Liver Abnormalities after Elimination of HCV Infection: Persistent Epigenetic and Immunological Perturbations Post-Cure

Chronic hepatitis C (CHC) is a major cause of hepatocellular carcinoma (HCC) worldwide. While directly acting antiviral (DAA) drugs are now able to cure virtually all hepatitis C virus (HCV) infections, even in subjects with advanced liver disease, what happens to the liver and progression of the disease after DAA-induced cure of viremia is only beginning to emerge. Several large-scale clinical studies in different patient populations have shown that patients with advanced liver disease maintain a risk for developing HCC even when the original instigator, the virus, is eliminated by DAAs. Here we review emerging studies derived from multiple, complementary experimental systems involving patient liver tissues, human liver cell cultures, human liver slice cultures, and animal models, showing that HCV infection induces epigenetic, signaling, and gene expression changes in the liver associated with altered hepatic innate immunity and liver cancer risk. Of critical importance is the fact that these virus-induced abnormalities persist after DAA cure of HCV. These nascent findings portend the discovery of pathways involved in post-HCV immunopathogenesis, which may be clinically actionable targets for more comprehensive care of DAA-cured individuals.

Liver immune abnormalities persist after cure of Hepatitis C Virus by antiviral therapy

Direct-Acting Antiviral (DAA) drugs are highly effective in eliminating HCV in most chronically infected subjects, but it is unclear whether the multiple mechanisms employed by HCV to disable both innate and adaptive immunity cease to function as soon as HCV is eliminated. To test this, we evaluated the capacity of human liver tissue to respond to TLR3 and TLR4 ligands using non-infected liver tissue, tissue with active HCV infection, and tissue from which HCV had been eliminated by DAA. We found that DAA-treated, formerly HCV-infected liver tissue manifested ongoing abnormalities of innate immunity that mapped to liver non-parenchymal cells (NPC). Hepatic innate immunity was not suppressed but enhanced in HCV-infected tissue, and these abnormalities were not corrected in the successfully DAA-treated liver tissue. In conclusion, ongoing immune activation persists in formerly HCV-infected but now DAA-cured liver.

Chaperone-Mediated Autophagy in the Liver: Good or Bad?

Hepatitis C virus (HCV) infection triggers autophagy processes, which help clear out the dysfunctional viral and cellular components that would otherwise inhibit the virus replication. Increased cellular autophagy may kill the infected cell and terminate the infection without proper regulation. The mechanism of autophagy regulation during liver disease progression in HCV infection is unclear. The autophagy research has gained a lot of attention recently since autophagy impairment is associated with the development of hepatocellular carcinoma (HCC). Macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) are three autophagy processes involved in the lysosomal degradation and extracellular release of cytosolic cargoes under excessive stress. Autophagy processes compensate for each other during extreme endoplasmic reticulum (ER) stress to promote host and microbe survival as well as HCC development in the highly stressed microenvironment of the cirrhotic liver. This review describes the molecular details of how excessive cellular stress generated during HCV infection activates CMA to improve cell survival. The pathological implications of stress-related CMA activation resulting in the loss of hepatic innate immunity and tumor suppressors, which are most often observed among cirrhotic patients with HCC, are discussed. The oncogenic cell programming through autophagy regulation initiated by a cytoplasmic virus may facilitate our understanding of HCC mechanisms related to non-viral etiologies and metabolic conditions such as uncontrolled type II diabetes. We propose that a better understanding of how excessive cellular stress leads to cancer through autophagy modulation may allow therapeutic development and early detection of HCC.

Dysregulated liver lipid metabolism and innate immunity associated with hepatic steatosis in neonatal BBdp rats and NOD mice

In a previous study we reported that prediabetic rats have a unique gene signature that was apparent even in neonates. Several of the changes we observed, including enhanced expression of pro-inflammatory genes and dysregulated UPR and metabolism genes were first observed in the liver followed by the pancreas. In the present study we investigated further early changes in hepatic innate immunity and metabolism in two models of type 1 diabetes (T1D), the BBdp rat and NOD mouse. There was a striking increase in lipid deposits in liver, particularly in neonatal BBdp rats, with a less striking but significant increase in neonatal NOD mice in association with dysregulated expression of lipid metabolism genes. This was associated with a decreased number of extramedullary hematopoietic clusters as well as CD68+ macrophages in the liver of both models. In addition, PPARɣ and phosphorylated AMPKα protein were decreased in neonatal BBdp rats. BBdp rats displayed decreased expression of antimicrobial genes in neonates and decreased M2 genes at 30 days. This suggests hepatic steatosis could be a common early feature in development of T1D that impacts metabolic homeostasis and tolerogenic phenotype in the prediabetic liver.

Glycine prevents metabolic steatohepatitis in diabetic KK-Ay mice through modulation of hepatic innate immunity

Strategies for prevention and treatment of nonalcoholic steatohepatitis remain to be established. We evaluated the effect of glycine on metabolic steatohepatitis in genetically obese, diabetic KK-Ay mice. Male KK-Ay mice were fed a diet containing 5% glycine for 4 wk, and liver pathology was evaluated. Hepatic mRNA levels for lipid-regulating molecules, cytokines/chemokines, and macrophage M1/M2 markers were determined by real-time RT-PCR. Hepatic expression of natural killer (NK) T cells was analyzed by flow cytometry. Body weight gain was significantly blunted and development of hepatic steatosis and inflammatory infiltration were remarkably prevented in mice fed the glycine-containing diet compared with controls. Indeed, hepatic induction levels of molecules related to lipogenesis were largely blunted in the glycine diet-fed mice. Elevations of hepatic mRNA levels for TNFα and chemokine (C-C motif) ligand 2 were also remarkably blunted in the glycine diet-fed mice. Furthermore, suppression of hepatic NK T cells was reversed in glycine diet-fed KK-Ay mice, and basal hepatic expression levels of NK T cell-derived cytokines, such as IL-4 and IL-13, were increased. Moreover, hepatic mRNA levels of arginase-1, a marker of macrophage M2 transformation, were significantly increased in glycine diet-fed mice. In addition, dietary glycine improved glucose tolerance and hyperinsulinemia in KK-Ay mice. These observations clearly indicate that glycine prevents maturity-onset obesity and metabolic steatohepatitis in genetically diabetic KK-Ay mice. The underlying mechanisms most likely include normalization of hepatic innate immune responses involving NK T cells and M2 transformation of Kupffer cells. It is proposed that glycine is a promising immunonutrient for prevention and treatment of metabolic syndrome-related nonalcoholic steatohepatitis.

Repetitive Manual Acupuncture Increases Markers of Innate Immunity in Mice Subjected to Restraint Stress

Objective To study the effects of repetitive manual acupuncture treatment on acute stress in mice and to explore its impact on the immune system. Methods Thirty-six mice were randomly allocated to one of four groups: control, acupuncture, stress and acupuncture+stress (n=9 each). Mice in the two acupuncture groups were given daily acupuncture treatment superficially (to skin depth) at CV6, CV12 and bilateral ST25, LR14, GB20, GB21, BL10, BL11, BL13, BL14, BL19, BL23 and BL25 for 7 days. On the eighth day mice in the stress and acupuncture+stress groups were exposed to acute stress for 2 h by confinement in a 50 mL centrifuge tube. Body temperature, blood glucose, the number and subpopulation ratios of leucocytes in the liver, spleen and thymus, natural killer (NK) cell percentage cytotoxicity and serum corticosterone and interferon gamma IFNγ were quantified. Results Mice exposed to stress (irrespective of acupuncture treatment) exhibited hypothermia and hyperglycaemia. However, the increase in glucose level was mitigated by repetitive acupuncture treatment (p<0.05). Percentage cytotoxicity and the level of corticosterone were significantly increased after stress but were unaffected by acupuncture. IFNγ levels did not differ between the groups. Hepatic innate immunity in the liver appeared to be stimulated by repetitive acupuncture treatment as proportions of extrathymic T cells, NK cells and NKT cells in the liver were greatest in the acupuncture+stress group and significantly increased relative to the control group. Conclusions Repetitive manual acupuncture mitigated stress-induced hyperglycaemia and enhanced markers of innate immunity in the liver within the range of normal homoeostasis. As long as acupuncture stimuli were appropriately applied, they did not appear to be stressful to the mice.