Exhaustion of antiviral CD8+ T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T‐cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct‐acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3‐NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8+ T‐cell response. However, these antiviral CD8+ T cells did not prevent persistent replication of DAA‐resistant HCV variants that emerged during treatment. Most vaccine‐induced CD8+ T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8+ T‐cell targeting‐conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8+ T‐cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8+ T‐cell expression of the inhibitory receptor programmed cell death 1 increased 25‐fold compared with those in circulation. Conclusion: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T‐cell response, primarily against nonconserved class I epitopes; exhaustion of liver‐infiltrating CD8+ T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants. (Hepatology 2016;63:1442‐1454)

Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T-cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)-free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct-acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T-cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T-cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape-prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection. Conclusions: The intrahepatic HCV-specific CD8+ T-cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T-cell responses and reduce the risk of a second persistent infection. (Hepatology 2014;60:1531–1540)

Obesity-related pathologies, such as nonalcoholic fatty liver disease, are linked to mitochondrial dysfunction and nitric oxide (NO) deficiency. Herein, we tested the hypothesis that a high-fat diet (HFD) modifies the liver mitochondrial proteome and alters proteins involved in NO metabolism, namely arginase 1 and endothelial NO synthase. Male C57BL/6 mice were fed a control or HFD and liver mitochondria were isolated for proteomics and reactive oxygen species measurements. Steatosis and hepatocyte ballooning were present in livers of HFD mice, with no pathology observed in the controls. HFD mice had increased serum glucose and decreased adiponectin. Mitochondrial reactive oxygen species was increased after 8 weeks in the HFD mice, but decreased at 16 weeks compared with the control, which was accompanied by increased uncoupling protein 2. Using proteomics, 22 proteins were altered as a consequence of the HFD. This cohort consists of oxidative phosphorylation, lipid metabolism, sulfur amino acid metabolism, and chaperone proteins. We observed a HFD-dependent increase in arginase 1 and decrease in activated endothelial NO synthase. Serum and liver nitrate + nitrite were decreased by HFD. In summary, these data demonstrate that a HFD causes steatosis, alters NO metabolism, and modifies the liver mitochondrial proteome; thus, NO may play an important role in the processes responsible for nonalcoholic fatty liver disease.

ABSTRACTThe RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8+T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8+T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8+T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.

Hepatitis C virus (HCV)-specific CD8+ T cells persist in the chronically infected liver but provide no apparent control of virus replication. Epitopes targeted by some of these T cells contain escape mutations while others remain intact. Studies in acutely infected humans suggest that circulating CD8+ T cells targeting the intact epitopes express more PD-1, a molecule associated with loss of effector function. Whether this is true in the persistently infected liver is unknown. CD8+ T cells targeting intact or mutated epitopes were visualized using tetramers in the liver of chronically infected chimpanzees. Unexpectedly, there was no obvious relationship between expression of PD-1 on the HCV-specific CD8+ T cells and the nature of the targeted epitope. More importantly all HCV-specific CD8+ T cells we examined displayed functional incapacity regardless of the nature of the targeted epitope and anergy could not be reversed by blockade of the PD-1/PD-L1 pathway. We conclude that decreased PD-1 expression previously observed on acute phase CD8+ T cells after escape of the targeted epitope is not necessarily maintained in the liver during chronic hepatitis C. Rather, our data suggest that mechanisms other than continuous TCR triggering might be responsible for PD-1 upregulation and exhaustion of CD8+ T cells, or that escaped epitopes are still able to induce TCR engagement without T cell activation resulting in exhaustion of viral specific CD8+ T cells.

S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

ABSTRACT Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of ∼5.5 log 10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Fatty liver disease associated with chronic alcohol consumption or obesity/type 2 diabetes has emerged as a serious public health problem. Steatosis, accumulation of triglyceride in hepatocytes, is now recognized as a critical “first-hit” in the pathogenesis of liver disease. It is proposed that steatosis “primes” the liver to progress to more severe liver pathologies when individuals are exposed to subsequent metabolic and/or environmental stressors or “second-hits.” Genetic risk factors can also influence the susceptibility to and severity of fatty liver disease. Furthermore, oxidative stress, disrupted nitric oxide (NO) signaling, and mitochondrial dysfunction are proposed to be key molecular events that accelerate or worsen steatosis and initiate progression to steatohepatitis and fibrosis. This review article will discuss the following topics regarding the pathobiology and molecular mechanisms responsible for fatty liver disease: (1) the “two-hit” or “multi-hit” hypothesis, (2) the role of mitochondrial bioenergetic defects and oxidant stress, (3) the interplay between NO and mitochondria in fatty liver disease, (4) genetic risk factors and oxidative stress-responsive genes, and (5) the feasibility of antioxidants for treatment.