid from Potentilla chinensis against alcoholic hepatitis. Rats were intragastrically fed with alcohol for 12 weeks to induce alcoholic hepatitis and then treated with various drugs for further 12 weeks. The results showed that Asiatic acid significantly alleviated liver injury caused by alcohol in rats, as evidenced by the improved histological changes and the lower levels of AST, ALT, and TBIL. Besides, Asiatic acid significantly enhanced the activity of ADH and ALDH, promoting alcohol metabolism. Asiatic acid suppressed CYP2E1 activity and NADP+/NADPH ratio, resulting in low ROS production. Further study revealed that Asiatic acid markedly reduced hepatocyte apoptosis by regulating the expression levels of apoptosis-related protein. Moreover, Asiatic acid could regulate the Nrf2 and NF-κB signaling pathway, attenuating oxidative stress and inflammation as a result. Interestingly, the comprehensive analysis of transcriptomics and metabolomics indicated that Asiatic acid inhibited the gene expression of Gpat3 and thereby affected the biosynthesis of the metabolites (1-acyl-Sn-glycerol-3-phosphocholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine), regulating the glycerophospholipid metabolism pathway and ultimately ameliorating hepatocyte damage. In conclusion, this study demonstrates that Asiatic acid can ameliorate alcoholic hepatitis by modulating the NF-κB and Nrf2 signaling pathways and the glycerophospholipid metabolism pathway, which may be developed as a potential medicine for the treatment of alcoholic hepatitis.
Asunto(s)
Hepatitis Alcohólica , Factor 2 Relacionado con NF-E2 , Animales , Etanol/farmacología , Glicerofosfolípidos/metabolismo , Glicerofosfolípidos/farmacología , Hepatitis Alcohólica/tratamiento farmacológico , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Hígado/metabolismo , Metabolómica , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Triterpenos Pentacíclicos , Ratas , TranscriptomaRESUMEN
This study aimed to develop a well-characterized mouse model of alcoholic hepatitis (AH) regression. Male C57BL/6J mice were fed a Lieber-DeCarli (LD) control diet or LD containing 5% ethanol for ten days followed by one binge, which is the chronic-binge model of AH developed by the National Institute on Alcohol Abuse and Alcoholism. To determine AH regression, mice previously exposed to ethanol were put on LD control diet and metabolic and inflammatory features were monitored weekly for three weeks. Serum alcohol, total cholesterol, and alanine transaminase levels were increased in ethanol-fed mice, which declined to those of no ethanol controls within one and three weeks after ethanol withdrawal, respectively. Serum malondialdehyde was increased with ethanol feeding, but it was restored to no ethanol control levels within one week. Ethanol-induced changes in the hepatic expression of genes involved in lipogenesis, fatty acid oxidation, ethanol metabolism, and antioxidant response were restored to those of no ethanol controls after 3 weeks of ethanol withdrawal. Also, ethanol-induced hepatic inflammation was gradually decreased during the 3 weeks of ethanol withdrawal. Hepatic nicotinamide adenine dinucleotide (NAD+) levels and the expression of enzymes involved in the NAD+ salvage pathway were decreased by ethanol feeding, which was mitigated after ethanol withdrawal. Ethanol significantly lowered hepatic sirtuin 1 expression, but its levels were restored with ethanol cessation. This study established a mouse model of AH regression, which can be used as a preclinical model to study the potential of dietary bioactives or therapeutic agents on AH regression.
Asunto(s)
Alcoholismo/complicaciones , Etanol/efectos adversos , Hígado Graso/metabolismo , Hepatitis Alcohólica/metabolismo , NAD/metabolismo , Estrés Oxidativo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/inmunología , Hepatitis Alcohólica/etiología , Hepatitis Alcohólica/genética , Hepatitis Alcohólica/inmunología , Humanos , Hígado/inmunología , Hígado/metabolismo , Masculino , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Alcohol-related liver disease (ALD) is characterized by accumulation of hepatic free fatty acids (FFAs) and liver injury. The present study aimed to investigate if mechanistic target of rapamycin complex 1 (mTORC1) plays a role in FFA-induced organelle dysfunction, thereby contributing to the development of ALD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated mTORC1 signaling in hepatocellular cell injury. C57BL/6J wild-type mice were subjected to chronic alcohol feeding with or without rapamycin to inhibit mTORC1 activation. We revealed that palmitic acid (PA)-induced ER stress and suppression of LAMP2 and autophagy flux were mTORC1-dependent as rapamycin reversed such deleterious effects. C/EBP homologous protein (CHOP) was downstream of ATF4 which partially modulated LAMP2. Supplementation with rapamycin to alcohol-fed mice attenuated mTORC1 activation and ER stress, restored LAMP2 protein, and improved autophagy, leading to amelioration of alcohol-induced liver injury. Induction of mTORC1 signaling and CHOP were also detected in the liver of patients with severe alcoholic hepatitis. This study demonstrates that hepatic FFAs play a crucial role in the pathogenesis of ALD by activating mTORC1 signaling, thereby inducing ER stress and suppressing LAMP2-autophagy flux pathway, which represents an important mechanism of FFA-induced hepatocellular injury.
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Autofagia , Estrés del Retículo Endoplásmico , Etanol/efectos adversos , Ácidos Grasos no Esterificados/farmacología , Hepatopatías/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Factor de Transcripción Activador 4/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Suplementos Dietéticos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ratones Endogámicos C57BL , Ácido Palmítico/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Factor de Transcripción CHOP/metabolismoRESUMEN
OBJECTIVES: In alcoholic hepatitis (AH), dysfunctional T lymphocytes may contribute to the high mortality from infections. T lymphocyte activation is governed by the expression of co-stimulatory receptors such as 4-1BB balanced by inhibitory receptors such as Programmed Death receptor 1 (PD-1). 4-1BB expression is unaccounted for in AH, while PD-1 is elevated. We characterized expression of 4-1BB and PD-1 and the associated T lymphocyte functional status in AH and investigated whether these were associated with short-term mortality. METHODS: Thirty-five patients with AH (at diagnosis and days 7 and 90) were compared with healthy controls (HC). Spontaneous and in vitro stimulated receptor expression were quantified by flow cytometry, and plasma proteins by ELISA. RESULTS: At diagnosis, the patients showed increased stimulated 4-1BB responses of CD4+ T lymphocytes. Also, the frequencies of PD-1+ T lymphocytes both with and without co-expressed 4-1BB were increased. Further, interferon-gamma was predominantly produced in T lymphocytes co-expressing 4-1BB. A decrease in the frequency of spontaneous 4-1BB+ T lymphocytes and an increase in soluble 4-1BB during the first week after diagnosis were associated with higher mortality at day 90 in AH. PD-1 expression showed no systematic dynamics related to mortality. CONCLUSIONS: We found an increased stimulated 4-1BB response of T lymphocytes in AH and early loss of these lymphocytes was associated with a higher short-term mortality. This suggests a role of T lymphocyte 4-1BB expression in the progression of AH.
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Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Galectinas/metabolismo , Hepatitis Alcohólica/mortalidad , Activación de Linfocitos/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Hepatitis Alcohólica/epidemiología , Hepatitis Alcohólica/inmunología , Hepatitis Alcohólica/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia , Factores de TiempoRESUMEN
Acute alcoholic liver injury (AALI) is a threat to human health. Dendrobium officinale polysaccharide (DOP) has the potential to protect the liver by enhancing the anti-oxidative system to maintain the relative balance of ROS (active oxygen species) and antioxidants in AALI mice. However, the dynamic improvement effect of DOP on AALI is still not clear and accurate medication guidance is not available, which limits the clinical application of DOP. Because of the advantages of high sensitivity, noninvasiveness, and visualization, near-infrared (NIR) fluorescence imaging has been widely studied in biochemistry and biomedicine. As the glutathione (GSH) level in the liver is closely related to the progression of AALI, herein, an NIR fluorescent probe for GSH, HCG was used to dynamically evaluate the effect of DOP on AALI mice. In this study, DOP was proven to maintain the relative balance of GSH content in the liver to protect it from damage. To the best of our knowledge, it is the first time to assess the effect of DOP on AALI mice through a NIR fluorescence imaging technique. This study may also provide a potential NIR imaging agent for the clinical research to improve the management of liver injury-related diseases.