Interleukin-11 drives human and mouse alcohol-related liver disease.

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.

PUFA-Induced Metabolic Enteritis as a Fuel for Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) globally emerges with Westernization of lifestyle and nutritional habits. However, a specific dietary constituent that comprehensively evokes gut inflammation in human inflammatory bowel diseases remains elusive. We aimed to delineate how increased intake of polyunsaturated fatty acids (PUFAs) in a Western diet, known to impart risk for developing CD, affects gut inflammation and disease course. We hypothesized that the unfolded protein response and antioxidative activity of glutathione peroxidase 4 (GPX4), which are compromised in human CD epithelium, compensates for metabolic perturbation evoked by dietary PUFAs. METHODS: We phenotyped and mechanistically dissected enteritis evoked by a PUFA-enriched Western diet in 2 mouse models exhibiting endoplasmic reticulum (ER) stress consequent to intestinal epithelial cell (IEC)-specific deletion of X-box binding protein 1 (Xbp1) or Gpx4. We translated the findings to human CD epithelial organoids and correlated PUFA intake, as estimated by a dietary questionnaire or stool metabolomics, with clinical disease course in 2 independent CD cohorts. RESULTS: PUFA excess in a Western diet potently induced ER stress, driving enteritis in Xbp1-/-IEC and Gpx4+/-IEC mice. ω-3 and ω-6 PUFAs activated the epithelial endoplasmic reticulum sensor inositol-requiring enzyme 1α (IRE1α) by toll-like receptor 2 (TLR2) sensing of oxidation-specific epitopes. TLR2-controlled IRE1α activity governed PUFA-induced chemokine production and enteritis. In active human CD, ω-3 and ω-6 PUFAs instigated epithelial chemokine expression, and patients displayed a compatible inflammatory stress signature in the serum. Estimated PUFA intake correlated with clinical and biochemical disease activity in a cohort of 160 CD patients, which was similarly demonstrable in an independent metabolomic stool analysis from 199 CD patients. CONCLUSIONS: We provide evidence for the concept of PUFA-induced metabolic gut inflammation which may worsen the course of human CD. Our findings provide a basis for targeted nutritional therapy.

Adipocyte GPX4 protects against inflammation, hepatic insulin resistance and metabolic dysregulation.

OBJECTIVES: Metabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism. METHODS: We studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4fl/fl mice with Adipoq-Cre+ (Gpx4-/-AT) or Fabp4-Cre+ (Gpx4+/-Fabp4) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80+ macrophages and TUNEL labelling. RESULTS: GPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1ß (IL-1ß), IL-6 and the IL-8 homologue CXCL1. Gpx4-/-AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4-/-AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4+/-Fabp4 mice. Gpx4-/-AT mice exhibited no signs of adipocyte death. CONCLUSION: Adipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future.

Alpha-1 antitrypsin governs alcohol-related liver disease in mice and humans.

OBJECTIVE: Alcohol-related liver disease (ALD) is a global healthcare problem with limited treatment options. Alpha-1 antitrypsin (AAT, encoded by SERPINA1) shows potent anti-inflammatory activities in many preclinical and clinical trials. In our study, we aimed to explore the role of AAT in ALD. DESIGN: An unselected cohort of 512 patients with cirrhosis was clinically characterised. Survival, clinical and biochemical parameters including AAT serum concentration were compared between patients with ALD and other aetiologies of liver disease. The role of AAT was evaluated in experimental ALD models. RESULTS: Cirrhotic ALD patients with AAT serum concentrations less than 120 mg/dL had a significantly higher risk for death/liver transplantation as compared with patients with AAT serum concentrations higher than 120 mg/dL. Multivariate Cox regression analysis showed that low AAT serum concentration was a NaMELD-independent predictor of survival/transplantation. Ethanol-fed wild-type (wt) mice displayed a significant decline in hepatic AAT compared with pair-fed mice. Therefore, hAAT-Tg mice were ethanol-fed, and these mice displayed protection from liver injury associated with decreased steatosis, hepatic neutrophil infiltration and abated expression of proinflammatory cytokines. To test the therapeutic capability of AAT, ethanol-fed wt mice were treated with human AAT. Administration of AAT ameliorated hepatic injury, neutrophil infiltration and steatosis. CONCLUSION: Cirrhotic ALD patients with AAT concentrations less than 120 mg/dL displayed an increased risk for death/liver transplantation. Both hAAT-Tg mice and AAT-treated wt animals showed protection from ethanol-induced liver injury. AAT could reflect a treatment option for human ALD, especially for alcoholic hepatitis.

Gastric banding-associated weight loss diminishes hepatic Tsukushi expression.

Obesity has emerged as a substantial global healthcare issue that is frequently associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD). Tsukushi (TSK), a liver-derived molecule, was recently identified as a major driver of NAFLD. Laparoscopic adjustable gastric banding (LAGB) has proven effective in reducing body weight and improving NAFLD. We therefore aimed to investigate the relation between LAGB-induced weight loss and TSK expression. Twenty-six obese patients undergoing LAGB were included in the study and metabolic parameters were assessed before (t0) and six months after LAGB (t6). The expression of TSK in liver and subcutaneous adipose tissue (AT) specimens was determined at both time points. To unravel regulatory mechanisms of TSK expression, human peripheral blood mononuclear cells (PBMCs) were stimulated with pro-inflammatory cytokines and TSK mRNA levels were analyzed by quantitative polymerase chain reaction. LAGB induced pronounced weight loss which was paralleled by amelioration of metabolic disturbances and histologically defined NAFLD. While hepatic TSK expression was markedly decreased after LAGB, adipose tissue TSK expression remained comparable to baseline. The decline in hepatic TSK expression after LAGB positively correlated with weight loss and the reduction in BMI, and negatively correlated with NAFLD activity score (NAS). In human PBMCs, pro-inflammatory cytokines such as IL-1ß and TNFα induced the expression of TSK. In conclusion, LAGB-induced weight loss reduces hepatic TSK expression. Inhibiting TSK might represent a promising target for treating NAFLD in the future.

Adipose type I interferon signalling protects against metabolic dysfunction.

OBJECTIVE: Low-grade chronic inflammation emerges as a potent driver of insulin resistance and glucose dysregulation in obesity and associated non-alcoholic fatty liver disease (NAFLD). The liver, subcutaneous fat and the immune system participate in disturbances of metabolism. Type I interferon (IFN) signalling initiated by innate and adaptive immunity modulates inflammatory responses consequent to infection. However, little is known about the role of type I IFN signalling in metabolic diseases and the development of NAFLD. DESIGN: We determined the impact of type I IFN signalling by tissue-specific deletion of interferon (α and ß) receptor 1 (Ifnar1) in hepatocytes (Ifnar1Δhep ), adipocytes (Ifnar1Δat ), intestinal epithelial cells (Ifnar1ΔIEC ) or myelocytes (Ifnar1Δmyel ) on glucose metabolism, obesity and hepatic disease in mice exposed to a high-fat or methionine-choline-deficient (MCD) diet. Furthermore, we investigated the expression of type I IFN-regulated genes in patients with obesity undergoing laparoscopic adjustable gastric banding (LAGB). RESULTS: Long chain fatty acids induce type I IFN responses in murine hepatocytes and macrophages and exposure to a high-fat diet elicited type I IFN-regulated gene expression in the liver of wild-type mice. Hepatocyte-specific, but not adipose tissue-specific deletion of Ifnar1 worsened steatosis and inflammation induced by the MCD diet. In contrast, adipose-specific, but not hepatocyte-specific deletion of Ifnar1 deteriorated metabolic dysregulation induced by a high-fat diet, indicated by increased weight gain, insulin resistance and an impaired glucose tolerance. Abrogated type I IFN signalling in myeloid or intestinal epithelial cells did not modulate susceptibility to metabolic or hepatic disease. Improved metabolic control in patients with obesity after LAGB was associated with increased expression of type I IFN-regulated genes in subcutaneous adipose tissue and liver. CONCLUSIONS: Our study implicates a role for adipose and hepatocyte type I IFN signalling in diet-induced metabolic dysregulation and hepatic disease. Further studies on type I IFN signalling in metabolic diseases are warranted.

Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease.

OBJECTIVE: Alcoholic liver disease (ALD) is a global health problem with limited therapeutic options. Intestinal barrier integrity and the microbiota modulate susceptibility to ALD. Akkermansia muciniphila, a Gram-negative intestinal commensal, promotes barrier function partly by enhancing mucus production. The aim of this study was to investigate microbial alterations in ALD and to define the impact of A. muciniphila administration on the course of ALD. DESIGN: The intestinal microbiota was analysed in an unbiased approach by 16S ribosomal DNA (rDNA) sequencing in a Lieber-DeCarli ALD mouse model, and faecal A. muciniphila abundance was determined in a cohort of patients with alcoholic steatohepatitis (ASH). The impact of A. muciniphila on the development of experimental acute and chronic ALD was determined in a preventive and therapeutic setting, and intestinal barrier integrity was analysed. RESULTS: Patients with ASH exhibited a decreased abundance of faecal A. muciniphila when compared with healthy controls that indirectly correlated with hepatic disease severity. Ethanol feeding of wild-type mice resulted in a prominent decline in A. muciniphila abundance. Ethanol-induced intestinal A. muciniphila depletion could be restored by oral A. muciniphila supplementation. Furthermore, A. muciniphila administration when performed in a preventive setting decreased hepatic injury, steatosis and neutrophil infiltration. A. muciniphila also protected against ethanol-induced gut leakiness, enhanced mucus thickness and tight-junction expression. In already established ALD, A. muciniphila used therapeutically ameliorated hepatic injury and neutrophil infiltration. CONCLUSION: Ethanol exposure diminishes intestinal A. muciniphila abundance in both mice and humans and can be recovered in experimental ALD by oral supplementation. A. muciniphila promotes intestinal barrier integrity and ameliorates experimental ALD. Our data suggest that patients with ALD might benefit from A. muciniphila supplementation.

Ethanol-mediated suppression of IL-37 licenses alcoholic liver disease.

BACKGROUND & AIMS: Chronic alcohol consumption and alcoholic liver disease (ALD) afflicts individuals with substantial morbidity and mortality with limited treatment options available. Hepatic inflammation, triggered by activated Kupffer cells, is a driving force in alcoholic liver disease. Interleukin 37 (IL-37) exerts anti-inflammatory effects in hepatic diseases, however, the impact of Interleukin 37 on alcoholic liver disease is unknown. In this study, we addressed the role of Interleukin 37 in alcoholic liver disease. METHODS: We utilized Interleukin 37 expressing transgenic mice and human recombinant Interleukin 37 in models of alcoholic liver disease. Interleukin 37 expression was measured in liver samples of 20 alcoholic steatohepatitis and 36 non-alcoholic fatty liver disease patients. RESULTS: Interleukin 37 transgenic mice are not protected against hepatic injury and inflammation in alcoholic liver disease. Ethanol suppressed Interleukin 37 expression in transgenic mice. Alcoholic steatohepatitis (ASH) patients similarly exhibited reduced Interleukin 37 expression when compared to non-alcoholic fatty liver disease (NAFLD) patients. Human recombinant Interleukin 37 ameliorated hepatic inflammation in a binge drinking model of alcoholic liver disease. CONCLUSION: We provide evidence for an exogenous noxae that suppresses Interleukin 37 expression which limits its anti-inflammatory effects in alcoholic liver disease.

Reversal of murine alcoholic steatohepatitis by pepducin-based functional blockade of interleukin-8 receptors.

OBJECTIVE: Alcoholic steatohepatitis is a life-threatening condition with short-term mortality up to 40%. It features hepatic neutrophil infiltration and blood neutrophilia, and may evolve from ethanol-induced breakdown of the enteric barrier and consequent bacteraemia. Signalling through CXCR1/2 G-protein-coupled-receptors (GPCRs), the interleukin (IL)-8 receptors, is critical for the recruitment and activation of neutrophils. We have developed short lipopeptides (pepducins), which inhibit post-ligand GPCR activation precisely targeting individual GPCRs. DESIGN: Experimental alcoholic liver disease was induced by administering alcohol and a Lieber-DeCarli high-fat diet. CXCR1/2 GPCRs were blocked via pepducins either from onset of the experiment or after disease was fully established. Hepatic inflammatory infiltration, hepatocyte lipid accumulation and overall survival were assessed as primary outcome parameters. Neutrophil activation was assessed by myeloperoxidase activity and liver cell damage by aspartate aminotransferase and alanine aminotransferase plasma levels. Chemotaxis assays were performed to identify chemoattractant signals derived from alcohol-exposed hepatocytes. RESULTS: Here, we show that experimental alcoholic liver disease is driven by CXCR1/2-dependent activation of neutrophils. CXCR1/2-specific pepducins not only protected mice from liver inflammation, weight loss and mortality associated with experimental alcoholic liver disease, but therapeutic administration cured disease and prevented further mortality in fully established disease. Hepatic neutrophil infiltration and triglyceride accumulation was abrogated by CXCR1/2 blockade. Moreover, CXCL-1 plasma levels were decreased with the pepducin therapy as was the transcription of hepatic IL-1ß mRNA. CONCLUSIONS: We propose that high circulating IL-8 in human alcoholic hepatitis may cause pathogenic overzealous neutrophil activation, and therapeutic blockade via pepducins merits clinical study.

Weight loss induced by bariatric surgery restores adipose tissue PNPLA3 expression.

BACKGROUND & AIMS: Obesity and its related co-morbidities such as non-alcoholic fatty liver disease (NAFLD) are increasing dramatically worldwide. The genetic variation in Patatin-like phospholipase domain-containing protein 3 (PNPLA3), which is also called adiponutrin (ADPN), in residue 148 (I148M, rs738409) has been associated with NAFLD. However, the regulation and function of PNPLA3 in metabolic diseases remains unclear. Laparoscopic gastric banding (LAGB) of severely obese patients reduces body weight, liver and adipose tissue inflammation. In this study, we investigated whether weight loss induced by LAGB affected PNPLA3 expression in hepatic and adipose tissue. METHODS: Liver and subcutaneous adipose tissue samples were collected from 28 severely obese patients before and 6 months after LAGB. PNPLA3 expression was assessed by quantitative real-time PCR. To understand whether inflammatory stimuli regulated PNPLA3 expression, we studied the effect of tumour necrosis factor alpha (TNFα) and lipopolysaccharide (LPS) on PNPLA3 expression in human adipocytes and hepatocytes. RESULTS: PNPLA3 was strongly expressed in the liver and clearly detectable in subcutaneous adipose tissue of obese patients. Weight loss induced by LAGB of severely obese patients led to significantly increased adipose, but not hepatic, tissue expression of PNPLA3. Subcutaneous PNPLA3 expression negatively correlated with body-mass-index, fasting glucose and fasting insulin. TNFα potently suppressed PNPLA3 expression in adipocytes but not hepatocytes. CONCLUSIONS: Weight loss induced by LAGB restored adipose tissue PNPLA3 expression which is suppressed by TNFα. Further studies will be required to determine the functional impact of PNPLA3 and its related genetic variation on adipose tissue inflammation and NAFLD.

Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease.

BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is characterised by neutrophil infiltration that contributes to hepatic injury and disease. Lipocalin-2 (LCN2) was originally identified as siderophore binding peptide in neutrophils, which exerted tissue protective effects in several disease models. Here we investigate the role of LCN2 in the pathogenesis of alcohol-induced liver injury. METHODS: We compared hepatic LCN2 expression in ASH patients, alcoholic cirrhosis patients without evidence of ASH and patients with non-alcoholic fatty liver disease (NAFLD; i.e. simple steatosis). To mechanistically dissect LCN2 function in alcohol-induced liver injury, we subjected wild-type (WT) and Lcn2-deficient (Lcn2(-/-)) mice to the Lieber-DeCarli diet containing 5% ethanol (EtOH) or isocaloric maltose. Adoptive transfer experiments were performed to track neutrophil migration. Furthermore, we tested the effect of antibody-mediated LCN2 neutralisation in an acute model of ethanol-induced hepatic injury. RESULTS: Patients with ASH exhibited increased hepatic LCN2 immunoreactivity compared to patients with alcoholic cirrhosis or simple steatosis, which mainly localised to neutrophils. Similarly, ethanol-fed mice exhibited increased LCN2 expression that mainly localised to leukocytes and especially neutrophils. Lcn2(-/-) mice were protected from alcoholic liver disease (ALD) as demonstrated by reduced neutrophil infiltration, liver injury and hepatic steatosis compared to WT controls. Adoptive transfers revealed that neutrophil-derived LCN2 critically determines hepatic neutrophil immigration and persistence during chronic alcohol exposure. Antibody-mediated neutralisation of LCN2 protected from hepatic injury and neutrophilic infiltration after acute alcohol challenge. CONCLUSIONS: LCN2 drives ethanol-induced neutrophilic inflammation and propagates the development of ALD. Despite a critical role for LCN2 in immunity and infection, pharmacological neutralisation of LCN2 might be of promise in ALD.

Adipose tissue and liver expression of SIRT1, 3, and 6 increase after extensive weight loss in morbid obesity.

BACKGROUND & AIMS: Severe obesity is associated with a state of chronic inflammation. Sirtuins (SIRT) are a family of conserved enzymes which are able to affect many metabolic and inflammatory pathways thereby potentially improving health and increasing lifespan. METHODS: We investigated the effect of weight loss on subcutaneous adipose tissue and liver mRNA and immunohistochemical expression of SIRT1, SIRT3, and SIRT6. Twenty-nine severely obese patients undergoing laparoscopic adjustable gastric banding (LAGB) were studied. Tissue samples were collected before and 6months after LAGB surgery. Tissue mRNA expression levels of SIRT1, SIRT3, and SIRT6 were correlated with clinical, biochemical, and histological parameters. In vitro, we studied sirtuin expression in native and stimulated monocytes, adipocytes, and hepatocytes. RESULTS: SIRT1, SIRT3, and SIRT6 mRNA expression was higher in the subcutaneous adipose tissue than in the liver. Weight loss resulted in a significant induction of SIRT1, SIRT3, and SIRT6 expression in the subcutaneous adipose tissue. In the liver, a significant increase after weight loss was observed, particularly for SIRT3 and SIRT6 mRNA expression; immunohistochemically, SIRT1 and SIRT3 expression was upregulated. Endotoxin and tumor necrosis factor-alpha suppressed SIRT1, SIRT3, and SIRT6 expression in human monocytes. The same stimuli suppressed total sirtuin deacetylase activity again, mainly in monocytes and less in adipocytes and hepatocytes. CONCLUSIONS: The relative abundance of adipose tissue mRNA expression of certain sirtuins exceeds its expression in the liver. Extensive weight loss increases sirtuin expression significantly both in adipose tissue and liver, probably as a consequence of reduced inflammation.

Apelin and the gut microbiome: Potential interaction in human MASLD.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease with increasing numbers worldwide. Adipokines like apelin (APLN) can act as key players in the complex pathophysiology of MASLD. AIMS: Investigating the role of APLN in MASLD. METHODS: Fecal and blood samples were collected in a MASLD cohort and healthy controls (HC). MASLD patients with liver fibrosis and MASLD-associated hepatocellular carcinoma (HCC) were included into the study. Systemic concentration of Apelin, Apelin receptor (APLNR) and circulating cytokines were measured in serum samples. RESULTS: Apelin concentration correlated with the Fib-4 score and was elevated in MASLD patients (mild fibrosis, mF (Fib-4 <3.25) and severe fibrosis, sF (Fib-4 >3.25)) as well as in MASLD-associated HCC patients compared to HC. In accordance APLNR and circulating cytokines were also elevated in mF and sF. In contrast apelin levels were negatively associated with liver survival at three and five years. Changes in taxa composition at phylum level showed an increase of Enterobactericae, Prevotellaceae and Lactobacillaceae in patients with sF compared to mF. We could also observe an association between apelin concentrations and bacterial lineages (phyla). CONCLUSIONS: Circulating apelin is associated with liver fibrosis and HCC. In addition, there might exist an interaction between systemic apelin and the gut microbiome.

A gut bacterial signature in blood and liver tissue characterizes cirrhosis and hepatocellular carcinoma.

BACKGROUND: HCC is the leading cause of cancer in chronic liver disease. A growing body of experimental mouse models supports the notion that gut-resident and liver-resident microbes control hepatic immune responses and, thereby, crucially contribute to liver tumorigenesis. However, a comprehensive characterization of the intestinal microbiome in fueling the transition from chronic liver disease to HCC in humans is currently missing. METHODS: Here, we profiled the fecal, blood, and liver tissue microbiome of patients with HCC by 16S rRNA sequencing and compared profiles to nonmalignant cirrhotic and noncirrhotic NAFLD patients. RESULTS: We report a distinct bacterial profile, defined from 16S rRNA gene sequences, with reduced α-and ß-diversity in the feces of patients with HCC and cirrhosis compared to NAFLD. Patients with HCC and cirrhosis exhibited an increased proportion of fecal bacterial gene signatures in the blood and liver compared to NAFLD. Differential analysis of the relative abundance of bacterial genera identified an increased abundance of Ruminococcaceae and Bacteroidaceae in blood and liver tissue from both HCC and cirrhosis patients compared to NAFLD. Fecal samples from cirrhosis and HCC patients both showed a reduced abundance for several taxa, including short-chain fatty acid-producing genera, such as Blautia and Agathobacter. Using paired 16S rRNA and transcriptome sequencing, we identified a direct association between gut bacterial genus abundance and host transcriptome response within the liver tissue. CONCLUSIONS: Our study indicates perturbations of the intestinal and liver-resident microbiome as a critical determinant of patients with cirrhosis and HCC.

Hepatic Meteorin-like and Krüppel-like Factor 3 are Associated with Weight Loss and Liver Injury.

OBJECTIVE: Laparoscopic adjustable gastric banding (LAGB) was found to be effective in reducing body weight and improving insulin resistance in patients with obesity and non-alcoholic fatty liver disease (NAFLD). The adipokine/myokine meteorin-like (METNRL) is an important regulator of whole-body energy expenditure. Krüppel-like factor 3 (KLF3), a regulator of METRNL expression in eosinophils, inhibits the beiging of adipose tissue in mice and therefore regulates adipose tissue development. METHODS: Thirty-three obese patients undergoing LAGB were included in the study. The hepatic and adipose tissue expression of METNRL and KLF3 was determined before (t0) and 6 months after (t6) LABG. The human liver cancer cell line (HepG2) was stimulated with cytokines and fatty acids and METNRL and KLF3 expressions were analyzed. RESULTS: LAGB-associated weight loss was correlated with decreased hepatic METNRL expression. The expression of METNRL and KLF3 in hepatic-and adipose tissues correlated before and after LAGB. Individuals with augmented LAGB-induced weight loss (>20 kg) showed lower hepatic METNRL and KLF3 expression before and after LAGB than patients with <20 kg weight loss. METNRL and KLF3 levels were higher in patients with higher NAFLD activity scores. HepG2 stimulation with interleukin-1ß, tumor necrosis factor-α, palmitic acid but not interleukin-6, oleic acid, or lipopolysaccharide, induced the expression of one or both investigated adipokines. CONCLUSIONS: The novel description of METRNL and KLF3 as hepatokines could pave the way to target their production and/or signaling in obesity, NAFLD, and related disorders. Both proteins may act as possible biomarkers to estimate weight loss after bariatric surgery.

Recovery of Bacteroides thetaiotaomicron ameliorates hepatic steatosis in experimental alcohol-related liver disease.

Alcohol-related liver disease (ALD) is a major cause of liver disease and represents a global burden, as treatment options are scarce. Whereas 90% of ethanol abusers develop alcoholic fatty liver disease (AFLD), only a minority evolves to steatohepatitis and cirrhosis. Alcohol increases lipogenesis and suppresses lipid-oxidation implying steatosis, although the key role of intestinal barrier integrity and microbiota in ALD has recently emerged. Bacteroides thetaiotaomicron (Bt) is a prominent member of human and murine intestinal microbiota, and plays important functions in metabolism, gut immunity, and mucosal barrier. We aimed to investigate the role of Bt in the genesis of ethanol-induced liver steatosis. Bt DNA was measured in feces of wild-type mice receiving a Lieber-DeCarli diet supplemented with an increase in alcohol concentration. In a second step, ethanol-fed mice were orally treated with living Bt, followed by analysis of intestinal homeostasis and histological and biochemical alterations in the liver. Alcohol feeding reduced Bt abundance, which was preserved by Bt oral supplementation. Bt-treated mice displayed lower hepatic steatosis and triglyceride content. Bt restored mucosal barrier and reduced LPS translocation by enhancing mucus thickness and production of Mucin2. Furthermore, Bt up-regulated Glucagon-like peptide-1 (GLP-1) expression and restored ethanol-induced Fibroblast growth factor 15 (FGF15) down-regulation. Lipid metabolism was consequently affected as Bt administration reduced fatty acid synthesis (FA) and improved FA oxidation and lipid exportation. Moreover, treatment with Bt preserved the mitochondrial fitness and redox state in alcohol-fed mice. In conclusion, recovery of ethanol-induced Bt depletion by oral supplementation was associated with restored intestinal homeostasis and ameliorated experimental ALD. Bt could serve as a novel probiotic to treat ALD in the future.

Adipose and liver expression of interleukin (IL)-1 family members in morbid obesity and effects of weight loss.

Morbid obesity is associated with a state of chronic inflammation. Interleukin-1 family (IL-1F) cytokine members are produced by human adipose tissue in obesity. Whereas certain IL-1F members such as IL-1ß or IL-18 are potently proinflammatory, others such as IL-1 receptor antagonist (IL-1Ra) or IL-37 (formerly IL-1F7) are antiinflammatory. The NLRP3 inflammasome plays a key role in the processing of bioactive IL-1ß and IL-18. We investigated the effect of excessive weight loss on subcutaneous adipose tissue and liver expression of IL-1α, IL-1ß, IL-18, IL-1Ra, IL-37 and NLRP3. Twenty-one severely obese patients undergoing laparoscopic adjustable gastric banding were studied. Tissue samples were collected before and 6 months after laparoscopic adjustable gastric banding surgery. mRNA expression of all studied IL-1F members, but especially of IL-37, was much higher in subcutaneous/visceral adipose tissue compared with their liver expression. Subcutaneous adipose tissue mRNA expression of IL-1ß decreased significantly after extensive weight loss; expression of IL-18 and IL-1Ra did not change, whereas IL-37 expression increased. Weight loss led to a significant reduction in liver IL-1ß, IL-18 and IL-1Ra expression, whereas hepatic IL-37 mRNA expression remained stable. Adipose/liver NLRP3 inflammasome and IL-1α expression were not affected by weight loss. Tissue expression of IL-1ß, IL-18 and IL-37 were significantly higher in subcutaneous/visceral adipose tissue compared with the liver. In conclusion, expression of IL-1F members is more pronounced in adipose compared with liver tissue in patients with severe obesity. Excessive weight loss changes the adipose and liver expression profile of IL-1F members toward a more antiinflammatory direction.

Increased Fecal Neopterin Parallels Gastrointestinal Symptoms in COVID-19.

INTRODUCTION: Coronavirus disease (COVID-19) has spread from Wuhan, China, and become a worldwide pandemic. Most patients display respiratory symptoms but up to 50% report gastrointestinal symptoms. Neopterin is a surrogate marker for viral inflammation, and its production by macrophages is driven by interferon-γ. METHODS: We measured fecal neopterin in 37 hospitalized COVID-19 patients not requiring intensive care measures and 22 healthy controls. RESULTS: Fecal neopterin was elevated in stool samples from COVID-19 patients compared with that in samples from healthy controls. Especially, patients reporting gastrointestinal symptoms exhibited increased fecal neopterin values. DISCUSSION: COVID-19 is associated with an inflammatory immune response in the gastrointestinal tract.

Role for hedgehog pathway in regulating growth and function of invariant NKT cells.

Lymphocyte accumulation is characteristic of chronic hepatitis, but the mechanisms regulating lymphocyte numbers and their roles in liver disease progression are poorly understood. The Hedgehog (Hh) pathway regulates thymic development and lymphopoeisis during embryogenesis, and is activated in fibrosing liver disease in adults. Our objective was to determine if Hh ligands regulate the viability and phenotype of NKT cells, which comprise a substantial sub-population of resident lymphocytes in healthy adult livers and often accumulate during liver fibrosis. The results demonstrate that a mouse invariant NKT cell line (DN32 iNKT cells), mouse primary liver iNKT cells, and human peripheral blood iNKT cells are all responsive to sonic hedgehog (Shh). In cultured iNKT cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the pro-fibrogenic cytokine, IL-13. Livers of transgenic mice with an overly active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that iNKT cells produce and respond to Hh ligands, and that Hh pathway activation regulates the size and cytokine production of liver iNKT cell populations. Therefore, Hh pathway activation may contribute to the local expansion of pro-fibrogenic iNKT cell populations during certain types of fibrosing liver damage.

The role of gut vascular barrier in experimental alcoholic liver disease and A. muciniphila supplementation.

The translocation of bacterial components from the intestinal lumen into the portal circulation is crucial in the pathogenesis of alcoholic liver disease (ALD). Recently the important role of the gut vascular barrier (GVB) was elucidated in alcoholic liver disease. Here we report about the influence of A. muciniphila supplementation in experimental ALD on the GVB. Ethanol feeding was associated with increased Pv-1, indicating altered endothelial barrier function, whereas A. muciniphila administration tended to restore GVB. To further investigate GVB in experimental ALD, ß-catenin gain-of-function mice, which display an enhanced GVB, were ethanol-fed. ß-catenin gain-of-function mice were not protected from ethanol-induced liver injury, suggest an alternative mechanism of ethanol-induced GVB disruption. The description of the GVB in ALD could pave the way for new therapeutic options in the future.