A. Cappon

MERTK rs4374383 polymorphism affects the severity of fibrosis in non-alcoholic fatty liver disease

BACKGROUND & AIMnHomozygosity for a common non-coding rs4374383 G>A polymorphism in MERTK (myeloid-epithelial-reproductive tyrosine kinase) has been associated with the protection against fibrosis progression in chronic hepatitis C. The main study objective was to assess whether MERTK AA genotype influences liver fibrosis, and secondarily MERTK expression in patients with non-alcoholic fatty liver disease (NAFLD). We also investigated whether MERTK is expressed in human hepatic stellate cells (HSC) and in murine models of fibrogenesis.nnnMETHODSnWe considered 533 consecutive patients who underwent liver biopsy for suspected non-alcoholic steatohepatitis (NASH) without severe obesity from two Italian cohorts. As controls, we evaluated 158 patients with normal liver enzymes and without metabolic disturbances. MERTK rs4374383 genotype was assessed by 5-nuclease assays. MERTK expression was analysed in mouse models of fibrosis, and the effect of the MERTK ligand GAS6 were investigated in human HSC.nnnRESULTSnClinically significant fibrosis (stage F2-F4) was observed in 19% of patients with MERTK AA compared to 30% in those with MERTK GG/GA (OR 0.43, CI 0.21-0.88, p=0.02; adjusted for centre, and genetic, clinical-metabolic and histological variables). The protective rs4374383 AA genotype was associated with lower MERTK hepatic expression. MERTK was overexpressed in the liver of NAFLD patients with F2-F4 fibrosis and in in vivo models of fibrogenesis. Furthermore, exposure of cultured human HSC to the MERTK ligand GAS6, increased cell migration and induced procollagen expression. These effects were counteracted by inhibition of MERTK activity, which also resulted in apoptotic death of HSC.nnnCONCLUSIONSnThe rs4374383 AA genotype, associated with lower intrahepatic expression of MERTK, is protective against F2-F4 fibrosis in patients with NAFLD. The mechanism may involve modulation of HSC activation.

HIV‐1 gp120 signaling through TLR4 modulates innate immune activation in human macrophages and the biology of hepatic stellate cells

Highly active antiretroviral therapy has significantly improved the prognosis of HIV‐infected subjects. However, patients treated long term still manifest increased mortality and, even with undetectable plasma viremia, often experience persistent immune activation. Furthermore, liver‐related mortality is now the most common cause of non‐AIDS‐related death in HIV‐infected individuals on highly active antiretroviral therapy through accelerated fibrosis progression. TLRs are the first line of the host response to pathogens and play an important role in human host defense against viruses through sensing of viral structural proteins. Growing evidence points to TLR4 as a key player in chronic immune activation, HIV recognition/replication, and liver fibrosis progression, suggesting that HIV triggering of TLR4 may dictate some aspects of the multifaceted AIDS pathogenesis. In this study, we provide evidence for an interplay between host TLR4 and HIV‐1 gp120 in human monocyte‐derived macrophages and hepatic stellate cells, leading to intracellular pathways and biologic activities that mediate proinflammatory and profibrogenic signals. Finally, we hypothesize that CCR5 and TLR4 are likely part of a common receptor cluster, as the blocking of CCR5 by specific antagonists impairs the macrophage capacity to produce chemokines in response to LPS. Chronic immune activation and liver fibrosis remain important obstacles for highly active antiretroviral therapy success. Thus, the identification of gp120‐TLR4 axis as a novel determinant of immune system and hepatic stellate cell biology opens new perspectives to the management of HIV infection and disease.

NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1β (interleukin 1β). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. Inxa0vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1β was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway inxa0vivo and inxa0vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.

Differential timing of oxidative DNA damage and telomere shortening in hepatitis C and B virus-related liver carcinogenesis.

In viral hepatitis, inflammation is correlated with chronic oxidative stress, one of the biological events leading to DNA damage and hepatocellular carcinoma (HCC) development. Aim of this study was to investigate the complex molecular network linking oxidative damage to telomere length and telomerase activity and regulation in hepatitis C and B virus-related liver carcinogenesis. We investigated 142 patients: 21 with HCC (in both tumor and peritumor tissues) and 121 with chronic viral hepatitis in different stages. We evaluated 8-hydroxydeoxyguanosine (8-OHdG), marker of oxidative DNA damage, OGG1 gene polymorphism, telomere length, telomerase activity, TERT promoter methylation, and mitochondrial TERT localization. In hepatitis C-related damage, 8-OHdG levels increased since the early disease stages, whereas hepatitis B-related liver disease was characterized by a later and sharper 8-OHdG accumulation (Pxa0=xa00.005). In C virus-infected patients, telomeres were shorter (Pxa0=xa00.03), whereas telomerase activity was higher in tumors than that in the less advanced stages of disease in both groups (Pxa0=xa00.0001, Pxa0=xa00.05), with an earlier increase in hepatitis C. Similarly, TERT promoter methylation was higher in tumor and peritumor tissues in both groups (Pxa0=xa00.02, Pxa0=xa00.0001). Finally, TERT was localized in mitochondria in tumor and peritumor samples, with 8-OHdG levels significantly lower in mitochondrial than those in genomic DNA (Pxa0=xa00.0003). These data describe a pathway in which oxidative DNA damage accumulates in correspondence with telomere shortening, telomerase activation, and TERT promoter methylation with a different time course in hepatitis B and C virus-related liver carcinogenesis. Finally, TERT localizes in mitochondria in HCC, where it lacks a canonical function.

P291 INHIBITION OF INDOLEAMINE 2,3-DIOXYGENASE AMELIORATES INFLAMMATION AND FIBROSIS IN EXPERIMENTAL STEATOHEPATITIS

s / Digestive and Liver Disease 46 (2014) e18–e42 e29 Our data provide new insights in p53 involvement in NAFLD pathogenesis, highlighting its role in the modulation of copper homeostasis. http://dx.doi.org/10.1016/j.dld.2014.01.066

1107 THE INFLAMMASOME COMPLEX IS A TARGET OF THE HIV ENVELOPE PROTEIN gp120 IN HUMAN HEPATIC STELLATE CELLS (HSC) AND MONONUCLEAR CELLS (MC)

s of the 46th A.I.S.F. Annual Meeting 2013 / Digestive and Liver Disease 45S (2013), S1–S48 S39 Aim: To investigate LS on TE in a population-based cohort of adults without any evidence of liver disease. Materials and methods: LS was performed in a cohort of 780 citizens, aged between 30–60 years, resident in Bagnacavallo (Italy) attending to a free medical check-up. Results: 689/780 subjects (88.3%), mean age 48.6±8.6, 328 male (47.6%), were considered for analysis; 91 were excluded because of missing data (n=21) or LSM failure (n=70). Subjects with LSM failure differed from the analysed group for age (51.3±8.7 vs. 48.6±8.6, p=0.012), body mass index (BMI) (29.4±5.6 vs. 25.1±3.8, p<0.001) and waist circumference (109.9±15.6 vs. 99.1±10.5, p<0.001). Mean LS value in the analysed group was 5.1±2.7 (range 3.1–8.3), higher in males respect to females (5.3±2.5 vs. 5±2.9, p=0.05) and in abnormal vs. normal BMI (5.5±2.5 vs. 4.7±2.8, p<0.001). After excluding subjects with virological positive test, alcohol consumption, MS, steatosis on ultrasonography and abnormal transaminases, 324 (47%) were identified and considered as healthy. Mean LS in this group was 4.6±1.3 (range 2.8–6.9). There was no difference in LS between males and females (4.7±1.4 vs. 4.6±1.3, p=0.63) and between BMI<25 and ≥25 (4.6±1.2 vs. 4.7±1.6, p=0.83). Conclusions: This study identifies the normal values of LS in the adult healthy population. In this setting, adequately characterized for the absence of any cause of chronic liver injury as well as of MS, the LS values are not influenced by sex and BMI.

1305 BERBERINE AMELIORATES HEPATIC INJURY IN MICE ACTING ON THE NALP3 INFLAMMASOME PATHWAY

4. Western blotting using anti-NFuB antibody for inflammatory signaling, anti-Acyl-CoA antibody to estimate the lipid metabolism, and anti-elF2, -Ire1 and -ATF6 antibodies to analyse the endoplasmic reticulum (ER) stress. Results: 1. Ratio of liver weight to body weight and blood ALT level were extremely decreased in MCDD with 0.1% WBPs treated group (Gr.3). 2. Blood albumin, total cholesterol and triglyceride levels increased gradually in proportion from MCDD with 0.2% WBPs treated group (Gr.2) to MCDD with 0.05% WBPs treated group (Gr.4). 3. Degree of steatosis, inflammation, ballooning of hepatocytes, Mallory-Denk hyaline bodies, and fibrosis improved in a WBPsdose dependent manner, and were significantly decreased in Gr.2 compared with MCDD with normal feed treated group (Gr.1). 4. In Western blotting, the expression of NFuB in liver tissues decreased, but that of Acyl-CoA, PERK, elF2 and ATF6 increased in proportion from Gr.1 to Gr.4. Conclusions: WBPs suppress ER stress, inflammation and enhance the suppressive effects of other pathogenetic factors in NASH. These peptides, which can be obtained easily, may be a useful therapy in patients with NASH.