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BACKGROUND AND AIMS: Patients with alcohol-associated hepatitis (AH) have an altered fecal metabolome, including reduced microbiota-derived tryptophan metabolites, which function as ligands for aryl hydrocarbon receptor (AhR). The aim of this study was to assess serum AhR ligand activity in patients with AH. APPROACH AND RESULTS: The study included 74 controls without AUD, 97 patients with AUD, and 330 patients with AH from 2 different multicenter cohorts (InTeam: 134, AlcHepNet: 196). Serum AhR activity was evaluated using an AhR reporter assay with HepG2-Lucia cells incubated with serum for 24 hours. Serum AhR activity was significantly higher in patients with AH compared with both controls (1.59 vs. 0.96-fold change, p < 0.001) and patients with AUD (1.59 vs. 0.93, p < 0.001). In both AH cohorts, patients with AhR activity ≥ 2.09 had significantly lower cumulative survival rates at 30, 60, 90, and 180 days compared to those with AhR activity < 2.09. When serum AhR activity was used to further stratify patients with severe AH, the cumulative 30, 60, 90, and 180-day survival rates for patients with severe AH and the AhR activity ≥ 2.09 group were all significantly lower than those with an AhR activity < 2.09 group. CONCLUSIONS: Serum AhR activity was significantly higher in patients with AH compared with controls and individuals with AUD, and this increased activity was associated with higher mortality. Consequently, serum AhR activity holds potential as a prognostic marker.
Assuntos
Hepatite Alcoólica , Receptores de Hidrocarboneto Arílico , Humanos , Receptores de Hidrocarboneto Arílico/sangue , Receptores de Hidrocarboneto Arílico/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Hepatite Alcoólica/mortalidade , Hepatite Alcoólica/sangue , Adulto , Estudos de Casos e Controles , Fatores de Transcrição Hélice-Alça-Hélice Básicos/sangue , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Taxa de Sobrevida , Células Hep G2 , Idoso , Biomarcadores/sangueRESUMO
Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.
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Bacteriófagos/fisiologia , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/virologia , Microbioma Gastrointestinal , Hepatite Alcoólica/microbiologia , Hepatite Alcoólica/terapia , Terapia por Fagos , Alcoolismo/complicações , Alcoolismo/microbiologia , Animais , Enterococcus faecalis/isolamento & purificação , Etanol/efeitos adversos , Fígado Gorduroso/complicações , Fígado Gorduroso/microbiologia , Fezes/microbiologia , Feminino , Vida Livre de Germes , Hepatite Alcoólica/complicações , Hepatite Alcoólica/mortalidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Perforina/metabolismoRESUMO
OBJECTIVE: Patients with alcohol-associated hepatitis (AH) have a high mortality. Alcohol exacerbates liver damage by inducing gut dysbiosis, bacterial translocation and inflammation, which is characterised by increased numbers of circulating and hepatic neutrophils. DESIGN: In this study, we performed tandem mass tag (TMT) proteomics to analyse proteins in the faeces of controls (n=19), patients with alcohol-use disorder (AUD; n=20) and AH (n=80) from a multicentre cohort (InTeam). To identify protein groups that are disproportionately represented, we conducted over-representation analysis using Reactome pathway analysis and Gene Ontology to determine the proteins with the most significant impact. A faecal biomarker and its prognostic effect were validated by ELISA in faecal samples from patients with AH (n=70), who were recruited in a second and independent multicentre cohort (AlcHepNet). RESULT: Faecal proteomic profiles were overall significantly different between controls, patients with AUD and AH (principal component analysis p=0.001, dissimilarity index calculated by the method of Bray-Curtis). Proteins that showed notable differences across all three groups and displayed a progressive increase in accordance with the severity of alcohol-associated liver disease were predominantly those located in neutrophil granules. Over-representation and Reactome analyses confirmed that differentially regulated proteins are part of granules in neutrophils and the neutrophil degranulation pathway. Myeloperoxidase (MPO), the marker protein of neutrophil granules, correlates with disease severity and predicts 60-day mortality. Using an independent validation cohort, we confirmed that faecal MPO levels can predict short-term survival at 60 days. CONCLUSIONS: We found an increased abundance of faecal proteins linked to neutrophil degranulation in patients with AH, which is predictive of short-term survival and could serve as a prognostic non-invasive marker.
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BACKGROUND AND AIMS: The prevalence of alcohol use disorder (AUD) and metabolic dysfunction-associated fatty liver disease (MAFLD) are increasing worldwide, leading to the increasing likelihood of both etiologies contributing to a patient's liver disease. However, the effects of modest alcohol use in NAFLD are controversial and more studies are needed. We compared the intestinal viromes of patients with AUD and NAFLD in order to evaluate the effect of alcohol consumption on the intestinal viromes of NAFLD patients by extracting virus-like particles and performing metagenomic sequencing. APPROACH AND RESULTS: Viral nucleic acids were extracted from fecal samples and subjected to metagenomic sequencing. We demonstrate significant differences in the intestinal viromes of NAFLD and AUD patients, and that alcohol use in NAFLD patients reclassified to MAFLD accounted for significant differences in the intestinal viromes. The relative abundance of several Lactococcus phages was more similar between AUD patients and alcohol-consuming MAFLD patients than non-alcohol-consuming MAFLD patients and control subjects, and multivariate modeling using the most discriminating Lactococcus phages could better predict alcohol use in the MAFLD population than the alcohol-associated liver disease/NAFLD Index. Significant differences in the viral composition and diversity were also seen between MAFLD patients with low and moderate alcohol consumption compared with no alcohol consumption. CONCLUSIONS: The intestinal virome of MAFLD patients who consume low to moderate amounts of alcohol are significantly different from those who do not, and many features of the intestinal virome of alcohol-consuming MAFLD patients resemble that of AUD patients.
Assuntos
Alcoolismo , Hepatopatias Alcoólicas , Hepatopatia Gordurosa não Alcoólica , Humanos , Viroma , Consumo de Bebidas Alcoólicas/efeitos adversos , EtanolRESUMO
BACKGROUND & AIMS: HEV genotype (gt) 3 infections are prevalent in high-income countries and display a wide spectrum of clinical presentations. Host - but not viral - factors are reported to be associated with worse clinical outcomes. METHODS: Demographic, clinical, and biochemical data laboratory-confirmed HEV infections (by PCR and/or a combination of IgM and IgG serology) at the Belgian National Reference Centre between January 2010 and June 2018 were collected using standardised case report forms. Genotyping was based on HEV open reading frame 2 sequences. Serum CXCL10 levels were measured by a magnetic bead-based assay. H&E staining was performed on liver biopsies. RESULTS: A total of 274 HEV-infected individuals were included. Subtype assignment was possible for 179/218 viraemic cases, confirming gt3 as dominant with an almost equal representation of clades abchijklm and efg. An increased hospitalisation rate and higher peak serum levels of alanine aminotransferase, bilirubin, and alkaline phosphatase were found in clade efg-infected individuals in univariate analyses. In multivariable analyses, clade efg infections remained more strongly associated with severe disease presentation than any of the previously identified host risk factors, being associated with a 2.1-fold higher risk of hospitalisation (95% CI 1.1-4.4, p = 0.034) and a 68.2% higher peak of bilirubin levels (95% CI 13.3-149.9, p = 0.010), independently of other factors included in the model. In addition, acute clade efg infections were characterised by higher serum CXCL10 levels (p = 0.0005) and a more pronounced liver necro-inflammatory activity (p = 0.022). CONCLUSIONS: In symptomatic HEV gt3 infections, clade efg is associated with a more severe disease presentation, higher serum CXCL10 levels, and liver necro-inflammatory activity, irrespective of known host risk factors. CLINICAL TRIAL REGISTRATION: The protocol was submitted to clinicaltrials.gov (NCT04670419). IMPACT AND IMPLICATIONS: HEV genotype (gt) 3 infections display a wide spectrum of clinical presentations currently ascribed to host factors. Here we examined the role of viral factors on liver disease outcomes by combining viral phylogeny with clinical, biochemical, cytokine, and histological data from 274 Belgian adults infected with HEV presenting between 2010 and 2018. HEV gt 3 clade efg infections were associated with a more severe disease presentation, higher serum CXCL10 levels and liver necro-inflammatory activity, irrespective of known host risk factors. HEV gt3 clade-dependent clinical outcomes call for broad HEV gt3 subtyping in clinical practice and research to help identify those at higher risk for worse outcomes and to further unravel underlying virus-host interactions.
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Vírus da Hepatite E , Hepatite E , Adulto , Humanos , Bélgica/epidemiologia , Bilirrubina , Genótipo , Hepatite E/diagnóstico , Hepatite E/epidemiologia , Filogenia , RNA Viral/análise , Protocolos de Ensaio Clínico como AssuntoRESUMO
BACKGROUND & AIMS: Studies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis. METHODS: We performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B. RESULTS: The presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis. CONCLUSIONS: Non-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NASH. LAY SUMMARY: Non-alcoholic fatty liver disease is one of the most common chronic liver diseases and is associated with changes in the fecal bacterial microbiome. We show that patients with non-alcoholic fatty liver disease and more severe disease stages have a specific composition of fecal fungi and an increased systemic immune response to Candida albicans. In a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis, we show that treatment with antifungals reduces liver damage.
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Microbioma Gastrointestinal , Micobioma , Hepatopatia Gordurosa não Alcoólica , Animais , Fezes/microbiologia , Humanos , Fígado , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologia , Saccharomyces cerevisiaeRESUMO
BACKGROUND & AIMS: Alterations in the gut microbiome have been associated with the severity of nonalcoholic fatty liver disease (NAFLD). Previous studies focused exclusively on the bacteria in the microbiome; we investigated changes in the viral microbiome (virome) in patients with NAFLD. METHODS: In a prospective, cross-sectional, observational study, we extracted RNA and DNA virus-like particles from fecal samples from 73 patients with NAFLD: 29 patients had an NAFLD Activity Score (NAS) of 0-4, 44 patients had an NAS of 5-8 or liver cirrhosis (LCI), 37 patients had F0-F1 fibrosis, and 36 patients had F2-F4 fibrosis. As controls, 9 individuals without liver disease and 13 patients with mild primary biliary cholangitis were included in the analysis. We performed shotgun metagenomic sequencing of virus-like particles. RESULTS: Patients with NAFLD and NAS 5-8/LCI had a significant decrease in intestinal viral diversity compared with patients with NAFLD and NAS 0-4 or control individuals. The presence of more advanced NAFLD was associated with a significant reduction in the proportion of bacteriophages compared with other intestinal viruses. Using multivariate logistic regression analysis with leave-1-out cross validation, we developed a model, including a viral diversity index and simple clinical variables, that identified patients with NAS 5-8/LCI with an area under the curve of 0.95 (95% confidence interval, 0.91-0.99) and F2-F4 fibrosis with an area under the curve of 0.88 (95% confidence interval, 0.80-0.95). Addition of data on viral diversity significantly improved multivariate models, including those based on only clinical parameters or bacterial diversity. CONCLUSIONS: In a study of fecal viromes from patients with NAFLD and control individuals, we associated histologic markers of NAFLD severity with significant decreases in viral diversity and proportion of bacteriophages. We developed a model based on fecal viral diversity and clinical data that identifies patients with severe NAFLD and fibrosis more accurately than models based only on clinical or bacterial data.
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Microbioma Gastrointestinal , Intestinos/virologia , Cirrose Hepática/virologia , Hepatopatia Gordurosa não Alcoólica/virologia , Viroma , Adulto , Idoso , Estudos de Casos e Controles , Estudos Transversais , Fezes/virologia , Feminino , Humanos , Cirrose Hepática/diagnóstico , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Estudos Prospectivos , Índice de Gravidade de Doença , Adulto JovemRESUMO
Chronic alcohol consumption causes increased intestinal permeability and changes in the intestinal microbiota composition, which contribute to the development and progression of alcohol-related liver disease. In this setting, little is known about commensal fungi in the gut. We studied the intestinal mycobiota in a cohort of patients with alcoholic hepatitis, patients with alcohol use disorder, and nonalcoholic controls using fungal-specific internal transcribed spacer amplicon sequencing of fecal samples. We further measured serum anti-Saccharomyces cerevisiae antibodies (ASCA) as a systemic immune response to fungal products or fungi. Candida was the most abundant genus in the fecal mycobiota of the two alcohol groups, whereas genus Penicillium dominated the mycobiome of nonalcoholic controls. We observed a lower diversity in the alcohol groups compared with controls. Antibiotic or steroid treatment was not associated with a lower diversity. Patients with alcoholic hepatitis had significantly higher ASCA levels compared to patients with alcohol use disorder and to nonalcoholic controls. Within the alcoholic hepatitis cohort, patients with levels of at least 34 IU/mL had a significantly lower 90-day survival (59%) compared with those with ASCA levels less than 34 IU/mL (80%) with an adjusted hazard ratio of 3.13 (95% CI, 1.11-8.82; P = 0.031). Conclusion: Patients with alcohol-associated liver disease have a lower fungal diversity with an overgrowth of Candida compared with controls. Higher serum ASCA was associated with increased mortality in patients with alcoholic hepatitis. Intestinal fungi may serve as a therapeutic target to improve survival, and ASCA may be useful to predict the outcome in patients with alcoholic hepatitis.
Assuntos
Disbiose/etiologia , Disbiose/imunologia , Hepatite Alcoólica/complicações , Hepatite Alcoólica/imunologia , Intestinos/microbiologia , Micobioma , Adulto , Idoso , Anticorpos Antifúngicos/sangue , Candida/imunologia , Estudos de Coortes , Disbiose/sangue , Feminino , Hepatite Alcoólica/sangue , Humanos , Fenômenos do Sistema Imunitário , Masculino , Pessoa de Meia-Idade , Saccharomyces cerevisiae/imunologiaRESUMO
BACKGROUND AND AIMS: Alcoholic hepatitis (AH) is a severe manifestation of alcohol-associated liver disease (ALD) with high mortality. Although gut bacteria and fungi modulate disease severity, little is known about the effects of the viral microbiome (virome) in patients with ALD. APPROACH AND RESULTS: We extracted virus-like particles from 89 patients with AH who were enrolled in a multicenter observational study, 36 with alcohol use disorder (AUD), and 17 persons without AUD (controls). Virus-like particles from fecal samples were fractionated using differential filtration techniques, and metagenomic sequencing was performed to characterize intestinal viromes. We observed an increased viral diversity in fecal samples from patients with ALD, with the most significant changes in samples from patients with AH. Escherichia-, Enterobacteria-, and Enterococcus phages were over-represented in fecal samples from patients with AH, along with significant increases in mammalian viruses such as Parvoviridae and Herpesviridae. Antibiotic treatment was associated with higher viral diversity. Specific viral taxa, such as Staphylococcus phages and Herpesviridae, were associated with increased disease severity, indicated by a higher median Model for End-Stage Liver Disease score, and associated with increased 90-day mortality. CONCLUSIONS: In conclusion, intestinal viral taxa are altered in fecal samples from patients with AH and associated with disease severity and mortality. Our study describes an intestinal virome signature associated with AH.
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Doença Hepática Terminal/virologia , Hepatite Alcoólica/virologia , Mucosa Intestinal/virologia , Cirrose Hepática/virologia , Viroma/genética , Adulto , Idoso , Animais , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Estudos de Casos e Controles , DNA Viral/isolamento & purificação , Doença Hepática Terminal/diagnóstico , Doença Hepática Terminal/mortalidade , Doença Hepática Terminal/terapia , Fezes/virologia , Feminino , Hepatite Alcoólica/diagnóstico , Hepatite Alcoólica/mortalidade , Hepatite Alcoólica/terapia , Herpesviridae/genética , Herpesviridae/isolamento & purificação , Humanos , Fígado/patologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/mortalidade , Cirrose Hepática/terapia , Masculino , Metagenômica , Pessoa de Meia-Idade , Parvoviridae/genética , Parvoviridae/isolamento & purificação , RNA Viral/isolamento & purificação , Índice de Gravidade de Doença , Taxa de SobrevidaRESUMO
Liver sinusoids are lined by liver sinusoidal endothelial cells (LSEC), which represent approximately 15 to 20% of the liver cells, but only 3% of the total liver volume. LSEC have unique functions, such as fluid filtration, blood vessel tone modulation, blood clotting, inflammatory cell recruitment, and metabolite and hormone trafficking. Different subtypes of liver endothelial cells are also known to control liver zonation and hepatocyte function. Here, we have reviewed the origin of LSEC, the different subtypes identified in the liver, as well as their renewal during homeostasis. The liver has the exceptional ability to regenerate from small remnants. The past decades have seen increasing awareness in the role of non-parenchymal cells in liver regeneration despite not being the most represented population. While a lot of knowledge has emerged, clarification is needed regarding the role of LSEC in sensing shear stress and on their participation in the inductive phase of regeneration by priming the hepatocytes and delivering mitogenic factors. It is also unclear if bone marrow-derived LSEC participate in the proliferative phase of liver regeneration. Similarly, data are scarce as to LSEC having a role in the termination phase of the regeneration process. Here, we review what is known about the interaction between LSEC and other liver cells during the different phases of liver regeneration. We next explain extended hepatectomy and small liver transplantation, which lead to "small for size syndrome" (SFSS), a lethal liver failure. SFSS is linked to endothelial denudation, necrosis, and lobular disturbance. Using the knowledge learned from partial hepatectomy studies on LSEC, we expose several techniques that are, or could be, used to avoid the "small for size syndrome" after extended hepatectomy or small liver transplantation.
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Células Endoteliais , Hepatectomia , Hepatócitos , Falência Hepática/patologia , Regeneração Hepática , Fígado , Animais , Células Endoteliais/citologia , Células Endoteliais/patologia , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Fígado/citologia , Fígado/patologiaRESUMO
Chronic alcohol consumption and alcohol-associated liver disease (ALD) represent a major public health problem worldwide. Only a minority of patients with an alcohol-use disorder (AUD) develop severe forms of liver disease (e.g., steatohepatitis and fibrosis) and finally progress to the more advanced stages of ALD, such as severe alcohol-associated hepatitis and decompensated cirrhosis. Emerging evidence suggests that gut barrier dysfunction is multifactorial, implicating microbiota changes, alterations in the intestinal epithelium, and immune dysfunction. This failing gut barrier ultimately allows microbial antigens, microbes, and metabolites to translocate to the liver and into systemic circulation. Subsequent activation of immune and inflammatory responses contributes to liver disease progression. Here we review the literature about the disturbance of the different host defense mechanisms linked to gut barrier dysfunction, increased microbial translocation, and impairment of liver and systemic inflammatory responses in the different stages of ALD.
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Disbiose/patologia , Microbioma Gastrointestinal , Hepatopatias Alcoólicas/complicações , Animais , Disbiose/microbiologia , Humanos , Hepatopatias Alcoólicas/microbiologiaRESUMO
BACKGROUND & AIMS: Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease. METHODS: C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin. RESULTS: The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the ß-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis. CONCLUSIONS: Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis. LAY SUMMARY: Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the ß-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.
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Candida albicans/metabolismo , Exotoxinas/metabolismo , Proteínas Fúngicas/metabolismo , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/microbiologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/microbiologia , Adulto , Idoso , Animais , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Exotoxinas/análise , Exotoxinas/farmacologia , Fezes/microbiologia , Feminino , Proteínas Fúngicas/análise , Proteínas Fúngicas/farmacologia , Microbioma Gastrointestinal , Hepatite Alcoólica/mortalidade , Hepatócitos/efeitos dos fármacos , Humanos , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Hepatopatias Alcoólicas/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Chronic liver diseases are characterized by the expansion of ductular reaction (DR) cells and the expression of liver progenitor cell (LPC) markers. In alcoholic hepatitis (AH), the degree of DR expansion correlates with disease progression and short-term survival. However, little is known about the biological properties of DR cells, their impact on the pathogenesis of human liver disease, and their contribution to tissue repair. In this study, we have evaluated the transcriptomic profile of DR cells by laser capture microdissection in patients with AH and assessed its association with disease progression. The transcriptome analysis of cytokeratin 7-positive (KRT7+ ) DR cells uncovered intrinsic gene pathways expressed in DR and genes associated with alcoholic liver disease progression. Importantly, DR presented a proinflammatory profile with expression of neutrophil recruiting C-X-C motif chemokine ligand (CXC) and C-C motif chemokine ligand chemokines. Moreover, LPC markers correlated with liver expression and circulating levels of inflammatory mediators such as CXCL5. Histologically, DR was associated with neutrophil infiltration at the periportal area. In order to model the DR and to assess its functional role, we generated LPC organoids derived from patients with cirrhosis. Liver organoids mimicked the transcriptomic and proinflammatory profile of DR cells. Conditioned medium from organoids induced neutrophil migration and enhanced cytokine expression in neutrophils. Likewise, neutrophils promoted the proinflammatory profile and the expression of chemokines of liver organoids. Conclusion: Transcriptomic and functional analysis of KRT7+ cells indicate that DR has a proinflammatory profile and promote neutrophil recruitment. These results indicate that DR may be involved in the liver inflammatory response in AH, and suggest that therapeutic strategies targeting DR cells may be useful to mitigate the inflammatory cell recruitment in AH.
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Hepatite Alcoólica/imunologia , Fígado/metabolismo , Infiltração de Neutrófilos , Quimiocinas/metabolismo , Estudos de Coortes , Feminino , Hepatite Alcoólica/metabolismo , Humanos , Inflamação/metabolismo , Fígado/citologia , Cirrose Hepática/metabolismo , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , TranscriptomaRESUMO
BACKGROUND: Fucosyltransferase 2 (Fut2)-mediated intestinal α1-2-fucosylation is important in maintaining a symbiotic host-microbiota relationship and can protect against several pathogens. Intestinal dysbiosis is an important factor for the progression of experimental ethanol (EtOH)-induced liver disease, but the role of Fut2 in modulating the intestinal glycocalyx during alcohol-associated liver disease is unknown. We investigated the role of Fut2-mediated intestinal α1-2-fucosylation for the development of alcohol-associated liver disease. METHODS: Immunohistochemistry staining was applied to evaluate α1-2-fucosylation in duodenal biopsies from patients with alcohol use disorder. Wild-type (WT) and Fut2-deficient littermate mice were subjected to Lieber-DeCarli models of chronic EtOH administration and the chronic-binge EtOH diet (NIAAA model). RESULTS: Intestinal α1-2-fucosylation was down-regulated in patients with alcohol use disorder. Lack of α1-2-fucosylation in Fut2-deficient mice exacerbates chronic EtOH-induced liver injury, steatosis, and inflammation without affecting EtOH metabolism. Dietary supplementation of the α1-2-fucosylated glycan 2'-fucosyllactose (2'-FL) ameliorates EtOH-induced liver disease in Fut2-deficient mice in the NIAAA model. Despite no direct effects on growth of Enterococcus faecalis in vitro, intestinal α1-2-fucosylation reduces colonization of cytolysin-positive E. faecalis in the intestine of EtOH-fed mice. CONCLUSIONS: Intestinal α1-2-fucosylation acts as a host-protective mechanism against EtOH-induced liver disease. 2'-FL is an oligosaccharide naturally present in human milk that could be considered as therapeutic agent for alcohol-associated liver disease.
Assuntos
Alcoolismo/metabolismo , Disbiose/genética , Fucosiltransferases/genética , Hepatopatias Alcoólicas/genética , Fígado/efeitos dos fármacos , Alcoolismo/genética , Alcoolismo/microbiologia , Animais , Depressores do Sistema Nervoso Central/toxicidade , Modelos Animais de Doenças , Disbiose/metabolismo , Disbiose/microbiologia , Etanol/toxicidade , Fucosiltransferases/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Glicocálix/efeitos dos fármacos , Glicocálix/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/microbiologia , Camundongos , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
OBJECTIVE: Antimicrobial C-type lectin regenerating islet-derived 3 gamma (REG3G) is suppressed in the small intestine during chronic ethanol feeding. Our aim was to determine the mechanism that underlies REG3G suppression during experimental alcoholic liver disease. DESIGN: Interleukin 22 (IL-22) regulates expression of REG3G. Therefore, we investigated the role of IL-22 in mice subjected to chronic-binge ethanol feeding (NIAAA model). RESULTS: In a mouse model of alcoholic liver disease, we found that type 3 innate lymphoid cells produce lower levels of IL-22. Reduced IL-22 production was the result of ethanol-induced dysbiosis and lower intestinal levels of indole-3-acetic acid (IAA), a microbiota-derived ligand of the aryl hydrocarbon receptor (AHR), which regulates expression of IL-22. Importantly, faecal levels of IAA were also found to be lower in patients with alcoholic hepatitis compared with healthy controls. Supplementation to restore intestinal levels of IAA protected mice from ethanol-induced steatohepatitis by inducing intestinal expression of IL-22 and REG3G, which prevented translocation of bacteria to liver. We engineered Lactobacillus reuteri to produce IL-22 (L. reuteri/IL-22) and fed them to mice along with the ethanol diet; these mice had reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain and upregulated expression of REG3G in intestine. However, L. reuteri/IL-22 did not reduce ethanol-induced liver disease in Reg3g-/- mice. CONCLUSION: Ethanol-associated dysbiosis reduces levels of IAA and activation of the AHR to decrease expression of IL-22 in the intestine, leading to reduced expression of REG3G; this results in bacterial translocation to the liver and steatohepatitis. Bacteria engineered to produce IL-22 induce expression of REG3G to reduce ethanol-induced steatohepatitis.
Assuntos
Disbiose , Etanol , Microbioma Gastrointestinal/fisiologia , Interleucinas/imunologia , Intestino Delgado/imunologia , Limosilactobacillus reuteri/imunologia , Hepatopatias Alcoólicas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Disbiose/complicações , Disbiose/etiologia , Disbiose/imunologia , Etanol/efeitos adversos , Etanol/metabolismo , Imunidade Inata , Ácidos Indolacéticos/metabolismo , Inflamação/metabolismo , Hepatopatias Alcoólicas/imunologia , Hepatopatias Alcoólicas/microbiologia , Hepatopatias Alcoólicas/terapia , Camundongos , Camundongos Knockout , Proteínas Associadas a Pancreatite/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Interleucina 22RESUMO
We investigated the migration of intestinal immune cells to the liver and their contribution to alcoholic liver disease. In mice fed ethanol, we found that an increased number of invariant natural killer T (iNKT) cells, which respond to the antigen presented by CD1d, migrated from mesenteric lymph nodes to the liver. iNKT cells react to lipid antigens, so we studied their activities in mice with intestinal epithelial cell-specific deletion of Pparg (PpargΔIEC) as a model for altering intestinal lipidomic profiles. Levels of CD1d increased in intestines of ethanol-fed PpargΔIEC mice, and in cell-tracking experiments, more iNKT cells migrated to the liver, compared with mice without disruption of Pparg. Livers of PpargΔIEC mice had increased markers of apoptosis and liver injury after ethanol feeding. iNKT cells isolated from livers of ethanol-fed PpargΔIEC mice induced apoptosis of cultured hepatocytes. An inhibitor of iNKT cells reduced ethanol-induced liver injury in PpargΔIEC mice. Duodenal tissues from patients with alcohol-use disorder have been found to have increased levels of CD1d compared with tissues from patients without alcohol overuse. Ethanol use, therefore, activates iNKT cells in the intestine to migrate to liver, where they-along with the resident hepatic iNKT cells-contribute to hepatocyte death and injury. NEW & NOTEWORTHY In this article, we studied migration of intestinal immune cells into the liver in response to ethanol-induced liver disease. We found that chronic ethanol feeding induces expression of CD1d by enterocytes, which activate invariant natural killer T (iNKT) cells in mesenteric lymph nodes; activation is further increased with loss of peroxisome proliferator-activated receptor gamma gene and altered lipid profiles. The activated iNKT cells migrate into the liver, where they promote hepatocyte apoptosis. Patients with alcohol use disorder have increased expression of CD1d in the small intestine. Strategies to block these processes might be developed to treat alcoholic liver disease.
Assuntos
Enterócitos , Etanol/farmacologia , Hepatócitos , Hepatopatias Alcoólicas , Células T Matadoras Naturais , Animais , Antígenos CD1d/metabolismo , Apoptose , Ensaios de Migração de Leucócitos/métodos , Movimento Celular , Depressores do Sistema Nervoso Central/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/imunologia , Enterócitos/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Ativação Linfocitária , Camundongos , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/metabolismoRESUMO
Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol-associated dysbiosis are largely unknown. The aim of this study was to identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over-representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)-15 protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)-7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine-restricted FXR agonist fexaramine, which protected mice from ethanol-induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF15, a nontumorigenic FGF19 variant-a human FGF15 ortholog-was overexpressed in mice using adeno-associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis. CONCLUSION: Taken together, alcohol-associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid-FXR-FGF15 signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol-induced liver disease in mice. (Hepatology 2018;67:2150-2166).
Assuntos
Ácidos e Sais Biliares/fisiologia , Etanol/administração & dosagem , Fatores de Crescimento de Fibroblastos/fisiologia , Microbioma Gastrointestinal/fisiologia , Hepatopatias Alcoólicas/etiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND/GOALS: To date, there is no consensus on optimal cut-off values and timing of transient elastography (TE, Fibroscan) for fibrosis staging and prediction of portal hypertension in alcoholic liver disease. We evaluated the accuracy of Fibroscan for the diagnosis of fibrosis and clinically significant portal hypertension in alcoholic patients. STUDY: Heavy drinkers admitted to our standardized alcohol withdrawal program were evaluated by Fibroscan, by transjugular hepatic venous pressure gradient (HVPG) measurement and liver biopsy if significant fibrosis was suspected and by upper gastrointestinal endoscopy. All investigations were performed within 3 days of admission. Patients who had remained abstinent for 2 weeks underwent a second Fibroscan. RESULTS: A total of 118 patients were included. Fibroscan correlated well with histology and HVPG. Negative predictive value of 92% and 93% for ruling out severe fibrosis (≥F3) and cirrhosis, and optimal cut-offs at ≥11.7, ≥15.2, and ≥21.2 kPa for F2, F3, and F4, respectively, were found. In abstinent patients, a mean decrease of 2.7 kPa improved concordance between Fibroscan and histology. A TE value of 30.6 kPa predicted a HVPG>10 mm Hg with 94% specificity and showed a good negative predictive value of 84% for ruling out the presence of varices at endoscopy. Steatosis, alcoholic hepatitis, sinusoidal fibrosis, cholestasis, and high transaminases did not influence TE values. CONCLUSIONS: Fibroscan is an accurate non-invasive method for the diagnosis of fibrosis in alcoholic patients. TE values below 11 and 30 kPa likely rule out significant fibrosis and varices, respectively.
Assuntos
Alcoolismo , Hipertensão Portal/diagnóstico , Cirrose Hepática/diagnóstico , Adulto , Idoso , Técnicas de Imagem por Elasticidade , Feminino , Humanos , Hipertensão Portal/patologia , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.
Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , Técnicas de Sonda Molecular , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA/métodos , Regiões 5' não Traduzidas , Sequência de Bases , Comércio , Genômica/métodos , Genótipo , Técnicas de Genotipagem/economia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Técnicas de Sonda Molecular/economia , Filogenia , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/economia , Estudos Retrospectivos , Análise de Sequência de RNA/economia , Centros de Atenção TerciáriaRESUMO
BACKGROUND: Alcohol-related liver disease is one of the most prevalent chronic liver diseases worldwide. Mechanisms involved in the pathogenesis of alcohol-related liver disease are not well understood. Oxylipins play a crucial role in numerous biological processes and pathological conditions. Nevertheless, oxylipins are not well studied in alcohol-related liver disease. AIMS: (1) To characterize the patterns of bioactive ω-3 and ω-6 polyunsaturated fatty acid metabolites in alcohol use disorder and alcoholic hepatitis patients and (2) to identify associations of serum oxylipins with clinical parameters in patients with alcohol-related liver disease. METHODS: We performed a comprehensive liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of serum and fecal oxylipins derived from ω-6 arachidonic acid, ω-3 eicosapentaenoic acid, and docosahexaenoic acid in a patient cohort with alcohol-related liver disease. RESULTS: Our results show profound alterations in the serum oxylipin profile of patients with alcohol use disorder and alcoholic hepatitis compared to nonalcoholic controls. Spearman correlation of the oxylipins with clinical parameters shows a link between different serum oxylipins and intestinal permeability, aspartate aminotransferase, bilirubin, albumin, international normalized ratio, platelet count, steatosis, fibrosis and model for end-stage liver disease score. Especially, higher level of serum 20-HETE was significantly associated with decreased albumin, increased hepatic steatosis, polymorphonuclear infiltration, and 90-day mortality. CONCLUSIONS: Patients with alcohol-related liver disease have different oxylipin profiles. Future studies are required to confirm oxylipins as disease biomarker or to connect oxylipins to disease pathogenesis.