Benedek Gyongyosi

Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice

BACKGROUND & AIMS The inflammasome is a well-characterized inducer of inflammation in alcoholic steatohepatitis (ASH). Inflammasome activation requires two signals for mature interleukin (IL)-1β production. Here we asked whether metabolic danger signals trigger inflammasome activation in ASH. METHODS Wild-type mice, ATP receptor 2x7 (P2rx7)-KO mice, or mice overexpressing uricase were fed Lieber-DeCarli ethanol or control diet. We also implemented a pharmacological approach in which mice were treated with probenecid or allopurinol. RESULTS The sterile danger signals, ATP and uric acid, were increased in the serum and liver of alcohol-fed mice. Depletion of uric acid or ATP, or lack of ATP signaling attenuated ASH and prevented inflammasome activation and its major downstream cytokine, IL-1β. Pharmacological depletion of uric acid with allopurinol provided significant protection from alcohol-induced inflammatory response, steatosis and liver damage, and additional protection was achieved in mice treated with probenecid, which depletes uric acid and blocks ATP-induced P2rx7 signaling. We found that alcohol-damaged hepatocytes released uric acid and ATP in vivo and in vitro and that these sterile danger signals activated the inflammasome in LPS-exposed liver mononuclear cells. CONCLUSIONS Our data indicate that the second signal in inflammasome activation and IL-1β production in ASH results from the endogenous danger signals, uric acid and ATP. Inhibition of signaling triggered by uric acid and ATP may have therapeutic implications in ASH.

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.

Alcohol-related changes in the intestinal microbiome influence neutrophil infiltration, inflammation and steatosis in early alcoholic hepatitis in mice

Background Alcohol-induced intestinal dysbiosis disrupts homeostatic gut-liver axis function and is essential in the development of alcoholic liver disease. Here, we investigate changes in enteric microbiome composition in a model of early alcoholic steatohepatitis and dissect the pathogenic role of intestinal microbes in alcohol-induced liver pathology. Materials and methods Wild type mice received a 10-day diet that was either 5% alcohol-containing or an isocaloric control diet plus a single binge. 16S rDNA sequencing defined the bacterial communities in the cecum of alcohol- and pair-fed animals. Some mice were treated with an antibiotic cocktail prior to and throughout alcohol feeding. Liver neutrophils, cytokines and steatosis were evaluated. Results Acute-on-chronic alcohol administration induced shifts in various bacterial phyla in the cecum, including increased Actinobacteria and a reduction in Verrucomicrobia driven entirely by a reduction in the genus Akkermansia. Antibiotic treatment reduced the gut bacterial load and circulating bacterial wall component lipopolysaccharide (LPS). We found that bacterial load suppression prevented alcohol-related increases in the number of myeloperoxidase- (MPO) positive infiltrating neutrophils in the liver. Expression of liver mRNA tumor necrosis factor alpha (Tnfα), C-X-C motif chemokine ligand 1 (Cxcl1) and circulating protein monocyte chemoattractant protein-1 (MCP-1) were also reduced in antibiotic-treated alcohol-fed mice. Alcohol-induced hepatic steatosis measured by Oil-Red O staining was significantly reduced in antibiotic treated mice. Genes regulating lipid production and storage were also altered by alcohol and antibiotic treatment. Interestingly, antibiotic treatment did not protect from alcohol-induced increases in serum aminotransferases (ALT/AST). Conclusions Our data indicate that acute-on-chronic alcohol feeding alters the microflora at multiple taxonomic levels and identifies loss of Akkermansia as an early marker of alcohol-induced gut dysbiosis. We conclude that gut microbes influence liver inflammation, neutrophil infiltration and liver steatosis following alcohol consumption and these data further emphasize the role of the gut-liver axis in early alcoholic liver disease.

Endoplasmic reticulum stress-induced hepatocellular death pathways mediate liver injury and fibrosis via Stimulator of Interferon Genes.

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-β (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4. Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4. In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease

The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol‐induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal‐regulated kinase 1 and 2‐mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol‐induced hepatic steatosis and interferon regulatory factor 3‐mediated apoptosis. Conclusion: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell‐driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis. (Hepatology 2016;64:1057‐1071)

Hepatocellular carcinoma is accelerated by NASH involving M2 macrophage polarization mediated by hif-1αinduced IL-10

ABSTRACT Obesity-related inflammation promotes cancer development. Tissue resident macrophages affect tumor progression and the tumor micro-environment favors polarization into alternatively activated macrophages (M2) that facilitate tumor invasiveness. Here, we dissected the role of western diet-induced NASH in inducing macrophage polarization in a carcinogen initiated model of hepatocellular carcinoma (HCC). Adult C57BL/6 male mice received diethyl nitrosamine (DEN) followed by 24 weeks of high fat–high cholesterol–high sugar diet (HF-HC-HSD). We assessed liver MRI and histology, serum ALT, AFP, liver triglycerides, and cytokines. Macrophage polarization was determined by IL-12/TNFα (M1) and CD163/CD206 (M2) expression using flow cytometry. Role of hif-1α-induced IL-10 was dissected in hepatocyte specific hif-1αKO and hif-1αdPA (over-expression) mice. The western diet-induced features of NASH and accelerated HCC development after carcinogen exposure. Liver fibrosis and serum AFP were significantly increased in DEN + HF–HC–HSD mice compared to controls. Western diet resulted in macrophage (F4/80+CD11b+) infiltration to liver and DEN + HF–HC–HSD mice showed preferential increase in M2 macrophages. Isolated hepatocytes from western diet fed mice showed significant upregulation of the hypoxia-inducible transcription factor, hif-1α, and livers from hif-1α over-expressing mice had increased proportion of M2 macrophages. Primary hepatocytes from wild-type mice treated with DEN and palmitic acid in vitro showed activation of hif-1α and induction of IL-10, a M2 polarizing cytokine. IL-10 neutralization in hepatocyte-derived culture supernatant prevented M2 macrophage polarization and silencing hif-1α in macrophages blocked their M2 polarization. Therefore, our data demonstrate that NASH accelerates HCC progression via upregulation of hif-1α mediated IL-10 polarizing M2 macrophages.

Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease

AIM To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD). METHODS Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA). RESULTS Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice. CONCLUSION We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC.

Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use

BACKGROUND & AIMS Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Herein, we studied NET formation and the process of neutrophil cell death (NETosis), as well as the clearance of NETs by macrophages (MΦ) (efferocytosis) in acute sepsis following binge drinking. METHODS Healthy volunteers consumed 2 ml of vodka/kg body weight, before blood endotoxin and 16 s rDNA were measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and intraperitoneal lipopolysaccharide (LPS) to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion. RESULTS Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation, but upon PMA stimulation alcohol attenuated NET formation. Binge alcohol in mice resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice receiving binge alcohol, suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETotic neutrophils and promoted expression of CD206 on MΦ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice. CONCLUSIONS Dysfunctional NETosis and efferocytosis following binge drinking exacerbate liver injury associated with sepsis. LAY SUMMARY Disease severity in alcoholic liver disease (ALD) is associated with a significant presence of neutrophils (a type of immune cell) in the liver. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection, resulting in increased liver injury during ALD.

Pharmacological inhibition of CCR2/5 signaling prevents and reverses alcohol‐induced liver damage, steatosis and inflammation in mice

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C‐C chemokine receptor type 2 (Ccr2) and C‐C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C‐C chemokine ligand types 2 (CCL2), and C‐C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as “prevention” throughout the alcohol feeding or as “treatment” started after the development of ALD. Alcohol‐induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen‐1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol‐related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi) and reduced proinflammatory Ly6Chi MØ in livers of alcohol‐fed mice. CVC increased liver T‐cell numbers and attenuated Il‐2 expression without an effect on CD69+ or CD25+ T‐cell expression. In vitro, CVC inhibited CCL2‐induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation‐related protein (Adrp), whereas it augmented acyl‐coenzyme A oxidase 1 (Acox‐1), proliferator‐activated receptor gamma co‐activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide‐induced liver injury (TNF‐α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP‐ribose polymerase [PARP] and caspase‐3 [CASP‐3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol‐induced steatohepatitis and liver damage.