Matrix stiffness regulate apoptotic cell death in HIV-HCV co-infected hepatocytes: Importance for liver fibrosis progression.

HIV-HCV co-infection causes rapid progression of liver fibrosis. These outcomes to liver cirrhosis can be improved, but not stopped by specific antiviral therapies. Due to high significance of HIV-HCV interactions for morbidity and mortality in co-infected patients, our attention was attracted to the multi-component pathogenesis of fibrosis progression as the transition to end-stage liver disease development. In this study, we hypothesize that increased matrix stiffness enhances apoptosis in HCV-HIV-co-infected hepatocytes and that capturing of apoptotic bodies (AB) derived from these infected hepatocytes by hepatic stellate cells (HSC) drives the fibrosis progression. As the source of viruses, JFH1 (HCV genotype 2a) and HIV-1ADA (either purified or containing in infected macrophage supernatants) were chosen. Using Huh7.5-CYP (RLW) cells and primary human hepatocytes mono-infected with HCV and HIV or co-infected, we have shown that both HCV and HIV RNA levels were increased in co-infected cells, which was accompanied by hepatocyte apoptosis. This apoptosis was attenuated by azidothymidine treatment. The levels of both infections and apoptosis were more prominent in primary hepatocytes cultured on substrates mimicking fibrotic stiffness (24 kPa-stiff) compared to substrates mimicking healthy liver (2.4 kPa-soft). The engulfment of AB from pathogen-exposed hepatocytes activated pro-fibrotic mRNAs in HSC. Overall, the increased matrix stiffness is not only a consequence of liver inflammation/fibrosis, but the condition that further accelerates liver fibrosis development. This is attributed to the switching of HSC to pro-fibrotic phenotype by capturing of excessive amounts of apoptotic HCV- and HIV-infected hepatocytes.

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1ß, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

Acetaldehyde accelerates HCV-induced impairment of innate immunity by suppressing methylation reactions in liver cells.

Alcohol exposure worsens the course and outcomes of hepatitis C virus (HCV) infection. Activation of protective antiviral genes is induced by IFN-α signaling, which is altered in liver cells by either HCV or ethanol exposure. However, the mechanisms of the combined effects of HCV and ethanol metabolism in IFN-α signaling modulation are not well elucidated. Here, we explored a possibility that ethanol metabolism potentiates HCV-mediated dysregulation of IFN-α signaling in liver cells via impairment of methylation reactions. HCV-infected Huh7.5 CYP2E1(+) cells and human hepatocytes were exposed to acetaldehyde (Ach)-generating system (AGS) and stimulated with IFN-α to activate IFN-sensitive genes (ISG) via the Jak-STAT-1 pathway. We observed significant suppression of signaling events by Ach. Ach exposure decreased STAT-1 methylation via activation of protein phosphatase 2A and increased the protein inhibitor of activated STAT-1 (PIAS-1)-STAT-1 complex formation in both HCV(+) and HCV(-) cells, preventing ISG activation. Treatment with a promethylating agent, betaine, attenuated all examined Ach-induced defects. Ethanol metabolism-induced changes in ISGs are methylation related and confirmed by in vivo studies on HCV(+) transgenic mice. HCV- and Ach-induced impairment of IFN signaling temporarily increased HCV RNA levels followed by apoptosis of heavily infected cells. We concluded that Ach potentiates the suppressive effects of HCV on activation of ISGs attributable to methylation-dependent dysregulation of IFN-α signaling. A temporary increase in HCV RNA sensitizes the liver cells to Ach-induced apoptosis. Betaine reverses the inhibitory effects of Ach on IFN signaling and thus can be used for treatment of HCV(+) alcohol-abusing patients.

FAT10 suppression stabilizes oxidized proteins in liver cells: Effects of HCV and ethanol.

FAT10 belongs to the ubiquitin-like modifier (ULM) family that targets proteins for degradation and is recognized by 26S proteasome. FAT10 is presented on immune cells and under the inflammatory conditions, is synergistically induced by IFNγ and TNFα in the non-immune (liver parenchymal) cells. It is not clear how viral proteins and alcohol regulate FAT10 expression on liver cells. In this study, we aimed to investigate whether FAT10 expression on liver cells is activated by the innate immunity factor, IFNα and how HCV protein expression in hepatocytes and ethanol-induced oxidative stress affect the level of FAT10 in liver cells. For this study, we used HCV(+) transgenic mice that express structural HCV proteins and their HCV(-) littermates. Mice were fed Lieber De Carli diet (control and ethanol) as specified in the NIH protocol for chronic-acute ethanol feeding. Alcohol exposure enhanced steatosis, induced oxidative stress and decreased proteasome activity in the liversof these mice, with more robust response to ethanol in HCV(+) mice. IFNα induced transcriptional activation of FAT10 in liver cells, which was dysregulated by ethanol feeding. Accordingly, IFNα-activated expression of FAT10 in hepatocytes (measured by indirect immunofluorescent of liver tissue) was also suppressed by ethanol exposure in both HCV(+) and HCV(-) mice. This suppression was accompanied with ethanol-mediated induction of lipid peroxidation marker, 4-HNE. All aforementioned effects of ethanol were attenuated by in vivo feeding of mice with the pro-methylating agent, betaine, which exhibits strong anti-oxidant properties. Based on this study, we hypothesize that FAT10 targets oxidatively modified proteins for proteasomal degradation, and that the reduction in FAT10 levels along with decreased proteasome activity may contribute to stabilization of these altered proteins in hepatocytes. In conclusion, IFNα induced FAT10 expression, which is suppressed by ethanol feeding in both HCV(+) and HCV(-) mice. Betaine treatment reverses HCV-ethanol induced dysregulation of protein methylation and oxidative stress, thereby restoring the FAT10 expression on liver cells.

Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism.

BACKGROUND & AIMS: Alcohol-induced progression of hepatitis C virus (HCV) infection is related to dysfunction of innate immunity in hepatocytes. Endogenously produced interferon (IFN)α induces activation of interferon-stimulated genes (ISGs) via triggering of the Janus kinase-signal transducer and activator of transcription 1 (STAT1) pathway. This activation requires protein methyltransferase 1-regulated arginine methylation of STAT1. Here, we aimed to study whether STAT1 methylation also depended on the levels of demethylase jumonji domain-containing 6 protein (JMJD6) and whether ethanol and HCV affect JMJD6 expression in hepatocytes. METHODS: Huh7.5-CYP (RLW) cells and hepatocytes were exposed to acetaldehyde-generating system (AGS) and 50 mmol/L ethanol, respectively. JMJD6 messenger RNA and protein expression were measured by real-time polymerase chain reaction and Western blot. IFNα-activated cells either overexpressing JMJD6 or with knocked-down JMJD6 expression were tested for STAT1 methylation, ISG activation, and HCV RNA. In vivo studies have been performed on C57Bl/6 mice (expressing HCV structural proteins or not) or chimeric mice with humanized livers fed control or ethanol diets. RESULTS: AGS exposure to cells up-regulated JMJD6 expression in RLW cells. These results were corroborated by ethanol treatment of primary hepatocytes. The promethylating agent betaine reversed the effects of AGS/ethanol. Similar results were obtained in vivo, when mice were fed control/ethanol with and without betaine supplementation. Overexpression of JMJD6 suppressed STAT1 methylation, IFNα-induced ISG activation, and increased HCV-RNA levels. In contrast, JMJD6 silencing enhanced STAT1 methylation, ISG stimulation by IFNα, and attenuated HCV-RNA expression in Huh7.5 cells. CONCLUSIONS: We conclude that arginine methylation of STAT1 is suppressed by JMJD6. Both HCV and acetaldehyde increase JMJD6 levels, thereby impairing STAT1 methylation and innate immunity protection in hepatocytes exposed to the virus and/or alcohol.